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University Of Sindh

Jamshoro

PH.D. Thesis

Biosynthesis of Pectolytic Enzyme by Plant Pathogenic

Fungi Using Agricultural Waste as a Carbon Source

BY

Ghulam Sughra Mangrio

ENZYME AND FERMENTATION RESEARCH

LABORATORY, INSTITUTE OF BIOTECHNOLOGY

AND GENETIC ENGINEERING, UNIVERSITY OF

SINDH, JAMSHORO, PAKISTAN

2014

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University Of Sindh

Jamshoro

PH.D. Thesis

Biosynthesis of Pectolytic Enzyme by Plant Pathogenic

Fungi Using Agricultural Waste as a Carbon Source

BY


THESIS SUBMITTED TO THE UNIVERSITY OF SINDH IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF THE DEGREE OF DOCTOR OF

PHILOSOPHY IN BIOTECHNOLOGY

ENZYME AND FERMENTATION RESEARCH LABORATORY, INSTITUTE OF BIOTECHNOLOGY AND GENETIC

ENGINEERING, UNIVERSITY OF SINDH, JAMSHORO, PAKISTAN

2014

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TABLE OF CONTENTS

Certificate і

Dedication іі

Acknowledgement ііі

List of Abbreviations iv

Summary vi

List of Figures ix

List of Tables xxi

CHAPTER 1 INTRODUCTION PAGE NO.

Introduction 1

Aim and objectives 9

CHAPTER 2 REVIEW OF LITERATURE 10

Review of literature 10

INTRODUCTION OF FUNGI 26

Aspergillus niger 26

Aspergillus fumigatus 27

Mucor geophillus 28

Penicillium lilacinum 28

CHAPTER 3 MATERIALS AND METHODS 30

Chemicals 30

Microorganism 30

Inoculum 30

Optimization of inoculum size 30

Mineral medium 31

Fermentation medium 31

Sample harvesting 31

Biomass 31

A- Optimization of culture conditions 32

i- Effect of fermentation time period 32

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ii- Effect of carbon source 32

iii- Effect of nitrogen source 32

iv- Effect of pH 32

v- Effect of Temperature 32

vi- Characterization of crude pectinase 33

Vii- Effect of Time of incubation 33

Viii- Effect of Substrate concentration 33

ix- Effect of Enzyme concentration 33

x- Effect of different pH 33

Xi- Effect of pH stability 33

Xii- Effect of temperature 34

Xiii- Effect of temperature stability 34

Xiv- Effect of Metal ions/compounds 34

B- Preparation of Enzyme 34

i- Ammonium sulphate fractionation 35

ii- Dialysis 35

iii- Preparation of gel Sephadex G-100 35

iv- Gel filtration chromatography 35

v- Ion exchange chromatography 36

C- Characterization of purified pectinase 36

i- Effect of temperature on pectinase activity and stability 36

ii- Effect of pH on pectinasee activity and stability 36

iii- Kinetic determinations 37

iv- Effect of metal ions/compounds 37

D- Analytical methods 37

i- Assay of pectinase activity 37

ii- Protein estimation 38

iii- Determination of total carbohydrate 40

iv- Determination of reducing sugars 40

v- Molecular mass determination 42

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CHAPTER 4 RESULTS AND DISCUSSION 44

A- Growth conditions and enzyme production 45

i- Effect of size of inoculums 45

ii- Fermentation mode 48

iii- Effect of incubation period 49

iv- Effect of agro-industrial wastes as carbon sources 55

v- Effect of sugars as carbon sources 91

vi- Effect of nitrogen sources 106

vii- Selection of the organism 126

viii- Effect of pH on pectinase production 127

ix- Effect of temperature on pectinase production 129

B- Characterization of crude pectinase Enzyme 130

i- Effect of time of incubation on crude pectinase 130

ii- Effect of substrate concentration on crude pectinase 131

iii- Effect of enzyme volume on crude pectinase 132

iv- Effect of different buffers on crude pectinase 133

v- Effect of pH on crude pectinase 134

vi- Effect of pH stability on crude pectinase 136

vii- Effect of temperature on crude pectinase 137

viii- Effect of temperature of crude Pectinase 139

ix- Effect of metal ions/compounds on crude pectinase 140

x- Effect of different concentration of CaCl2 as activator 141

xi- Effect of thermostability with and without activator 142

C- Purification of enzyme 144

i- Removal of microbial cells and other solid matter 145

ii- Concentration by precipitation 145

iii- Ammonium sulphate fractionation 145

iv- Dialysis 146

D- Chromatography 146

i- Gel filtration chromatography 146

ii- Ion exchange chromatography 147

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iii- Homogeneity 149

iv- Molecular weight 149

E- Characterization of purified pectinase 151

i- Effect of substrate specificity 151

ii- Effect of substrate concentration on pectinase activity 152

iii- Effect of pH on pectinase activity produced by Aspergillus niger 153

iv- Effect of pH stability on pectinase activity produced by Aspergillus niger 155

v- Effect of temperature on pectinase activity produced by Aspergillus niger 156

vi- Effect of temperature stability on pectinase activity produced by

Aspergillus niger 158

vii- Effect of activators and inhibitors 159

CONCLUSION 163

Further Suggestions 164

REFERENCES 166

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LIST OF TABLES

4.1 Composition of working resolving and stacking gels. 43

5.1 Effect of size of inoculums on growth and pectinase production by A. fumigates. 46

5.2 Effect of size of inoculum on growth and pectinase production by A. niger.

46

5.3 Effect of size of inoculum on growth and pectinase production by a mixed culture of A. niger + A. fumigatus. 47

5.4 Effect of size of inoculum on growth and pectinase production by M . geophillus. 47

5.5 Effect of size of inoculum on growth and pectinase production by P. lilacinum.

47

5.6 Effect of fermentation mode for the growth and biosynthesis through different filamentous fungi. 48

5.7 A. fumigatus was grown on mineral medium without glucose at 30 ± 2º C pH was adjusted at 6.5. 56

5.8 A mixed culture of A. fumigatus + A. niger was grown on mineral without glucose at 30 ± 2 ºC pH was adjusted at 6.5. 56

5.9 A. niger was grown on mineral medium without glucose at 30± 2ºC pH was adjusted at 6.5. 57

5.10 M. geophillus was grown on mineral medium without glucose at 30 ± 2 ºC pH was adjusted at 6.5. 57

5. 11 P. lilacinum was grown on mineral medium without glucose at 30 ± 2 ºC pH was adjusted at 6.5. 58

5.12 A. fumigatus was grown on mineral medium supplemented with 1 % glucose at 30 ± 2 ºC pH was adjusted at 6.5. 60

5.13 A mixed culture of A. fumigatus + A. niger was grown on mineral medium supplemented with 1 % glucose at 30 ± 2 ºC pH was adjusted at 6.5. 60

5.14 A. niger was grown on mineral medium supplemented with 1 % glucose at 30 ± 2 ºC pH was adjusted at 6.5. 61

5.15 M. geophillus was grown on mineral medium supplemented with 1 % glucose at 30 ± 2 ºC pH was adjusted at 6.5. 61

5.16 P. lilicinum was grown on mineral medium supplemented with 1 % glucose at 30 ± 2 ºC pH was adjusted at 6.5. 62

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5.17 Effect on growth and pectinase production by different filamentous fungi when grown on mineral medium supplemented with 1% glucose and without glucose at 30± 2 ºC and the initial pH was adjusted at 6.5. 63

5.18 A. fumigatus was grown on mineral medium supplemented with 2.5% date syrup at 30 ± 2ºC and pH was adjusted to 6.5. 64

5.19: A mixed culture of A. niger + A. fumigatus was grown on mineral medium supplemented with 2.5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5. 65

5.20 A. niger was grown on mineral medium supplemented with 2.5% date syrup at 30 ± 2ºC and pH was adjusted to 6.5. 65

5.21 M. geophillus was grown on mineral medium supplemented with 2.5% date syrup at 30 ± 2ºC and pH was adjusted to 6.5. 66

5.22 P. lilacinum was grown on mineral medium supplemented with 2.5% date syrup at 30 ± 2ºC and pH was adjusted to 6.5. 66

5.23 A. fumigatus was grown on mineral medium supplemented with 5% date syrup at 30 ± 2ºC and pH was adjusted to 6.5. 67

5.24 A mixed culture of A. niger + A. fumigatus was grown on mineral medium supplemented with 2.5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5. 68

5.25 A. niger was grown on mineral medium supplemented with 5% date syrup at 30 ± 2ºC and pH was adjusted to 6.5. 68

5.26 M. geophillus was grown on mineral medium supplemented with 5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5. 69

5.27 P. lilacinum was grown on mineral medium supplemented with 5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5. 69

5.28 Effect on growth and pectinase production by different fungi when grown on mineral medium supplemented with 2.5 and 5% date Syrup at 30 + 2 ºC and pH was adjusted 6.5. 70

5.29 A. fumigatus was grown on mineral medium supplemented with 2.5 % molasses at 30 + 2 ºC and pH was adjusted at 6.5. 71

5.30 A mixed culture of A. niger + A. fumigatus was grown on mineral medium supplemented with 2.5 % molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 72

5.31 A. niger + A. fumigatus was grown on mineral medium supplemented with 2.5 % molasses when incubated at3 0± 2 ºC and pH was adjusted at 6.5. 72

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5.32 M. geophillus was grown on mineral medium supplemented with 2.5 % molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 73

5.33 P. lilacinum was grown on mineral medium supplemented with 2.5 % molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 73

5.34 A. fumigatus was grown on mineral medium supplemented with 5 % molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 74

5.35 A mixed culture of A. fumigatus + A. niger was grown on mineral medium supplemented with 5 % molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 75

5.36 A. niger was grown on mineral medium supplemented with 5 % molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 75

5.37 M. geophillus was grown on mineral medium supplemented with 5 % molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 76

5.38 P. lilacinum was grown on mineral medium supplemented with 2.5 % molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 76

5.39 Effect on growth and pectinase production by different fungi when grown on mineral medium supplemented with 2.5 and 5% molasses at 30 + 2 ºC and pH was adjusted 6.5. 77

5.40 A. fumigatus was grown on mineral medium supplemented with 2.5 % citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 78

5.41 A mixed culture of A. niger + A. fumigatus was grown on mineral medium supplemented with 2.5 % citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 78

5.42 A. niger was grown on mineral medium supplemented with 2.5 % citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 79

5.43 M.geophilus was grown on mineral medium supplemented with 2.5% citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 79

5.44 P. lilacinum was grown on mineral medium supplemented with 2. 5 % citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 80

5.45 A. fumigatus was grown on mineral medium supplemented with 5 % citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 81

5.46 A mixed culture of A. niger +A. fumigatus was grown on mineral medium supplemented with 5 % citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 81

5.47 A. niger was grown on mineral medium supplemented with 5 %citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 82

5.48 M. geophilus was grown on mineral medium supplemented with 5 % citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 82

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5.49 P. lilacinum was grown on mineral medium supplemented with 5 % citrus pectin at 30 ± 2 ºC and pH was adjusted at 6.5. 83

5.50 Effect on growth and pectinase production by different fungi when grown on mineral medium supplemented with 2.5 and 5% citrus pectin at 30 + 2 ºC and pH was adjusted 6.5. 84

5.51 A. fumigatus was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 85

5.52 A mixed culture of A. fumigatus + A. niger was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 85

5.53 A. niger was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 86

5.54 M. geophillus was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 86

5.55 P. lilacinum was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 87

5.56 A. fumigatus was grown on mineral medium supplemented with 5% CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 88

5.57 A mixed culture of A. fumigatus + A. niger was grown on mineral medium supplemented with 5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 88

5.58 A. niger was grown on mineral medium supplemented with 5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 89

5.59 M. geophillus was grown on mineral medium supplemented with 5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 89

5.60 P. lilacinum was grown on mineral medium supplemented with 5 % CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 90

5.61 Effect on growth and pectinase production by different fungi when grown on mineral medium supplemented with 2.5 and 5% CCP (commercial citrus pectin) at 30 ± 2 ºC and pH was adjusted at 6.5. 91

5.62 A. niger was grown on mineral medium supplemented with 2.5% fructose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 93

5.63 A. niger was grown on mineral medium supplemented with 5% fructose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 93

5.64 P. lilacinum was grown on mineral medium supplemented with 2.5% fructose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 94

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5.65 P. lilacinum was grown on mineral medium supplemented with 5% fructose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 94

5.66 A. niger was grown on mineral medium supplemented with 2.5% maltose and 5% molasses as carbon source at 30 ±2 ºC and pH was adjusted at 6.5. 95

5.67 A. niger was grown on mineral medium supplemented with 5% maltose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 96

5.68 P. lilacinum was grown on mineral medium supplemented with 2.5% maltose and 5% molasses at 30 ± 2 ºC and pH was adjusted at 6.5. 96

5.69 P. lilacinum was grown on mineral medium supplemented with 5% maltose and 5% molasses as carbon source at 30±2 ºC and pH was adjusted at 6.5. 97

5.70 A .niger was grown on mineral medium supplemented with 2.5% sucrose and 5% molasses as carbon source at 30 ±2 ºC and pH was adjusted at 6.5. 98

5.71 A. niger was grown on mineral medium supplemented with 5% sucrose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 98

5.72 P. lilacinum was grown on mineral medium supplemented with 2.5% sucrose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 99

5.73 P. lilacinum grown on mineral medium supplemented with 5% sucrose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 99

5.74 A. niger was grown on mineral medium supplemented with 2.5%galactose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 100

5.75 A niger was grown on mineral medium supplemented with 5% galactose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 100

5.76 P. lilacinum was grown on mineral medium supplemented with 2.5% galactose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 101

5.77 P. lilacinum was grown on mineral medium supplemented with 5%galactose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 101

5.78 A. niger was grown on mineral medium supplemented with 2.5% starch and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 103

5.79 A. niger was grown on mineral medium supplemented with 5% starch and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 103

5.80 P. lilacinum was grown on mineral medium supplemented with 2.5% starch and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 104

5.81 P. lilacinum was grown on mineral medium supplemented with 5 % starch, 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 104

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5.82 Effect on growth and pectinase production by A. niger grown on mineral medium supplemented with 5% molasses and sugars (2.5% and 5%) as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 105

5.83 Effect on growth and pectinase production by P.lilacinum grown on mineral medium supplemented with 5% molasses and sugars (2.5% and 5%) as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. 106

5.84 A niger was grown on mineral medium supplemented with 5% sucrose and 5% molasses as carbon source and 0.2% corn steep liquor at 30 ± 2 ºC and pH was adjusted at 6.5. 107

5.85 A. niger was grown on mineral medium supplemented with 5% sucrose and 5% molasses as carbon source and 0.4% corn steep liquor at 30 ± 2 ºC and pH was adjusted at 6.5 108

5.86 P. lilacinum was grown on mineral medium supplemented with 5% sucrose and 5% molasses as carbon source and 0.2% corn steep liquor at 30 ± 2 ºC and pH was adjusted at 6.5. 108

5.87 P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.4% corn steep liquor at 30 ± 2 ºC and pH was adjusted at 6.5. 109

5.88 A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses as carbon source and 0.2% urea at 30 ± 2 ºC and pH was adjusted at 6.5. 110

5.89 A. niger was grown on mineral medium supplemented with 5%sucrose, 5% molasses as carbon source and 0.4% urea at 30 ± 2 ºC and pH was adjusted at 6.5. 110

5.90 P. lilacinum was grown on mineral medium supplemented with 5%sucrose, 5% molasses as carbon source and 0.2% urea at 30 ± 2 ºC and pH was adjusted at 6.5. 111

5.91 P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses as carbon source and 0.4% urea at 30 ± 2 ºC and pH was adjusted at 6.5. 111

5.92 A. nigar was grown on mineral medium supplemented with 5% sucrose, 5%

molasses and 0.2% NaNO3 at 30 ± 2 ºC and pH was adjusted at 6.5. 112

5.93 A. niger was grown on mineral medium supplemented with 5% sucrose, 5%


5.94 P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5%





molasses and 0.2% KNO3 at 30 ± 2 ºC and pH was adjusted at 6.5. 114

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molasses and 0.4% KNO3 at 30 ± 2 ºC and pH was adjusted at 6.5. 114


molasses and 0.2% KNO3 at 30 ±2 ºC and pH was adjusted at 6.5 115

5.99 P. lilacinum was grown on mineral medium supplemented with 5% Sucrose, 5%

molasses and 0.4% KNO3 at 30 ± 2 ºC and pH was adjusted at 6.5 115


molasses and 0.2% NH4NO3 at 30 ± 2 ºC and pH was adjusted at 6.5 116

5.101 A. nigar was grown on mineral medium supplemented with 5% sucrose, 5%

molasses and 0.4% NH4NO3 at 30 ± 2 ºC and pH was adjusted at 6.5 116

5.102 P. lilacinum was grown on mineral medium supplemented with 5% Sucrose, 5%

Molasses and 0.2% NH4NO3 at 30± 2 ºC and pH was adjusted at 6.5. 117

5.103 P.lilacinam was grown on mineral medium supplemented with 5% sucrose, 5%

molasses and 0.4% NH4NO3 at 30 ± 2 ºC and pH was adjusted at 6.5. 117

5.104 A. niger was grown on mineral medium supplemented with 5%sucrose, 5% molasses and 0.2% peptone at 30 ± 2 ºC and pH was adjusted at 6.5. 118

5.105 A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.4% Peptone at 30 ± 2 ºC and pH was adjusted at 6.5. 118

5.106 P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% peptone at 30 ± 2 ºC and pH was adjusted at 6.5. 119

5.107 P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.4% peptone at 30 ±2 ºC and pH was adjusted at 6.5. 119

5.108 A. niger was grown on mineral medium supplemented with 5%sucrose, 5%

molasses and 0.2% (NH4)2SO4 at 30 ± 2ºC and pH was adjusted at 6.5. 121

5.109 A.niger was grown on mineral medium supplemented with 5% sucrose, 5%

molasses and 0.4% (NH4)2SO4 at 30 ± 2 ºC and pH was adjusted at 6.5. 121


molasses and 0.2% (NH4)2SO4 at 30 ± 2 ºC and pH was adjusted at 6.5. 122


molasses and 0.4% (NH4)2SO4 at 30 ± 2 ºC and pH was adjusted at 6.5 122

5.112 Effect of nitrogen sources on growth and pectinase production by A. niger 124

5.113 Effect of nitrogen sources on growth and pectinase production by P. lilacinum 125

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5.114 Effect of pH on Biosynthesis of Pectinase by A. niger grown on mineral medium

supplemented with 5% sucrose, 5% molasses and 0.4% (NH4)2SO4 at 30 ± 2 ºC for 72 hours 128

5.115 Effect of Temperature on Biosynthesis of Pectinase by A. niger grown on mineral

medium containing 5% molasses 5% sucrose and 0.4% (NH4)2SO4 while pH was adjusted 6.00 for 72 hours 130

5.116 Purification steps of Pectinase produced by Aspergillus niger 148

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LIST OF FIGURES

1.1 Structure (main chain) of low and high methylated pectic substances and site of action of enzymes involved in their degradation 05

4.1 Standard graph for Galacturonic acid 38

4.2 Standard Graph for total protein 39

4.3 Standard Graph for total carbohydrate 40

4.4 Standard curve for reducing sugar 41

5.1 A. fumigatus, A. niger , A. niger+ A. fumigatus , M.geophilus, P. lilacinum were grown on mineral medium containing 1% glucose as carbon source at 30 ± 2ºC pH was adjusted at 6.5 51

5.2 A. fumigatus, A. niger , A. niger+ A. fumigatus, M.geophilus, P. lilacinum were grown on mineral medium containing 2.5 % date sugar as carbon source at 30 ± 2 ºC pH was adjusted at 6.5 51

5.3 A. fumigatus, A. niger , A. niger+ A. fumigatus , M.geophilus, P. lilacinum were grown on mineral medium containing 5 % date sugar as carbon source at 30 ± 2 ºC pH was adjusted at 6.5 52

5.4 A. fumigatus, A. niger, A. niger+ A. fumigatus, M.geophilus, P. lilacinum were grown on mineral medium containing 2.5 % Molasses as carbon source at 30 ± 2 ºC pH was adjusted at 6.5. 52

5.5 A. fumigatus, A. niger , A. niger+ A. fumigatus ,M.geophilus, P. lilacinum were grown on mineral medium containing 5 % Molasses as carbon source at 30 ± 2 ºC pH was adjusted at 6.5. 53

5.6 A. fumigatus, A. niger, A. niger+ A. fumigatus , M.geophilus, P. lilacinum were grown on mineral medium containing 2.5 % crude citrus pectin as carbon source at 30 ± 2ºC pH was adjusted at 6.5. 53

5.7 A. fumigatus, A. niger, A. niger+ A. fumigatus , M.geophilus, P. lilacinum were grown on mineral medium containing 5 % Crude citrus pectin as carbon source at 30 ± 2 ºC pH was adjusted at 6.5. 54

5.8 A. fumigatus, A. niger , A. niger + A. fumigatus , M.geophilus, P. lilacinum were grown on mineral medium containing 5 % Crude citrus pectin as carbon source at 30 ± 2 ºC pH was adjusted at 6.5 54

5.9 A. fumigatus, A. niger, A. niger+ A. fumigatus , M.geophilus, P. lilacinum were grown on mineral medium containing 5 % crude citrus pectin as carbon source at 30 ± 2 ºC pH was adjusted at 6.5. 55

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5.10 Comparison of pectinase production by different organisms grown on 5 % Molasses as a carbon source 92

5.11 Comparison of Pectinase production produced by Aspergillus niger and Penicillium lilacinum 126

5.12 Effect of time of incubation on crude Pectinase 131

5.13 Effect of substrate concentration on crude Pectinase 132

5.14 Effect of enzyme concentration on crude Pectinase 133

5.15 Effect of different buffers on crude Pectinase 133

5.16 Effect of pH on crude Pectinase 135

5.17 Effect of pH stability on crude Pectinase 137

5.18 Effect of temperature on crude Pectinase 138

5.19 Effect of temperature stability on crude pectinase 139

5.20 Effect of metal ions/ compounds on pectinase activity 141

5.21 Effect of different concentrations of CaCl2 142

5.22 Effect of themostability at 60°C on different time periods with and without activator

CaCl2 (15mM) on pectinase Activity produced by Aspergillus niger 143

5.23 Effect of themostability at 70 °C on different time periods with and without activator

CaCl2 (15mM) on pectinase Activity produced by Aspergillus niger 143

5.24 Gel Chromatography 147

5.25 Purifiction of Pectinase (F-3) on ion exchange chromatography 148

5.26 SS-PAGE (10% Polyacrylamide) of the purified enzymes. Lane 1, low Mw Marker; Lane 2, Fraction 1; Lane 3, Fraction 2; Lane 4,Fraction 3; Lane 5, Fraction 4; Lane 6, Crude enzyme 150

5.27 SDS-PAGE (10% Polyacrylamide) of the purified enzymes. Lane 1, low Mw Marker; Lane 2, Fraction 3a; Lane 3, Fraction 3b; Lane 4, Crude enzyme 150

5.28 Effect of substrate specificity on pectinase produced by Aspergillus niger 152

5.29 Effect of substrate concentration on Pectinase activity produced by Aspergillus niger 153

5.30 Effect of pH on pectinase activity produced by Aspergillus niger 155

5.31 Effect of pH stability on pectinase activity produced by Aspergillus niger 156

5.32 Effect of temperature on pectinase activity produced by Aspergillus niger 157

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5.33 Effect of thermostability on pectinase activity produced by Aspergillus niger 159

5.34 Effect of activators & inhibitors (F-1) 161


5.36 Effect of activators & inhibitors (F-3a) 162

5.37 Effect of activators & inhibitors (F-3b) 162


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CERTIFICATE

This is to certify that research work entitled “Biosynthesis of pectolytic enzyme

by plant pathogenic fungi using agricultural waste as a carbon source” has

been carried out by Miss Ghulam Sughra Mangrio under my supervision in the

Enzyme and Fermentation Research Laboratory, Institute of Biotechnology and

Genetic Engineering, University of Sindh, Jamshoro, Pakistan. The work

reported in this thesis is genuine and distinct. This dissertation is worthy of

presentation to the University of Sindh for the award of degree of Doctor of

Philosophy in Biotechnology.

Signature of the supervisor Prof. Dr. Muhammad Umar Dhot Professor and Ex Director and Founder

Institute of Biotechnology and Genetic Engineering,

University of Sindh, Jamshoro

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DEDICATION

I Dedicate

This Little Effort To

My Father

Late Prof. Haji Khan Mangrio

The First Inspiration Towards Life

ACKNOWLEDGEMENTS

All praise for the, “Allah SWT” Who is the only supreme Authority, my

countless thanks to Him for accrediting me to accomplish this important task in

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my life. All my inspiration and greatest respect to the Prophet of Islam (peace be

upon him) who is a greatest and matchless teacher for human kind. In view of

his saying: “He who does not thank to people is not thankful to Allah”.

I am highly grateful in paying deepest thanks to my highly respected

teacher and supervisor Prof. Dr. Muhammed Umar Dahot Ex- Director and

Founder of Institute of Biotechnology and Genetic Engineering, University of

Sindh, Jamshoro for his kindness, valuable guidance, encouragement and

cooperation. His enthusiastic inspiration and affection enabled me to attain the

objectives without any difficulty.

I feel pleasure to express my sincere gratitude to my colleague Dr. Altaf

Ahmed Simair for his valuable suggestions during research and in writing the

dissertation and most gratefully I want to express my deep appreciation to all the

staff members of the IBGE, University of Sindh for their nice behavior and co-

operation throughout the work and especially I want to say thanks to Mr.

Gulbahar who remained with us even on holidays.

I wish to express my thanks to Mr. Gain Chand Lab. Assistant Department

of Biotechnology, Sindh Agriculture University, Tandojam for his sincere

cooperation and moral support during compilation of my work.

Also, thanks for the generosity of HEC Islamabad and IBGE, University of

Sindh for providing funds for completing this project.


LIST OF ABBREVIATIONS

E Activation energy (kJmol-1)

ATCC American Type of Culture Collection

22

BSA Bovine Serum Albumin

CCP Commercial citrus pectin

conc. Concentration

CSL Corn Steep Liquor

Da Dalton

DNS Dinitrosalicylic acid

endo-PGLLs EndoPolygalacturonate lyase

exo-PGLs Exo Polygalacturonate lyase

g Gram

h Hour

kDa Kilo-Dalton

k0 Frequency factor (min-1)

Km Michaelis-Menten constant (equilibrium constant)

L Litre

M Molar

M.O Microorganism

M.W Molecular weight

min minutes

Ml Millilitre

mm Mili meter

mM Millimolar

OD Optical Density

PG Polygalacturonase

PGA Polygalacturonic Acid

PE Pectin esterases

PL Pectin Lyase

PG Polygalacturonase

PMG Polymethylgalacturonases

Endo-PMG Endopolymethylgalacturonases

Exo-PMG Exopolymethylgalacturonases

Exo-PG Exopolygalacturonase

PME Pectin Methyl Esterase

PME Pectin Methyl Esterase

PMG Polymethylgalacturonase

23

PMGL Polymethylegalacturonate lyase

Endo-PMGL EndoPolymethylegalacturonate lyase

Exo-PMGL ExoPolymethylegalacturonate lyase

PGL Polygalacturonate lyase

SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel

Electrophoresis

(sp.) Species

nm Nanometer

No. Number

rpm Revolutions per minute

SMF Submerged fermentation

SSF Solid state fermentation

t1/2 Half-life time of enzyme (min)

Vm Maximum Forward Velocity of the

U Unit

μm Micrometer

v/v Volume per volume

w/v Weight per volume

w/w weight per weight

μg micrograms

μl microliters

Endo-PGL poly (1,4-α-D-galacturonide) lyase,

Exo-PGL poly (1,4-α-D-galacturonide) exolyase

24

SUMMARY

Present study was carried out in the Enzyme and Fermentation Research

laboratory, Institute of Biotechnology and Genetic Engineering, University of

Sindh, Jamshoro. In this study Agricultural waste was used as a carbon source.

New enzymes have been focused by researchers due to their commercial

applications with desirable biochemical and physico-chemical characteristics and

a low cost of production. Research on the selection of suitable substrates for

fermentation has mainly been centered on agro-industrial residues due to their

potential advantages for filamentous fungi. The utilization of these agro-

industrial wastes, on the one hand, provides alternative substrates and, on the

other, helps in solving pollution problems, which otherwise may cause big

problem for their disposal.

In this study various concentration (2.5% and 5%) of natural sugars were

used and 5% molasses was investigated best substrate/carbon source, the best

pectinase producers were selected as A. niger and P. lilacinum. Different carbon

sources (2.5 and 5% fructose, maltose, sucrose galactose and starch) were

incorporated with 5% (v/v) molasses. The experiments were conducted in

triplicates and the results presented are the mean values. Synthetic sugars along

with 5% molasses were used as a carbon source for the growth and production of

pectinase by submerged fermentation process. Different synthetic and natural

nitrogen sources were also used and optimization of temperature and pH was

carried out to maintain a maximum production of pectinase enzyme.

After screening best substrate (carbon source) which was 5% molasses,

fermentation medium was supplemented with 5% molasses and various sugars 2.5

and 5% (fructose, maltose, sucrose, galactose and starch) were tested to find out

optimum carbon sources. The addition of 5.0% sucrose as carbon source induced

the pectinase production while low production of pectinase was recorded with

25

carbon sources other than sucrose. In this study, A. niger and P. lilacinum exhibit

high pectinase production when grown on media supplemented with 5%

molasses. Aspergillus niger is most efficient among filamentous fungi and results

reveal that enzyme to be produced is highly depended upon substrate and

microorganism. An overview of results obtained show that 5% sucrose, 5%

molasses and 0.4% (NH4)2SO4 the best carbon and nitrogen sources for the

production of pectinase by A. niger. The maximum production of pectinase (26.87

U/ml) was observed at pH 6.0 after 72 h incubation. The optimum temperature for

the maximum production of pectinase was achieved at 35 ºC when maximum

production of pectinase was obtained as 28.25 U/ml. The ammonium sulphate

was selected as a best nitrogen source and Aspergillus niger was selected best

organism, which has produced higher amount of pectinase when cultivated in

comparison to Penicillium lilacinum on same optimal conditions.

The crude Pectinase enzyme was characterized on the basis of various

parameters such as incubation time, substrate concentration, enzyme volume,

pH, pH stability, temperature, temperature stability, and effect of various metal

ions or compounds. The Pectinase activity was noted maximum at 15 minutes of

incubation time, 1.5% citrus pectin and 1ml enzyme volume. The highest enzyme

activity was found at pH 5, whereas pectinase exhibited stability in the range of

pH 4.0 to 7.0. The optimum Pectinase activity was noted at 40˚C temperature

while crude pectinase was 100% stable up to 40 °C but activity declined and

retains more than 30% activity up to 80 °C. CaCl2 (1.5 mM) stimulated the

Pectinase activity as compare to other metal ions /compounds.

Pectinase enzyme was purified with ammonium sulphate precipitation and

dialyzed sample was finally applied on gel filtration chromatography (Sephadex G-

100) and Ion Exchange DEAE A-50. The enzyme was purified 2.5 fold by gel

chromatography on Sephadex G-100 and 2.19 fold by Ion Exchange DEAE A-50.

Four fractions were obtained, Fraction 1, 2, 4 showed single bands while Fraction -3

26

showed multiple bands on SDS Page electrophoresis. Fraction -3 was pooled,

dialyzed and separated on Sephdex A-50 and two fractions 3a and 3b showed single

band. The molecular weights of the purified fractions were detected in the range of

found to be 33000 ± 2000 and 38000± 2000 Daltons. The purified enzyme was

specifically most active with pure pectin, while crude pectin, Lemon pectin and

orange peel given lower activity as compared to (control) i-e pure pectin. The

optimum pH and temperature for pectinase activity for different fractions were

between pH 5.0 and 6.0 and 40°- 50 °C, respectively. The enzyme was stable over the

pH range 3.0-8.0. More than 30 % activity was retained when purified pectinase was

incubated with pH 8.0. The thermostability of each fraction was determined and it

was observed that the pectinase activity in all fractions is heat stable up to

temperatures ranging from 50 to 60 °C and activity decreased as the incubation

temperature was increased above 60 °C. The temperature profile showed that

purified pectinase retained maximum activity up to 60 °C and retain activity more

than 40% when incubated at 90°C for 10 minutes. The pectinase activity of different

fractions (FI, F2, F3a, F3b and, F4) was increased with different metal ions. The

Pectinase activity was stimulated in the presence of CaCl2 in all fractions in the effect

of 110-130. ZnSO4, MnSO4 and Mg SO4 shown higher activity in fractions ( F3a, F3b

and F4), while in fractions F1 and F2 ZnSO4 and MnSO4 shown slight inhibition

effect on pectinase activity, which indicates that the pectinase belongs to metalo-

enzymes.

It is concluded that Aspergillus niger is capable to produce pH stable and

thermostable pectinase for industrial purposes. Pectinase from Aspergillus niger

could convert orange peel pectin successfully, and thus the enzyme could not

only act as an agent for bioconversion but also could replace the use of highly

expensive commercial pectin in food industry.

27

CHAPTER NO.1

INTRODUCTION

Human civilization has been using a variety of enzymes in food and in

other processes since long time. Historically, the enzyme industry was developed

through the use of plant and animal materials and industrial use of enzyme is

now an integral part of a wide variety of commercial processes. The applications

of enzymes ranging from small to complex or large scale in the manufacturing of

chemicals, processed foods and many supermarket products. Due to the increase

in the cost of energy and contaminated water, enzyme technology play

important role in science and technology and their use will be enhanced in

future. The development of stirring tank fermented and the genetic manipulation

of microbial cultures used for overproduction of the desired product. Nowadays,

microorganisms are the major sources of enzymes both in volume and variety.

Genetic engineering technology has helped in accelerating the development and

production of enzyme in both bacteria and fungi. Bacteria have the advantage of

fast growth and short fermentation cycle while fungi are preferred to produce

large quantities of desirable enzymes (Gupta and Mukerji, 2001).

It is most likely that the greatest variety of traditional biotechnological

processes are found in the area of food and nutrition, particularly in the

manufacture of foodstuffs and beverages. These processes can be improved, their

efficiency and yield through the selection of more productive microbial strain,

the control of culture condition, and through the adaptation of fermentation

products to the evaluation of food habits and to the consumers’ changing tastes

(Vibha and Neelam 2010, Gurung et al., 2013).

28

According to Global Industry Analysts, Inc (2011) and Norus (2006) in the

field of Biotechnology enzymes are a very well recognized products and the

production of food and brewing enzymes in the world market is estimated to

spread about $ 1.3 billion by 2015 with the highest sales occurred in the milk and

dairy market, BBC Research (2011).

Pectinases production enjoys about 10% of total enzyme production.

Many microorganisms like yeast, protozoan, bacteria, fungi, insects, nematodes

and plants produce Pectinolytic enzymes, but microbial pectinases are more

significant due to their involvement in the Phytopathology plant– microbe

association and the decay of deceased plant materials as reported by Pedrolli et

al., (2009). Enzymes of Microbial origin are good biocatalysts for different

industrial applications (Hasan et al., 2006 ).

Among the various types of fermentation, which aim of providing a

higher nutritional and economic value to agricultural products and by-products

of the food industries, fermentations in solid medium comprise a number of

biotechnological processes and these are of great interest to developing countries

to adopt a fermentation process for the production of daily used goods. Today,

pectinase enzymes are one of the forthcoming enzymes of commercial zone and

the pectinases of microbial origin account for 25% of the international food

enzymes sales (Jayani et al., 2005). This enzyme has an excessive impact with

extraordinary prospective to be offered to food industry specially to process

fruits and vegetables (Whitaker, 1990). The pectinases are very powerful and

continuously upcoming for the commercial sector, especially for food and juice

industry (Kashyap et al., 2001) these enzymes play a major role in the pulp and

paper industry (Beg et al., 2001 and Vikari et al., 2001).

Enzymes are capable to catalyze and degrade all synthetic products of

living creatures. Enzymes first time reported in the nineteenth century. Since

then their use has highly increased in various industries and laboratories. In the

recent years their use is rapidly increasing in the field of biotechnology,

29

especially in the fields of protein and genetic engineering. There are many

exciting research studies involving enzymes with the development of new

commercial and industrially important processes. Enzymes are widely used in

various emerging industries because of their high catalytic power, a specific

mode of action, stereo specificity, and eco-friendly and capable to reduce energy

requirements etc (Vikari et al., 2001)

The biodiversity of microbes is very significant for many reasons,

starting from aesthetic concern to its usefulness, especially in biotechnology.

The excessive emergent sectors are enzymes for the production of the food and

fuel. The potential of the white biotechnology has an environmental advantage and this

economically beneficial technology is beyond all the questions. Enzymes are biocatalysts

with high selectivity and are utilized in the food industry for centuries and play a

significant role in various other industries such as a detergents, textile, pharmaceuticals,

paper and pulp (Vibha and Neelam, 2010).

Huge amounts of industrial waste residues are produced worldwide by

processing raw agricultural ingredients for foodstuffs. These, in turn, carry out

an excessive BOD load on the atmosphere when discarded. These industrial

wastes produced from the processing of sugar cane, orange, coffee and

rice, which provide proper feed stocks for bioconversion into chemicals as well

as enzymes under fermentation techniques. The other waste produces from

agriculture arises from citrus fruits belong to a significant group of fruit crops

grown all over the globe (Giese et al., 2008).

Fruit processing industries produce a large amount of waste material in

the form of peel, pulp, seeds, etc. Some fresh orange peel is used in shredded

form in the preparation of orange-marmalade. Dried citrus peel is rich in

carbohydrates, proteins and pectin with small amount of fat (Vibha and Neelam,

2011).

Several micro bial conversions have been suggesting the use of

processing food waste to produce valued products like biogas, citric acid,

30

ethanol, chemicals, different enzymes, volatile flavoring agents, fatty acids and

microbial biomass. Citrus skin comprises a considerable quantity of pectin and

that may be utilized as an inducer for the production of pectinase enzyme by

various microorganisms. Exploitation of microorganisms for the production of

enzymes is beneficial and advantageous as climate and seasonal influences

cannot affect them, may also be subjected to genetic and ecological managements

to improve production. To reduce the production cost at the industrial level,

extremely productive strains of micro-organisms are required. Several microbes

are used for enzyme biosynthesis. Pectinolytic enzymes are synthesized by

a huge number of microbes including bacteria and fungi for instance

Bacillus Spp., Clostridium Spp., Pseudomonas Spp., Aspergillus Spp., Monilla laxa,

Fusarium Spp., Verticillium Spp., Penicillium Spp., Sclerotinia libertiana,

Coniothyrium diplodiella, Thermomyces lanuginosus, Polyporus squamosus,. etc

(Vibha and Neelam, 2010).

Pectinase enzymes mostly exist in various living beings like plants,

bacteria, fungi, yeast, insects, nematodes and protozoa. These are negatively

charged acidic glycosidic macromolecules having bigger molecular mass. Pectic

substances occur in the plants as the main components of the middle lamella in

the shape of calcium pectate and magnesium pectate. Pectic material comprises

pectins, pectinic acids, protopectins, and pectic acids. The foremost chain of

pectin is partially methyl esterified 1, 4- D- galacturonan. Demethylated pectin is

known as pectic acid (pectate) or polygalacturonic acid (Alkorta et al., 1998).

31

Figure:-1 Structure (main chain) of low and high methylated pectic substances

and site of action of enzymes involved in their degradation (Sieiro et al., 2012).

The pectinase enzymes attack in several ways on the pectin and these

are widely used in processing of fruit juices, extraction of vegetable oil, processing

of alcoholic drinks and a range of uses in food industries. The commercial

pectinolytic enzyme usually might usually be stimulated at pH 5.0 and 45 to 55

ºC. Pectinase producing microorganisms are broadly dispersed in soil, rotten

fruits, vegetables, decomposed leaves, wood and can also be found in samples

of water taken from decomposing coconut peelings, especially in coastal

areas. Duodenal flora of humans also comprise pectinolytic microbes,

predominantly bacteria, since pectin the dietary fiber have been the substrate

for them (Vibha and Neelam, 2010). Traditionally, pectin of citrus peel and apple

pomace is the commercial source because these sources contain high pectin

content with excellent color properties, citrus peel has often been and perfect

source for pectin manufacturers. Sugar beet and sunflower are most recent

sources for the pectin, the quantity of pectin from various sources differs

significantly like apple pomace 10- 15%, and citrus peel 25-35 %, sugar beet 10-20

% and sunflower 15- 25% (Vibha and Neelam, 2010).

32

Classification:

The classification of pectinase enzymes is established in their mode of

action on the galacturonan chain of the molecules of pectic substances. Primarily,

there are three types of pectinase enzymes (Sakai 1992; Palomaki and Saarilahti

1997).

1) De-esterifying enzymes (pectin esterase): These enzymes produce pectic acid

and methanol by catalyzing the hydrolysis of methyl

2) Depolymerizing enzymes: Depolymerizing enzymes comprise hydrolases

and lyases. Lyases enzymes are called transeliminases, those torn apart the

glycosidic linkages of each pectate (polygalacturonate) or pectin (polymethyl-

galacturonate).These are also sectioned into endo- if its configuration of action

is haphazard or exo if its configuration of action is at the terminal end (Rexova-

Bencova and Markovic 1976; Fogarty and Kelly 1983; Whitaker, 1990; Sakai

1992 and Jayani et al., 2005 ).

3) Protopectinases: This enzyme accelerates the change of protopectin into

soluble pectin or pectinic acids with the consequential parting of plant cells from

one another. Pectinase enzymes are also divided into two types, acidic pectinase

and alkaline pectinase (Kashyap et al ., 2001 )

Acidic pectinase:

The pectinase enzymes used in fruit juices and wine making are known

as acidic pectinases. These pectinases are commonly isolated from fungal

sources. To produce glittering clear juices with the help of enzymes to increase

the yield of juice during pressing, draining and also to eliminate suspending

particles to make particles to make the juice sparkling and clear (Kashyap et al.,

2001) and it also reduces filtration time up to 50% (Blanco et al., 1999). According

to Kashyap et al., (2001) this principle is applied in the following processes:

Preparation of purees and nectars.

Pear juice processing and preparation of purees and nectars.

Strawberry, blackberry, raspberry, apple, orange juice and wine clarification.

33

In cloudy juices more amount of polygalacturonases is complemented to fruit

juices to resolve the cloud some examples are –

Cloud stabilization of orange juice.

Lemon juice clarification, recovery of citrus peel oils, preparation of citrus

salads and dried animal feed from citrus fruits.

Processing of fruits like mango, apricot, guava, papaya, pineapple, banana, etc.

The integral plant cells are preserved by selectively polysaccharides

hydrolysis of the middle lamella (Kashyap et al., 2001). Unicellular goods (Cells

suspension material) are substances produced by the conversion of arranged

tissues, which are utilized as basic material for nectars and pulpy juices for

infant foods, as components for the dairy foodstuffs such as desserts and yogurt

and as protoplasts for several biotechnological uses. The enzymes exploited in

this manner are mentioned as 'macerases'. This practice is written as

maceration. The best enzyme used for the maceration consists of cellulases and

hemicellulases in accumulation of the pectinase enzymes for the maceration of

plant tissues, saachariffication and liquefaction of biomass and isolation of

protoplasts. Filamentous fungi, especially Aspergillus niger is most often used

for industrial synthesis of pectinase enzymes (Kotzekidov, 1991; Barnby et al.,

1990; Naidu and Panda, 1998). The enzymes of fungal origin are used in food

industry as fungi are very powerful producers of pectinase enzymes and the

plus point is their pH (ranges from 3 - 5.5) which is equivalent to various fruit

juices. A number of researchers have conveyed that banana pulp can efficiently

be clarified by depectinazation using pectinase enzyme (Viquez et al., 1981;

Koffi et al., 1991; Yusof and Ibrahim, 1994; Brasil et al., 1995; Alvarez et al., 1998;

Ceci and Lozano, 1998; Vaillant et al., 1999; Lee et al., 2006)

Alkaline pectinases:

These enzymes are mostly utilized in treatment of fiber crops for

pectolytic pretreatment of waste water in food industry particularly fruit juices,

production of paper and pulp, oil extraction and coffee and tea fermentation.

34

Pectic enzymes are also involved in wood preservation; here enzyme

preparations or specific bacteria that produce these enzymes are used Ward and

Fogarty, (1973).

In view of the diverse applications of these acidic and alkaline

pectinases, they form the backbone of the biotechnology industry. The various

ongoing researches are likely to find the application of these extremely important

enzymes.

35

Aims and Objectives:

Pectinases are industrially important enzymes and their demand is

increasing in line with the emerging markets of processed food especially in

processed fruits vegetables, juices and wines industry. Keeping in view the

commercial significance of pectinase and biomass proteins, present project was

chalked out with following objectives:

The major object of this study was to discover the potent producer for

pectinase enzyme by various filamentous fungi.

To grow filamentous fungi on agro-industrial waste for the biosynthesis of

pectinase to explore the cost effective production protocols.

To compare the production rate of pectinase among fungi on natural and

synthetic media with optimal conditions.

To check the final pH and thermostable conditions for pectinase enzyme.

To purify the pectinase enzyme and characterize the enzyme in terms of

optimal pH, temperature, molecular weight and Michaels constant.

36

CHAPTER NO.2

REVIEW OF LITRARURE

Microorganisms under appropriate cultural condition synthesize many

enzymes (including pectinase) and other useful products. The effect of

various factors or different cultural conditions must be optimized before the

production of the final product. Different research workers have used various

microorganisms for the production of pectinase and some of the work done

is as under.

Aguilar and Huitron (1986, 1987) reported the results of many important

aspects of fungal pectinase production by Aspergillus niger. Aspergillus was

isolated from Mexican soil and experiments were conducted through fed

batch culture fermentation. The effect of galacturonic acid, glucose, and the

influence of pH on the production of pectinase was determined through strain

Aspergillus sp CH-Y- 1043.

Solis-Pereyra et al., (1993, 1996) reported a comparative study in which the

effect of high initial concentration of glucose and the effect of different

carbon sources was analyzed for the production of pectinase enzyme by

Aspergillus niger in the solid state fermentation (SSF) and submerged

fermentation (SmF).

Maldonado and Strasser (1998) have conducted research and reported a

study to compare solid state fermentation (SSF) and submerged fermentation

(SmF) for the production of pectin estrases and poly galaturonase by A.niger .

This study showed that through solid state fermentation more pectinase was

produced.

Kashyap et al., (2000) have used Bacillus sp. DT7 (isolated from soil) isolate

bacteria a potent producer of extracellular pectinase enzyme, characterized as

37

pectin lyase (PL) enzyme. In optimized conditions Bacillus sp. DT7 produced 53

units/ml of pectin lyase, which was higher as compared to reported in the

literature. The enzyme was purified through gel filtration and ion exchange

Chromatography. The molecular mass of the purified enzyme was determined

as about 106 kDa. Highest enzyme activity was recorded at temperature 60 ºC

and pH 8.0. Calcium chloride (CaCl2) 100 mM and mercaptoethanol boosted the

activity of the purified enzyme.

Teixeira et al., (2000) have used Aspergillus japonicus 586 for the production of

pectinase estrases, endo and exo-polygalacturonases and tested the effect of

various carbon sources (different concentrations) in a liquid media (Manachini

solutions). The medium was inoculated with 5× 106 spoers /ml and kept under

(140 rpm) agitation at 30 °C for 122 hours. After every 24 hours the culture

broth was separated by filtration and evaluation of pectinestrase was carried

out from Culture broth (Enzyme extract) of A. japonicus 586, which showed

best activity in presence of 0.5% pectin. The higher endopolygalacturonase was

observed in presence of 0.2% pectin and 0.2%, glycerol while 0.5% pectin was

used along with 0.5% glucose showed highest activity of exopolygalacturonase.

Concentration of carbon sources significantly affected the pectinestrase, endo-

and exo-polygalacturonase activities. A repression effect was exhibited on all

the analyzed enzymes when high concentrations of pectin, saccharose and

glucose were used in the culture medium.

Shubakov and Elkina, (2002) have compared the production of

polygalacturonases (PGs) through fungal species like Aspergillus niger ACM

F-1119 and Penicillium dierckxii ACIM F- 152. Polygalacturonases (PGs)

produced from both of the microorganisms and it was found that for both

microorganisms sugar beet pectin showed a powerful inducing effect and

zosteran pectin was also an active inducer for P. dierckxii. The most effective

nitrogen source was found ammonium sulphate (2.2g/l) for both organisms.

A. niger shown highest PG production in a medium with preliminary pH value

38

3.0-4.0 while P. dierckxii was unsuccessful to depend significantly on

preliminary pH value of the medium. However, Penicillium dierckxii was

observed to be more active producer of PG as compared to Aspergillus niger.

Malvessi and da Silveira (2004) have investigated a liquid media

supplemented with wheat bran, salts and inducer (pectin) and found that it was

suitable to produce exo and endopolygalacturonases by Aspergillus oryzae CCT

3940.The higher production of polygalacturonase enzyme was observed in

comparison to rinds of citrus fruits used as inducer. The highest enzyme

activities were recorded at initial pH 4.0 (control) when it reduced somewhat

lower than pH 3.0 the enzyme produced 159 Units of endopolygalacturonase

mL-1 at 83 hours and 45 units of exopolygalacturonase mL-1 at 64 h .The

optimal values of pH and temperature for the production of

exopolygalacturonase (4.5/57 ºC) and endopolygalacturonase (4.3 /40 ºC) were

recorded respectively.

Martin et al., (2004) have produced pectinases from newly isolated strains of

fungal origin under solid state fermentation process; the Penicillium sp EGC5

and Moniliella SB9 synthesized pectin lyase (PL) and polygalacturonase strain

Penicillium (PG) respectively on a medium contained combination of orange

bagasse and wheat bran as a substrate. The strain Moniliella produced

Polygalacturonase and pectin lyase showing optimal activity at pH 4.5 and

10 at 55 °C and 45 °C respectively, when same enzymes were produced by

Penicillium EGC5 shown an optimal activity at pH 4.5 - 5 and 9 at 40 °C,

respectively.

Phutela et al., (2005) have screened 120 different isolates of thermophilic

fungal strain for the production of pectinase and polygalacturonase. The fungal

strain was recognized as Aspergillus fumigatus Fres. MTCC 4163. Various

optimum parameters for pectinase and polygalacturonase (PG) production were

determined under solid state fermentation (SSF) system. Maximum enzyme

activities were obtained in cultures when grown in a medium comprising wheat

39

bran, sucrose, yeast extract and ammonium sulphate after 2-3 days of incubation

when temperature was maintained as 50 ºC. Maximum enzyme activities of 1116

U/g-1 for pectinase and 1270 Ug-1 for polygalacturonase were acquired at pH

4.0 and 5.0 respectively.

Joshi et al., (2006) have compared pectin methyl esterase (PME) production by

Aspergillus niger using apple pomace as a substrate under solid state

fermentation (SSF) and submerged fermentation (SmF). Optimal temperature

25 °C and pH 4.0 were found for maximum enzyme production was recorded

within 96 hours under both solid state and submerged fermentation systems.

0.2% ammonium sulphate under solid state fermentation (SSF) and 0.2%

diammonium hydrogen phosphate in submerged fermentation contributed the

maximum production of Pectin Methyl Esterase (PME). The 0.5% Sodium

chloride in solid state fermentation and 2.0% manganese sulphate in submerged

fermentation as an additive contributed the maximum production of pectin

methyl esterase (PME). Solid state fermentation produced as about 2.3 times

higher pectin methyl estrase (PME) activity as compared to submerged

fermentation, under optimized parameters of fermentation.

Patil and Dayanand (2006 a) have assessed pectin rich agro-wastes, which were

locally available as lemon rind, sorghum stalk and sunflower head for the

production of pectinase by Aspergillus niger DMF 27 and Aspergillus niger DMF

45 under submerged fermentation (SmF) and solid state fermentation (SSF)

systems. Maximum patience was produced when Agro wastes were combined

with carbon and nitrogen sources. By adding ammonium sulphate the synthesis

level of pectinase was increased increased with all the substrates in both

submerged fermentation (SmF) and solid state fermentation (SSF) systems.

Kabli (2007) exploited Kluyveromyces marxianus for the production of

pectinases and enzymes were partially purified through fractional precipitation

with ammonium sulphate and at 65% ammonium sulphate the most active

fractionation was obtained. In the next step the purification was done through

40

gel filtration on Sephadex G- 75 followed by ion exchange chromatography on

CM- Sephdex C- 50. Through separation 4 peaks of protopectinase were

obtained, the second peak containing most of the recovered protein and highest

protopectinase activity. The enzyme allowed reacting with propectin sources

and the enzyme showed different hydrolytic activities. Enzyme concentration,

effect of substrate, pH and temperature was determined to characterize the

enzyme. The enzyme showed stability up to 50 ºC at pH 4 and 7, the effect of

metal ions on enzyme activity was also tested.

Maller et al., (2007) have reported that pectinolytic enzymes are mostly

produced by many Aspergillus sp. but no reports are available on Aspergillus

niveus. The objectives were to optimize culture conditions and physico-chemical

parameters for the enzyme production by A. niveus and the partial purification

of the enzyme through ion exchange chromatography. The assays were carried

out with 1% Polygalacturonic acid in 100 mM sodium acetate buffer pH. DNS

method was used to quantify the reducing sugar. Czapeck medium was

optimized when optimized when supplemented with 1% pectin (Sigma) at 30 ºC

for 9 days under stationary conditions, or 2 days under agitation. The citrus fruit

peels shown to be good inducers for polygalacturonases and also low levels of

pectin and pectate lyases polygalacturonase shown highest activity at 55ºC

when pH was adjusted at 4.0 while enzyme was thermostable for 90 min at 60ºC.

The enzyme was activated by 1mM Mn++ (17%) and EDTA (10%). 80%

ammonium sulphate precipitation was used to purify the polygalacturonase and

elution done in DEAE cellulose followed by Biogel P 100.

Arotupin et al., (2008) have isolated Aspergillus repens from cultivated soils,

which synthesized pectin methyl esterase (PME) in the liquid culture medium.

The enzyme was partially purified by ammonium sulphate precipitation

and dialyzed. The dialysate fraction of the enzyme was isolated by molecular

exclusion and ion exchange chromatography.The molecular mass of pectin

methyl esterase (PME) was found to be 141.3 9 kDa.The optimum pH and

41

temperature of the enzyme activity were 6.5 and 30 ºC respectively.The

enzyme activity was stimulated by Na+, K+, Ca2+, Mg2+ and Zn2+, whereas

EDTA, PbCl2, HgCl2 and IAA showed inhibition effect to the enzyme

activity.With the increase of substrate concentration up to 4 mg/ml enzyme

activity was also increased. The Line weaver-Burk plot of pectin hydrolysis

showed approximately 1.3 mg/ml.

Li et al., (2008) have isolated Bacillus gibsonii, designated as S-2 (CGMCC1215)

to produce alkaline pectinases using sugar beet pulp as a substrate. Three endo

PGs were purified through ultra-filtration, ammonium sulphate precipitation

and ion– exchange chromatography followed by characterization of the enzyme

. The three purified alkaline endo PGs, designated as S-I, S-II, and S-III and

their molecular weights were detetmined as 38 kDa on SDS-PAGE. The Km

value and optimal temperature for optimal enzyme activities of S-I, S-II and S-III

were 1.2 mg/ mL and 60 °C, 0.9 mg /mL and 55 °C, 1.1mg/mL and 60°C

respectively. Maximum enzyme activity was found at pH 10.5, metal ions

such as Mg 2+ and Ca 2+ enhanced the activities of S-I, S- II while S -III was

suppressed by Ca 2+, and Mn 2+ while Zn 2+ ions inhibited the activity of all

three alkaline enzymes.

Rashmi et al., (2008) have isolated 34 strains of Aspergillus niger and screened

for pectinase production, the best producers to be found as Aspergillus niger

isolate JGIm2, Aspergillus niger isolate JGIm3 and Aspergillus niger isolate

JGIm5. Optimal synthesis of the enzyme found in a medium incorporated with

5% pectin at 48 hours. The enzyme was partially purified through ethanol

precipitation at optimum temperature (45 °C) and pH (4.0). The Km and Vmax

values were calculated as about 0.178 g/dl and 11.621 U/mg proteins

respectively.

Reda et al., (2008) have worked with Bacillus firmus-1-4071 and found that this

bacteria is capable to synthesize very high amount of the polygalacturonase

enzyme under solid state fermentation (SSF) system. Fifty one isolates of

42

bacteria from fermented clayed Solanum tuberosum (potatoes) were screened

for their potential to synthesize pectinase using pectin as a substrate under

(SSF) conditions. The results showed that all the isolates were producing

pettiness' and out of which twenty isolates showed good pectinase production

by using agro- industrial wastes viz Solanum tuberosum, Solanum mélange and

Echoria cresips on citrus peels mixtures at 30 ºC and pH was adjusted at 6 by

the technique of pectin clearing zones (PCZ).

Prodanović and Anton (2008) have investigated the opportunity of the

purification and partitioning of pectinase enzymes obtained from Penicillium

cyclopium by partitioning in polymer/polymer and polymer/salt aqueous two-

phase systems. In the system with 10% (w/w) polyethylene glycol, 1500/5%

(w/w) dextran, 500,000/85% (w/w) crude enzyme, the highest values

for partitioning factors were obtained. The partitioning constant, top phase yield

and purification factor were obtained as about 2.11, 85.68% and 11.28

respectively for the endo-pectinase activity. On the other hand partitioning

constant was 1.89 followed by the , top phase yield of 84.28% purification factor

3.82 for the exo- pectinase activity. In the system with 10% (w/w)

polyethylene glycol 6000/15% (w/w) ammonium sulphate 75% (w/w) crude

enzyme, purification factor 37.85 and 19.52 for exo- and endo- pectinase

repectively in the bottom phase were achieved.

Martínez-Trujillo et al., (2009) have evaluated growth and pectinase

synthesis by a fungal strain Aspergillus flavipes FP-500 at various initial pH

values using various carbon sources like pectin, galacturonic acid,

polygalacturonic acid arabinose, rhamnose, xylose, glycerol and glucose.

Aspergillus flavipes FP-500 has shown the production of exo-pectinases, endo-

pectinases and pectin lyases. Exo-pectinases and pectin lyase (PL) were

produced at basal level as constitutive enzymes.Endo-pectinases are inducible

enzymes and only can be produced when pectin is present as an inducer.

43

Results revealed that pectinases produced in an intensive mode by A. flavipes

FP- 500.

Mohsen et al., (2009) have purified polygacturonase (PG) obtained from

Aspergillus niger U-86 through ammonium sulphate fractionation followed by

gel filtrationon sephadex G.75.SDS-PAGE of the purified enzyme revealed

two bands having molecular mass of 35000 and 38000 DA. The purified

enzyme was stable at the pH (3.0-6.0) and at 30 ºC. The Km value was

calculated as about 1.42 mg/ml.

Pedrolli et al., (2009) have reported that pectinase enzymes act upon pectic

polysaccharides producing simple molecules. The enzyme has long been used to

clarify along with the increase in the amount of fruit juices. These enzymes are

particularly distributed into two major groups that attack on pectin “smooth”

sections or on pectin “hairy” sections. Pectinases are one of the most broadly

dispersed enzymes in fungi, bacteria as well as in plants.

Banu et al., (2010) have selected ten molds, which were isolated from

metropolitan waste samples and screened for the production of pectinase

enzyme. These molds were grown on solid media (YPSS) containing pectin. The

Penicillium chrysogenum was selected on the basis of the clear zone and the

production of pectinase was conducted in submerged fermentation conditions.

The higher amount of the enzyme production was obtained by Penicillium

chrysogenum using sucrose and ammonium per sulphate as carbon and

nitrogen source respectively at pH 6.5 and 35 °C. The maximum activity of the

pectinase enzyme by Penicillium chrysogenum was recorded at pH 6.5 and 50 °C.

The enzyme was thermostable up to 40 °C. Magnesium chloride (MgCl2) and

calcium chloride (CaCl2) ions had a slight effect on the activity pectinase

enzyme. Km and Vmax values of the enzyme were 1.0 mg/ml and 85 U/mg

protein, respectively. The molecular mass of the enzyme was found as a bout of

31000 Da on SDS-PAGE.

44

Martin et al., (2010) have isolated 34 thermophilic and thermotolarent strains

of fungi from soil, organic manure and industrial waste heap based on their

aptitude to be grown at 45 ºC in pectin containing liquid medium. About 50%

of these fungi were recognized such as Aspergillus Monascus, Thermomyces,

Thermomucor, Chaetomium, Neosartia and Scopilariopsis. All the strains synthesized

pectinase under the solid state fermentation system. Maximum activity of the

enzyme was achieved in a culture medium inoculated with thermophilic strain

N31, which was recognized as Thermomucor Indicae –seudaticae cultured on a

medium incorporated with a blend of orange bagasse and wheat bran (1:1) with

70% preliminary moisture using solid state fermentation (SSF) system. This

fungus synthesized the highest amount of Exo-polygalacturonase 120 U/ml in

solid state fermentation (SSF) system while in submerged fermentation (SmF) it

was capable to produce only 13.6 U/ml of the enzyme. When crude

polygalacturonase was characterized produced under SSF and and SmF, it was

found that the crude enzyme from SmF was more thermostable than the enzyme

produced under SSF system and revealed maximum stability in acidic pH.

Patil and Chaudhari (2010) isolated pectinase producing organisms from pectin

industry waste using the selective isolation technique. On the morphological

basis culture was identified as Penicillium sp. Which was found a potential

producer of patience and it has produced a significant amount of extracellular

pectinase enzyme under submerged fermentation process. The produced

enzyme was identified as Polygalacturonase (PG). On partial optimization,

highest production of enzymes was achieved at 35 ºC in a medium comprising

pectin at pH 6.0 within 72 hours. The enzyme purification was done by

ammonium sulphate precipitation, size exclusion and ion exchange

chromatography and then its molecular weight were determined as 35000 DA

by SDS-PAGE. Under optimized conditions activity of purified

Polygalacturonase (PG) has shown as 98.66 U/ml which is almost 12 fold

45

higher than crude. Pectinase production was very cost effective and orange

bagasse gave 64.50 units/gm that was higher than the other natural substrate.

Suresh and Viruthagiri (2010) have produced pectinase through solid-state

fermentation (SSF) process by Aspergillus niger using sugar cane bagasse and

wheat bran as a substrate. Media and fermentation parameters were optimized

for the highest yield of pectinase enzyme. Different combinations of substrate

were used for highest production of pectinase. About 90% of wheat bran and

10% of sugarcane bagasse gave highest production of pectinase within 96 hours.

The optimum pH was found as 5 and temperature 40 °C. The kinetics study of

the pectinase showed Km 294.12 and Vmax 2.33 U/ml.

Thakur et al., (2010) have worked with Mucor circineloides for the synthesis of

extracellular pectinase enzymes. The enzyme biosynthesis was boosted when

different synthesis factors were optimized. High pectinase activity was

achieved within 48 hours at 30 ºC when pH was maintained at 4.0. In this

study (1% w/v) methyl ester and (0.1% w/v) casein hydrolysate respectively,

were used as carbon and nitrogen sources. The pectinase enzyme was purified

to homogeneity (13.3 fold) by Sephacryl S-100 gel filtration Chromatography.

The molecular mass of the enzyme was determined as 66 kDa on SDS-PAGE.

Km and Vmax values were determined as 2.2 mM and 4.81 U/ml at

0.1% and 0.5% (w/v) substrate concentration phenolic acids (0.05 mm), metal

ions such as Mn+2, Co+2, Mg+2, Fe+3, Al+3, Hg+2, and Cu+2, and thiols showed

inhibitory influence to the enzyme activity whereas (0.1% w/v)

polygalacturonic acid at pH 5.5 and 42 ºC showed highest enzyme activity.

Damásio et al., (2011) have isolated fungi from decaying plants and soil of

Brazil for pectinase enzyme production. The best producer was Rhizopus

microsporus var. rhizopodiformis, evaluated for the pectinase production under

various environmental and nutritional situations. The production of pectinase

enzyme was examined at optimum temperature of 40 ºC. The medium was

supplemented with 28 different carbon sources. The inducer influence of

46

different agro-industrial wastes like sugar cane bagasse, wheat flour and

corncob on polygalacturonase (PG) enzyme activity was found as 4-, 3- and 2-

fold greater respectively in comparison to control (pectin). In a medium

supplemented with glucose a constitutive Pectin lyase (PL) activity observed.

Rhizopus microsporus showed maximum production 6of PG (57. 7U /mg) and

PL (88. 6U /mg) respectively in a medium containing lemon rind. PG showed

optimal temperature at 65 ºC and total thermostability at 55 ºC for 90 min. Half-

life of enzyme at 70 ºC was 68 min. These results indicated the great usefulness

of the different agro- industrial wastes by R. microsporus for the production of

pectinase enzyme. The enzyme can be helpful for cost effective in production

and could be helpful related to the waste disposal.

Hendges et al., (2011) have produced endo-polygalacturonase by a

strain Aspergillus niger T0005/007-2 in solid medium within 96 h using a

cylindrical double surface bioreactor with 170 mm of height. In the standard

conditions (static ) the cell concentration nearly 292 mg.g-¹dm (mg per g of dry

medium) was acquired, while in experiments under enforced aeration of 21.4

and 2.8 L.min-1. Kg-1mm (L of air per minute per Kg of moist medium) and with

the central shaft the fungal biomass achieved around 100 mg. g-1dm. Maximum

endopolygalacturonase activity was acquired with the central-shaft

system, 78U .g-1dm (units per g of dry medium). Enforced aeration and

pressurepulse exhibited no optimistic effect on the synthesis of endo-PG, 45U .

g-1dm and 28U .g-1dm, respectively. The enzyme showed thermostability up to

40 ºC, 50% activity decreased after 120 minutes at 50 ºC.

Janani et al., (2011) isolated bacteria from agricultural waste dump soils in

Vellore, Tamilnadu and South India to screen them out for pectinase production.

Out of total ten bacterial strains only 3 strains were positive in pectinase

depolymerization assay plates. The enzyme The partial purification of enzyme

was carried out through ammonium sulphate precipitation following by

dialysis. The strains were identified as Bacillus sp. and they were capable to

47

synthesize high amounts of pectinase under submerged and semi-solid

fermentation systems. Maximum enzyme production was acquired in the

medium supplemented with wheat bran as substrate compared to rice bran.

The optimal temperature for enzyme synthesis was observed as 30° C.

Joshi et al., (2011) have used solid state fermentation system for the optimized

parameters to produce pectinase (Pectin methyl esterase) by Aspergillus niger.

It was found that It was found that the partially purified enzyme by ammonium

sulphate fractionation (20-80% concentration) was stable up to 60 days and its

thermostability was up to 50 ºC and became totally deactivated at 90 ºC. The

partially purified PME presented maximum activity at pH 3.5. The enzyme

was tested for extraction and clarification of juice of different fruits like plum,

peach, pear and apricot. It was determined that the pectin esterase enzyme

produced from apple pomace had required activity and it enhanced the quality

of evaluated fruit juices.

Maller et al., (2011) have grown A. niveus on liquid or solid media incorporated

with agro- industrial trashes as carbon source. For this purpose, Czapeck media

was complemented with a number of 28 carbon sources and amongst those

orange rind was the top polygacturonase inducing substrate. While in

submerged fermentation lemon rind was found the best as polygalacturonase

inducer. When the results of submerged fermentation and soild state

fermentation were compared, it was detected that polygalacturonase level were

4.4-fold higher under solid state fermentation (SSF) system, when both SSF

and SmF were supplemented with lemon peel. Highest PG activity was

recorded when temperature was 55 °C and pH 4.0 whereas enzyme stability

was found at 60 °C for 90 min at pH 3.0-5.0.

Murad and Azzaz (2011) have observed that pectinases are distributed in

microbes and higher plants. Those are highly popular and upcoming enzymes

for the commercial sector and microbial pectinases are estimated to be 25% of

sales among overall food enzyme. Microbial pectinase enzymes may be

48

synthesized from bacteria, yeast and fungi. Aspergillus niger among fungal

species is exploited at industrial level for the production of pectinase enzyme.

Praveen et al., (2011) have studied the synthesis of pectinase through

Aspergillus niger NCIM 548 under solid state fermentation process with a

high concentration of nutrients, micronutrients along with a large surface area

and using Ficus religiose leaves as a substrate. Size of inoculum, pH,

temperature, particle size and moisture content were optimized to acquire

the highest production of the enzyme. The highest production of pectinase was

recorded at pH 5.0 and at 30 ºC under solid state fermentation (SSF) system.

Optimized carbon and nitrogen sources were 4% glucose and 0.3% ammonium

sulphate respectively. Highest production of pectinase was 34. 12U /ml in solid

state fermentation

Rajendran et al., (2011) have used Fusarium sp., to produce novel pectinase,

which was isolated from the natural environment. The isolate was subjected to

varying parameters of incubation time, substrate concentration, pH and

temperature for optimal production of the enzyme. The maximum production

of pectinase was observed up to 40 U/ml when the culture was maintained at

27 °C. At pH 6 and at an initial substrate concentration of 0.5% whilst the

productivity was about 46 U/ml and 40 U/ml of pectinase enzyme

respectively. The enzyme was produced at optimized conditions, and enzyme

was purified using acetone precipitation. After characterization it was found

that the crude enzyme was capable to retain its activity at 48 °C when the pH

range was in-between 4 and 8 respectively.

Geetha et al., (2012) have utilized fruit rind waste (one of the contaminating

solid trashes) for the production of pectinase enzymes, and also isolated

microorganisms like bacterial and fungal origin from the fruit peel waste. The

microorganisms were recognized as Bacillus sp. and Pseudomonas sp. and the

fungal species were recognized as Aspergillus niger, Aspergillus flavus and

Penicillium chrysogenum. About all the identified microorganisms were able to

49

produce sufficient amount of pectinolytic enzymes like pectin estrase and

pectate lyase. Various levels of pectin inducer were observed and all the fungal

and bacterial species produced the maximum amount of the enzyme when 1%

pectin was used in the media. Highest amount of pectate lyase and pectin

esterase were produced by Bacillus sp. and A. niger when grown in the medium

supplemeted with 1% pectin. A. flavus was grown in submerged fermentation;

it produced higher pectin esterase and pectate lyase. Maximum enzyme was

produced under solid state fermentation in contrast to submerged fermentation.

Aspergillus performed better as compared to all other organisms.

Kumar and Sharma (2012) have used Bacteria for the production of various

commercial enzymes along with pectinase enzyme. The pectinase producing

bacteria were isolated from two decomposing fruit materials like apple and

oranges and were screened for pectinolytic activities. The best producer (O1,

i.e. Orange 1) was Cocci sp. The optimized temperature was 35 ºC while

optimum pH was found 8.0 with 120 rpm agitation (supporting aerobic

conditions) using orange as substrate and incubation time was 72 h. This process

requires surfactant for achieving maximum enzyme activity 13.96 U/ml in

crude enzyme extracts. The study suggested that orange substrate is strong

bacterial candidate for industrial production of pectinase enzymes.

Ogunlade and Oluwayemisi (2012) have isolated three strains of A. niger

from banana peels metropolis of Ibadan, Nigeria, and used them to

depolymerize citrus pectin. Best pectinolytic produced on the medium

supplemented with pectin as substrate under the solid state fermentation and

submerged fermentation process. Fermentation process were compared higher

amount of pectinase enzyme produced in solid state fermentation than

submerged fermentation. Different treatments were applied to banana peel

used as carbon source, higher amount of pectinase was produced when banana

peel was treated as compared to control and it proved that pretreatement of

agro-waste increases pectinase production.

50

Panda et al., (2012) have isolated twenty five fungal strains from the soil of

Similipal Bioreserve Forest and screened for cellulytic and pectinolytic activity

and potential producers found as Aspergillus sp. In the samples. Aspergillus

niger and Aspergillus flavus showed high pectinase and cellulase activity

which showed Potency index of IFcel and IFpect respectively. The other

parameters like temperature and pH were optimized from the predominant

strains and predicted from the study that both the fungal strains may be utilized

for the industrial purpose of large scale production of the enzyme.

Patil et al., (2012) screened vegetable to obtain pectinase producing

microorganisms from carrot. The best isolate was identified as a unique strain of

Bacillus sp. based on morphological, biochemical tests and using 16S rRNA

gene sequencing studies. This isolate was selected for further bulk production

using the optimized conditions of pH 9 and temperature of 50 ºC to produce

maximum yield of 49.58 % pectinase from carrot waste.

Tariq and Reyaz (2012) used Penicillium chrysogenum strain MTCC *160 for

pectinase production under solid state fermentation system and enzyme activity

was found 15U/ mL . The pectinase activity was increased when carbon sources

were added and it showed 82.5 U/mL in sucrose, 67.5 U/mL in glucose and

79.1U/mL in lactose. The pectinase activity was also increased in presence of

nitrogen sources like ammonium sulphate 65 U/mL, peptone 37.5 U/mL and

yeast extract 31.6 U/mL. The pectinase activity was maximum at substrate

concentration of 7g and activity found 25 U/mL.

Vasanthi and Meenakshisundaram (2012) have reported that citrus fruit

processing industries yield a huge quantity of waste material, which create

difficulties of waste disposal and finally produces contamination. During

process of citrus fruits a huge amount of waste consist peels , pulp and seeds are

produced. Dried citrus rind is rich in pectin and could be utilized pectin inducer

by microorganisms. The orange peel used for the biosynthesis of pectinase

enzyme by A. niger under solid state fermentation system. The parameters were

51

optimized as 4% substrate concentration, temperature 30 ºC, time period 48 h,

pH 5, nitrogen source 0.3% ammonium sulphate whereas moisture holding

capacity was optimized as 50 %. The pectinase synthesized by Aspergillus niger

was purified by using acetone, ammonium sulphate precipitation and dialysis.

The molecular weight of the purified enzyme determined by SDS- PAGE in the

range of 35- 60 KDa .

52

INTRODUCTION OF FUNGI

In nature, microorganisms have been proficient with enormous potential

for the production of a range of daily used goods including enzymes, which have

been commercially exploited over the years. Pectinase enzymes are known to

produce by many organisms and are beneficial for invading host tissues.

Additionally these enzymes are indispensable in the deterioration of deceased

plant materials by microorganisms and accordingly help to reutilize carbon

compounds on the globe (Alaea et al., 1989). Pectinases are important for the

plants as they assist in cell wall extension and tempering of some plant tissues

throughout maturation and storage period. Pectinase enzymes also maintain

environmental equilibrium by reutilizing of waste materials of plants and

degrade pectin via depolymerization and desertification reactions (Wood and

Kellogg, 1988 and Hölker et al., 2004). This is inducible enzyme, which is

produced by microorganisms throughout their growth on cellulosic materials

(Lee and Koo, 2001). Pectinases widely studied and sold in enormous volumes

for their usage in various industrial applications (Ogle et al., 2001).

Aspergillus niger

The black Aspergilli are perhaps more common than any other group

within the genus. They are worldwide in dissemination and occur in and upon

the utmost range of substrates, including grains, forage products, rotten fruits

and vegetables, exposed cotton textiles and fabrics, leather, dairy products and

other protein rich substrate, and decomposing vegetation in the field. These are

numerous in soils from tropical and subtropical zones. Because of their

cosmopolitan and their roles in natural processes of decomposition and their

extensive use in physiological and nutritional studies of the fungi, and their

important applications in industry they are very useful. Colony of A. niger grows

53

on the Czapek's medium at 24-26 °C, slow growing, attaining diameters of 4.0 to

5.0 cm in 3-4 days, deeply velvety plane or nearly somewhat zonate. It consist

fairly compact white basal mycelium, which may extend 2 to 3 m beyond the

central area of abundant sporulation and remains white even in age at contigious

margins of adjacent colonies, conidial heads are in slightly grayish black brown

shades, borne on long conidiophores, which commonly have a metallic sheen at

low magnification reverse white; exudates and odor lacking. Conidial heads

globose to radiate, mostly 200 to 300\1 in diameter, but ranging up to 500\x

globose heads of a single strains, spores of lighter color terminal in the chains,

conidiophores smooth, long and coarse, commonly 2 to 3 mm but up to 5 to 6

mm high with diameters that reach 30u but are mostly 15 to 20u usually lightly

colored in brown shades, comparatively thin walled with wall thickness

generally 1 U and only rarely reaching 2. Vesicles globose rather variable in size,

most-commonly 40 to 60 p in diameter ranging from 20 m to 80u, fertile over

their entire surface,, sterigmata in two rows, primaries mostly 15 to 30 u long but

ranging from 20 u to 80 \1 fertile over their entire surface- sterigmata in two

rows, primaries mostly 15 to 30u, long but ranging up to 50/1-, conidia globose

with hyaline echinulations when first formed, becoming progressively darker

androugher and finally appearing longitudinally striate from conspicuous bars

of coloring material. Sclerotia produced in some strains, occasionally dominating

the colony appearance, globose to subglobose, cream colored at first then pinkish

buff and black in age. A. niger produce enzymes that could degrade rutin. It

produces citric acid, antibiotics, amylolytic, pectolytic and lipolitic enzymes.

Aspergillus fumigatus

It can grow even at higher temperatures (the most predominant mold

present in moist plant materials undergoing rapid decomposition). Colonies of A.

fumigatus spread broadly over Czapek's solution, with superficial character

changing from silky to deeply felted, while at first, becoming green with the

54

expansion of conidial heads, and it is colorless from the reverse, in other strains

showing changing amounts of yellow, green or even dark red brown shades

produced abundantly and distributed in mass like fume giving the characteristic

name of the fungus A. fumigatus. A. fumigatus is helpful in enzyme production,

decomposing resins, antibiotic production like fumigation and spinulosin.

Mucor geophillus

This is one of the largest genera of the order include number of species. It

is thermophilic fungus and its name was proposed by oudemans (Oudemans

and Koning, 1902). The genus belongs to the kingdom thallophyta and it is

included in order mucorales and family mucroacease (Gilman, 1998). It was

found from the soil of Holland, United States and lowa (Oudemans and Koning,

1902, Abbott, 1923, 1926), Mycelium snow white, very tardily gray, finally pales

olive. Sporangiophores simple or branched in cymes, carrying two to three

branches sporangiaglobes, at first yellow, then olivaceous, leaving acollars after

the destruction of membrane, 50-350µ m diameter, wall small warts. It is

columella globose, voluminous and pale gray. Chlamydospores on the branches

of the mycelium 20µ in diameter, at a time in a more or less extended series

zygospore very like chlamydospores about 30µ in diameter (Gilman, 1957). The

phialides had the shape of typical Purpureocillium lilacinum phialides, or were

very long (up to 30 µm) and Acremonium - like. Conidia, which were primarily

cylindrical and contains 1verticillate branches with whorls of two to four

phialides.

Penicillium lilacinum

(Purpureocillium lilacinum) (Thom, 2010) (Luangsa- ard et al., 2011)

Colonies after 7 days at 25 ℃ 24 ~26 mm in diameter, pentagonal sulcation in the

center, with radial sulcation, white to pale vinaceous, and reverse buff to pale

luteous. Conidiophores were erect, arising mainly from sub-merged hyphae,

with occasional formation of synnemata. Formation of typical conidial structures

55

was observed near the agar, with either solitary phialides or 2~4 in verticils,

which varied in length and occasionally slightly curved or ellipsoidal, measuring

3~17 (~20) × 1.2~2.5 µm, formed in 'slimy heads' on these Acremonium-like

structures with variable size. Chlamydospores were absent Samson (1974).

56

CHAPTER NO.3

MATERIALS AND METHODS

Chemicals: The chemicals of analytical grade used in this study were

purchased from Merck Chemicals, ICN chemicals, Sigma chemicals, Fluka

chemicals and Oxoid etc. All solutions were prepared in double distilled water.

Microorganism: Mucor geophillus was obtained from Research laboratory Shah

Abdul Latif University of Khairpur, whereas Aspergillus niger, Aspergillus

fumigatus and Penicillium lilacinum were isolated and identified in Fermentation

and Enzyme Laboratory, Institute of Biotechnology and Genetic Engineering,

University of Sindh, Pakistan. The slants were maintained on medium

comprising (g/l) glucose 20, peptone 10, agar 20 and distilled water. The

components of stock culture were thoroughly mixed and kept in culture vessels

sterilized at 15 pounds /cm² for 20 minutes at 121 ºC. The sterilized slants

were inoculated with Aspergillus niger, A. fumigatus, Mucor geophillus and

Penicillium lilacinum and incubated at 37 ºC to obtain luxuriant growth.

Inoculum: Other than Mucor geophillus, the rest of fungi (Aspergillus niger ,

Aspergillus fumigatus and Penicillium lilacinum) used in this work were isolated

from soil as these are potent producers of pectinase enzymes and were

maintained on agar slants. Sterilized distilled water was added to each slant of

3-4 days old to scrap spores. The spore suspension was adjusted to a final

concentration by adding sterilized water to stock culture to obtain 5 x 106

spores/ml.

Optimization of inoculum size: The Erlenmeyer flasks (250 ml) comprising 50

ml growth medium (pH 6.5) were plugged with cotton and autoclaved. After

sterilization, spore suspension in the series of 0. 5 ml to 2.0 ml (0.5, 1.0, 1.5 and

57

2.0) was added under aseptic conditions to each Erlenmeyer flasks and

incubated in orbital shaker (129 rpm) at 30 ± 2 °C for the optimization of

inoculum proportion for the synthesis of pectinase. After adjusting the inoculum

size in the subsequent study the growth media were inoculated with 1.0ml

inoculum to optimize the different parameters for the production of pectinase.

Mineral medium: The mineral medium as reported by Burrel et al., (1966) was

slightly modified and used for the growth of fungi and production of pectinase,

which contains (g/L) glucose 10.0 g, KH2PO4 1.0 g, MgSO4 0.25 g, FeSO4 .7H2O

6.32 mg, ZnSO4 . 7H2O 1.1 mg, MnCl2. 2H2O 3.5 mg, CaCl2.2H2O 46.7 mg and

NH4NO3 2.4 g or (NH4) 2 SO4 2.12 g. The pH of medium was adjusted at 6.5.

Fermentation medium: Submerged fermentation was carried out in 250 ml

Erlenmeyer flasks comprising 50 ml of mineral medium with and without 1%

glucose, molasses, date syrup, pectin (raw and synthetic), sucrose, fructose,

maltose, glucose, starch, corn steep liquor, urea, sodium nitrate, potassium

nitrate, ammonium nitrate and ammonium sulphate was taken in a 250 ml

flask. The pH of the medium was adjusted 6.5. These flasks were plugged with

cotton wool and sterilized at 121 ºC for 25 minutes at 15 pounds/cm 2 after

cooling at room temperature. The sterilized medium was inoculated with 1.0 ml

inoculum of different filamentous fungi such as A. niger, A. fumigatus, P.

lilacinum, and M. geophillus in separate flasks. The inoculated flasks were

incubated in an orbital shaking incubator (Gallenkamp) at 30 ± 2 ºC.

Sample harvesting: The samples were harvested after 24 hour interval up to

240 hours and filtered through Whatman No.1 filter paper. The filtrate was then

centrifuged at 5000 rpm for 10 minutes to remove undissolved matter and

impurities. The supernatant was separated carefully with the help of

autopippitte. The spore free filtrate thus obtained was assayed for pectinase

activity, the total and reducing sugars.

Biomass: Mycelial mass obtained after filtration was washed with distilled

water, dried in oven at 80 °C till constant weight.

58

A- Optimization of culture conditions: Growth medium was prepared to study

different parameters for the optimization of pectinase production.

i- Effect of fermentation time period

The Fermentation time period was optimized for the production of Pectinase

by A. niger, A. fumigatus, and a mixed culture of A. niger + A. fumigatus, M.

geophillus and P. lilacinum grown on different carbon sources like 1% glucose

and 2.5 and 5.0% date sugar, molasses, citrus pectin and pure sugars.

ii- Effect of carbon source: i) The results of control (1% glucose) was compared

with 2.5 and 5 % of date syrup, molasses, citrus pectin (synthetic pectin) were

used as carbon sources. ii) Different sugars like fructose, maltose, glucose,

galactose, sucrose and starch were used in two different concentrations 2.5 and 5

% along with selected agricultural waste (5 % molasses) incorporated in

fermentation medium to check their effect on extracellular pectinase production

while the inoculated flasks were incubated at 30 ± 2 ºC.

iii- Effect of nitrogen source: After optimizing carbon source five different

nitrogen sources like corn steep liquor, urea, sodium nitrate, potassium nitrate,

ammonium nitrate and ammonium sulphate were used in the range of 0.2 and

0.4% and selected carbon source was supplemented in the fermentation

medium.

iv- Effect of pH: pH was optimized in order to achieve maximum production

of pectinase. Optimized medium adjusted with various pH values ranging from

2.0 to 11.0 before sterilization and were inoculated with desired microorganism

and incubated on orbital shaker at 120 rpm for 96 hours at 30 ± 2 °C. The final

pH was checked after completion of fermentation process by pH meter and

maximum production of pectinase was found at pH 6.0.

v- Effect of Temperature : The production of pectinase was checked at different

temperatures rangimg from 20 to 45 °C under the similar culture conditions as

discussed above and 35 °C was optimum temperature for the maximum

synthesis of pectinase enzyme.

59

vi- Characterization of crude pectinase : The crude enzyme was characterized

on the basis of different parameters such as incubation time, substrate

concentration, enzyme concentration, pH, pH stability, temperature,

temperature, temperature stability and the effect of metal ions on Pectinase

activity.

vii- Effect of time of incubation : The pectinase activity was observed and

recorded at different time periods (10 – 60 minutes) with 5 minutes difference

by using 1.0 ml of pectinase enzyme (from Broth) and 1.0 ml citrus pectin (1.0%)

was used as substrate. The reaction mixture was incubated at 37 ºC for different

time periods.

viii- Effect of substrate concentration: The effect of substrate concentration was

observed for the rate of enzymatic reaction of pectinase by using citrus pectin

as a substrate with different concentration ranging from 0.5 - 2.5%. The reaction

mix consists of 1. 0 ml of culture broth and 1. 0 ml of substrate of different

concentration and the reaction mixture was incubated at 37 °C for 15 minutes.

ix- Effect of enzyme concentration : The effect of enzyme concentration (0.2–

1.4 ml culture broth) on the rate of enzyme reaction was studied by incubating

with 1.0 ml citrus pectin (1.5%) and the reaction mixture was incubated at 37

°C for 15 minutes.

x- Effect pH : The effect of pH on pectinase activity was tested by evaluating the

enzyme activity at various pH in the range of (3 –10) using 1.0 ml sample of

crude enzyme samples and 1.0 ml citrus pectin (1.5% dissolved in different

range of buffers).

xi- Effect of pH stability: The effect of pH on stability of pectinase enzyme

was checked by measuring % of relative activity at 37 °C when 1.0 ml enzyme

was mixed with 0.2 ml citrus pectin (1.5 %) pH ranging between 3–10 using,

sodium citrate buffer 0.1M incubated for 10 minutes. After 10 minutes 0.5 ml

substrate was added and again incubated for 15 minutes.

60

xii- Effect of temperature: The pectinase activity of culture broth was studied at

different temperatures in the range of 20 °C to 100 °C. The enzyme activity

carried out by reported assay method.

xiii- Effect of temperature stability: The thermo stability of Pectinase enzyme

was tested from culture broth samples by measuring the % of rmaining activity

after heating the enzyme 1.0 ml in the presence of 0.2 ml Citrus pectin (1.5%

dissolved in sodium citrate buffer pH 5.0) at different temperatures ranging

between 20° C to 100 °C for 10 minutes. After 10 minutes the assay was carried

out by adding 1.0 ml 1.5 % citrus pectin as substrate and incubated at 40 °C for

15 minutes.

xiv- Effect of Metal ions / Compounds on crude pectinase: Various metal ions

and compounds in 5mM concentrations were reacted with enzyme (1.0 ml) in

sodium citrate buffer pH 5.0 for 10 minutes at optimum temperature prior to

addition of substrate and remaining activities were determined by adding 1.0 ml

citrus pectin (1.5%) as substrate and incubated at 40 °C for 15 minutes. The

thermo stability of enzyme was also studied at different time periods (10-60

minutes) with and without activator (15 mM CaCl2) at 60 and 70°C.

B- Preparation of enzyme: Erlenmeyer flask (500 ml) containing mineral

medium along with 5% molasses, 5% sucrose and 0.4% ammonium sulphate

and media was sterilized, cooled and inoculated with pre-grown culture of

the Aspergillus niger. The flasks were incubated at 35 °C. The culture was

harvested after 96 hours. The broth was filtered through Whatman No.1 filter

paper, vortexed thoroughly and centrifuged at 6 ,000 rpm for 15 min, 4 °C. The

enzyme was precipitated by the method of Saxena et al., (2003) from the culture

supernatant by adding ammonium sulphate to 60% saturation. This was left

overnight and the precipitates were collected by centrifugation at 6,000

rpm for 15 min. The precipitate obtained were dissolved in sodium citrate buffer

(pH 5 .0) and dialyzed against the same buffer for 24 h. Dialysis was carried out

using cellulose tubing (molecular weight cut off 10, 000 Da). Pectinase enzyme

61

from Aspergillus niger was purified according to the method reported by

Guessous et al., (2001). The technique adopted included fractionation by

ammonium sulphate followed by dialysis and gel filtration chromatography.

Purity was checked using polyacrylamide gel electrophoresis.

i- Ammonium sulphate fractionation: The culture broth at optimized culture

condition was obtained and centrifuged at 6 ,000 rpm for 20 minutes in a

refrigerated condition. Solid ammonium sulphate (GR grade MERCK) was

slowly added to the supernatant up to 60 % saturation. Addition of ammonium

sulphate was carried out with continuous stirring in an ice bath, and then it

was kept at 4. 0 °C for overnight. The precipitates were obtained by

centrifugation at 6 ,000 rpm for 20 minutes at 4. 0 °C. The precipitates were

dissolved in 30 ml of sodium citrate buffer (0.1 M, pH 5.0). The protein content

of the fraction was determined by the method of Lowry et al., (1951).

ii- Dialysis: The precipitates obtained after treatment with ammonium sulphate

were dialysed against 0.1M sodium citrate buffer (pH 5.0) for overnight. Dialysis

was carried out using cellulose tubing (molecular weight cut off 10 ,000 Da).

After dialysis, the sample was concentrated by polyethylene glycol. The

dialyzed sample was concentrated to 20.0 ml by PEG.

iii- Preparation of gel Sephadex G-100: 10.0 grams of Sephadex G-100 (Sigma

Chemicals, USA) was suspended in 150 ml distilled water and placed for

overnight. The solution was stirred occasionally. Then 0.1M solution of NaOH

and 0.1 N HCl was used to suspend the gel after soaking in acid and base. The

gel was again soaked in distilled water for 3 hours and then distilled water was

decanted. Finally the gel was equilibrated with 0.1 M sodium citrate buffer,

pH 5 .0

iv- Gel filtration chromatography: The dialyzed enzyme (10 ml) was applied

on Sephadex G-100 (Sigma Chemicals, USA) column (65 x 1.5cm). Elution of the

62

enzyme was carried out with 0. 1 M sodium citrate buffer (pH 5.0) At a flow rate

of 2 ml /min and the fraction of 5.5 ml was collected by a fraction collector

(Tokyo, Rikakikai Co. Ltd) using EYELA 9900, UV-Vis. Detector. The absorbanc

of protein was monitored at 280 NM. The fractions were collected and the four

active pools were obtained. The pool fractions were stored at 4°C for further

analysis.

v- Ion exchange chromatography: Concentrated enzyme (5 ml) of fraction -3

was loaded into an anion exchange DEAE Sephadex A-50 (Sigma

Chemicals, USA) column (30 x 1.5 cm) at a flow rate of 0.5 ml /min.

Equilibration and elution were performed first with 0. 1 M sodium citrate buffer

to remove unbound proteins and then with a linear salt gradient from 0. 0 to 1.0

N NaCl was used to elute pectinase enzyme. Fractions of 5.5 ml were collected

and analyzed for Pectinase activity and protein content. The active fractions

were pooled and used for the studies described below.

C- Characterization of purified Pectinase

i- Effect of Temperature on Pectinase activity and stability: The optimal

temperature for the purified pectinase was obtained by assaying the enzyme

activity at different temperatures increasing from 20 ºC to 100 ºC. In order to

assess the stability, the enzyme solution (1.0mL) was heated at 20 °C, 30 °C, 40

°C, 50 °C, 60 °C, 70 °C, 80 ºC, 90 ºC and 100 ºC for 10 minutes intervals over the

period of incubation. The residual enzyme activity was measured following the

procedure described above.

ii- Effect of pH on Pectinase activity and stability: The relative pectinase

activity using 1. 5 % (w/v) citrus pectin was determined at various pH. The

range of pH varied from 3 to 11. To test the pH stability, the purified enzyme

using respective buffer having pH ranging from 3 to 10 as described above and

were incubated for 80 minutes at room temperature. The residual enzyme

activity was estimated at 10 minutes interval during the 80 minute period of

incubation.

63

iii- Kinetic determinations: Initial reaction rates Pectin hydrolysis were

determined at different substrate concentrations ranging from 0. 4 g to 2.4 g/100

ml in 0.1 M citrate buffer (pH 5.0) at 40 °C for 15 minutes.

iv- Effect of Metal ions / Compounds: Various metal ions and compounds with

5mM concentrations were reacted with enzyme (1.0 ml) and 1.5 ml sodium

citrate buffer pH 5.0 for 10 minutes at optimum temperature prior to addition of

substrate and remaining activities were determined using 1.0 ml (1.5 %) Citrus

pectin as substrate and incubated at 60 °C for 15 minutes.

D- Analytical methods

i- Assay of Pectinase Activity: Pectinase activity was determined by the

spectrophotometer method as reported by Bailey et al., (1992). A brief

description of the method is given below.

One unit of Pectinase activity was described as the amount of Pectinase

producing 1 μmole of reducing sugar per 1 ml under standard test conditions.

Reagents:

Sodium citrate Buffer (0.1 M. pH 5.0): Dissolve citric acid monohydrate,

C6 H8 O7 . H2O (M. wt. 210.14) 0.1M-solution contains 21.01 g/l. Trisodium

citrate dihydrate, C6 H5 O7 Na3.2H2O, (M. wt. 294.12) 0.1M-solution contains

29.41 g/l, and then mixed to the desired pH.

Substrate-1% pectin: Dissolve 1.0 % citrus pectin (Fluka) in about 100 ml of

citrate buffer pH 5.0, stirrer to dissolve the substrate and continued stirring to

room temperature. Dilute to volume in a 100 ml volumetric flask with citrate

buffer and Store at 4 ºC for a maximum of one week. DNS Reagent: Potassium

sodium tartrate solution: 300 grams of Potassium sodium tartrate were

dissolved in 500 ml of distilled water.

Dinitro salicylic acid reagent: 10 grams of Dinitro salicylic acid were dissolved

in 200 ml of 2.0 M sodium hydroxide.

Dinitro salicylic acid solution: This solution was prepared by mixing solution

1 and 2 and the volume was making up to 1 liter with distilled water. Procedure:

64

1ml of broth was added in 1.0 ml 1% Citrus pectin as a substrate in a test tube

and after mixing thoroughly, incubated in a water bath for 15 minutes at 60 ºC.

After incubation 1.0 ml DNS solution was added and the solution was heated in

a boiling water bath for 5 minutes, according to the Miller method (1959).

After 5 minutes the test tubes were cooled under tap water and color intensity

was observed against reagent blank at 540 nm. Reducing sugar concentration

was calculated from standard curve was calculated from galacturonic acid as

shown in Figure-4.1.

y = 0.3884x R2 = 0.9989

Figure-4.1: Standard graph for Galacturonic acid

ii- Protein Estimation

Total Protein estimation was carried out from the filtrate after harvesting the

biomass according to Lowry et al., method (1951). A brief description of the

method is given below:

0

0.1

0.2

0.3

0.4

0.5

0.6

0.2 0.4 0.6 0.8 1

5

4

0

nm

Concentration of Galacturonic acid

mg/mL

A

B

S

O

R

B

A

N

C

E

65

Reagents:

01. Alkaline sodium carbonate solution: 20.0 grams of sodium carbonate was

dissolved in one liter of 0.1M sodium hydroxide.

02. Copper sulphate- sodium potassium tartrate solution: 5.0 grams of copper

sulphate (CuSO4..5H2O) and 10.0 grams of sodium, potassium tartrate

(NaKC4H4O6. 4H2O) were dissolved in one liter distilled water.

03. Alkaline solution: Prepared on day of use by mixing 50 ml of reagent (1)

and 1.0 ml of reagent (2)

04. Folin‘s reagent (C10H5NaO5S): Commercially available by E. Merck, BD.H

Procedure: 0.5 ml test solution was added to 2.5 ml alkaline copper reagent.

Regents were mixed thoroughly and allow standing at room temperature for

10 minutes. 0.25 ml diluted Folin’s reagent (1:1 V/V with water) was added in

each tube and incubated for further 30 minutes. After 30 minutes, the

absorbance was noted against blank (blank containing water plus all reagents)

at 750 nm. Standard protein BSA (Bovine albumin) solutions were prepared in

100, 200, 300, 400 and 500 micrograms per ml for calibration curve. The graph of

absorption vs. concentration was plotted and the concentrations of test solutions

were calculated from protein calibration curve (Figure-4.2).

Figure-4.2 Standard Graph for total protein

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

100 200 300 400 500

Concentration of Albumin ug/ml

Ab

sorb

an

ce a

t 750n

m.

66

iii- Determination of Total Carbohydrate: The carbohydrate concentration in

the culture broth was carried out by phenol sulphuric acid method as described

by Montgomery (1961); a brief description of the method is given below:

Reagents: 01. Concentrated H2SO4.

02. 80% Phenol: 80.0 grams of phenol was dissolved in 100 ml of distilled water.

Procedure: 0.5 ml of test solution was added in 2.5 ml concentrated

sulphuric acid and 0.05 ml 80% phenol solution. After thoroughly mixing, it was

stand at room temperature for 15 minutes. The blank was prepared by

substituting distilled water for the test solution. The absorbance was monitored

against the blank at 485 nm. The concentration curve prepared by same manner

as test sample using glucose as standard (Figure-4.3).

Figure-4.3 Standard Graph for total carbohydrate

iv- Determination of Reducing Sugars: The concentration of reducing sugar

from culture broth was determined by dinitrosalicylic acid (DNS) method as

described by Miller (1959) and a brief description of the method is given below:

0

0.2

0.4

0.6

0.8

1

1.2

0.05 0.1 0.15 0.2 0.25 0.3

Concentration of Glucose mg/ml

Ab

sorb

an

ce a

t 485 n

m.

67

Reagents:

01. Potassium sodium tartrate (NaKC4H4O6. 4H2O) solution: 300 grams of

Potassium sodium tartrate was dissolved in 500 ml of distilled water.

02. Dinitrosalicylic acid reagent: 10 grams of Dinitrosalicylic acid was

dissolved in 200 ml of 2 M Sodium hydroxide.

03. Dinitrosalicylic acid solution: This solution was prepared by mixing

Solution 1 and 2 and the volume was making up to 1 liter with distilled

water. Procedure: 2.0 ml of test solution was added in 2.0 ml dinitrosalicylic

acid in a test tube and after mixing thoroughly heat it in a boiling water

bath for 5 minutes. After 5 minutes the test tubes were cooled under tap

water and color intensity was observed against reagent blank at 540 nm.

The concentration, reducing sugar was calculated from the standard curve

(Figure -4.4)

Figure-4.4 Standard curve for reducing sugar

0

0.2

0.4

0.6

0.8

1

1.2

0.06 0.12 0.18 0.24 0.3

Concentration of Glucose mg/ml

Ab

sorb

an

ce a

t 5

40

nm

.

68

v- Molecular Mass Determination:

Preparation of gel Solution

A. Acrylamide-bisacrylamide solution (30:0.8): 30.0 grams of acrylamide and 0.8

grams of bisacrylamide were dissolved in 100 ml distilled water. The stock of

acrylamide solution was then stored at 4 °C.

B. Resolving gel buffer (Tris- HCl pH 8.8): 36.6 gram of tris and 48.0 ml 1.0 M

HCl were mixed and volume was made up to 100 ml with distilled

water. This buffer solution was then stored at 4° C.

C. Stacking gel buffer (0.5M Tris–HCl pH 6.8): 6.0 gram of Tris was dissolved

in 40 ml distilled water and the pH was adjusted to 6.8 by titrating with 1.0 M

HCl. This buffer was stored in refrigerator at 4°C.

D. Reservoir buffer (0.25 M Tris and 1.92 M glycine pH 8.3): 3.03 grams of Tris,

14.4 grams of glycine and 1.0 gram of SDS were dissolved in 1 litre of distilled

water. The solution was stored at 4°C.

E. 1.0% SDS solution: 1.0 gram of sodium dodecyl sulphate was dissolved in 100

ml distilled water.

F. 1.5% Ammonium per sulphate solution: 1.5 gram of ammonium per sulphate

was dissolved in 100 ml distilled water. This solution was prepared freshly each

time.

G. Sample buffer: Tris - HCl buffer pH 6.8 (starching gel buffer) contains 1%

mercaptoethanol, 40% sucrose or 2% glycerol and 1% sodium dodecyl sulphate.

H. Staining solution: 0.1 gram of Coomassie blue R-250 was dissolved in water-

methanol- aceticacid (5:5:2 v/v/v/).

I. Destaining solution: Destaining solution contains methanol -acetic- water (30-

10-60 v/v/v).

69

Table–4.1 Composition of working resolving and stacking gels

Stock solutions Stacking gel ml

Resolving gel 10.0%

Acrylamide-bisacrylamide ml

Acrylamide-bisacrylamide 1.25 5.0

Stacking gel buffer - 2.0

Resolving gel buffer 2.50 -

1.0%SDS 1.00 1.5

1.5 Ammonium per sulphate 0.50 0.75

Distilled water 5.50 6.5

TEMED 0.005 0.005

Preparation of sample and their application: 20ul of tracking dye (0.05%

Bromophenol blue in water) 50 uL of test sample and 50ul of sample buffer were

mixed. The sample protein was denatured by heating at 100°C for 2 minutes

before applying to the gel. 50ul of denatured sample was applied on the surface

of gel without disturbing the buffer layer. Power supply was adjusted at 5 mA

current per plate for 180 minutes.

Development of protein bands: The gels after electrophoresis were stained

with Coomassie blue R 250 for 45 minutes. The gels were stained with

Coomassie blue R 250 for 45 minutes. The gels were then destained in methanol-

- water-acetic acid (30.60: 10 v/v/v) reported by (Hames and Rickwood, 1986)

Molecular weight determination: After purification the enzyme was subjected

to electrophoretic studies to confirm purity. Molecular mass was determined by

using SDS-PAGE electrophoresis (Vertical Gel Electrophoresis system Wealtec

Corp. USA). SDS-PAGE (10%) was performed as reported by Hames and

Rickwood (1986) using size markers SDS-6H (Pharmacia, Uppsala, Sweden)

comprising phosphorylase b (MW 94,000) bovine serum albumin (MW 67,000)

ovalbumin (MW 43,000) carbonic anhydrase (MW 30,000) soybean trypsin

inhibitor (MW 20,100) and lactalbumin (MW 14,400) were used as reference

proteins. Proteins were envisaged by staining with Coomassie brilliant blue.

70

CHAPTER NO.4

RESULTS AND DISCUSSION

In this study an agro-industrial waste and sugars were used as a

carbon source. New enzymes have been focused by researchers for low cost

production due to their commercial applications. The utilization of agro-

industrial wastes, on one hand, provides alternative substrates and, on the other

helps in solving pollution problems, which otherwise may cause their disposal

problems. Pectinases are industrially very important enzymes and are used in

different industries as processing aids for extraction, clarification and maceration

of fruits and vegetables. A number of fungal strains have shown great potential

to produce different types of pectinolytic enzymes (Junwei et al., 1992; Junwei et

al., 2000; Nitninkumar and Bhushan, 2010; Poonpairoj et al., 2001; Silva et al.,

1993; Solis et al., 2009).Pectinases can be produced by fermentation. Aspergillus

sp. are most frequently exploited filamentous fungi to produce pectinase

(Blandino et al., 2001; Patil and Dayanand, 2006 a and b; Rodríguez-Fernández et

al., 2011).

Microbial pectin degradation is important for the decomposition of the

plant material, digestion of plant food and the retting process. Pectin degrading

enzymes have been extensively used to improve the stability of fruit and

vegetable nectar and in the clarification of fruit juices and wines (Bailey and

Pessa, 1990, Fogarty and Kelly, 1983, Ros, et al., 1993, Sreekantian, et al., 1971;

Stressler and Joslyn, 1971). Currently, they are widely used in industries for

setting of natural fibers and the extraction of oils from vegetables and citrus peels

(Federici and Petruccioli, 1885, Fernandes-Salomão, et al., 1996). The enzyme

preparations used in the food industry are of fungal origin because fungi are

71

potent producers of pectic enzyme and the optimal pH of many fruit juices,

which ranges from pH 3–5.5 (Fonseca and Said, 1995). Furthermore, due to the

relatively low temperature stability of the fungal enzyme preparation maceration

needs to be carried out at a temperature not exceeding 45°C, necessitating the

incorporation of a pasteurization step to limit the growth of mesophillic

microorganisms (Silley, 1986).

In this study different concentration like 2.5% and 5% of the sugars were

used as a carbon source for the growth of fungi and production of pectinase

through the submerged fermentation process. Different nitrogen sources were

also used and optimized, optimization of temperature and pH was carried out to

acquire a maximum production of pectinase enzyme.

A- Growth conditions and enzyme production:

Culture conditions were optimized for optimum pectinase production by

fungi, changing one variable at a time while keeping the other constant.

i- Effect of size of the inoculum:

The effect of the size of the inoculum was studied by adding five days old culture

with different concentrations ranging from 0.5 to 2.5 ml in 50 ml of fermentation

medium and incubated in orbital shaker (120 rpm) at 30 ± 2 °C. Results are

shown in Table-5.1 to 5.5. 1ml inoculum size was optimum for maximum

enzyme production by different fungi as A. niger, A. fumigatus, and a mixed

culture of A. niger + A. fumigatus , M. geophillus and P. lilacinum. In the

subsequent 1.0 ml inoculum of micropropagation was used for the production of

pectinase.

72

Table-5.1 Effect of size of inoculums on growth and pectinase production by

A.fumigatus grown on mineral medium containing 1 % glucose as

carbon source at 30 ± 2 ºC pH was adjusted at 6.5.

Size of the inoculum Biomass Pectinase Activity

(ml) g/50 ml Broth U/ml

0.5 0.421±0.004 5.75±0.08 1.0 0.457±0.002 8.271±0.002 1.5 0.436±0.005 5.56±0.07

2.0 0.433±0.008 4.74±0.09

2.5 0.417±0.009 4.66±0.04

Table-5.2 Effect of size of inoculums on growth and pectinase production by

A.niger grown on mineral medium containing 1 % glucose as carbon

source at 30 ± 2 ºC pH was adjusted at 6.5.



0.5 0.432±0.003 5.98±0.06 1.0 0.439±0.009 8.973±0.001 1.5 0.349±0.003 5.58±0.03

2.0 0.344±0.004 5.21±0.06 2.5 0.434±0.006 4.93±0.07

Table-5.3 Effect of size of inoculums on growth and pectinase production by a

mixed culture of A.fumigatus + A.niger grown on mineral medium containing

1% glucose as carbon source at 30 ± 2 ºC pH was adjusted at 6.5.



0.5 0.332±0.003 2.02±0.01 1.0 0.419±0.003 7.37±0.09

1.5 0.449±0.004 5.98±0.03 2.0 0.423±0.002 5.21±0.02

2.5 0.434±0.006 5.93±0.06

73

Table-5.4 Effect of size of inoculums on growth and pectinase production by M. geophillus grown on mineral medium containing 1 % glucose

ascarbon source at 30 ± 2 ºC pH was adjusted at 6.5.



0.5 0.564±0.001 6.19±0.04 1.0 0.412±0.01 7.54±0.03

1.5 0.451±0.003 6.58±0.03 2.0 0.456±0.008 6.21±0.02

2.5 0.434±0.009 6.13±0.05

Table-5.5 Effect of size of inoculums on growth and pectinase production by P. lilacinum grown on mineral medium containing 1 % glucose as




0.5 0.432±0.005 5.98±0.01 1.0 0.469±0.005 8.79±0.02

1.5 0.449±0.002 5.58±0.06 2.0 0.440±0.001 6.21±0.06

2.5 0 .434±0.006 5.93±0.05

74

ii- Fermentation mode

Fermentation medium (Table-5.6) was inoculated with five days old 1 ml inoculum to each flask, incubated to optimize the

fermentation mode.

Table.5.6 Effect of fermentation mode for the growth and biosynthesis through different filamentous fungi

Fermentatin (Smf) after

72 hours

Biomass g/50 ml

Pectinase activity U/ml

Still culture

Semi shaking

Continuous

shaking

A.

fum

igat

us

A.

nig

er+

A.

fum

igat

us

A.

nig

er

M.g

eoph

illo

us

P. l

ilac

inu

m

A.

fum

igat

us

A.

nig

er+

A.

fum

igat

us

A.

nig

er

M.g

eoph

illo

us

P. l

ilac

inu

m

0.49±0.02

0.43±0.03

0.45±0.04

0.5±0.1

0.39±0.04

0.41±0.02

0.51±0.02

0.4±0.1

0.43±0.02

0.48±0.04

0.39±0.03

0.41±0.03

0.45±0.03

0.42±0.03

0.41±0.03

7.32±0.04

7.4±0.3

8.27±0.04

6.8±0.3

7.7±0.2

8.02±0.01

7.3±0.2

7.9±0.3

8.97±0.02

6.74±0.04

6.82±0.02

7.54±0.02

7.42±0.03

7.95±0.02

8.79±0.03

76

In this study submerged fermentation system was used as liquid culture

which was usually preferable to solid state culture not only due to it allowing

better aeration and proper agitation , but also the separation of the enzyme from

the solid substrate is more difficult than submerged fermentation (Alazard and

Raimbault, 1981). Submerged fermentation and solid state fermentation have

been used successfully in the production of pectinase by fungi (Dinu et al., 2007,

Pedrolli et al., 2008, Castilho et al., 2000). However, according to Sunnotel and

Nigam (2002) submerged fermentation is technically easier as compared to solid

state fermentation. It is a well developed system used in industrial scale to

synthesize a large variety of microbial metabolites. This system was strongly

developed from the 1940s onward to produce large scale of antibiotics. On the

other hand, despite the advantages, the application of SSF at industrial is hard to

imagine. There is difficult scale-up, the often unfeasible biomass determination

and complicated product purification by downstream processes resulting from

the use of heterogeneous organic growth substrates Sunnotel and Nigam (2002).

About 90% of all Industrially important enzymes are synthesized in the

submerged fermentation system Murad and Azzaz (2011). Table-5.6 shows that

continuous shaking was best for maximum growth and Pectinase production,

while result reveals that incubation period varies from organism to organism and

carbon to carbon sources but according to different experiments 72 hours were

optimum for the higher yield of pectinase enzyme. Acuna-Arguelles et al., (1995)

stated that the type of culture methods used influence the kinetic and

physiochemical properties of these enzymes. All the organisms have produced

maximum pectinase when continuous shaking was applied for the experiment.

iii- Effect of Incubation Period:

The optimum incubation time for the synthesis of pectinase was studied by A.

niger, A. fumigatus, and a mixed culture of A. niger + A. fumigatus , M. geophillus

and P. lilacinum grown on different carbon sources like 1% glucose and 2.5 and

5.0% date sugar, molasses, crude citrus pectin, and commercial citrus pectin as

77

shown in Fig- 5.1 to 5.9. The maximum pectinase production was achieved at at

72 hours. There was no fair activity for pectinase on the first day (up to 24 hours) .

After that Pectinase activity increased slowly and reached the peak on the 3rd day.

A decrease in activity was occurred just before and after the optimum period for

pectinase. The decline of pectinase production after reaching its maximum level

may be due to catabolic repression, cessation of enzyme synthesis, or to the

increased proteolysis in culture (Sakellaris et al., 1988). As the incubation period

for pectinase production by all the fungi used in this study were 72 hours, which

is against the results of pectinase production by other workers (Phutela et al.,

2005, Said et al., 1991). But these results are in agreement of Fujio and Eledago

(1993), who have reported 72 hours incubation time for polygalacturonase

production by Rhizopus oryzae. The incubation time less than 72 hours was

reported for the highest activity of pectinase enzyme from the cultures of

Aspergillus niger by Patil and Dayanand, (2006 b) and from Coriolus versicolor by

Freixo et al., (2008 a). It is obvious from the observation of the present study that

the addition of different carbon sources were enhanced the Pectinase production

and fungi produced highest enzyme amounts after 72 hours of fermentation

period. Results presented by other workers also reveal that incubation period

varies from organism to organism and carbon to carbon source (Botella et al.,

2007; Gummadi and Kumar, 2007; Hours et al., 1988; Jacob et al., 2008; Joshi et

al., 2006; Kashyap et al., 2000, 2003; Maria et al., 2002; Shivakumar and

Krishnanand (1995); Silva et al (2005; Pande, 1991; Patil and Dayanand, 2006b ;

Solis-Pereyra et al., 1996; Taragano et al., 1997; Teixeria, et al., (2000). Similar

results were also observed by Mrudula and Anitharai (2011) during pectinase

production by Penicillium sp.

78

Fig- 5.1: A. fumigatus, A. niger , A. niger + A. fumigatus , M. geophillus, P.

lilacinum were grown on mineral medium containing 1 % glucose

as carbon source at 30 ± 2 ºC pH was adjusted at 6.5.

Fig- 5.2 :A. fumigatus, A. niger , A. niger + A. fumigatus , M.geophillus, P. lilacinum

were grown on mineral medium containing 2.5 % date sugar as carbon source

at 30 ± 2 ºC pH was adjusted at 6.5.

0

1

2

3

4

5

6

7

8

24 48 72 96 120 144 168 192 216 240

Time (hours)

Pe

ctin

ase

Act

ivit

y U

/ml

A.fumigatus

A.niger

A.niger+A.fumigatus

M.geophllus

P.lilacinum

0

1

2

3

4

5

6

24 48 72 96 120 144 168 192 216 240

Time (hours)

Pe

ctin

ase

Act

ivit

y U

/mL

A.fumigatus

A.niger

A.niger+ A.fumigatus

M.geophillus

P.lilacinum

79

Fig- 5.3:A. fumigatus, A. niger , A. niger + A. fumigatus , M. geophillus,

P. lilacinum were grown on mineral medium containing 5 % date

sugar as carbon source at 30 ± 2 ºC pH was adjusted at 6.5.

Fig- 5.4: A. fumigatus, A. niger , A. niger + A. fumigatus , M.geophillus, P.lilacinum

were grown on mineral medium containing 2.5 % molasses as carbon


0

1

2

3

4

5

6

7

8

9

24 48 72 96 120 144 168 192 216 240

Time (hours)

Pecti

nas

e A

cti

vit

y I

U/m

L

A.fumigatus

A.nigar

A.nigar+A.fumigatus

M.geophilus

P.lilacinum

0

2

4

6

8

10

12

24 48 72 96 120 144 168 192 216 240

Time

Pecti

nas

e A

cti

vit

y I

U/m

L

A.fumigatus

A.nigar

A.nigar+A.fumigatus

M.geophillus

P.lilacinum

80

Fig- 5.5:A. fumigatus, A. niger , A. niger + A. fumigatus , M.geophillus, P. lilacinum

were grown on mineral medium containing 5 % molasses as carbon


Fig- 5.6: A. fumigatus, A. niger, A. niger + A. fumigatus , M.geophillus, P. lilacinum

were grown on mineral medium containing 2.5 % crude citrus pectin as


0

1

2

3

4

5

6

7

8

24 48 72 96 120 144 168 192 216 240

Time (hours)

Pe

ctin

ase

Act

ivit

y U

/mL

A.fumigatus

A.niger

A.niger+A.fumigatus

M.geophillus

P.lilacinum

0

1

2

3

4

5

6

7

8

24 48 72 96 120 144 168 192 216 240

Time (hours)

Pect

inase

Act

ivit

y U

/mL

A.fumigatus

A.niger

A.niger + A.fumigatus M.geophillus

P.lilacinum

81

Fig -5.7: A. fumigatus, A. niger , A. niger + A. fumigatus , M. geophillus, P. lilacinum

were grown on mineral medium containing 5 % crude citrus pectin


Fig-5.8:A. fumigatus, A. niger , A. niger + A. fumigatus , M.geophillus, P. lilacinum

were grown on mineral medium containing 5 % commercial citrus pectin


0

1

2

3

4

5

6

7

8

24 48 72 96 120 144 168 192 216 240

Time (hours)

Pecti

nas

e A

cti

vit

y I

U/m

L

A.fumigatus

A.niger )

A.niger+ A.fumigatus , M.geophillus

P.lilacinum

0

1

2

3

4

5

6

7

8

9

24 48 72 96 120 144 168 192 216 240

Time (hours)

Pect

inase

Act

ivit

y U

/mL

A.fumigatus A.niger A.niger+ A.fumigatus M.geophillus P.lilacinum

82

Fig -5.9: A. fumigatus, A. niger , A. niger + A. fumigatus , M. geophillus, P.lilacinum

were grown on mineral medium containing 5% commercial citrus

pectin as carbon source at 30 ± 2 º C pH was adjusted at 6.5.

iv- Effect of agro-industrial waste as carbon sources:

The use of low cost substrates for the production of industrial enzymes is one of

the ways to reduce production costs significantly. This can be achieved using

solid agricultural waste materials after treatment as substrates, Agricultural

based carbon sources are more appropriate and these are very cost effective,

renewable and available in huge quantities Yugandhar et al., (2008).

Table.5.7-5.11. Shows the results of pectinase produced by A. fumigatus, mixed

culture of A. niger + A. fumigatus, A. niger, M. geophillus and P. lilacinum when

grown on medium without sugar. The maximum production of pectinase 0.87

U/ml was achieved by A.fumigatus at 120 hours and then pectinase was

decreased with the increase of the time period. The concentration of total sugar

and reducing sugar was absent at the initial period Aspergillus niger produced

0.92 U/ml at 120 and then pectinase was decreased with the increase of the time

period. The fluctuation was noted in the final pH of the culture broth of A. niger,

M. geophillus and P. lilacinum produced 0.91 U/ml, 0.95 U / ml and 0.89 U/ml

respectively after 120 hours. The increase of the time period, pectinase

0

2

4

6

8

10

12

24 48 72 96 120 144 168 192 216 240

Time (hours)

Pe

ctin

ase

Act

ivit

y U

/mL

A.fumigatus

A.niger

A.niger+ A.fumigatus

M.geophillus

P.lilacinum

83

production was decreased in each case. From above results it can be understood

that in the absence of any carbon source the filamentous fungi are capable to

produce very low or negligible amount of pectinase enzyme but it took more

time to produce enzymes.

Table- 5.7: A. fumigatus was grown on mineral medium without glucose at

30± 2º C pH was adjusted at 6.5.

Time Hours

Final pH Biomass g/50 ml Broth

Total Proteins (mg/ml)

Total Sugar (mg/ml)

Reducing Sugars (mg/ml)

Pectinase Activity (U/ml)

24 6.30±0.03 0.005±0.003 0.0013±0.003 - - -

48 6.21±0.03 0.01±0.001 0.0013±0.001 - - -

72 6.26±0.05 0.01±0.005 0.017±0.005 0.045±0.004 0.039±0.003 0.26±0.03

96 6.12±0.02 0.01±0.006 0.018±0006 0.067±0.005 0.059±0.002 0.43±0.05

120 6.22±0.05 0.01±0.001 0.018±0.001 0.093±0.005 0.08±0.05 0.87±0.05

144 6.35±0.04 0.03±0.02 0.014±0.02 0.098±0.003 0.08±0.02 0.77±0.07

168 5.94±0.05 0.03±0.01 0.012±0.01 0.12±0.05 0.10±0.04 0.74±0.06

192 5.89±0.02 0.03±0.02 0.019±0.02 0.15±0.04 0.12±0.06 0.38±0.03

216 5.64±0.02 0.02±0.01 0.018±0.01 0.19±0.06 0.14±0.02 0.36±0.05

240 6.51±0.06 0.02±0.01 0.01±0.01 0.2±0.1 0.18±0.05 0.23±0.04

Table-5.8: A mixed culture of A. fumigatus + A. niger was grown on mineral

medium without glucose at 30 ± 2 ºC pH was adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 6.26±0.07 0.005±0.003 0.005±0.002 - - -

48 6.15±0.05 0.01±0.005 0.008±0.003 0.058±0.003 0.050±0.003 0.18±0.04

72 6.17±0.05 0.01±0.002 0.03±0.02 0.069±0.005 0.061±0.004 0.32±0.03

96 6.19±0.04 0.01±0.008 0.03±0.01 0.093±0.005 0.082±0.003 0.53±0.03

120 6.17±0.06 0.01±0.005 0.005±0.004 0.105±0.003 0.095±0.004 0.92±0.06

144 6.32±0.03 0.03±0.02 0.015±0.007 0.11±0.04 0.098±0.003 0.81±0.04

168 6.22±0.04 0.03±0.01 0.035±0.004 0.113±0.005 0.10±0.04 0.73±0.03

192 6.28±0.04 0.06±0.02 0.037±0.004 0.12±0.03 0.10±0.06 0.70±0.04

216 6.41±0.05 0.03±0.02 0.04±0.02 0.12±0.06 0.10±0.07 0.36±0.04

240 6.41±0.01 0.02±0.01 0.041±0.002 0.135±0.004 0.12±0.06 0.29±0.02

84

Table-5.9: A. niger was grown on mineral medium without glucose at 30 ± 2 ºC pH was adjusted at 6.5.

Time Hours

Final pH Biomass g/50 ml

Broth

Total Proteins

mg/ml

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 6.23±0.06 0.01±0.003 0.005±0.002 - - -

48 6.21±0.06 0.01±0.001 0.015±0.008 - - -

72 6.11±0.02 0.01±0.006 0.015±0.006 0.08±0.03 0.05±0.04 0.28±0.01

96 6.25±0.03 0.01±0.002 0.1±0.01 0.091±0.004 0.088±0.003 0.49±0.02

120 6.22±0.03 0.01±0.005 0.12±0.04 0.13±0.05 0.12±0.03 0.91±0.02

144 6.32±0.01 0.03±0.02 0.14±0.02 0.135±0.006 0.133±0.008 0.87±0.04

168 6.17±0.05 0.04±0.02 0.11±0.04 0.154±0.004 0.12±0.03 0.77±0.05

192 5.70±0.04 0.03±0.02 0.01±0.002 0.167±0.002 0.139±0.005 0.70±0.03

216 6.05±0.04 0.02±0.01 0.004±0.003 0.16±0.02 0.15±0.04 0.40±0.06

240 6.50±0.03 0.02±0.01 0.05±0.03 0.21±0.06 0.15±0.03 0.29±0.03

Table- 5.10: M. geophillus was grown on mineral medium without glucose at 30 ± 2 ºC pH was adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 6.27±0.04 0.01±0.006 0.025±0.1333 0.05±0.04 0.045±0.003 -

48 6.25±0.04 0.01±0.004 0.043±0.004 0.051±0.005 0.046±0.003 0.19±0.0

72 6.27±0.02 0.01±0.007 0.04±0.0213 0.069±0.002 0.061±0.005 0.37±0.0

96 6.25±0.06 0.01±0.003 0.04±0.03 0.071±0.006 0.069±0.004 0.51±0.0

120 6.21±0.06 0.01±0.006 0.045±0.004 0.093±0.006 0.08±0.04 0.95±0.02

144 6.04±0.03 0.03±0.01 0.075±0.002 0.099±0.004 0.085±0.007 0.84±0.03

168 6.18±0.04 0.03±0.02 0.084±0.008 0.12±0.08 0.10±0.05 0. 81±0.084

192 5.73±0.02 0.05±0.03 0.078±0.004 0.154±0.003 0.14±0.03 0.55±0.04

216 6.13±0.05 0.10±0.03 0.011±0.005 0.164±0.004 0.151±0.006 0.31±0.04

240 6.53±0.0 0.02±0.01 0.006±0.003 0.187±0.0062 0.166±0.003 0.27±0.04

85

Table 5. 11. P. lilacinum was grown on mineral medium without glucose at 30± 2 ºC pH was adjusted at 6.5.

Time Hours


Broth


Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 6.28±0.03 0.005±0.003 0.00013±0.00002 - - -

48 6.23±0.04 0.01±0.007 0.00014±0.00002 - - -

72 6.25±0.02 0.01±0.006 0.0112±0.0003 0.04±0.03 0.031±0.004 0.31±0.0

96 6.22±0.03 0.01±0.002 0.0114±0.0007 0.061±0.004 0.053±0.006 0.54±0.0

120 6.33±0.02 0.01±0.006 0.013±0.004 0.09±0.06 0.079±0.007 0.89±0.07

144 6.10±0.04 0.01±0.005 0.0014±0.0004 0.11±0.05 0.087±0.004 0.81±0.04

168 5.71±0.03 0.02±0.01 0.009±0.002 0.11±0.02 0.09±0.05 0.57±0.06

192 0.65±0.03 0.03±0.01 0.019±0.006 0.125±0.006 0.099±0.003 0.39±0.08

216 5.20±0.04 0.02±0.01 0.018±0.003 0.14±0.05 0.12±0.04 0.35±0.06

240 5.21±0.02 0.03±0.02 0.01±0.002 0.152±0.005 0.126±0.003 0.32±0.02

The enzyme synthesis has been greatly influenced by the addition of different

carbon sources. The carbon sources affect not only the mode of enzyme formation,

but also the rate by which carbohydrates are metabolized (Dubey, 2000; Abdullah

et al., 2003). It is reported by Teixeira et al., (2000) that concentration of carbon

sources affects the pectinase enzyme production.

Table-5.12-5.16 shows the results of pectinase biosynthesis by different

filamentous fungi as A. fumigatus, mixed culture of A. niger + A. fumigatus , A.

niger, M. geophillus and P. lilacinum when grown on a culture medium

supplemented with 1% glucose as a carbon source. The maximum production of

pectinase 5.89 U/ml by A. fumigatus was achieved at 72 hours and then

production was decreased with the increase of the time period. The concentration

of total sugar and reducing sugar decreases with increase of the time period. The

fluctuation was noted in the final pH of the culture broth. Mixed culture of A.

niger + A. fumigatus produced 5.52 U/ml of pectinase produced in 72 hours when

cultured on the same medium. A. niger when inoculated on a culture medium

containing 1% glucose as carbon source produced 7.16 U / ml of pectinase at 72

hours. M. geophillus and P. lilacinum when inoculated on the same medium,

86

synthesized 5.76 U / ml and 6.79 U/ml of pectinase respectively at 72 hours. It is

observed that pectinase production was higher at 72 hours of incubation and

later it was decreased with the passage of time. In most of fungi glucose shows a

catabolic repression when used as a carbon source and many genes turned off in

its presence and metabolize other carbon sources (Ronne, 1995, Apel et al., 1993,

Tonukari et al., 2002).In this study glucose has exhibited repression, which is supported

by many early researchers , in their opinion, this may be due to lowering of pH

during incubation time period . Another cause may be catabolic repression of pectinase

production due to high initial glucose/sugar concentration. The lower amount of

enzyme produced when glucose was used as a carbon source as reported by

Zeilinger et al., (1996), Wang et al., (1992) and Southerton et al., (1993). All the

above researchers carried out studies on application of various carbon sources for different

microbes and strains are in agreement with Kunte and Shastri, (1980) who worked with

Atlemaria altemata, Sakellaris et al., (1988) in Lactobacillus plantarum. Macfarlane et al.,

(1990) in Bacteroides ovatus, Said et al., 1991) in Penicillium frequentans, Solis- Pereyra et

al., (1993) in Aspergillus niger, Bahkali , (1995) in verticillium tricorpus, Acuna-Arguelles et

al., (1995) in production was and Kapoor et al., (2000) in Bacillus species , have reported

that pectinase activities were inhibited by the presence of glucose and other sugars .

Catabolic repression has been expressed in many microorganisms as reported by

Fraissinet and Fevre (1996), Runco, et al., (2001) and Panda et al., (2004).

Pedrolli et al., (2008) pointed out that A. giganteus did not produce

polygalacturonase in a media supplemented with only glucose as a substrate

probably be due to catabolic repression, same findings were also presented by

Runco et al., (2001) and Fawole and Odunfa 2003) in Aspergillus terreus and

Aspergillus niger, respectively.

87

Table-5.12: A. fumigatus was grown on mineral medium supplemented with 1 %

glucose at 30 ± 2 ºC pH was adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total sugar

(mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 5.7±0.2 0.052±0.001 0.13±0.02 13.735±0.004 12.485±0.004 3.91±0.08

48 5.52±0.20 0.251±0.003 0.09±0.02 19.225±0.003 18.8±0.1 4.77±0.02

72 4.9±0.3 0.285±0.004 0.103±0.002 12.8±0.3 10.5±0.3 5.89±0.02

96 4.6±0.3 0.325±0.003 0.225±0.002 4.4±0.3 3.15±0.02 4.46±0.02

120 4.35±0.03 0.25±0.02 0.09±0.03 1.2±0.1 0.98±0.03 3.97±0.03

144 4.2±0.2 0.26±0.02 0.208±0.001 1.2±0.2 0.86±0.01 3.9±0.2

168 3.7±0.2 0.345±0.003 0.2±0.1 1.1±0.01 0.81±0.03 1.8±0.1

192 3.6±0.2 0.28±0.01 0.1±0.03 1.1±0.03 0.75±0.02 1.63±0.04

216 4.4±0.3 0.295±0.004 0.078±0.002 0.96±0.03 0.72±0.03 1.5±0.2

240 4.47±0.01 0.27±0.02 0.068±0.003 0.89±0.02 0.71±0.01 0.8±0.3

Table -5.13: A mixed culture of A. fumigatus + A. niger was grown on mineral

medium supplemented with 1 % glucose at 30± 2 ºC pH was adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 2.9±0.2 0.245±0.004 0.385±0.003 10.1±0.1 9.79±0.02 4.1±0.1

48 2.40±0.03 0.315±0.003 0.397±0.002 8.45±0.02 8.12±0.03 4.9±0.3

72 1.77±0.02 0.385±0.004 0.418±0.004 6.94±0.03 6.91±0.03 5.52±0.03

96 1.69±0.03 0.458±0.003 0.429±0.003 5.86±0.03 5.7±0.2 5.3±0.3

120 4.17±0.01 0..499±0.002 0.434±0.004 4.31±0.03 4.29±0.02 4.21±0.02

144 2.18±0.03 0.516±0.003 0.417±0.003 3.85±0.03 3.79±0.05 3.6±0.3

168 1.89±0.03 0.525±0.004 0.412±0.003 2.59±0.04 2.53±0.02 3.2±0.2

192 0.14±0.03 0.524±0.001 0.41±0.02 1.96±0.04 1.76±0.03 2.9±0.4

216 4.42±0.03 0.635±0.003 0.412±0.003 1.79±0.04 1.7±0.2 1.11±0.02

240 6.05±0.04 0.722±0.004 0.403±0.002 0.52±0.02 0.49±0.03 0.95±0.05

88

Table -5.14 : A. niger was grown on mineral medium supplemented with 1 % glucose at 30 ± 2 ºC pH was adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 3.8±0.2 0.32±0.01 0.18±0.04 11.2±0.2 10.9±0.2 4.92±0.01

48 4.4±0.3 0.47±0.03 0.275±0.004 9.52±0.03 9.41±0.03 5.11±0.03

72 4.7±0.2 0.69±0.02 0.297±0.002 8.25±0.04 8.1±0.1 7.16±0.03

96 4.7±0.5 0.71±0.05 0.205±0.004 6.65±0.03 6.43±0.04 4.95±0.04

120 4.5±0.2 0.73±0.02 0.252±0.004 4.7±0.4 4.6±0.4 4.50±0.5

144 4.1±0.1 0.75±0.02 0.197±0.004 3.8±0.4 3.5±0.2 3.29±0.04

168 4.2±0.2 0.74±0.02 0.155±0.003 2.5±0.3 2.44±0.05 2.57±0.05

192 3.9±0.3 0.73±0.04 0.171±0.004 1.34±0.06 1.22±0.05 2.3±0.2

216 3.8±0.2 0.73±0.06 0.179±0.002 0.85±0.04 0.8±0.4 1.1±0.1

240 4.1±0.1 0.7±0.2 0.177±0.005 0.7±0.2 0.68±0.02 0.93±0.05

Table -5.15:M. geophillus was grown on mineral medium supplemented with 1 %

glucose at 30 ± 2 ºC pH was adjusted at 6.5.

Time Hours

Final pH

Biomass g/50 ml

Broth

Total Proteins

(mg/ml)

Total Sugar (mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 5.8±0.4 0.706±0.003 0.915±0.002 12.485±0.004 11.525±0.004 4.1±0.1

48 5.4±0.2 0.812±0.003 0.995±0.002 9.913±0.006 9.625±0.002 4.34±0.03

72 5.7±0.3 0.842±0.005 1.237±0.004 7.642±0.005 7.635±0.004 5.76±0.03

96 6.1±0.1 0.878±0.001 1.242±0.005 4.1±0.1 3.9±0.2 5.4±0.2

120 5.6±0.3 0.885±0.004 1.397±0.005 3.6±0.3 3.1±0.1 4.7±0.2

144 5.5±0.4 0.965±0.003 1.178±0.005 2.37±0.05 2.085±0.002 4.5±0.2

168 5.3±0.3 0.974±0.002 1.159±0.003 1.72±0.02 1.629±0.006 3.13±0.02

192 5.2±0.1 0.98±0.01 1.19±0.02 1.126±0.002 1.048±0.004 2.8±0.2

216 4.9±0.2 1.011±0.003 1.184±0.003 0.845±0.002 0.699±0.002 1.2±0.1

240 4.8±0.5 1.135±0.002 1.176±0.003 0.615±0.004 0.591±0.003 1.1±0.1

89

Table 5.16: P.lilicinum was grown on mineral medium supplemented with 1 % glucose at 30 ± 2 ºC pH was adjusted at 6.5.

Time Hours

Final pH

Biomass g/50 ml

Broth

Total Proteins

(mg/ml)

Total Sugar (mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 5.8±0.2 0.276±0.005 0.17±0.04 13.882±0.002 12.96±0.03 3.87±0.04

48 5.1±0.3 0.369±0.002 0.13±0.03 9.977±0.002 9.91±0.01 4.92±0.02

72 5.6±0.3 0.377±0.005 0.21±0.01 6.5±0.3 6.49±0.04 6.79±0.03

96 5.2±0.2 0.392±0.002 0.28±0.02 5.621±0.001 5.558±0.002 5.16±0.02

120 5.1±0.1 0.416±0.003 0.19±0.03 4.431±0.003 4.35±0.02 3.82±0.02

144 4.9±0.3 0.421±0.001 0.23±0.03 2.3±0.1 2.28±0.05 3.6±0.3

168 4.6±0.2 0.438±0.003 0.17±0.03 2.25±0.02 2.168±0.004 2.92±0.01

192 4.3±0.2 0.317±0.004 0.23±0.02 1.75±0.02 1.629±0.003 2.3±0.3

216 4.1±0.1 0.36±0.02 0.23±0.02 1.72±0.02 1.24±0.03 1.86±0.02

240 3.9±0.6 0.308±0.002 0.29±0.03 0.948±0.005 0.82±0.01 1.2±0.1

Table.5.17 shows the results of pectinase synthesis by A. fumigatus, ( A. niger + A.

fumigatus), A. niger, M. geophillus and P. lilacinum when grown in mineral

medium without glucose in comparison to a medium contains 1% glucose as a

carbon source. The maximum production of pectinase 0.87/ml, 0.91 was

achieved at 120 hours by A. fumigatus and mixed culture of A. niger +A. fumigatus

respectively, then decreased with increase of time period. On the other hand A.

fumigatus and mixed culture of A. niger + A. fumigatus produced 5.89 U/ml and

5.52 U/ml, respectively at 72 hours, when it was inoculated on a culture medium

supplemented with 1% glucose as a carbon source. The maximum production of

pectinase 0.97 U /ml was achieved at 120 hours on medium with and without

glucose while pectinase 7.16 U/ml was produced by A.niger in 72 hours, the

medium supplemented with 1% glucose. (Table.5.17). The maximum production

of pectinase 0.95 U/ml by M. geophillus was achieved at 120 hours in medium

without glucose and then decreased with the increase of the time period, but it

produced an amount of pectinase 5.76 U/ ml in 1% glucose medium at 72 hours

and then decreased with the increase of the time period. The maximum

production of pectinase 0.89U/ml was achieved at 120 hours by P.lilacinum in

90

without glucose medium. The maximum production of pectinase 6.79 U/ml was

achieved at 72 hours in medium containing 1% glucose and with the increase of

the time period pectinase production decreased. The concentration of total sugar

and reducing sugar decreased with the increase of the growth period after 72

hours. Teixeira et al., (2000) reported that high concentrations of carbon sources

have an inhibition effect on enzyme production.

Table-5.17: Effect on growth and pectinase production by different filamentous

fungi when grown on mineral medium supplemented with 1% glucose and

without glucose at 30± 2 ºC and pH was adjusted at 6.5.

Filamentous fungi Biomass g/50 ml Broth

Pectinase Activity U/ml

Biomass g/ 50 ml Broth


Without Glucose With 1% Glucose

A. fumigates 0.04±0.02 0.87±0.02 0.352±0.001 5.89±0.02

A. niger + A. fumigates 0.03±0.02 0.91±0.01 0.415±0.001 5.52±0.02

A. niger 0.03±0.01 0.97±0.03 0.43±0.03 7.16±0.03

M.geophillus 0.02±0.01 0.95±0.01 0.65±0.03 5.76±0.02

P. lilacinum 0.01± 0.89±0.03 0.398±0.001 6.79±0.03

Table-5.18 to 5.22 shows the results of Pectinase production when A. fumigatus

(A. niger + A. fumigatus), A. niger, M. geophillus and P. lilacinum grown on a

culture medium supplemented with 2.5% date sugar as a carbon source. The date

syrup is a liquid by-product of date palm industry containing 75% carbohydrates

w/w small amount of fats and proteins along with other micro and macro

elements (Al-Farsi et al., 2007; Al-Hooti et al., 2002).The maximum production of

Pectinase 3.82 U/ml by A. fumigatus was achieved at 72 hours and then

decreased with the increase of the time period. (Table-5.18)

Table-5.19 to 5.21 shows the results of pectinase production by, A. niger ,

mixed culture of A. niger + A. fumigatus and M. geophillus as 3.47 U/ml, 4.67

U/ml and 3. 9 U/ ml, respectively at 72 hours. Table-5.22 shows the results of

Pectinase production when P. lilacinum was grown on a culture medium

91

supplemented with date sugar at 2.5 % at 30 ± 2 ºC and pH was maintained as

6.5. The highest production 4.32 U /ml was recorded at 72 hours fermentation

period. The decrease in pectinase production after 72 hours may be due to

change in pH during the incubation period (Sampriya et al., 2012) or may be due

to denaturation of enzyme inhibition or interaction with other components of

medium (Soares et al., 1999). The low level of production could be also due to

depletion of nutrients in the medium (Palaniyappan et al., 2009).

Table-5.18: A. fumigatus was grown on mineral medium supplemented with

2.5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 5.35±0.03 0.609±0.003 1.0978±0.0002 7.248±0.004 6.32±0.02 2.23±0.02

48 5.55±0.04 0.642±0.001 0.9448±0.0003 4.858±0.002 4.758±0.006 3.36±0.03

72 5.35±0.03 0.717±0.004 0.894±0.003 3.830±0.01 3.792±0.002 3.82±0.01

96 4.9±0.3 0.732±0.001 0.726±0.003 2.99±0.06 2.57±0.04 2.51±0.01

120 4.8±0.3 0.802±0.002 0.679±0.004 2.54±0.03 2.43±0.02 2.19±0.03

144 4.5±0.3 0.837±0.003 0.687±0.005 1.967±0.002 1.95±0.03 1.53±0.02

168 4.8±0.6 0.874±0.002 0.666±0.005 1.863±0.003 1.81±0.01 1.32±0.01

192 4.2±0.2 0.926±0.001 0.638±0.003 1.616±0.003 1.63±0.02 1.3±0.3

216 4.1±0.1 0.942±0.002 0.628±0.005 1.516±0.001 1.41±0.01 0.82±0.05

240 3.8±0.3 0.979±0.003 0.619±0.001 1.120±0.004 0.99±0.06 0.74±0.03

92

Table- 5.19: A mixed culture of A. niger + A. fumigatus was grown on mineral medium supplemented with 2.5% date syrup at 30 ± 2 ºC and pH was

adjusted to 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 5.4±0.2 0.530±0.002 1.04±0.03 13.66±0.05 6.167±0.003 2.74±0.03

48 5.9±0.2 0.611±0.001 1.06±0.03 7.53±0.02 4.07±0.05 3.18±0.05

72 6.1±0.1 0.624±0.003 1.04±0.02 5.212±0.02 3.112±0.002 3.47±0.04

96 6.45±0.04 0.63±0.02 0.99±0.04 4.069±0.06 1.891±0.001 2.98±0.05

120 6.15±0.02 0.65±0.03 0.92±0.02 3.75±0.03 1.567±0.002 2. 78±0.056

144 6.35±0.03 0.65±0.02 0.90±0.03 3.06±0.02 1.367±0.004 1.82±0.02

168 7.25±0.01 0.66±0.04 0.89±0.03 0.84±0.03 1.0728±0.0005 1.4±0.2

192 6.35±0.02 0.67±0.06 0.87±0.03 2.22±0.02 1.0592±0.0002 0.97±0.04

216 7.05±0.04 0.66±0.01 0.7±0.4 1.9±0.2 0.609±0.002 0.86±0.02

240 7.2±0.2 0.65±0.03 0.7±0.2 1.98±0.03 0.551±0.001 0.58±0.04

T able-5.20: A. niger was grown on mineral medium supplemented with 2.5%

date syrup at 30 ± 2 ºC and pH was adjusted to 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 3.9±0.3 0.542±0.001 1.026±0.002 8.229±0.006 7.973±0.002 2.94±0.02

48 3.6±0.2 0.627±0.002 1.022±0.001 5.985±0.002 5.329±0.003 3.243±0.002

72 3.75±0.04 0.637±0.005 0.855±0.004 4.950±0.002 4.77±0.03 4.67±0.03

96 4.00±0.3 0.667±0.004 0.776±0.004 2.826±0.003 2.719±0.005 3.37±0.04

120 4.2±0.2 0.674±0.001 0.663±0.002 2.184±0.003 1.461±0.001 2.98±0.04

144 4.5±0.3 0.707±0.003 0.547±0.004 1.727±0.004 1.029±0.003 1.82±0.02

168 4.8±0.3 0.752±0.001 0.425±0.004 1.560±0.02 0.965±0.004 1.78±0.04

192 5.4±0.2 0.773±0.003 0.493±0.002 1.500±0.2 0.675±0.002 1.64±0.03

216 5.5±0.3 0.907±0.003 0.535±0.002 1.301±0.001 0.569±0.003 0.57±0.03

240 5.7±0.4 0.887±0.005 0.582±0.002 0.723±0.002 0.391±0.001 0.5±0.01

93

Table-5.21:M. geophillus was grown on mineral medium supplemented with 2.5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 5.4±0.2 0.51±0.04 0.81±0.03 9.4±0.3 8.76±0.03 2.44±0.04

48 5.65±0.4 0.60±0.05 0.85±0.03 7.18±0.06 6.94±0.07 3.19±0.06

72 5.5±0.6 0.61±0.04 0.82±0.07 6.54±0.06 6.21±0.07 3.9±0.4

96 6.25±0.03 0.74±0.07 0.83±0.02 5.98±0.06 5.73±0.07 2.82±0.06

120 6.25±0.04 0.78±0.04 0.84±0.07 4.76±0.03 4.49±0.05 1.77±0.04

144 6.55±0.06 0.81±0.05 0.83±0.04 3.72±0.04 3.59±0.04 1.46±0.03

168 6.8±0.5 0.81±0.02 0.82±0.02 2.21±0.04 2.16±0.05 0.78±0.03

192 6.5±0.4 0.82±0.01 0.81±0.04 1.92±0.04 1.86±0.08 0.69±0.04

216 6.6±0.4 0.83±0.04 0.75±0.02 1.18±0.03 1.15±0.03 0.66±0.02

240 7.1±0.2 0.84±0.07 0.71±0.03 0.91±0.04 0.76±0.04 0.56±0.05

Table-5.22: P. lilacinum was grown on mineral medium supplemented with

2.5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 5.55±0.06 0.749±0.006 0.7512±0.0005 8.69±0.07 7.997±0.006 2.18±0.04

48 5.57±0.06 0.822±0.009 0.747±0.003 6.8±0.3 5.76±0.03 2.67±0.02

72 5.95±0.03 0.857±0.002 0.679±0.004 5.77±0.09 4.63±0.06 4.32±0.03

96 5.25±0.02 0.873±0.007 0.692±0.007 4.6±0.3 4.34±0.07 2.73±0.08

120 5.45±0.06 0.889±0.006 0.632±0.004 3.942±0.003 3.649±0.002 1.84±0.08

144 5.7±0.4 0.922±0.006 0.598±0.003 2.983±0.008 2.86±0.04 1.65±0.08

168 6.35±0.04 0.981±0.005 0.567±0.004 2.84±0.09 2.67±0.04 1.44±0.07

192 6.5±0.3 1.021±0.002 0.546±0.008 2.25±0.02 2.167±0.008 0.97±0.05

216 7.15±0.06 1.1±0.4 0.518±0.005 1.86±0.11 1.78±0.06 0.78±0.09

240 7.45±0.04 1.127±0.006 0.513±0.005 0.84±0.03 0.63±0.05 0.71±0.04

94

Table-5.23-5.27 show the results of pectinase production when filamentous fungi

like A. fumigatus, A. niger + A. fumigatus, A. niger, M. geophillus and P. lilacinum

were grown on a culture medium supplemented with 5% date syrup as a

carbon source. A. fumigatus and a mixed culture of A. niger+A. fumigatus,

produced pectinase 4.87U/ml and 4.21 U/ml respectively, and production

decreased with the passage of time. Table - 5.25 shows the results of highest

(5.68 U/ml) of [ectinase production by A. niger when grown on mineral medium

supplemented with 5 % date syrup as carbon source. The increase of time

period decreased the rate of enzyme production and concentration of sugar

and proteins. Tables-5.26 and 5.27 shows the results of Pectinase production by

M. geophillus and P. lilacinum.The highest Pectinase production was recorded as

4.51U/ml and 5.16 U/ml respectively, while with the passage of time period

decreased Pectinase production, concentration of total sugar, reducing sugar and

total protein. The pH of the medium also changed gradually from acidic to

alkaline except in A. niger .

Table-5.23:.A. fumigatus was grown on mineral medium supplemented with 5%

date syrup at 30 ± 2 ºC and pH was adjusted to 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 5.4±0.3 1.542±0.005 1.17±0.03 9.67±0.04 9.17±0.04 2.53±0.04

48 5.9±0.4 1.442±0.006 1.23±0.03 7.56±0.04 6.14±0.08 3.97±0.05

72 6.1±0.4 1.367±0.005 1.23±0.04 5.97±0.05 5.46±0.07 4.87±0.03

96 6.45±0.08 1.817±0.004 1.24±0.06 5.69±0.06 4.93±0.04 3.96±0.04

120 6.15±0.03 1.817±0.006 1.24±0.04 3.75±0.07 3.67±0.05 2.84±0.03

144 6.85±0.04 1.732±0.005 1.24±0.05 3.56±0.04 3.51±0.04 2.97±0.04

168 7.35±0.06 1.852±0.009 1.36±0.04 2.84±0.03 2.71±0.05 1.95±0.04

192 7.3±0.4 1.817±0.005 1.39±0.04 2.22±0.03 2.17±0.06 1.69±0.04

216 7.1±0.3 1.792±0.003 1.47±0.04 1.19±0.04 0.96±0.03 1.26±0.08

240 7.8±0.6 1.057±0.003 1.48±0.04 0.61±0.07 0.57±0.04 0.8±0.04

95

Table-5.24: A mixed culture of A. niger + A. fumigatus was grown on mineral

medium supplemented with 5% date syrup at 30 ± 2 ºC and pH was

adjusted to 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 5.65±0.04 1.522±0.005 1.085±0.007 11.946±0.004 11.188±0.003 2.98±0.07

48 5.7±0.4 1.857±0.006 1.206±0.003 7.47±0.06 7.091±0.005 3.22±0.05

72 5.8±0.3 1.667±0.004 1.138±0.003 5.125±0.07 4.969±0.004 4.21±0.02

96 6.45±0.4 1.737±0.03 0.995±0.04 4.252±0.004 4.012±0.007 3.98±0.04

120 6.8±0.5 1.847±0.05 0.991±0.04 3.25125±0.0001 3.180±0.003 2.64±0.03

144 6.75±0.4 1.957±0.07 0.987±0.04 3.157±0.005 3.079±0.003 1.54±0.03

168 7±0.2 1.957±0.008 0.979±0.002 2.467±0.002 2.415±0.006 1.24±0.07

192 7.7±050 1.542±0.006 0.974±0.004 1.962±0.005 1.853±0.003 0.95±0.08

216 7.95±0.02 1.307±0.005 0.973±0.005 1.763±0.004 1.587±0.002 0.72±0.03

240 8.2±0.2 1.387±0.006 0.974±0.003 0.942±0.005 0.837±0.006 0.54±0.03

Table-5.25: A. niger was grown on mineral medium supplemented with 5% date

syrup at 30 ± 2 ºC and pH was adjusted to 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 5.2±0.2 1.113±0.006 1.348±0.003 9.246±0.003 8.719±0.006 2.89±0.04

48 4.45±0.03 1.2902±0.0002 1.382±0.006 7.973±0.004 7.815±0.006 3.46±0.08

72 4.55±0.04 1.342±0.003 1.55±0.03 5.547±0.004 4.296±0.008 5.68±0.04

96 4.7±0.4 1.357±0.005 1.673±0.003 3.951±0.008 3.598±0.004 3.65±0.04

120 4.45±0.03 1.352±0.006 1.567±0.006 2.29±0.08 2.181±0.005 2.3±0.2

144 6.35±0.03 1.355±0.004 1.584±0.002 2.212±0.004 1.174±0.003 1.99±0.05

168 6.4±0.3 1.392±0.006 1.256±0.005 1.987±0.006 1.859±0.004 1.32±0.03

192 6.6±0.4 1.417±0.004 1.189±0.007 1.546±0.004 1.424±0.007 1.2±0.2

216 6.65±0.07 1.547±0.004 1.161±0.004 1. 3±0.2828 0.98±0.04 0.9±0.3

240 6.85±0.02 1.52±0.03 0.998±0.005 0.87±0.06 0.81±0.03 0.62±0.03

96

Table-5.26: M. geophillus was grown on mineral medium supplemented with 5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 5.65±0.06 1.113±0.006 1.018±0.005 8.59±0.06 7.87±0.04 2.54±0.03

48 5.6±0.3 1.117±0.003 0.997±0.005 8.17±0.08 7.53±0.06 3.14±0.07

72 6.05±0.05 1.122±0.004 0.988±0.004 6.8±0.4 5.95±0.08 4.51±0.03

96 6.6±0.2 1.072±0.003 0.986±0.003 6.5±0.3 5.38±0.06 3.29±0.02

120 6.5±0.2 1.812±0.006 0.981±0.004 5.51±0.04 5.14±0.02 2.34±0.07

144 6.95±0.06 1.452±0.005 0.979±0.006 4.81±0.05 4.19±0.04 2.16±0.08

168 6.35±0.03 1.567±0.004 0.893±0.005 3.9±0.2 3.37±0.04 1.85±0.04

192 7.15±0.03 1.692±0.004 0.884±0.003 1.43±0.04 1.23±0.06 1.03±0.02

216 7.55±0.04 1.427±0.004 0.835±0.003 1.2±0.2 0.97±0.04 0.87±0.03

240 7.7±0.4 0.777±0.005 0.825±0.008 0.7±0.5 0.58±0.03 0.81±0.04

Table -5.27: P. lilacinum was grown on mineral medium supplemented with 5% date syrup at 30 ± 2 ºC and pH was adjusted to 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 5.6±0.2 0.857±0.004 1.123±0.006 13.404±0.003 8.39±0.06 2.86±0.03

48 6.15±0.06 0.987±0.006 1.931±0.004 9.879±0.006 6.038±0.003 3.7±0.3

72 6.05±0.01 1.122±0.003 1.905±0.002 7.549±0.005 5.209±0.005 5.16±0.03

96 6.05±0.03 1.129±0.007 1.722±0.007 5.729±0.003 3.766±0.004 4.18±0.06

120 6.5±0.2 1.134±0.003 1.698±0.005 3.623±0.006 2.13±0.04 2.9±0.2

144 6.25±0.06 1.259±0.003 1.569±0.006 2.640±0.004 2.021±0.004 2.21±0.03

168 7.15±0.07 1.27±0.04 1.537±0.006 1.775±0.003 1.181±0.002 1.76±0.04

192 7.45±0.04 1.331±0.002 1.013±0.004 1.073±0.006 0.819±0.006 1.33±0.04

216 7.35±0.02 1.34±0.01 0.982±0.005 0.9667±0.0002 0.959±0.004 1.3±0.2

240 8.3±0.4 1.37±0.03 0.916±0.001 0.888±0.007 0.831±0.004 0.49±0.06

97

Table-5.28 shows the comparative results of pectinase synthesis by different

filamentous fungi like A. fumigatus, (A. niger + A. fumigatus) , A. niger, M.

geophillus and P. lilacinum when grown on medium supplemented (2.5% and 5%)

of date sugar as carbon source. Results of pectinase production indicate that A.

niger produces a higher level of pectinase when the culture medium containing

2.5% and 5% date syrup as a carbon source in comparison to other fungi.

Table-5.28: Effect on growth and pectinase production by different fungi when

grown on mineral medium supplemented with 2.5 and 5% date syrup at 30 ± 2

ºC and pH was adjusted 6.5.

Filamentous fungi Biomass g/50 ml Broth




2.5% Date syrup 5% Date syrup

A. fumigatus 0.717±0.006 3.82±0.02 1.167±0.002 4.87±0.02

A.niger+

A. fumigatus 0.624±0.001 3.47±0.06 1.208±0.003 4.21±0.04

A. niger 0. 637±0.002 4.67±0.01 1.667±0.005 5.68±0.05

M. geophillus 0.612±0.001 3.96±0.04 1.122±0.001 4.5±0.2

P. lilacinum 0.857±0.002 4.32±0.03 1.2±0.1 5.16±0.01

The results of pectinase synthesis by A. fumigatus and a mixed culture of A. niger

+ A. fumigatus are shown in Table-5.29 and 5.30, when these fungi were

inoculated on a fermentation culture medium supplemented with 2.5 % molasses

as a carbon source and incubated at 30 + 2 ºC and initial pH was adjusted at 6.5.

The maximum production of pectinase 6.69 U/ml and 6.16 U/ml respectively,

were recorded at 72 hours and with the passage of time sugars concentration

decreased. This indicates an enzymatic repression may be due to some kind of

metabolites which act as enzyme inhibitors.

98

Tables-5.31 and 5.33 shows the results of pectinase synthesis by A. niger,

M. geophillus and P.lilacinum when inoculated on a culture medium incorporated

with 2.5% molasses as a carbon source. The maximum production of Pectinase

7.76 U/ml and 6.58 U/ ml and 6.98 U/ml by above reported fungi respectively

was found respectively at 72 hours and then pectinase activity was decreased

with the increase of the time period. The concentration of total sugars, reducing

sugars and protein in culture broth was decreased with the increase of the

fermentation time period. The pH value was also changed with increase of

fermentation time and becomes basic after 192 and 216 hours in case of mixed

culture and M. geophillus .

Table-5.29: A. fumigatus was grown on mineral medium supplemented with

2.5 % molasses at 30 + 2 ºC and the pH was adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 5.4±0.3 0.985±0.007 1.01±0.001 8.67±0.06 8.1±0.2 4.8±0.4

48 5.9±0.4 0.990±0.004 1.11±0.03 7.95±0.04 6.96±0.05 4.58±0.04

72 6.1±0.2 1.089±0.004 1.13±0.04 7.2±0.3 6.12±0.03 6.69±0.04

96 6.45±0.03 1.033±0.002 1.14±0.03 6.61±0.04 5.92±0.06 5.9±0.2

120 6.15±0.04 1.045±0.008 1.2±0.2 5.45±0.04 4.68±0.05 4.63±0.07

144 6.2±0.2 1.042±0.005 1.29±0.06 3.85±0.02 3.27±0.04 3.37±0.04

168 6.25±0.02 1.205±0.002 1.2±0.1 2.8±0.4 2.28±0.04 3.2±0.4

192 6.35±0.03 1.206±0.006 1.17±0.02 1.62±0.02 1.49±0.04 2.21±0.03

216 6.5±0.4 1.203±0.002 1.16±0.02 1.25±0.07 0.99±0.03 1.81±0.04

240 6.75±0.04 1.165±0.003 1.15±0.06 0.98±0.05 0.75±0.07 0.789±0.006

99

Table-5.30: A mixed culture of A. niger +A. fumigatus was grown on mineral medium supplemented with 2.5 % molasses when incubated at 30 + 2ºC

and pH was adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 5.4±0.2 0.819±0.005 0.918±0.005 9.1±0.01 8. 32±0.01 3.942±0.002

48 5.65±0.04 0.922±0.002 0.925±0.004 8. 18±0.05 7.74±0.02 4.523±0.002

72 5.5±0.2 0.962±0.001 0.898±0.004 7.9±0.3 6.87±0.02 6.16±0.03

96 6.25±0.03 1.057±0.003 0.872±0.002 6.74±0.03 6.23±0.02 5.82±0.01

120 6.25±0.02 1.122±0.002 0.853±0.002 5.7±0.3 5.17±0.03 3.98±0.04

144 6.55±0.01 1.189±0.004 0.842±0.001 4.72±0.01 3.8±0.3 3.16±0.02

168 6.8±0.4 1.197±0.003 0.831±0.001 2.97±0.03 2.5±0.2 2.76±0.03

192 7.6±0.3 1.242±0.001 0.818±0.005 2.37±0.05 1.85±0.04 2.43±0.02

216 8.15±0.04 1.32±0.02 0.811±0.001 1.27±0.03 1.16±0.03 1.76±0.04

240 7.45±0.02 1.447±0.004 0.802±0.001 0.95±0.03 0.73±0.03 1.22±0.02

Table-5.31: A. niger was grown on mineral medium supplemented with 2.5 %

molasses at 30 + 2 ºC and pH was adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

U/ml)

24 4.57±0.05 1.113±0.002 0.995±0.002 8.87±0.04 7.93±0.07 4.67±0.04

48 5.52±0.02 1.124±0.004 1.192±0.006 8.62±0.04 7.21±0.04 5.36±0.04

72 5.13±0.04 1.276±0.003 1.253±0.005 7.8±0.3 7.1±0.2 7.76±0.03

96 6.95±0.04 1.325±0.006 1.25±0.04 6. 7±0.4243 5.915±0.006 5.71±0.03

120 6.75±0.03 1.25±0.08 1.219±0.004 5.53±0.04 4.98±0.03 5.1±0.3

144 5.2±0.3 1.26±0.08 1.212±0.006 4.2±0.3 3.71±0.04 4.28±0.03

168 5.8±0.3 1.345±0.004 1.195±0.007 3.6±0.4 2.82±0.04 3.13±0.03

192 3.62±0.04 0.28±0.03 1.189±0.006 2.31±0.04 1.75±0.04 2.5±0.2

216 4.405±0.003 0.295±0.007 1.167±0.004 1.36±0.03 1.1±0.2 2.25±0.08

240 4.475±0.004 0.27±0.04 1.155±0.003 0.93±0.06 0.64±0.07 1.4±0.2

100

Table-5.32:M. geophillus was grown on mineral medium supplemented with 2.5 % molasses at 30 + 2ºC and pH was adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 5.55±0.04 0.87±0.02 1.119±0.006 8.248±0.003 7.67±0.03 3.298±0.004

48 5.57±0.03 0.89±0.05 1.142±0.001 7.5687±0.0005 7.17±0.03 4.967±0.003

72 5.95±0.03 0.97±0.01 1.218±0.004 6.98±0.02 6.21±0.01 6.58±0.03

96 5.45±0.02 1.1±0.1 1.225±0.003 5.91±0.01 4.86±0.02 5.673±0.002

120 5.45±0.01 1.11±0.04 1.228±0.004 3.85±0.04 3.44±0.03 3.576±0.004

144 6.35±0.02 1.14±0.03 1.239±0.004 3.49±0.05 3.12±0.01 3.535±0.002

168 6.35±0.03 1.17±0.02 1.241±0.001 2.78±0.04 2.21±0.01 3.446±0.003

192 6.5±0.1 1.2±0.1 1.249±0.003 1.63±0.02 1.26±0.03 2.765±0.003

216 7.15±0.04 1.21±0.01 1.232±0.001 1.98±0.02 0.89±0.03 1.869±0.003

240 7.45±0.03 1.22±0.01 1.116±0.002 0.89±0.04 0.653±0.002 0.783±0.003

Table-5.33: P. lilacinum was grown on mineral medium supplemented with 2.5 % molasses at 30 + 2ºC and pH was adjusted at 6.5.

Time

Hours

Final

pH

Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 3.8±0.2 0.967±0.002 0.915±0.002 8.46±0.002 7.59±0.04 4.154±0.002

48 3.4±0.2 0.988±0.005 1.198±0.003 8.17±0.003 6.84±0.03 5.54±0.03

72 2.7±0.2 1.029±0.003 1.212±0.001 7.85±0.001 6.3±0.02 6.98±0.05

96 2.8±0.4 1.078±0.003 1.317±0.004 6.1±0.004 5.94±0.1 5.76±0.05

120 2.6±0.2 1.132±0.001 1.32±0.02 5.98±0.02 5.51±0.04 5.13±0.02

144 3.1±0.1 1.137±0.003 1.336±0.003 5.3±0.003 4.76±0.2 4.35±0.03

168 3.8±0.6 1.145±0.004 1.342±0.001 4.72±0.001 4. 25±0.02 3.3±0.1

192 3.7±0.3 1.205±0.003 1.415±0.004 3.54±0.004 3.2±0.01 2.82±0.01

216 4.8±0.3 1.255±0.004 1.245±0.002 2.45±0.002 2.39±0.05 1.4±0.3

240 4.9±0.3 1.358±0.002 1.225±0.003 1.3±0.003 0.62±0.2 1.2±0.2

Table - 5.34-5.36. reveal the results of A. fumigatus , (A. niger + A. fumigatus ) and

A. niger after 72 hours of incubation pectinase production 8.62 U/ml, 8.54 U/ml

and 10.35 U/ml respectively. The same trend has been noticed in the presence of

the same substrate i-e when the concentration of 2.5% molasses was used. With

the passage of time sugars concentration decreased and results were also

indicative of an enzymatic repression, may be due to some kind of metabolites

101

which act as enzyme inhibitors. pH values fluctuated as incubation time was

increased. The change of pH towards the acidic range during 72-96 hours of

fermentation, Probably due to microbial production of organic acids, when the

concentration of reducing sugars was very low, the pH increased, may be due to

microbial assimilation of organic acids (Botella et al., 2007). Similar pH trends

have been reported by many other researchers (Blandino et al., (2001); Roque

and Takuo (1994); Yoshikawa et al., (1995); Domenguez, (2002).

Tables 5.37 and 5.38 show that best amount of pectinase as 8.14 U/ml and 9.89

U /ml respectively, was obtained at 72 hours when M. geophillus and P.

lilacinum were grown on a medium supplemented with 5% molasses and

incubated at 30+2 ºC and the pH was adjusted to 6.5. With the passage of time

sugars decreased in amount, may be due to some kind of metabolites which

perform as enzyme inhibitors (Teixeira et al., 2000).

Table-5.34: A. fumigatus was grown on mineral medium supplemented with 5%


Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 4.9±0.3 0.997±0.004 2.18±0.05 8.145±0.002 7.98±0.05 5.89±0.04

48 4.4±0.2 1.206±0.003 2.25±0.03 7.78±0.06 6.74±0.04 6.76±0.04

72 4.3±0.3 1.295±0.004 1.8±0.3 6.98±0.07 5.87±0.04 8.62±0.02

96 4.7±0.3 1.285±0.003 1.98±0.04 6.32±0.01 5.31±0.01 6.78±0.03

120 4.9±0.5 1.338±0.003 1.97±0.04 5.38±0.04 4.78±0.02 6.27±0.05

144 5.3±0.2 1.360±0.003 1.96±0.03 4.89±0.03 4.24±0.03 5.785±0.004

168 5.4±0.2 1.378±0.006 1.95±0.03 3.6±0.2 3.29±0.06 4.38±0.06

192 5.45±0.04 1.391±0.001 1.9±0.5 2.62±0.02 2.35±0.02 3.58±0.01

216 5.85±0.03 1.316±0.004 1.88±0.05 1.94±0.03 1.31±0.01 2.16±0.03

240 6.4±0.3 1.408±0.002 1.86±0.05 0.78±0.03 0.62±0.01 1.71±0.01

102

Table-5.35: A mixed culture of A. fumigatus + A. niger was grown on mineral medium supplemented with 5 % molasses at 30 + 2 ºC


Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 4.2±0.2 1.165±0.003 1.476±0.005 8.96±0.04 7.94±0.03 4.43±0.02

48 4.75±0.04 1.656±0.002 1.97±0.05 8.49±0.02 7.32±0.02 6.1±0.1

72 5.4±0.2 1.684±0.003 1.97±0.02 7.96±0.01 6.83±0.03 8.54±0.01

96 6.15±0.02 1.68±0.06 1.84±0.04 6.88±0.03 6.15±0.04 7.87±0.05

120 6.25±0.02 1.875±0.002 1.83±0.02 5.97±0.02 4.96±0.03 6.49±0.05

144 6.45±0.03 1.78±0.03 1.79±0.06 4.61±0.01 3.52±0.01 5.44±0.03

168 6.6±0.5 1.78±0.06 1.75±0.03 3.95±0.03 3.27±0.05 4.77±0.04

192 6.7±0.4 1.75±0.04 1.7±0.3 2.9±0.2 2.47±0.04 2.26±0.03

216 6.5±0.5 1.77±0.05 1.65±0.05 1.68±0.05 1.43±0.03 1.19±0.02

240 6.1±0.1 1.77±0.01 1.6±0.3 1.23±0.02 0.84±0.02 0.99±0.03

Table-5.36:A. niger was grown on mineral medium supplemented with 5 %


Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 5.4±0.3 1.142±0.002 1.19±0.06 9.66±0.05 8.13±0.03 5.53±0.02

48 5.9±0.3 1.19±0.05 1.27±0.03 8.53±0.02 7.1±0.1 6.2±0.1

72 6.1±0.1 1.22±0.01 1.295±0.002 8.12±0.02 6.94±0.03 10.35±0.03

96 6.4±0.2 1.23±0.03 1.29±0.03 7.9±0.4 6.23±0.01 8.16±0.03

120 6.1±0.2 1.25±0.03 1.31±0.01 5.75±0.03 4.98±0.01 6.45±0.02

144 6.8±0.3 1.3±0.2 1.33±0.02 4.18±0.05 3.43±0.03 5.19±0.05

168 7.35±0.02 1.33±0.01 1.33±0.03 3.84±0.02 2.95±0.05 4.37±0.02

192 7.3±0.2 1.41±0.01 1.39±0.07 2.23±0.02 1.76±0.03 2.61±0.01

216 7.1±0.1 1.42±0.01 1.44±0.03 1.91±0.01 1.69±0.04 1.76±0.05

240 7.8±0.2 1.45±0.03 1.45±0.05 0.98±0.02 0.57±0.02 1.18±0.02

103

Table-5.37:M. geophillus was grown on mineral medium supplemented with 5 % molasses at 30 + 2 ºC and pH was adjusted at 6.5

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 5.65±0.03 1.37±0.04 1.921±0.001 9.9±0.4 8. 97±0.02 4.54±0.03

48 5.6±0.2 1.452±0.002 1.929±0.003 9.29±0.07 7.8±0.3 6.54±0.02

72 6.05±0.02 1.512±0.001 1.922±0.001 7.861±0.001 7.59±0.03 8.14±0.03

96 6.6±0.3 1.677±0.004 1.876±0.004 6.28±0.04 5.83±0.03 6.9±0.7

120 6.5±0.3 1.681±0.001 1.865±0.004 4.97±0.04 4.74±0.04 5.34±0.03

144 6.95±0.05 1.705±0.002 1.857±0.003 3.9±0.3 3.48±0.05 4.63±0.02

168 6.35±0.04 1.737±0.005 1.854±0.002 3.87±0.04 2.93±0.02 3.7±0.5

192 7.15±0.03 1.782±0.002 1.8±0.4 3.3±0.2 2.8±0.4 2.67±0.04

216 7.55±0.05 1.79±0.06 1.85±0.04 1.9±0.4 1.5±0.1 2. 9±0.5

240 7.7±0.5 1.823±0.002 1.88±0.05 1.16±0.03 0.831±0.001 1.15±0.04

Table-5.38: P. lilacinum was grown on mineral medium supplemented with 5 %

molasses at 30 + 2 ºC and pH was adjusted at 6.5

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 5.8±0.5 0.970±0.002 2.013±0.002 8.52±0.02 7.93±0.03 5.13±0.02

48 6.1±0.1 0.975±0.002 2.59±0.03 8.13±0.02 7.38±0.03 6.83±0.03

72 6.15±0.04 1.083±0.003 1.96±0.01 7.86±0.03 6.89±0.02 9.89±0.04

96 6.0±0.3 1.04±0.02 1.86±0.03 6.97±0.04 5.57±0.04 7.14±0.03

120 5.8±0.6 1.151±0.001 1.86±0.04 5.65±0.02 4.55±0.01 5.84±0.02

144 5.4±0.2 1.17±0.03 1.77±0.04 4.6±0.2 3.97±0.02 4.12±0.02

168 5.75±0.04 1.22±0.01 1.84±0.04 3.69±0.06 2.94±0.03 3.2±0.1

192 6.4±0.1 1.25±0.03 1.85±0.03 2.48±0.04 2.13±0.02 2.74±0.02

216 6.65±0.02 1.3±0.2 1.82±0.02 1.86±0.03 1.7±0.5 2. 5±0.3

240 6.8±0.3 1.32±0.01 1.88±0.03 0.97±0.03 0.69±0.05 1.12±0.02

Table- 5.39 shows the results of pectinase synthesis by A. fumigatus, (A. fumigatus

+ A. niger ), A. niger, M. geophillus, P. lilacinum.When these fungi were inoculated

on a fermentation culture medium supplemented with 2.5 % and 5% molasses as

a carbon source produces pectinase 8.62 U/ml, biomass 1.295 g/50 ml, 5.97

U/ml, biomass 1.68 g/50 ml, 10.35 U/ml, biomass 1.226 g /50 ml, 8.14 U/ml,

104

biomass 1.512 g/50ml and 9.89 U/ml, biomass 1.083 g/50 ml respectively was

recorded at 72 hours.These results reveal that maximum amount of pectinase

was achieved by A. niger and P. lilacinum as 10.132 U/ml and 9.89 U/ml

respectively, when the same amount of molasses was used in a medium as a

carbon source.

Table-5.39: Effect on growth and pectinase production by different fungi when

grown on mineral medium supplemented with 2.5 and 5% molasses at 30 ± 2 ºC

and pH was adjusted 6.5.

Filamentous Fungi

Biomass g/50 ml Broth




2.5% Molasses 5% Molasses

A. fumigates 1.089±0.005 6.69±0.03 1.295±0.004 8.62± 0.02

A. niger and A.

fumigates 0.962±0.002 6.16±0.03 1.68± 0.06 5.97±0.03

A. niger 1.27±0.04 7.76±0.01 1.226 ±0.003 10.35±0.03

M. geophillus 0.998±0.005 6.58±0.02 1.512±0.003 8.14±0.03

P. lilacinum 1.029±0.002 6.98±0.02 1.083±0.002 9.89±0.03

Table- 5.40 indicated that the production of pectinase 5.4 U/ml was obtained by

A. fumigatus after 72 incubation hours when grown on 2.5% citrus pectin where

as mixed culture of A. niger + A. fumigatus and A. niger produced 5.17 U/ml and

6.59 U/ml respectively after 72 hours (Table-5.41-5.42). The pH value shown

fluctuation as incubation time was increased. The results represented in Table-

5.43 and 5.44 show that production of pectinase 4.65 U/ml and 6.35 U/ml

respectively, at 72 hours incubation when M. geophillus and P. lilacinum were

grown on a medium supplemented with 2.5 % citrus pectin. The activity was

reduced after 96 hours of incubation period that may be due to some metabolites

which act as enzyme inhibitors. Stutzenberger, (1992) investigated that limitations of

105

the microorganisms to use pectin as the carbon and energy source is might be due to their

inability to make pectin methyl esterase. Tsuymu, (1979) reported that this type of

phenomenon may be a catabolic repression of galacturonic acid or one of the metabolite

produced undergoing self catabolite repression. Palaniyappan et al., (2009) has reported

similar findings of enzyme inhibition when higher concentrations of pectin was used as a

substrate.

Table-5.40: A. fumigatus was grown on mineral medium supplemented with 2.5 % citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 4.1±0.1 0.99±0.06 1.43±0.03 9.68±0.04 8.23±0.02 3.75±0.04

48 3.35±0.04 1.06±0.03 1.56±0.03 8.82±0.02 7.54±0.03 4.19±0.01

72 3.55±0.02 1.31±0.01 1.83±0.02 7.55±0.02 7.13±0.03 5.4±0.3

96 3.05±0.03 1.53±0.02 1.87±0.03 7.12±0.01 6.93±0.03 4.32±0.01

120 3.5±0.3 1.57±0.05 1.69±0.04 5.54±0.02 4.66±0.04 3.97±0.02

144 3.8±0.4 1.67±0.03 1.682±0.002 4.7±0.1 3.54±0.02 3.28±0.05

168 3.95±0.04 1.69±0.04 1.587±0.006 3.98±0.05 2.9±0.3 2.95±0.04

192 4.55±0.02 1.82±0.01 1.565±0.003 2.89±0.02 2.4±0.2 2.6±0.2

216 4.15±0.01 1.89±0.03 1.517±0.006 2.7±0.1 2.1±0.1 1.42±0.02

240 4.2±0.1 2.26±0.02 1.466±0.003 1.6±0.3 1.29±0.04 1.34±0.03

Table-5.41: A mixed culture of A. niger + A. fumigatus was grown on mineral medium supplemented with 2.5 % citrus pectin at 30 + 2ºC and pH was

adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 4.15±0.04 0.985±0.002 2.176±0.003 12.637±0.005 9.784±0.003 3.67±0.02

48 3.6±0.2 0.991±0.001 2.3779±0.0004 101.249±0.006 9.394±0.002 3.97±0.03

72 3.45±0.03 1.112±0.002 2.426±0.003 90.622±0.001 8.734±0.001 5.17±0.06

96 3.65±0.01 1.136±0.003 2.476±0.004 9.183±0.003 7.556±0.003 3.94±0.01

120 3.55±0.05 1.243±0.002 2.396±0.005 7.159±0.002 6.498±0.003 3.2±0.1

144 3.85±0.04 1.383±0.002 2.297±0.003 5.996±0.003 4.454±0.004 2.53±0.03

168 4.00±0.3 1.446±0.004 2.278±0.004 4.986±0.004 3.975±0.002 1.97±0.03

192 4.15±0.03 1.651±0.002 2.259±0.003 3.739±0.003 2.928±0.004 1.53±0.05

216 4.15±0.02 1.682±0.002 2.208±0.004 2.185±0.004 1.987±0.005 1.327±0.0089

240 4.2±0.1 1.921±0.003 1.979±0.004 0.959±0.007 0.670±0.003 1.3±0.2

106

Table-5.42: A. niger was grown on mineral medium supplemented with 2.5 % citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 4.15±0.03 0.886±0.003 1.376±0.005 9.237±0.004 8.63±0.02 3.97±0.05

48 3.6±0.2 1.219±0.006 1.779±0.004 8.497±0.001 8.1±0.1 4.47±0.03

72 3.45±0.04 1.223±0.001 1.826±0.003 8.64±0.04 7.734±0.002 6.57±0.01

96 3.65±0.05 1.237±0.005 1.877±0.006 7.844±0.006 7.13±0.02 4.6±0.2

120 3.55±0.01 1.346±0.003 1.899±0.001 6.817±0.006 5.49±0.04 4.13±0.02

144 3.85±0.02 1.383±0.002 1.687±0.004 4.69±0.05 4.15±0.04 3.76±0.04

168 4.00±0.3 1.4465±0.0003 1.649±0.007 3.98±0.02 3.75±0.03 3.15±0.02

192 4.15±0.06 1.551±0.001 1.597±0.002 3.83±0.03 3.28±0.05 2.74±0.03

216 4.15±0.03 1.682±0.002 14208±0.0004 2.58±0.04 2.46±0.03 2.18±0.03

240 4.2±0.2 1.8215±0.0004 1.1055±0.0002 1.39±0.03 1. 2±0.2 1.89±0.01

Table-5.43:M. geophillus was grown on mineral medium supplemented with 2.5%

citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 4.55±0.03 0.978±0.004 2.5397±0.003 13.007±0.003 8.101±0.001 3.15±0.03

48 3.55±0.05 1.036±0.001 2.606±0.003 10.859±0.003 8.979±0.002 4.13±0.02

72 3.35±0.01 1.27±0.04 2.435±0.002 9.317±0.006 7.912±0.002 4.65±0.02

96 3.65±0.02 1.379±0.001 2.449±0.001 8.887±0.001 6.398±0.003 3.58±0.02

120 3.75±0.04 1.4395±0.0004 2.857±0.002 6.338±0.006 5.497±0.002 3.18±0.03

144 3.95±0.06 1.5885±0.0003 2.758±0.003 5.315±0.002 5.308±0.001 2.62±0.01

168 4.15±0.03 1.4865±0.0003 2.489±0.004 4.033±0.002 3.71±0.01 1.98±0.03

192 4.25±0.01 1.4866±0.0003 2.357±0.004 2.785±0.002 2.196±0.003 1.57±0.02

216 4.25±0.05 1.7705±0.0004 2.322±0.002 2.178±0.004 1.679±0.001 1.19±0.03

240 4.15±0.02 1.957±0.003 2.309±0.001 1.301±0.001 0.7225±0.0004 1.17±0.06

107

Table-5.44: P. lilacinum was grown on mineral medium supplemented with 2. 5 % citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 4.55±0.04 0.978±0.004 1.26±0.03 9.97±0.02 7.819±0.003 3.75±0.02

48 3.55±0.03 1.036±0.003 1.507±0.001 9.766±0.004 7.097±0.005 4.81±0.01

72 3.35±0.01 1.27±0.05 1.968±0.003 8.527±0.006 6.912±0.002 6. 35±0.03

96 3.65±0.05 1.379±0.001 1.893±0.002 7.489±0.001 6.76±0.04 4.66±0.03

120 3.75±0.02 1.439±0.004 1.878±0.006 6.857±0.003 5.49±0.05 3.48±0.07

144 3.95±0.01 1.588±0.002 1.768±0.002 4.752±0.002 3.35±0.04 3.22±0.01

168 4.15±0.03 1.486±0.002 1.748±0.004 3.859±0.003 2.71±0.01 2.8±0.2

192 4.25±0.06 1.486±0.001 1.658±0.003 1.78±0.05 2.19±0.01 2.17±0.03

216 4.25±0.08 1.775±0.003 1.596±0.002 1.62±0.01 1.34±0.03 1.45±0.03

240 4.15±0.06 1.957±0.003 1.603±0.002 1.43±0.02 1.2±0.1 1.3±0.2

The higher amount of pectinase 6.15 U/ml was obtained after 72 incubation

hours when a culture of A. fumigatus was grown on a medium supplemented

with 5 % citrus pectin as carbon source and incubated at 30 + 2 ºC when pH was

adjusted at 6.5 ( Table-5.45). Pectinase activity started to reduce after 96 hours of

incubation period. A mixed culture of A. niger + A. fumigatus produced the

higher amount of pectinase 5.97 U/ml after 72 hours of incubation when a

mixed culture was grown on a medium supplemented with 2.5 % citrus pectin

Table-5.46).

Tables 5.47and 5.48 represent the highest amount of pectinase 6.86 U/ml and

5.81 U/ml respectively produced by A. niger and M. geophillus when grown on

a medium supplemented with 5 % citrus pectin .Table - 5.49. shows the highest

amount of pectinase 6.71 U/ml obtained by P. lilacinum after 72 hours, grown on

a medium supplemented with 5 % citrus pectin. Teixeira et al., (2000) reported

the best production of pectic enzymes in the presence of pectin. With the

passage of time sugars concentration decreased, which is showing an enzymatic

suppression and the reason of some kind of metabolites which act as enzyme

inhibitors, reduction in pectinase activity by pectin may be due to the presence of

108

catabolic repression formed by the high galacturonic acid concentration by pectin

degradation. The present t results are in agreement with observations reported by

Aguilar and Huitron, (1987); Maldonado et al., (1989) and Teixeira et al., (2000).

Catabolic repression can occur not only due to pectin degradation, but also due to the

neutral sugars attached to the pectin molecule.

Table-5.45: A. fumigatus was grown on mineral medium supplemented with 5 % citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 4.1±0.1 0.98±0.04 2.06±0.03 11.61±0.01 8.23±0.02 4.75±0.04

48 3.35±0.02 1.06±0.03 2.56±0.05 11.21±0.01 7. 63±0.03 5.89±0.03

72 3.55±0.04 1.31±0.01 2.29±0.05 8.58±0.04 6.93±0.02 6.15±0.02

96 3.05±0.03 1.53±0.02 2.27±0.05 8.12±0.01 5.93±0.01 5.5±0.1

120 3.5±0.1 1.57±0.03 2.256±0.002 6.54±0.04 4.66±0.03 5.177±0.003

144 3.8±0.3 1.67±0.05 2.25±0.03 4.713±0.002 4.14±0.03 4.85±0.04

168 3.65±0.03 1.69±0.04 2.233±0.002 3.798±0.004 2.77±0.03 3.345±0.003

192 3.25±0.05 1.82±0.01 2.225±0.002 3.351±0.001 2.29±0.05 2.955±0.001

216 3.15±0.04 1.89±0.04 2.179±0.005 2.83±0.02 1.98±0.03 2.65±0.05

240 3.2±0.1 2.26±0.02 2.116±0.003 1.11±0.01 0.993±0.002 1.425±0.004

Table-5.46:A mixed culture of A. niger + A. fumigatus was grown on mineral

medium supplemented with 5 % citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 3.8±0.4 0.995±0.003 1.08±0.04 9.152±0.001 8.12±0.01 3.31±0.01

48 3.55±0.04 1.107±0.004 1.19±0.05 8.47±0.02 7.38±0.04 4.46±0.03

72 3.15±0.02 1.26±0.03 1. 2±0.1 7.961±0.001 6.74±0.02 5.97±0.03

96 3.1±0.1 1.215±0.004 1.27±0.04 7.78±0.04 6.15±0.04 4.48±0.02

120 2.8±0.3 1.273±0.002 1.31±0.01 5.987±0.004 4.25±0.03 3.84±0.03

144 2.4±0.2 1.366±0.002 1.34±0.02 4.796±0.003 3.99±0.06 3.49±0.04

168 2.75±0.04 1.398±0.003 1.32±0.01 3.884±0.002 3.39±0.01 2.42±0.01

192 3.45±0.02 1.418±0.002 1.3±0.2 2.481±0.001 2.13±0.02 2.14±0.03

216 3.65±0.03 1.425±0.004 1.3±0.1 1.868±0.003 1.9±0.3 1.78±0.04

240 3.8±0.5 1.539±0.003 1.2±0.2 1.592±0.001 0.98±0.02 1.2±0.1

109

Table-5.47: A. niger was grown on mineral medium supplemented with 5 % citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 4.2±0.1 0.989±0.001 2.101±0.001 11.675±0.003 9.792±0.001 3.913±0.002

48 3.75±0.03 1.022±0.001 2.187±0.002 9.98±0.03 7.469±0.004 5.235±0.004

72 3.4±0.2 1.19±0.03 2.235±0.004 8.69±0.03 6.762±0.001 6.86±0.05

96 3.15±0.02 1.125±0.004 2.243±0.002 7.93±0.02 6.019±0.003 5.14±0.03

120 2.95±0.01 1.242±0.001 1.998±0.002 5.71±0.01 4.198±0.004 4.29±0.05

144 3.15±0.02 1.129±0.005 1.987±0.002 4.93±0.02 3.998±0.003 3.9±0.3

168 3.45±0.03 1.231±0.001 1.982±0.001 3.69±0.03 2.712±0.001 3.2±0.1

192 3.8±0.3 1.337±0.003 1.978±0.002 2.98±0.02 2.259±0.003 2.9±0.6

216 3.85±0.04 1.392±0.001 1.867±0.002 1.68±0.03 1.131±0.001 2.1±0.1

240 4.0±0.03 1.4±0.2 1.869±0.005 1.11±0.01 0.998±0.004 1.97±0.02

Table-5.48: M. geophillus was grown on mineral medium supplemented with

5 % citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 5.15±0.03 1.09±0.04 2.14±0.03 11.99±0.01 9.256±0.003 3.3±0.2

48 5.0±0.4 1.16±0.03 2.15±0.01 11.69±0.05 9.667±0.005 4.45±0.04

72 4.85±0.04 1.22±0.01 2.27±0.05 10.31±0.01 9.36±0.02 5.81±0.01

96 4.3±0.2 1.23±0.01 2.27±0.04 9.17±0.02 7.85±0.03 5.32±0.01

120 4.05±0.03 1.24±0.03 2.27±0.01 6.98±0.05 6.19±0.05 4.25±0.03

144 4.3±0.2 1.28±0.03 1.22±0.01 4.88±0.01 4.24±0.03 3.45±0.01

168 4.4±0.2 1.39±0.03 2.1±0.1 3.32±0.02 3.14±0.01 3. 15±0.02

192 4.45±0.02 1.54±0.02 2.1±0.2 2.69±0.08 2.16±0.03 2.85±0.05

216 4.55±0.05 1.57±0.02 1.99±0.04 1.82±0.01 1.13±0.02 1.97±0.01

240 5.5±0.1 1.58±0.04 1.98±0.02 1.1±0.1 0.98±0.03 1.59±0.06

110

Table-5.49: P. lilacinum was grown on mineral medium supplemented with 5 % citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 4.92±0.01 0.998±0.004 2.275±0.004 9.97±0.01 8.149±0.004 3.66±0.03

48 3.75±0.01 1.113±0.002 2.592±0.001 8.13±0.03 7.24±0.03 4.96±0.03

72 3.55±0.04 1.24±0.02 2.603±0.002 7.56±0.03 6.86±0.02 6.71±0.01

96 3.35±0.03 1.417±0.003 2.325±0.004 7. 26±0.02 6.75±0.03 5.45±0.01

120 3.0±0.2 1.498±0.002 2.291±0.001 6.87±0.01 5.48±0.02 4.81±0.01

144 2.8±0.3 1.518±0.002 2.264±0.002 4.89±0.04 3.353±0.002 3.21±0.03

168 2.95±0.02 1.632±0.002 2.249±0.002 4.16±0.02 3.167±0.002 2.84±0.03

192 3.3±0.2 1.819±0.002 2.437±0.005 3.6±0.3 2.13±0.02 2.23±0.02

216 3.55±0.03 1.861±0.001 2.218±0.004 1.72±0.01 1.32±0.02 1.98±0.02

240 3.8±0.1 1.911±0.003 2.125±0.002 1.32±0.02 1.1±0.1 1.27±0.01

Table - 5.50 shows the results of pectinase synthesis by different filamentous

fungi like A. fumigatus, (A. niger + A. fumigatus), A. niger, M. geophillus and P.

lilacinum.when grown on 2.5% and 5% of crude citrus pectin . Results show that

highest pectinase production (6.86 U/ml) and growth ( biomass 1.22 g/50ml )

was obtained after 72 of incubation period with A. niger and on a medium

supplemented with 5% crude citrus pectin. P. lilacinum produced pectinase 6.71

U/ml and biomass 1.24 g/50ml when grown on with 5% crude citrus pectin as

a sole carbon source at 30 + 2 ºC and pH was adjusted at 6.5. Pectinase activity

was reduced after 120 hours of incubation period, with the passage of time.

Naidu and Panda (1998) and Pedrolli et al., (2009) have reported that high

concentrations of carbon source create repression in the biosynthesis of enzymes.

111

Table-5.50: Effect on growth and pectinase production by different fungi when grown on mineral medium supplemented with 2.5 and 5% citrus pectin at

30 ± 2 ºC and pH was adjusted 6.5.

Filamentous Fungi

Biomass g/50 ml Broth




2.5% Crude citrus pectin 5% Crude citrus pectin

A. fumigatus 1.31±0.01 5.4±0.2 1.31±0.02 6.15±0.02

A. niger and

A. fumigatus 1.11±0.01 5.17±0.03 1.26±0.01 5.97±0.01

A. niger 1.225±0.002 6.57±0.03 1.19±0.03 6.86±0.02

M. geophillus 1.28±0.07 4.65±0.03 1.22±0.01 5.81±0.01

P. lilacinum 1.27±0.01 6.35±0.02 1.24±0.03 6.71±0.03

Table- 5.51 to 5.53 reveal the activity of pectinase obtained after 72 hours 6.47

U/ml, 6.12 U /ml , 7.36 U/ml respectively by A. fumigatus, mixed culture of A.

niger + A. fumigatus and A. niger when grown on a medium supplemented with

2.5 % pure citrus pectin at 30 + 2 ºC and pH was adjusted at 6.5. Tables 5.54 and

5.55 shows the results of pectinase synthesis by M. geophillus and P. lilacinum

when inoculated on a culture medium incorporated with 2.5% citrus pectin as a

carbon source. The maximum production of pectinase was achieved 6.27 U/ml

; 7.14 U / ml after 72 hours respectively, and then started to decrease with the

increase of incubation time period .The results show the amount of pectinase

produced by P. lilacinum having similarity with the results of A. niger produced

more or less same amount of pectinase. The concentration of total sugars,

reducing sugars and total protein were decreased with the increase of

fermentation time period. pH was also changed with increase of fermentation

time it becomes basic after 240 hours.

112

Table-5.51: A. fumigatus was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 + 2 ºC and pH was

adjusted at 6.5

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 4.3±0.2 0.955±0.007 1.898±0.004 10.165±0.002 8.878±0.005 4.71±0.03

48 4.0±0.4 0.991±0.004 1.9055±0.0002 9.893±0.006 8.134±0.003 5.12±0.04

72 3.85±0.03 1.154±0.005 2.0708±0.0004 8.819±0.003 7.513±0.002 6.47±0.05

96 3.35±0.04 1.219±0.006 2.183±0.004 7.66±0.02 6.554±0.004 5.31±0.07

120 3.1±0.01 1.271±0.004 2.2429±0.0006 6.786±0.003 6.209±0.006 3.97±0.05

144 3.35±0.03 1.323±0.004 2.2599±0.0005 5.329±0.004 4.983±0.002 3.58±0.04

168 3.45±0.02 1.365±0.003 2.3186±0.0004 4.965±0.002 3.8252±0.0004 2.75±0.06

192 3.5±0.5 1.422±0.004 1.952±0.003 4.279±0.0004 3.749±0.005 2.7±0.2

216 3.54±0.06 1.45±0.06 1.948±0.004 2.416±0.003 1.21420±0.0002 2.62±0.03

240 3.7±0.4 1.549±0.002 1.877±0.006 0.788±0.006 0.569±0.006 1.87±0.02

Table-5.52: A mixed culture of A. fumigatus + A. niger was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 + 2 ºC


Time Hours



Total Sugar (mg/ml)



24 3.85±0.02 0.996±0.001 1.056±0.003 11.16±0.02 8.83±0.04 4.87±0.06

48 3.5±0.3 1.07±0.04 1.159±0.004 10.35±0.06 8.27±0.06 5.25±0.03

72 3.35±0.04 1.16±0.03 1.236±0.005 9.37±0.05 7.67±0.04 6.23±0.02

96 3.15±0.02 1.27±0.05 1.282±0.003 7.98±0.05 6.48±0.02 6.12±0.05

120 2.8±0.4 1.29±0.04 1.210±0.04 5.34±0.03 5.69±0.03 5.89±0.06

144 2.9±0.4 1.31±0.03 1.191±0.003 5.19±0.06 4.8±0.4 3.63±0.05

168 3.00±0.2 1.33±0.02 1.174±0.004 3.98±0.04 3.18±0.03 3.57±0.04

192 3.45±0.04 1.37±0.03 1.166±0.004 2.93±0.06 2.55±0.05 1.97±0.05

216 3.5±0.2 1.36±0.03 1.153±0.002 1.99±0.02 1.41±0.04 1.35±0.02

240 4.05±0.04 1.3±0.2 1.139±0.007 1.037±0.004 0.94±0.05 1.11±0.04

113

Table-5.53: A. niger was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 + 2 ºC and pH was

adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 3.95±0.06 0.899±0.004 2.1±0.1 9.74±0.03 8.54±0.02 5.67±0.04

48 3.4±0.3 1.116±0.003 2.11±0.04 9.14±0.05 8.37±0.06 6.43±0.03

72 3.35±0.03 1.182±0.003 2.13±0.07 8.49±0.04 7.94±0.03 7.36±0.03

96 3.5±0.4 1.198±0.004 2.18±0.04 6.67±0.04 6.13±0.03 6.89±0.07

120 3.95±0.07 1.256±0.001 2.21±0.02 6.33±0.03 6.04±0.01 4.97±0.02

144 4.5±0.3 1.204±0.002 2.25±0.03 5.62±0.04 4.74±0.02 4.83±0.03

168 4.25±0.01 1.281±0.004 2.28±0.05 4.291±0.003 3.45±0.05 3.89±0.04

192 4.4±0.2 1.322±0.004 2.28±0.03 3. 25±0.02 2.87±0.02 3.86±0.02

216 4.47±0.06 1.416±0.003 2.27±0.02 2.49±0.06 1.65±0.03 2.85±0.04

240 4.85±0.05 1.508±0.004 2.63±0.03 0.98±0.04 0.67±0.05 2.27±0.03

Table-5.54: M. geophillus was grown on mineral medium supplemented with 2.5 % CCP (commercial citrus pectin) at 30 + 2 ºC and pH was

adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 5.4±0.3 1.542±0.003 1.067±0.002 13.98±0.03 11.179±0.002 4.58±0.03

48 5.9±0.5 1.442±0.001 1.179±0.006 9.536±0.004 7.329±0.004 5.97±0.04

72 6.1±0.5 1.367±0.003 1.197±0.001 5.212±0.003 4.887±0.004 6.27±0.06

96 6.45±0.03 1.411±0.003 1.229±0.002 4.791±0.004 3.939±0.003 5.67±0.02

120 6.15±0.04 1.487±0.004 1.263±0.003 3.89±0.03 2.98±0.02 4.14±0.01

144 6.5±0.2 1.527±0.002 1.286±0.004 3.584±0.001 2. 58±0.02 3.75±0.02

168 6.35±0.02 1.552±0.002 1.337±0.002 2.846±0.002 1.976±0.002 2.541±0.002

192 6.3±0.2 1.587±0.003 0.995±0.001 2.22±0.02 1.696±0.003 1.96±0.03

216 7.1±0.4 1.592±0.002 0.976±0.002 1.99±0.04 1.579±0.004 1.765±0.003

240 7.3±0.3 1.599±0.003 0.964±0.001 0.97±0.04 0.773±0.003 1.577±0.002

114

Table-5.55: P. lilacinum was grown on mineral medium supplemented with 2.5% CCP (commercial citrus pectin) at 30 + 2ºC and pH was

adjusted at 6.5.

Time

Hours

Final pH Biomass

g/100 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 3.95±0.01 1.542±0.002 1.0236±0.003 9.2298±0.004 8.073±0.002 4.412±0.004

48 3.6±0.3 1.627±0.002 1.0202±0.003 6.858±0.003 5.949±0.004 5.485±0.004

72 3.75±0.03 1.637±0.004 1.154±0.001 4.505±0.004 4.772±0.002 7.14±0.01

96 4.00±0.2 1.663±0.003 1.176±0.001 4.263±0.001 3.199±0.006 5.158±0.003

120 4.2±0.2 1.674±0.002 1.213±0.003 3.848±0.003 3.978±0.007 4.963±0.003

144 4.5±0.3 1.697±0.002 1.324±0.001 3.727±0.004 3.098±0.002 3.827±0.002

168 4.85±0.02 1.752±0.002 1.346±0.003 2.568±0.003 2.163±0.001 2.886±0.005

192 5.4±0.3 1.827±0.004 1.395±0.001 1.971±0.001 1.668±0.003 2.484±0.004

216 5.95±0.03 1.887±0.003 1.425±0.005 1.301±0.001 0.569±0.003 1.972±0.004

240 7.00±0.1 1.894±0.001 1.582±0.001 0.723±0.003 0.391±0.001 1.259±0.007

The results of pectinase synthesis by A. fumigatus, (A. niger + A. fumigatus),

A. niger, M. geophillus and P. lilacinum are presented in Tables - 5.56 to 5.60 when

M.O grown in a culture medium with 5% CCP (commercial citrus pectin) as a

carbon source.The maximum production of pectinase 8.95 U / ml ; 8.76 U / ml ;

10.19 U /ml ; 8.92 U / ml and 10.1 U / ml respectively obtained after incubation

at 72 hours and then decreased with the increase of time period . A. niger and P.

lilacinum produced more or less the same amount of Pectinase, and reduction in

activity by pectin may be due to the presence of catabolic repression formed by the

high galacturonic acid concentration by pectin degradation (Aguilar and Huitron,

(1987); Maldonado and Callieri, (1989). Catabolic repression can occur not only due to

pectin degradation, but also due to the natural sugars attached to the pectin molecule.

Siddiqui et al., (2013 ) reported pure pectin as a best carbon source while working with

Rhizomucor pusillus , pectin as a good carbon source also reported by (Maria et al., 2002)

for Mucor sp., for Mucor ramosissimus Marques et al., ( 2006) and for Mucor circinelloides

Thakur et al. (2010) . Arijit et al., (2013) observed that maximum pectinase activity was

achieved by using pectin as a carbon source.

115

Table-5.56: A. fumigatus was grown on mineral medium supplemented with 5% CCP (commercial citrus pectin) at 30 + 2 ºC and pH was

adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 4.75±0.02 1.11±0.04 1.753±0.001 13.126±0.004 11.975±0.003 5.86±0.01

48 4.55±0.05 1.202±0.002 1.892±0.002 10.028±0.003 9.37±0.05 7.608±0.003

72 4.0±0.2 1.233±0.002 1.923±0.003 7.526±0.005 6.23±0.03 8.95±0.01

96 4.1±0.1 1.278±0.003 2.094±0.003 6.455±0.005 6.01±0.01 7.35±0.05

120 3.8±0.3 1.286±0.003 2.18±0.03 6.303±0.002 5.85±0.05 6.17±0.05

144 3.5±0.2 1.313±0.00 2.187±0.001 3.874±0.001 3.127±0.006 4.96±0.01

168 3.7±0.1 1.425±0.003 2.242±0.002 2.229±0.007 1.895±0.002 3.92±0.05

192 4.15±0.04 1.519±0.008 2.22±0.04 1.265±0.002 1.41±0.05 2.85±0.04

216 4.2±0.2 1.567±0.005 0.16±0.03 1.806±0.002 1.261±0.002 1.53±0.03

240 5.85±0.03 0.105±0.004 0.19±0.03 1.97±0.05 0.884±0.002 0.48±0.05

Table-5.57:A mixed culture of A. fumigatus + A. niger was grown on mineral medium supplemented with 5 % CCP (commercial citrus pectin) at 30 + 2 ºC and

pH was adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 4.2±0.2 0.99±0.04 1.64±0.03 11.9±0.5 9.18±0.04 6.64±0.05

48 4.1±0.3 1.19±0.02 1.964±0.001 9.42±0.04 8.32±0.02 7.28±0.03

72 4.0±0.3 1.29±0.02 2.168±0.003 9.83±0.03 7.46±0.03 8.76±0.01

96 5.15±0.04 1.30±0.07 2.281±0.005 6.47±0.01 5.93±0.03 7.1±0.1

120 5.45±0.02 1.29±0.04 2.285±0.005 4.62±0.02 4.15±0.02 6.87±0.05

144 5.5±0.3 1.39±0.03 2.172±0.004 3.57±0.02 3.44±0.01 5.97±0.02

168 5.25±0.04 1.4±0.1 2.164±0.002 2.86±0.05 2.73±0.01 3.26±0.03

192 5.1±0.1 1.44±0.04 2.089±0.004 2.56±0.03 2.42±0.04 2.62±0.02

216 4.9±0.4 1.49±0.07 2.039±0.007 1.48±0.03 1.34±0.03 1.55±0.04

240 4.4±0.2 1.56±0.01 2.015±0.005 0.94±0.01 0.67±0.03 0.85±0.02

116

Table-5.58: A. niger was grown on mineral medium supplemented with 5 % CCP (commercial citrus pectin) at 30 + 2 ºC and pH was

adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 5.5±0.3 1.078±0.002 1.219±0.004 11.896±0.003 9.987±0.005 7.83±0.02

48 4.85±0.02 1.125±0.002 1.233±0.003 9.978±0.004 8.358±0.005 8.64±0.01

72 4.15±0.02 1.249±0.005 1.317±0.004 7.62±0.02 6.986±0.004 10.06±0.04

96 5.85±0.01 1.298±0.005 1.385±0.003 6.541±0.003 5.987±0.001 8.31±0.04

120 5.4±0.1 1.313±0.003 1.382±0.004 4.868±0.002 4.119±0.002 7.35±0.06

144 5.75±0.07 1.356±0.006 1.392±0.007 4.60±0.03 3.86±0.01 5.03±0.02

168 6.25±0.06 1.387±0.005 1.376±0.003 3.766±0.003 3.319±0.006 4.76±0.01

192 5.0±0.2 1.391±0.001 1.265±0.004 2.894±0.003 2.316±0.005 3.49±0.03

216 4.4±0.2 1.411±0.005 1.263±0.003 1.982±0.002 1.345±0.005 3.51±0.01

240 5.8±0.1 1.523±0.002 1.252±0.004 0.914±0.001 0.772±0.001 2.75±0.02

Table-5.59 : M. geophillus was grown on mineral medium supplemented with 5 % CCP (commercial citrus pectin) at 30 + 2 ºC and pH was

adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 4.9±0.2 1.022±0.003 1.19±0.06 12.03±0.04 10.80±0.04 6.98±0.03

48 4.7±0.2 1.19±0.04 1.72±0.04 10.90±0.02 8.72±0.02 7.1±0.1

72 6.4±0.1 1.213±0.003 1.79±0.04 6.17±0.04 5.97±0.04 8.92±0.01

96 6.3±0.1 1.134±0.004 1.790±0.007 5.8±0.05 4.57±0.05 7.14±0.03

120 5.5±0.3 1.285±0.002 1.783±0.003 4.117±0.010 3.949±0.001 6.28±0.06

144 3.2±0.1 1.305±0.003 1.83±0.05 3.116±0.004 3.461±0.004 4.96±0.03

168 5.85±0.05 1.335±0.004 1.8±0.3 3.985±0.005 3.127±0.005 4.6±0.5

192 6±0.2 1.413±0.005 1.721±0.004 2.863±0.02 2.23±0.02 3.0±0.3

216 6.1±0.1 1.449±0.008 1.714±0.001 1.982±0.03 1.56±0.03 2.2±0.1

240 5.9±0.3 1.510+0.004 1.68±0.03 0.869±0.002 0.397±0.002 1.8±0.2

117

Table-5.60: P. lilacinum was grown on mineral medium supplemented with 5 % CCP (commercial citrus pectin) at 30 + 2 ºC and pH was

adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 4.1±0.1 1.085±0.003 1.737±0.005 11.76±0.03 9.94±0.01 7.985±0.001

48 4.3±0.2 1.11±0.01 1.84±0.03 11.2±0.1 8.76±0.06 8.83±0.05

72 4.5±0.2 1.135±0.004 1.816±0.003 9.85±0.01 8.36±0.06 9.87±0.1

96 4.35±0.05 1.281±0.003 1.874±0.001 8.931±0.004 8.13±0.03 7.91±0.04

120 4.75±0.03 1.209±0.003 1.784±0.002 6.69±0.04 6.35±0.01 7.57±0.04

144 5.2±0.2 1.295±0.002 1.775±0.006 5.58±0.01 5.143±0.003 6.13±0.02

168 5.0±0.3 1.355±0.001 1.767±0.006 3.67±0.04 3.19±0.02 4.33±0.02

192 4.8±0.5 1.395±0.003 1.728±0.006 2.41±0.03 2.12±0.03 3.19±0.04

216 4.65±0.01 1.485±0.001 1.633±0.007 1.40±0.06 1.18±0.03 2.85±0.05

240 4.38±0.03 1.548±0.004 1.612±0.003 0.831±0.002 0.65±0.04 1.16±0.02

Table-5.61 shows the results of pectinase synthesis by different filamentous fungi

like A. fumigatus, (A. niger + A. fumigatus ), A. niger, M. geophillus and P.lilacinum

when grown on 2.5% and 5% of CCP (commercial citrus pectin) incorporated

with mineral medium as carbon source. The highest Pectinase activity and

growth were obtained in comparison to other organisms by A. niger 10.06 U/ml

and 1.249 g/50 ml and P. lilacinum 9.87 U/ml and 1.135 g/50 ml respectively.

The results are in agreement with Akhilesh et al., (2010) and Favela-Torres et al.,

(2006) indicate that pectin can induce the polygalacturonase activity.

118

Table-5.61:Effect on growth and pectinase production by different fungi when grown on mineral medium supplemeted with 2.5 and 5% CCP (commercial

citrus pectin) at 30 + 2 ºC and pH was adjusted at 6.5. Filamentous Fungi

Biomass g/50ml Broth




2.5% Synthetic pectin 5% Synthetic pectin

A. fumigatus 1.154±0.003 6.47±0.004 1.233±0.005 8.9±0.2

A. niger+ A. fumigatus

1.16±0.03 6.12±0.001 1.198±0.005 8.76±0.03

A. niger 1.182±0.004 7.36±0.002 1.249±0.006 10.06±0.01

M. geophillus 1.367±0.001 6.27±0.002 1.213±0.002 8.92±0.01

P. lilacinum 1.637±0.006 7.142±0.0001 1.135±0.005 9.87±0.1

v- Effect of sugars as carbon sources:

In concern with the use of molasses which is an agro-industrial

byproduct in the present study suggested high pectinase production by

Aspergillus niger and Penicillium lilacinum with molasses as sole carbon source. It

is economic to use molasses for the production of pectinase as compared to pure

pectin. In Pakistan molasses is a byproduct of sugar industry and it is used to

produce ethanol; however it can be successfully used in enzyme production. Its

careless dumping in nature can causes environmental pollution, hence it can be

eco-friendly used as a good substrate for enzyme production. After selection of

molasses as a carbon source different sugars were added to molasses to

investigate their effect as a carbon source with selected microorganisms i-e A.

niger and P. lilacinum

119

Fig # 5.10 Comparison of pectinase production by different organisms grown on

5 % Molasses as a carbon source.

Tables -5.63 and 5.64 show the results of pectinase synthesis by A. niger when

grown on mineral medium containing (2.5 % and 5%) fructose and 5% molasses

as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. The maximum

production of pectinase 12.00 U/ml and 13.89 U/ ml respectively, were obtained

at 72 hours. The concentration of total sugars, reducing sugars and total protein

was decreased with the increase of the fermentation time period. Our results are

supported by previous work as reported that polygalacturonase production in

Geotrichum candidum (Shastri et at., 1988).

Tables-5.64 and 5.65 represents the production of pectinase by P. lilacinum

grown on mineral medium containing (2.5% and 5%) fructose and 5% molasses

incubated at 30 ± 2 ºC and the pH was adjusted at 6.5. The higher amount of

pectinase was produced by P. lilacinum 10.73 U/ml and 12.11 U/ml respectively.

pectinase production by A. niger, A. alliaceus, Geotrichum lactis, Neurospora crassa,

induced by fructose ( McKay, 1988; Pardo et al., 1991).

0

2

4

6

8

10

12

Pect

ina

se A

ctiv

ity

U/

ml

120

Table-5.62: A. niger was grown on a mineral medium supplemented with 2.5%

fructose and 5% molasses as carbon source at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar (mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

U/ml

24 5.3±0.2 0.879±0.008 1.591±0.006 14.154±0.003 13.078±0.003 5.53±0.01

48 5±0.2 0.879±0.001 1.599±0.003 12.463±0.003 11.197±0.004 6.96±0.01

72 4.85±0.05 0.897±0.005 1.607±0.003 10.128±0.003 9.625±0.005 12.00±0.2

96 4.35±0.02 0.996±0.001 1.686±0.003 8.783±0.002 5.513±0.002 7.17±0.04

120 4.1±0.1 1.137±0.004 1.612±0.004 5.0375±0.0006 3.202±0.001 5.96±0.03

144 4.35±0.06 1.269±0.004 1.599±0.005 3.098±0.005 2.918±0.005 4.53±0.01

168 4.45±0.03 1.365±0.007 1.573±0.003 1.987±0.001 1.825±0.002 3.48±0.04

192 4.5±0.2 1.452±0.005 1.567±0.002 1.882±0.004 1.862±0.001 3.06±0.04

216 4.5±0.4 1.464±0.001 1.556±0.002 1.339±0.002 1.214±0.003 2.95±0.02

240 4.7±0.2 1.517±0.004 1.544±0.001 1.196±0.005 1.059±0.002 1.62±0.01

Table-5.63: A. niger was grown on a mineral medium supplemented with 5%

fructose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50ml Broth

Total

Proteins (mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity U/ml

24 5.2±0.1 0.875±0.002 1.768±0.004 17.997±0.001 15.169±0.004 6.05±0.04

48 4.45±0.02 0.937±0.003 1.688±0.003 12.978±0.006 9.919±0.002 6.59±0.06

72 4.7±0.1 1.192±0.005 1.726±0.005 9.988±0.005 7.251±0.001 13.89±0.04

96 4.7±0.3 1.115±0.009 1.687±0.004 7.842±0.001 6.159±0.002 5.87±0.06

120 5.45±0.05 1.138±0.003 1.677±0.001 6.580±0.004 5.079±0.002 4.67±0.04

144 5.75±0.07 2.285±0.002 1.638±0.002 4.782±0.003 3.487±0.004 4.13±0.02

168 6.35±0.02 1.318±0.003 1.616±0.003 4.017±0.004 3.102±0.001 3.72±0.02

192 6.4±0.2 1.367±0.004 1.524±0.004 3.603±0.001 2.276±0.005 2. 05±0.03

216 6.65±0.03 1.466±0.001 1.513±0.002 2.17±0.04 1.395±0.006 1.28±0.03

240 6.9±0.1 1.592±0.002 1.484±0.001 1.037±0.006 0. 952±0.005 1.16±0.03

121

Table-5.64: P. lilacinum was grown on a mineral medium supplemented with 2.5% fructose and 5% molasses as carbon source at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ ml)

Pectinase

Activity (U/ ml)

24 5.15±0.02 1.025±0.004 1.47±0.0005 16.899±0.006 14.165±0.0002 6.14±0.03

48 5.0±0.1 1.317±0.004 1.598±0.001 13.395±0.005 11.45±0.001 8.52±0.02

72 4.85±0.02 1.365±0.002 1.706±0.003 9.501±0.001 7.083±0.005 10.73±0.02

96 4.2±0.1 1.421±0.004 1.741±0.003 8.894±0.003 6.385±0.0003 7.03±0.02

120 4.15±0.01 1.592±0.006 1.755±0.003 7.825±0.002 5.67±0.05 6.32±0.04

144 4.3±0.4 1.87±0.04 1.76±0.0003 5.778±0.002 4.326±0.001 5.87±0.04

168 4.4±0.1 2.196±0.004 1.799±0.006 3.351±0.003 2.81±0.003 5.22±0.05

192 4.45±0.07 2.24±0.01 1.816±0.003 2.259±0.004 1.83±0.003 3.85±0.01

216 4.6±0.3 2.48±0.02 1.803±0.002 1.627±0.005 1.50±0.001 2.29±0.04

240 5.8±0.1 2.512±0.001 1.76±0.04 1.076±0.002 0.917±0.001 2.02±0.01

Table-5.65: P. lilacinum was grown on a mineral medium supplemented with 5%

fructose and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 4.7±0.3 1.015±0.004 1.566±0.003 17.458±0.003 1.623±0.002 4.94±0.01

48 4.8±0.3 1.225±0.002 1.629±0.004 15.842±0.002 12.766±0.003 5.94±0.07

72 5.75±0.02 1.339±0.002 1.723±0.002 13.541±0.004 11.528±0.003 12.11±0.06

96 5.2±0.1 1.371±0.003 1.784±0.002 9.875±0.002 8.176±0.001 8.58±0.02

120 5.8±0.3 1.386±0.003 1.684±0.003 7.634±0.003 6.943±0.002 7.85±0.04

144 6.05±0.03 1.391±0.004 1.678±0.002 5.917±0.002 3.395±0.002 5.74±0.05

168 6.15±0.04 1.403±0.002 1.615±0.002 3.257±0.002 3.146±0.002 4.87±0.04

192 6.3±0.2 1.434±0.003 1.607±0.002 3.171±0.003 2.68±0.03 3.04±0.03

216 5.85±0.06 1.497±0.004 1.598±0.005 1.998±0.007 1.518±0.005 3.00±0.2

240 6.15±0.02 1.529±0.004 1.591±0.001 1.319±0.002 0.792±0.002 1.62±0.05

122

Tables–5.66 to 5.69 show the results of pectinase synthesis by A. niger and P.

lilacinum were grown on mineral medium containing 2.5% and 5% maltose and

5% molasses as carbon source at 30 ± 2 ºC and the pH was adjusted at 6.5.The

maximum production of pectinase achieved 12.00 U/ml and 12.75U/ml by A.

niger and 11.04 U/ml and 11.20 U/ml by P. lilacinum respectively. Pectinase

production increased with fermentation duration up to 72 hours beyond that, the

production of the enzymes decreased gradually may be due to the depletion of

nutrients. Singh and Mandal (2012) reported that fermentation time period

depends on the growth rate of the microorganism and its pattern of enzyme

synthesis.

Table-5.66: A.niger was grown on mineral medium supplemented with 2.5% maltose and 5% molasses as carbon source at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 5.7±0.2 1.129±0.003 1.647±0.004 17.514±0.003 14.179±0.003 6.30±0.07

48 4.6±0.4 1.285±0.005 1.701±0.001 15.298±0.003 13.465±0.002 7.15±0.04

72 4.5±0.2 1.31±0.04 1.739±0.002 12.743±0.002 9.158±0.004 12.00±0.5

96 4.35±0.02 1.425±0.001 1.743±0.002 10.151±0.003 8.916±0.003 8.31±0.02

120 4.4±0.3 1.525±0.004 1.751±0.003 7.033±0.002 5.958±0.003 5.32±0.06

144 4.5±0.2 1.58±0.04 1.774±0.001 6.913±0.002 5.165±0.004 4.85±0.02

168 4.8±0.3 1.625±0.002 1.771±0.003 5.952±0.004 4.179±0.006 4.40±0.03

192 5.05±0.03 1.63±0.05 1.759±0.002 4.138±0.001 3.509±0.004 3.44±0.01

216 5.25±0.04 1.644±0.003 1.717±0.003 2.496±0.003 1.692±0.001 2.31±0.04

240 5.5±0.1 1.753±0.003 1.696±0.003 1.312±0.001 0.866±0.003 1 .13±0.02

123

Table-5.67: A.niger was grown on mineral medium supplemented with 5% maltose and 5% molasses as carbon source at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 5.4±0.3 1.098±0.005 1.622±0.005 19.482±0.006 14.795±0.001 7.32±0.01

48 5.05±0.02 1.206±0.0004 1.695±0.002 16.355±0.004 13.215±0.007 8.20±0.03

72 4.85±0.01 1.409±0.004 1.791±0.006 12.734±0.001 10.198±0.003 12.75±0.03

96 4.4±0.3 1.568±0.0003 1.786±0.003 11.706±0.004 9.165±0.001 10.21±0.03

120 3.85±0.06 1.568±0.0003 1.769±0.002 8.761±0.0004 6.169±0.007 7.20±0.07

144 3.55±0.02 1.626±0.0007 1.765±0.001 6.656±0.003 5.417±0.004 7.02±0.01

168 3.41±0.04 1.631±0.004 1.75±0.04 4.927±0.0003 3.242±0.002 6.90±0.04

192 3.5±0.4 1.656±0.003 1.851±0.003 3. 615±0.002 2.269±0.004 4.18±0.03

216 3.6±0.2 1.639±0.003 1.825±0.002 1.869±0.004 1.197±0.004 2.92±0.01

240 3.9±0.4 1.617±0.004 1.806±0.001 1.476±0.002 0.875±0.001 2.01±0.01

Table-5.68: P. lilacinum was grown on mineral medium supplemented with 2.5%

maltose and 5% molasses at 30 ± 2 ºC and pH was adjusted at 6.5.

Time Hours



Total Sugar (mg/ml)



24 4.90±0.04 1.105±0.004 1.10±0.03 17.618±0.003 13.439±0.003 5.44±0.03

48 4.88±0.03 1.162±0.002 1.11±0.07 15.429±0.006 13.534±0.003 6.85±0.07

72 4.82±0.04 1.205±0.001 1.125±0.004 12.858±0.002 9.180±0.0003 11.04±0.02

96 4.74±0.01 1.273±0.001 1.143±0.005 10.995±0.002 7.034±0.0005 8.07±0.02

120 4.45±0.05 1.336±0.005 1.164±0.001 8.935±0.004 5.454±0.0002 5.427±0.006

144 4.4±0.2 1.355±0.003 1.174±0.006 7.448±0.004 5.240±0.0002 5.17±0.04

168 4.55±0.07 1.425±0.002 1.181±0.005 5.216±0.003 3.816±0.003 5.91±0.04

192 4.85±0.02 1.483±0.005 1.185±0.002 4.737±0.002 2.448±0.001 4.47±0.04

216 5.05±0.03 1.498±0.005 1.182±0.006 1.447±0.004 1.065±0.003 2.83±0.04

240 5.20±0.03 1.561±0.005 1.18±0.03 0.869±0.006 0.703±0.002 1.13±0.02

124

Table-5.69: P. lilacinum was grown on mineral medium supplemented with 5% maltose and 5% molasses as carbon source at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time

Hours

Final

pH

Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 4.6±0.1 1.11±0.05 1.30±0.06 23.38±0.06 22.83±0.01 6.02±0.01

48 4.5±0.4 1.12±0.01 1.28±0.07 21.05±0.03 18.02±0.01 7.47±0.04

72 4.2±0.1 1.125±0.002 1.29±0.02 17.70±0.04 16.76±0.03 11.20±0.07

96 3.85±0.03 1.13±0.08 1.29±0.04 13.61±0.04 12.72±0.01 8.12±0.03

120 3.65±0.01 1.18±0.02 1.29±0.07 11.47±0.06 9.93±0.05 7.32±0.01

144 3.25±0.04 1.36±0.05 1.30±0.01 9.30±0.02 8.86±0.04 6.51±0.05

168 4.15±0.02 1.44±0.04 1.30±0.04 6.54±0.03 5.457±0.005 5.11±0.06

192 4.45±0.06 1.47±0.04 1.31±0.06 5.17±0.04 4.70±0.03 3.68±0.05

216 4.5±0.2 1.53±0.02 1.35±0.02 3.86±0.04 3.44±0.02 2.64±0.02

240 4.15±0.04 1.55±0.01 1.40±0.03 2.47±0.02 2.12±0.01 1.27±0.03

Tables-5.70 to 5.73 show the results of pectinase synthesis by A. niger and

P. lilacinum when grown on mineral medium containing 2.5% and 5% sucrose

and 5% molasses as carbon source at 30 ± 2 ºC and the pH was adjusted at 6.5.

The maximum production of pectinase was achieved 13.66 U/ml and 16.16 U/ml

by A.niger but 12.29 U/ml and 15.54 U/ml by P.lilacinum respectively after

incubation of 72 hours. By A. niger and then its concentration was decreased

with the increase of the time period. The present study is in full agreement with

Tariq and Reyaz ( 2012), Reda et al., (2008) and Solis-Pereyra et al., (1993), who

reported that the presence of sucrose increases pectinase production. Similar

results were presented by Hoa and Hung (2013).

125

Table-5.70: A.niger was grown on mineral medium supplemented with 2.5 % sucrose and 5 % molasses as carbon source at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 5.40±0.04 0.93±0.04 1.56±0.05 17.24±0.01 10.47±0.04 7.82±0.04

48 4.95±0.04 1.05±0.03 1.62±0.04 15.80±0.04 10.41±0.04 8.04±0.02

72 4.45±0.01 1.18±0.05 1.62±0.02 12.32±0.02 9.25±0.04 13.66±0.03

96 4.35±0.01 1.29±0.03 1.62±0.07 9.54±0.03 7.27±0.04 7.39±0.04

120 3.75±0.06 1.24±0.01 1.63±0.02 8.18±0.03 5.93±0.06 7.30±0.08

144 3.85±0.03 1.35±0.04 1.62±0.04 5.36±0.05 3.99±0.04 6.17±0.04

168 3.65±0.01 1.36±0.02 1.58±0.01 4.79±0.03 3.12±0.02 5.95±0.01

192 3.45±0.02 1.48±0.06 1.55±0.07 3.84±0.06 2.16±0.03 3.40±0.04

216 3.15±0.07 1.55±0.02 1.48±0.04 1.98±0.03 1.13±0.02 2.13±0.01

240 3.10±0.06 1.58±0.08 1.48±0.06 1.02±0.01 0.87±0.01 1.23±0.02

Table-5.71: A.niger was grown on mineral medium supplemented with 5% sucrose and 5% molasses as carbon source at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 3.75±0.04 0.84±0.01 1.46±0.03 20.73±0.01 12.33±0.05 6.54±0.03

48 4.05±0.02 1.14±0.03 1.51±0.04 18.23±0.03 11.97±0.06 8.22±0.05

72 3.5±0.3 1.25±0.07 1.58±0.03 15.15±0.02 11.49±0.01 16.16±0.03

96 3.25±0.08 1.30±0.04 1.64±0.01 12.84±0.02 8.07±0.02 10.56±0.04

120 2.85±0.07 1.32±0.06 1.62±0.05 9.50±0.03 6.19±0.04 7.04±0.03

144 4.15±0.02 1.33±0.1 1.57±0.02 7.25±0.02 4.70±0.07 6.29±0.01

168 3.85±0.02 1.48±0.03 1.53±0.01 6.85±0.04 4.23±0.01 5.13±0.08

192 3.75±0.01 1.55±0.02 1.53±0.06 4.53±0.04 3.09±0.04 3.93±0.06

216 3.7±0.2 1.59±0.02 1.52±0.01 3.26±0.04 2.81±0.05 1.27±0.04

240 3.95±0.04 1.61±0.01 1.51±0.07 1.28±0.01 0.73±0.02 1.18±0.03

126

Table-5.72: P. lilacinum was grown on mineral medium supplemented with 2.5% sucrose and 5% molasses as carbon source at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time Hours


Broth

Total Protein

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 4.8±0.3 1.00±0.3 1.20±0.05 17.91±0.04 11.30±0.05 7.61±0.04

48 5.4±0.2 1.18±0.05 1.58±0.06 16.12±0.02 10.99±0.08 8.04±0.02

72 4.85±0.04 1.23±0.01 1.68±0.03 14.94±0.03 9.36±0.05 12.20±0.07

96 4.75±0.07 1.32±0.05 1.67±0.01 12.28±0.03 8.96±0.03 7.04±0.03

120 4.15±0.02 1.46±0.03 1.57±0.04 9.09±0.03 6.66±0.04 6.15±0.06

144 4.45±0.01 1.49±0.04 1.55±0.01 7.48±0.05 4.07±0.02 5.64±0.02

168 4.75±0.06 1.51±0.02 1.55±0.06 5.23±0.06 5.15±0.02 5.43±0.02

192 5.05±0.02 1.52±0.01 1.53±0.02 4.75±0.04 3.56±0.02 2.93±0.05

216 5.25±0.04 1.53±0.06 1.52±0.02 2.92±0.06 1.53±0.05 2.35±0.03

240 5.35±0.02 1.55±0.02 1.44±0.02 1.47±0.05 1.02±0.01 1.74±0.01

Table-5.73: P. lilacinum grown on mineral medium supplemented with 5% sucrose and 5% molasses as carbon source at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time Hours


Total Protein (mg/ml)

Total Sugar (mg/ml)



24 4.8±0.2 0.94±0.07 1.65±0.03 28.87±0.05 13.67±0.04 6.97±0.05

48 4.15±0.02 1.03±0.02 1.67±0.02 22.14±0.03 21.87±0.02 8.90±0.07

72 3.85±0.07 1.30±0.05 1.68±0.04 16.15±0.06 15.08±0.04 15.54±0.04

96 3.65±0.01 1.32±0.01 1.68±0.07 12.62±0.05 11.53±0.05 9.80±0.02

120 3.5±0.1 1.34±0.08 1.69±0.01 10.31±0.02 9.55±0.02 7.63±0.01

144 3.6±0.3 1.38±0.03 1.63±0.03 8.88±0.06 8.59±0.03 6.56±0.03

168 3.75±0.01 1.54±0.06 1.61±0.03 6.42±0.03 6.41±0.05 5.63±0.06

192 3.95±0.02 1.56±0.04 1.58±0.06 3.89±0.03 2.97±0.04 2.06±0.04

216 4.3±0.1 1.57±0.06 1.57±0.04 3.15±0.02 2.62±0.01 1.46±0.04

240 4.15±0.07 1.58±0.02 1.53±0.05 1.59±0.03 1.07±0.02 1.19±0.04

The pH values fluctuate during fermentation and remain acidic. Pectinase synthesis

by A.niger when grown on mineral medium containing 2.5% and 5% galactose and

5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5. Tables -5.74

and 5.75 clearly show that when A.niger was grown on above described cultural

conditions at 72 h incubation showed the maximum pectinase activity 12.22 and

127

13.98 U/ml respectively. Both tables -5.76 and 5.77 show more or less same trend for

pectinase production by P.lilacinum in the presence of the same substrate. Galactose

induced pectinase production in Bacteroides ovatus rather than pectin as reported by

Macfarlane et al., (1990). The observation indicates the constitutive nature of the

enzymes and also the stimulating capacity of carbon source for the production of

pectinase enzymes, and also varies from strain to strain (Ramachandran and Kurup,

2013).

Table-5.74: A. niger was grown on mineral medium supplemented with 2.5% galactose and 5% molasses as carbon source at 30 ± 2 ºC and


Time Hours



Total Sugar (mg/ml)



24 4.35±0.06 0.98±0.05 1.61±0.03 16.13±0.02 13.26±0.05 6.19±0.06

48 4.2±0.01 0.99±0.04 1.49±0.03 15.77±0.04 12.75±0.02 6.95±0.03

72 3.65±0.01 1.11±0.05 1.47±0.04 13.52±0.04 10.90±0.05 12.22±0.07

96 3.85±0.03 1.12±0.03 1.44±0.03 8.19±0.02 6.41±0.04 9.19±0.05

120 3.65±0.02 1.13±0.06 1.46±0.02 5.96±0.04 4.33±0.02 8.27±0.04

144 3.45±0.02 1.14±0.03 1.44±0.01 4.84±0.05 3.24±0.01 6.24±0.03

168 3.15±0.04 1.18±0.03 1.43±0.05 4.10±0.05 3.04±0.01 4.08±0.06

192 2.8±0.2 1.18±0.03 1.42±0.04 3.33±0.01 2.38±0.04 2.82±0.04

216 3.2±0.2 1.20±0.07 1.40±0.05 2.86±0.01 2.02±0.01 2.66±0.01

240 3.6±0.3 1.23±0.02 1.36±0.03 1.38±0.07 0.65±0.02 1.84±0.04

Table-5.75: A niger was grown on mineral medium supplemented with 5% galactose

and 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 5.00±0.3 1.08±0.05 1.57±0.02 17.95±0.02 14.69±0.04 4.167±0.004

48 5.25±0.02 1.12±0.01 1.72±0.02 14.95±0.04 11.196±0.003 8.69±0.02

72 5.05±0.01 1.14±0.05 1.31±0.05 11.78±0.03 8.95±0.04 13.98±0.05

96 4.9±0.4 1.21±0.06 1.36±0.04 10.93±0.04 7.94±0.04 9.92±0.03

120 2.95±0.02 1.26±0.04 1.43±0.04 9.24±0.04 6.72±0.05 8.93±0.06

144 2.65±0.07 1.27±0.04 1.44±0.02 7.73±0.01 5.20±0.05 6.52±0.02

168 3.4±0.1 1.36±0.01 1.45±0.05 5.01±0.01 3.93±0.02 5.07±0.01

192 3.65±0.03 1.39±0.08 1.51±0.03 4.28±0.05 2.92±0.06 2.39±0.08

216 6.1±0.1 1.47±0.04 1.10±0.06 2.44±0.03 2.02±0.01 2.34±0.01

240 6.2±0.1 1.47±0.01 1.43±0.06 1.18±0.03 0.83±0.05 1.13±0.02

128

Table-5.76: P. lilacinum was grown on mineral medium supplemented with 2.5% galactose and 5% molasses as carbon source at 30 ± 2 ºC and


Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 4.00±0.3 0.976±0.003 1.21±0.08 17.19±0.02 13.68±0.03 5.51±0.03

48 4.45±0.01 0.99±0.08 1.27±0.02 16.94±0.03 13.48±0.04 8.64±0.03

72 4.15±0.02 1.22±0.05 1.39±0.02 15.41±0.04 12.27±0.06 11.14±0.01

96 3.9±0.2 1.22±0.03 1.40±0.06 12.46±0.03 10.20±0.05 7.16±0.05

120 3.65±0.04 1.24±0.02 1.40±0.03 9.80±0.02 8.68±0.06 7.11±0.04

144 3.75±0.02 1.25±0.04 1.42±0.02 8.30±0.06 6.27±0.05 6.07±0.04

168 3.3±0.3 1.26±0.02 1.43±0.01 5.46±0.04 4.13±0.02 5.86±0.04

192 2.95±0.06 1.42±0.02 1.44±0.05 4.07±0.04 3.88±0.05 2.69±0.06

216 2.65±0.04 1.47±0.04 1.44±0.01 1.54±0.03 1.27±0.02 1.87±0.04

240 2.85±0.02 1.51±0.02 1.45±0.04 1.08±0.05 0.96±0.03 1.25±0.01

Table-5.77: P. lilacinum was grown on mineral medium supplemented with 5% galactose and 5% molasses as carbon source at 30 ± 2 ºC and


Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 4.70±0.06 0.89±0.07 1.47±0.04 18.38±0.01 15.14±0.03 6.85±0.03

48 4.75±0.02 1.20±0.07 1.49±0.04 16.15±0.01 13.29±0.04 9.66±0.03

72 3.15±0.02 1.22±0.01 1.41±0.04 13.28±0.06 11.06±0.03 12.13±0.02

96 2.95±0.05 1.38±0.03 1.46±0.02 12.84±0.04 10.45±0.04 7.25±0.01

120 2.70±0.03 1.43±0.03 1.45±0.03 10.80±0.09 7.57±0.01 5.52±0.04

144 2.8±0.2 1.49±0.04 1.44±0.03 9.94±0.01 6.88±0.03 5.51±0.08

168 4.30±0.01 1.51±0.08 1.32±0.05 8.34±0.04 5.88±0.07 4.16±0.03

192 4.15±0.02 1.57±0.02 1.30±0.07 5.70±0.07 4.57±0.04 3.24±0.03

216 5.10±0.07 1.60±0.02 1.28±0.03 3.36±0.04 2.19±0.02 2.47±0.01

240 5.25±0.02 1.63±0.02 1.24±0.02 1.47±0.06 1.11±0.05 2.31±0.04

129

Tables-5.78 -5.79 showed the result of A. niger grown on mineral medium

containing 2.5 % and 5% starch, 5% molasses at 30 ± 2 ºC and pH was adjusted at

6.5. Like other carbon sources when starch was used as a carbon source in above

defined media A. niger produces greater 12.85 U/ml and 15.64 U/ml quantity of

pectinase respectively at 72 h incubation. Total and reducing sugars were

decreased with the increase of the time period. The pH values continuously

changed during the fermentation period

Tables-5.80 and 5.81 shows the P. lilacinum growth pattern and enzyme

production on mineral medium containing 2.5% and 5% starch , 5% molasses at

30 ± 2 ºC and pH was adjusted at 6.5. It shows a maximum production of

pectinase 12.21 U/ml and 15.17 U/ml respectively, at 72 hours and then its

concentration was decreased with the increase of the time period. The

concentration of total and reducing sugars was decreased with the increase of

fermentation time period. Total protein concentration increased during

fermentation. The present study was supported by the work of Singh and

Mandal (2012) who reported that starch was identified as best carbon source for

the production of Pectinolytic enzymes by a mixed culture of Aspergillus species

like A. fumigatus and A. sydowii. It is also reported by Singh and Mandal (2012)

that with the increase in concentration of carbon source the decrease in enzyme

activity starts due to substrate inhibition and catabolic repression, probably due

to the presence of high concentration of galacturonic acid. These investigations

are also supported by Aguilar and Huitron, (1987) and Maldonado et al., (1989).

130

Table-5.78: A. niger was grown on mineral medium supplemented with 2.5% starch and 5% molasses as carbon source at 30 ± 2 ºC and


Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 5.00±0.3 0.81±0.04 1.61±0.03 18.97±0.05 10.90±0.02 5.23±0.01

48 5.25±0.02 0.83±0.02 1.62±0.02 14.30±0.04 10.13±0.03 8.07±0.02

72 5.90±0.02 0.89±0.06 1.78±0.07 11.35±0.04 8.12±0.05 12.85±0.04

96 5.00±0.1 0.89±0.04 1.79±0.03 9.93±0.03 6.51±0.05 9.65±0.02

120 4.05±0.02 1.17±0.06 1.87±0.05 6.74±0.01 5.32±0.04 7.38±0.06

144 3.90±0.04 1.22±0.03 1.89±0.04 5.64±0.06 3.92±0.02 6.96±0.03

168 3.65±0.02 1.25±0.03 1.65±0.02 3.99±0.06 2.22±0.01 5.41±0.04

192 3.85±0.04 1.27±0.04 1.62±0.03 2.88±0.06 1.76±0.04 4.14±0.03

216 4.25±0.03 1.29±0.02 1.55±0.01 2.34±0.03 1.63±0.02 2.95±0.01

240 4.60±0.01 1.32±0.04 1.54±0.03 2.00±0.3 1.08±0.03 1.71±0.03

Table-5.79: A .niger was grown on mineral medium supplemented with 5% starch and 5% molasses as carbon source at 30 ± 2 ºC and


Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

U/ml

24 5.2±0.1 1.04±0.03 1.76±0.04 21.13±0.05 12.98±0.08 6.89±0.04

48 4.45±0.02 1.19±0.06 1.85±0.04 19.15±0.04 12.16±0.03 9.89±0.07

72 4.7±0.2 1.22±0.04 1.92±0.05 17.69±0.04 11.65±0.03 15.64±0.03

96 4.7±0.3 1.25±0.03 1.95±0.02 14.75±0.04 9.161±0.003 10.24±0.01

120 5.45±0.05 1.28±0.02 1.98±0.06 11.98±0.05 9.246±0.003 8.43±0.04

144 5.75±0.02 1.31±0.01 1.97±0.02 7.98±0.01 5.88±0.04 7.64±0.02

168 6.35±0.03 1.33±0.01 1.90±0.04 4.60±0.04 3.30±0.05 5.80±0.04

192 6.4±0.4 1.37±0.06 1.80±0.03 2.86±0.04 2.13±0.02 4.65±0.02

216 6.55±0.01 1.39±0.04 1.71±0.03 2.29±0.02 1.98±0.01 2.67±0.06

240 8.50±0.02 1.40±0.07 1.69±0.04 1.95±0.01 0.82±0.03 2.54±0.01

131

Table-5.80: P. lilacinum was grown on mineral medium supplemented with 2.5% starch and 5% molasses as carbon source at 30 ± 2 ºC and


Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 4.95±0.01 0.89±0.02 1.39±0.08 21.9±0.2 18.99±0.04 6.02±0.01

48 5.15±0.02 0.72±0.04 1.48±0.04 16.60±0.02 14.91±0.04 8.14±0.03

72 4.85±0.03 0.97±0.04 1.54±0.02 12.53±0.02 11.98±0.05 12.21±0.05

96 4.3±0.2 0.99±0.04 1.55±0.01 9.89±0.04 8.88±0.06 7.23±0.01

120 4.45±0.04 1.15±0.02 1.58±0.08 6.823±0.002 5.07±0.02 6.09±0.01

144 4.6±0.1 1.30±0.05 1.77±0.04 3.770±0.004 3.43±0.03 5. 93±0.02

168 4.8±0.1 1.38±0.06 1.83±0.04 3.451±0.003 3.28±0.03 5.82±0.04

192 4.65±0.02 1.47±0.04 1.89±0.06 3.157±0.004 2.64±0.03 4.86±0.04

216 4.6±0.4 1.49±0.04 1.89±0.04 2.626±0.004 2.50±0.05 2.98±0.03

240 5.1±0.1 1.51±0.03 1.70±0.05 1.73±0.02 1.62±0.01 1.47±0.03

Table-5.81: P. lilacinum was grown on mineral medium supplemented with 5 %

starch, 5% molasses as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 4.40±0.03 0.907±0.003 1.52±0.05 21.58±0.03 15.19±0.02 6.32±0.05

48 4.95±0.02 0.922±0.003 1.60±0.05 17.26±0.03 14.59±0.06 7.70±0.05

72 5.65±0.02 0.935±0.004 1.66±0.03 16.54±0.03 14.33±0.02 15.17±0.04

96 5.40±0.01 0.97±0.04 1.69±0.02 12.77±0.04 9.17±0.06 9.65±0.03

120 5.65±0.03 1.038±0.006 1.72±0.02 10.57±0.02 7.43±0.02 8.72±0.03

144 6.05±0.01 1.255±0.002 1.79±0.05 9.77±0.02 6.93±0.05 6.27±0.04

168 6.15±0.04 1.278±0.006 1.83±0.01 5.72±0.04 3.62±0.02 5.80±0.04

192 6.30±0.03 1.295±0.001 1.82±0.03 5.18±0.05 3.30±0.04 4.74±0.01

216 5.85±0.02 1.305±0.002 1.80±0.03 2.68±0.03 2.13±0.03 2.25±0.03

240 6.15±0.02 1.355±0.003 1.82±0.04 1.68±0.01 1.06±0.02 1.76±0.05

Table-5.82 and 83 show the results of two different concentrations (2.5% and 5%)

of additional carbon sources incorporated in mineral medium to screen out the

best productivity and cost effective secondary carbon source. The secondary

132

carbon sources were used like fructose, maltose, sucrose, galactose and starch

and both selected organisms A. niger and P. lilacinum were grown on these

carbon sources. The results compiled in the Table-5.82 shown that most of the

carbon source when added in optimized medium, all of those have produced a

good amount of pectinase, but sucrose proved as a best inducer with highest

activity and growth 16.16 U/ml, while second most promising substrate was

starch with 15.64 U/ml. This induced effect of sucrose has been reported by

Hours et al., (1988) and different isoenzymes production was affected by the type

and concentration of the substrate present in the culture medium (Leone and Van

Den Heuvel, 1987). The present study is also in agreement with Phutela et al.,

(2005), Crotti et al.,(1998), Baracat-Pereira et al., (1994) and Minussi et al., (1996).

Table-5.82: Effect on growth and pectinase production by Aspergillus niger when grown on mineral medium supplementedwith 5% molasses and sugars (2.5%

and 5%) as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5.

Carbon sources Biomass Pectinase Activity

% g/50 ml Broth U/ml

Fructose 2.5 0.897 ±0.004 12.00±0.3 5 1.192 ±0.004 13.89 ±0.03

Maltose 2.5 1.339 ±0.006 12.00±0.2 5 1.31 ±0.05 12.75 ±0.03 Sucrose 2.5 1.18 ±0.03 13.66±0.02

5 1.25 ±0.04 16.16±0.03 Galactose 2.5 1.11 ±0.03 12.22±0.02

5 1.14 ±0.02 13.98±0.03 Starch 2.5 0.89 ±0.03 12.85±0.02

5 1.22 ±0.01 15.64±0.03

133

Table-5.83: Effect on growth and pectinase production by Penecillium lilacinum grown on mineral medium supplemented with 5% molasses and sugars (2.5%

and 5%) as carbon source at 30 ± 2 ºC and pH was adjusted at 6.5.

Carbon sources Biomass Pectinase Activity


Frutose 2.5 1.365± 0.006 10.73±0.04 5 1.339 ±0.003 12.0±0.1

Maltose 2.5 1.205±0.002 11.04±0.02 5 1.125± 0.002 11.2±0.1

Sucrose 2.5 1.23±0.03 12.178±0.005 5 1.30±0.03 15.54 ±0.03 Galactose 2.5 1.22±0.01 11.14±0.01

5 1.22±0.04 12.13±0.03 Starch 2.5 0.97±0.01 12.21±0.04

5 0.935±0.002 15.17 ±0.03

vi- Effect of nitrogen source:

Nitrogen source is an important nutrient in fermentation medium and

has a significant role in the growth, development and production of metabolites

by microorganisms. Most of the industrially useful microbes are grown on

organic and inorganic sources, (Hunter, 1972).

Nutritional Factors such as carbon and nitrogen have always been of great

attention to the investigators to design cost effective media in the enzyme

production. About 30–40% of the manufacturing cost of industrial enzymes are

predictable as the cost of growth medium. Therefore, it is of enormous worth to

optimize the conditions for low cost enzyme production (Palaniyappan et al.,

2009). Nonetheless, a study on the impact of carbon and nitrogen sources had

shown that not all carbon and nitrogen supplements would proceed as boosters

for the concurrent production of enzymes in a single fermentation system (Negi

and Banerjee, 2010).

Organic nitrogen may be used as amino acids, proteins and peptone and urea

where as inorganic nitrogen source used like ammonium sulphate, potassium

nitrate, sodium nitrate etc. In this study ammonium sulphate was used as

control in culture media to grow fungi and best results were achieved with

134

inorganic nitrogen source which is in contrast with observations of Narasimha et

al., (2006) who reported that organic nitrogen sources maintained the superior

growth of fungi more than the nitrogen from inorganic sources. Vahidi et al.,

(2004) also reported same statement that good growth was achieved by using

complex nitrogen sources like yeast extract, peptone as compared to inorganic

nitrogen sources. Addition of yeast extract in media increase pectic lyase

synthesis (Phutela et al., 2005). Fadel ( 2000 ) has reported that irrespective of

substrate used the amount of enzyme production is influenced due to nitrogen

source as well as other supplements used in the media. The source of nitrogen in

a culture medium plays a substantial role for the growth of fungi and production

of enzyme (Juwon and Emmanuel, 2012).

Tables 5.84 to 5.87 showed the results of A. niger and P. lilacinum grown

on optimized mineral medium containing 0.2% and 0.4% corn steep liquor, the

maximum production of pectinase was achieved 23.29 U/ml and 23.23 U/ml

respectively.

Table-5.84: A niger was grown on mineral medium supplemented with 5%

sucrose and 5% molasses as carbon source and 0.2% corn steep liquor at 30 ± 2 ºC and pH was adjusted at 6.5.

Time Hours



Total Sugar (mg/ml)



24 6.10±0.03 1.05±0.02 1.45±0.02 23.92±0.03 17.57±0.05 7.2±0.1

48 5.85±0.02 1.17±0.04 1.58±0.03 20.63±0.05 15.20±0.04 8.97±0.04

72 6.20±0.03 1.27±0.04 1.58±0.07 18.15±0.04 14.28±0.05 21.19±0.06

96 6.10±0.01 1.31±0.01 1.67±0.04 15.86±0.04 11.53±0.03 11.86±0.04

120 5.75±0.02 1.36±0.01 1.69±0.02 13.73±0.02 9.25±0.03 9.87±0.02

144 5.25±0.03 1.38±0.07 1.70±0.01 9.88±0.03 6.59±0.06 6.90±0.01

168 5.55±0.02 1.39±0.02 1.74±0.01 5.41±0.03 3.31±0.03 4.68±0.03

192 5.70±0.01 1.41±0.02 1.79±0.03 3.13±0.02 2.33±0.03 3.66±0.05

216 5.15±0.01 1.41±0.01 1.78±0.03 2.29±0.04 1.62±0.05 2.45±0.02

240 5.35±0.02 1.43±0.05 1.70±0.03 1.70±0.04 0.67±0.05 1.87±0.01

135

Table-5.85: A. niger was grown on mineral medium supplemented with 5% sucrose and 5% molasses as carbon source and 0.4% corn steep

liquor at 30 ± 2 ºC and pH was adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 6.15±0.01 0.98±0.05 1.62±0.04 22.73±0.02 16.23±0.05 8.73±0.03

48 6.05±0.04 1.17±0.04 1.71±0.02 19.13±0.02 14.75±0.02 9.81±0.03

72 5.55±0.03 1.20±0.03 1.75±0.02 14.94±0.02 11.49±0.04 23.39±0.02

96 5.25±0.02 1.21±0.06 1.77±0.02 13.93±0.04 11.07±0.04 13.56±0.04

120 4.85±0.02 1.28±0.06 1.79±0.07 9.50±0.07 6.10±0.02 8.04±0.01

144 5.15±0.03 1.30±0.02 1.82±0.02 8.58±0.03 6.98±0.05 6.29±0.04

168 5.35±0.03 1.33±0.02 1.84±0.03 6.35±0.03 4.23±0.01 5.13±0.02

192 4.90±0.03 1.35±0.02 1.88±0.03 4.21±0.04 3.89±0.06 4.94±0.01

216 4.70±0.02 1.44±0.04 1.79±0.06 1.98±0.05 1.21±0.06 2.27±0.03

240 3.95±0.02 1.47±0.02 1.79±0.02 1.31±0.03 0.98±0.05 1.18±0.03

Table 5.86: P. lilacinum was grown on mineral medium supplemented with 5%

sucrose and 5% molasses as carbon source and 0.2% corn steep liquor at 30 ± 2 ºC and pH was adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugar (mg/ml)

Pectinase

Activity (U/ml)

24 5.75±0.02 1.21±0.04 1.52±0.04 22.73±0.05 15.34±0.03 6.935±0.004

48 5.65±0.03 1.24±0.01 1.57±0.04 20.23±0.03 15.25±0.03 9.217±0.004

72 5.5±0.1 1.30±0.04 1.65±0.03 19.95±0.03 13.99±0.06 21.172±0.004

96 5.25±0.03 1.36±0.04 1.66±0.03 14.83±0.05 11.67±0.02 13.264±0.002

120 4.85±0.02 1.38±0.05 1.72±0.02 9.99±0.04 6.90±0.07 8.039±0.002

144 4.15±0.01 1.39±0.02 1.74±0.03 8.35±0.02 6.29±0.07 7.294±0.004

168 3.95±0.03 1.42±0.03 1.78±0.03 6.25±0.02 4.23±0.06 4.126±0.004

192 4.75±0.02 1.45±0.02 1.83±0.05 4.21±0.04 3.19±0.06 3.428±0.003

216 4.40±0.02 1.56±0.04 1.89±0.06 3.33±0.01 1.81±0.03 2.27±0.01

240 3.90±0.01 1.67±0.06 1.89±0.04 2.07±0.04 1.12±0.03 1.18±0.04

136

Table-5.87: P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.4% corn steep liquor at 30 ±2 ºC and


Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugar (mg/ml)

Pectinase

Activity (U/ml)

24 5.55±0.01 0.94±0.03 1.55±0.02 23.69±0.07 16.167±0.005 7.97±0.02

48 5.75±0.02 1.23±0.03 1.57±0.05 20.74±0.03 15.27±0.02 9.91±0.02

72 5.85±0.02 1.30±0.04 1.58±0.03 17.95±0.02 12.68±0.05 23.23±0.04

96 5.65±0.03 1.37±0.04 1.58±0.01 14.55±0.04 10.23±0.05 12.83±0.02

120 4.5±0.1 1.38±0.03 1.63±0.04 12.13±0.03 8.95±0.02 7.27±0.05

144 4.6±0.4 1.41±0.04 1.66±0.03 11.88±0.06 7.99±0.01 6.17±0.04

168 4.75±0.02 1.54±0.02 1.68±0.03 9.41±0.03 6.92±0.02 5.84±0.04

192 4.95±0.03 1.56±0.01 1.72±0.03 6.13±0.08 4.37±0.04 3.91±0.06

216 4.3±0.3 1.59±0.01 1.74±0.01 3.97±0.05 2.62±0.01 2.89±0.06

240 4.1±0.2 1.62±0.03 1.75±0.05 1.82±0.02 1.07±0.02 1.86±0.04

Urea was used as nitrogen source more or less same the result 22.16 U/ml

and 23.94 U/ml respectively obtained when A.niger and P. lilacinum grown on

0.4% urea at 72 hours as shown in Tables 5.88 to 5.91. This type of shorter

incubation time period can be advantageous for industrial production. Said et al.,

(1991) have reported maximum pectinase production by Penicillium frequentans

after 48 h in media containing urea and trace elements Rashmi et al., (2008) and

Neeta et el., (2011) reported that urea was a good nitrogen source enhancing

pectinase production after peptone. Hoa and Hung (2013) reported urea as a best

nitrogen source for the production of pectinase by A. oryzae .

137

Table-5.88: A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses as carbon source and 0.2% urea at 30 ± 2 ºC and


Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 6.05±0.02 1.11±0.05 1.57±0.02 20.38±0.04 14.51±0.03 6.70±0.02

48 5.55±0.03 1.13±0.04 1.62±0.02 17.87±0.04 13.25±0.02 8.94±0.04

72 5.1±0.1 1.16±0.01 1.63±0.06 14.56±0.01 11.36±0.04 21.29±0.02

96 5.05±0.01 1.18±0.05 1.67±0.04 13.96±0.03 11.14±0.03 11.54±0.03

120 5.25±0.02 1.21±0.03 1.68±0.06 9.52±0.02 7.21±0.02 7.99±0.04

144 5.5±0.3 1.23±0.01 1.69±0.04 7.46±0.04 5.22±0.01 6.27±0.04

168 5.55±0.03 1.24±0.03 1.71±0.02 6.30±0.03 4.18±0.03 5.44±0.03

192 5.75±0.01 1.28±0.03 1.74±0.03 4.45±0.04 3.18±0.01 3.18±0.03

216 5.87±0.04 1.44±0.05 1.77±0.04 3.23±0.03 2.49±0.06 1.39±0.02

240 5.95±0.01 1.56±0.04 1.81±0.03 2.67±0.04 1.14±0.02 1.34±0.01

Table-5.89: A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses as carbon source and 0.4% urea at 30 ± 2 ºC and


Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar (mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 5.8±0.3 1.071±0.003 1.56±0.05 22.65±0.02 16.27±0.04 7.99±0.06

48 6.05±0.02 1.132±0.002 1.57±0.04 17.92±0.05 13.83±0.03 9.90±0.03

72 5.85±0.03 1.193±0.003 1.59±0.02 16.69±0.04 12.48±0.03 23.94±0.03

96 5.65±0.01 1.219±0.002 1.62±0.03 15.89±0.03 11.54±0.01 11.90±0.02

120 5.5±0.2 1.242±0.004 1.73±0.02 12.87±0.05 9.15±0.04 8.86±0.02

144 5.1±0.2 1.2825±0.0002 1.75±0.01 8.69±0.06 6.60±0.02 7.40±0.03

168 4.55±0.02 1.2905±0.0004 1.79±0.02 5.67±0.01 3.27±0.05 5.73±0.03

192 4.45±0.03 1.3605±0.0002 1.79±0.06 4.13±0.02 3.96±0.03 4.18±0.03

216 4.3±0.2 1.37±0.05 1.83±0.03 2.69±0.04 1.43±0.02 3.71±0.03

240 4.15±0.01 1.41±0.04 1.84±0.02 1.15±0.02 0.88±0.04 1.23±0.03

138

Table-5.90: P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses as carbon source and 0.2% urea at 30 ± 2 ºC and


Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

activity (U/ml)

24 6.10±0.02 0.92±0.03 1.58±0.04 22.34±0.02 15.97±0.02 7.82±0.02

48 5.95±0.02 1.49±0.02 1.60±0.02 19.39±0.02 15.41±0.04 11.18±0.03

72 5.45±0.04 1.21±0.03 1.62±0.02 16.32±0.02 11.12±0.04 20.14±0.03

96 5.35±0.03 1.23±0.02 1.65±0.02 15.77±0.02 10.89±0.07 12.54±0.03

120 5.75±0.02 1.23±0.03 1.67±0.02 13.18±0.03 9.92±0.03 8.15±0.07

144 5.25±0.05 1.24±0.01 1.68±0.01 10.56±0.05 7.20±0.05 7.97±0.04

168 4.85±0.03 1.26±0.04 1.70±0.06 7.28±0.06 5.13±0.02 5.95±0.03

192 4.45±0.01 1.32±0.03 1.75±0.02 4.24±0.04 3.96±0.01 2.40±0.03

216 4.20±0.01 1.42±0.04 1.79±0.02 2.98±0.05 1.69±0.06 2.13±0.03

240 4.10±0.01 1.48±0.04 1.83±0.03 1.12±0.02 0.69±0.01 1.24±0.02

Table-5.91: P. lilacinum was grown on mineral medium supplemented with 5%

sucrose, 5% molasses as carbon source and 0.4% urea at 30 ± 2 ºC and pH was adjusted at 6.5.

Time Hours



Total Sugar (mg/ml)



24 6.10±0.02 1.10±0.03 1.43±0.06 22.94±0.02 14.61±0.05 6.96±0.03

48 5.8±0.1 1.12±0.07 1.46±0.03 18.72±0.06 13.91±0.04 10.84±0.02

72 5.55±0.03 1.14±0.03 1.52±0.02 14.94±0.03 9.85±0.04 22.16±0.01

96 5.75±0.02 1.22±0.04 1.56±0.04 11.39±0.02 8.79±0.01 13.64±0.03

120 5.15±0.01 1.32±0.02 1.58±0.03 9.69±0.06 6.82±0.07 9.75±0.07

144 4.95±0.02 1.35±0.02 1.65±0.03 9.36±0.05 7.04±0.02 7.14±0.05

168 4.75±0.02 1.40±0.01 1.68±0.03 6.74±0.02 4.15±0.01 4.24±0.02

192 4.05±0.03 1.46±0.05 1.75±0.02 3.04±0.03 2.86±0.03 3.77±0.04

216 4.25±0.01 1.54±0.02 1.78±0.07 2.98±0.05 1.54±0.03 1.65±0.01

240 4.65±0.04 1.61±0.05 1.83±0.03 1.42±0.05 1.13±0.03 1.29±0.06

Tables 5.92 to 5.95 indicates that A. niger and P. lilacinum was grown on optimized

culture medium with addition to 0.2% and 0.4% NaNO3 and the maximum pectinase

was obtained 22.88 U/ml and 22.90 U/ml respectively. The present results are in

agreement with the findings of Arijit et al., (2013). Neeta et al., ( 2011) reported that

next to peptone NaNO3 exhibited higher pectinase production from A. niger.

139

Table-5.92: A. nigar was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% NaNO3 at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 6.30±0.03 1.05±0.03 1.39±0.06 23.17±0.04 15.86±0.01 7.23±0.04

48 5.90±0.04 1.21±0.02 1.42±0.02 21.84±0.02 15.74±0.03 9.14±0.01

72 6.65±0.01 1.24±0.01 1.49±0.06 17.98±0.03 14.61±0.01 21 .11±0.01

96 5.2±0.1 1.26±0.05 1.50±0.04 12.90±0.03 9.13±0.02 9.73±0.05

120 4.75±0.02 1.28±0.01 1.56±0.04 11.72±0.02 7.24±0.06 8.94±0.07

144 4.25±0.04 1.32±0.02 1.69±0.04 9.92±0.03 6.39±0.03 6.80±0.04

168 4.55±0.03 1.34±0.03 1.70±0.02 8.68±0.05 6.17±0.04 5.39±0.02

192 4.7±0.2 1.42±0.05 1.72±0.02 4.42±0.02 3.98±0.06 2.13±0.02

216 4.15±0.02 1.43±0.02 1.78±0.05 3.80±0.07 3.13±0.06 1.27±0.05

240 4.2±0.2 1.46±0.01 1.79±0.04 1.67±0.05 1.11±0.02 1.13±0.03

Table-5.93: A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.4% NaNO3 at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 6.30±0.03 0.99±0.04 1.59±0.01 31.13±0.02 27.33±0.01 7.61±0.05

48 5.65±0.02 1.05±0.03 1.63±0.03 25.97±0.04 24.12±0.01 9.98±0.05

72 5.55±0.03 1.09±0.04 1.67±0.04 23.94±0.02 22.30±0.07 22.90±0.07

96 5.20±0.01 1.14±0.03 1.69±0.03 16.73±0.03 15.81±0.03 11.61±0.04

120 5.10±0.02 1.18±0.03 1.69±0.02 11.32±0.04 11.00±0.3 8.61±0.06

144 4.90±0.04 1.29±0.04 1.73±0.02 9.85±0.03 8.92±0.06 7.69±0.02

168 4.85±0.02 1.32±0.02 1.76±0.01 7.98±0.05 7.22±0.05 5.77±0.01

192 4.75±0.01 1.36±0.03 1.79±0.06 5.32±0.04 5.17±0.04 4.92±0.02

216 4.50±0.02 1.43±0.03 1.80±0.05 3.81±0.01 3.52±0.03 2.10±0.04

240 3.95±0.02 1.49±0.05 1.80±0.01 1.68±0.03 1.56±0.05 1.71±0.08

140

Table-5.94 : P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% NaNO3 at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugarss (mg/ml)

Pectinase

Activity (U/ml)

24 6.25±0.02 1.16±0.03 1.62±0.05 18.13±0.03 13.13±0.06 6.12±0.05

48 6.05±0.01 1.14±0.02 1.67±0.05 17.12±0.04 13.21±0.01 9.67±0.04

72 6.10±0.01 1.21±0.01 1.75±0.02 14.62±0.04 11.20±0.06 20.39±0.02

96 5.75±0.02 1.25±0.04 1.79±0.04 11.12±0.01 8.81±0.03 11.40±0.06

120 5.35±0.03 1.29±0.04 1.80±0.03 11.00±0.5 7.89±0.04 9.61±0.01

144 5.15±0.03 1.30±0.03 1.88±0.05 7.15±0.06 5.61±0.05 7.82±0.05

168 4.85±0.01 1.32±0.05 1.84±0.03 5.26±0.03 4.12±0.02 6.34±0.03

192 4.75±0.02 1.36±0.04 1.87±0.01 4.72±0.02 2.62±0.04 5.72±0.04

216 4.3±0.2 1.37±0.02 1.89±0.03 2.91±0.08 1.72±0.05 3.72±0.01

240 3.95±0.03 1.39±0.03 1.90±0.01 1.12±0.01 0.98±0.06 2.91±0.06

Table- 5.95 : P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.4% NaNO3 at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 6.40±0.03 1.10±0.03 1.65±0.02 21.79±0.06 15.79±0.06 6.19±0.03

48 6.10±0.02 1.12±0.01 1.67±0.05 19.00±0.3 14.81±0.01 8.16±0.03

72 5.85±0.02 1.17±0.04 1.71±0.04 15.45±0.04 12.22±0.03 22.50±0.06

96 5.65±0.03 1.22±0.02 1.76±0.03 11.87±0.05 8.10±0.06 12.85±0.03

120 5.10±0.01 1.29±0.04 1.80±0.04 10.74±0.03 7.21±0.03 8.88±0.04

144 4.60±0.07 1.31±0.04 1.83±0.03 8.91±0.03 6.25±0.03 7.98±0.05

168 4.25±0.01 1.34±0.03 1.84±0.02 6.19±0.04 4.97±0.02 5.12±0.02

192 3.95±0.02 1.36±0.03 1.87±0.02 4.92±0.02 4.13±0.02 2.81±0.04

216 3.80±0.02 1.49±0.07 1.88±0.01 2.98±0.06 1.70±0.07 2.16±0.05

240 3.70±0.03 1.52±0.02 1.89±0.09 1.27±0.05 0.53±0.02 1.94±0.02

Tables 5.96 to 5.99 shows the growth pattern and pectinase production when A. niger

and P. lilacinum was grown on 5% sucrose, 5% molasses ,0.2%and 0.4% KNO3 as

nitrogen source at 30 ± 2 ºC and pH was adjusted at 6.5. The maximum production

141

of pectinase 20.67 U/ml and 19.14 U/ml respectively was observed at 72 hours and

then its concentration was decreased with the increase of time period.

Table-5.96: A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses & 0.2% KNO3 at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 6.15±0.02 0.91±0.04 1.52±0.02 22.95±0.02 16.86±0.04 7.16±0.05

48 5.90±0.01 1.06±0.03 1.53±0.05 20.93±0.05 15.71±0.03 11.13±0.05

72 5.80±0.02 1.11±0.05 1.56±0.05 16.16±0.03 11.63±0.03 19.22±0.07

96 5.25±0.02 1.19±0.06 1.59±0.02 13.87±0.05 11.24±0.01 10.48±0.04

120 5.85±0.03 1.22±0.01 1.62±0.04 12.47±0.04 9.79±0.05 8.42±0.02

144 5.15±0.02 1.24±0.04 1.64±0.02 9.99±0.06 7.16±0.04 7.37±0.05

168 4.85±0.01 1.27±0.05 1.69±0.06 6.90±0.02 5.28±0.06 4.16±0.03

192 475±0.01 1.28±0.03 1.70±0.03 3.82±0.02 2.29±0.08 2.14±0.03

216 4.40±0.02 1.34±0.01 1.72±0.03 1.92±0.05 1.22±0.06 1.36±0.07

240 4.10±0.01 1.37±0.03 1.79±0.06 1.18±0.05 0.76±0.03 1.02±0.02

Table-5.97:A. niger was grown on mineral medium supplemented with 5%

sucrose, 5% molasses and 0.4% KNO3 at 30 ± 2 ºC and pH was adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 6.30±0.01 0.98±0.04 1.46±0.05 22.94±0.04 15.17±0.04 5.73±0.04

48 6.25±0.02 1.01±0.01 1.48±0.03 18.99±0.06 14.45±0.02 11.24±0.03

72 6.10±0.01 1.17±0.04 1.57±0.04 17.43±0.02 12.91±0.05 20.67±0.05

96 5.70±0.02 1.20±0.01 1.59±0.03 14.83±0.02 11.64±0.03 9.17±0.04

120 5.6±0.3 1.21±0.04 1.62±0.03 10.19±0.05 7.69±0.06 8.92±0.02

144 5.30±0.03 1.26±0.04 1.68±0.06 7.49±0.05 5.19±0.05 7.97±0.04

168 5.15±0.01 1.24±0.03 1.70±0.02 6.46±0.04 4.32±0.02 6.61±0.05

192 4.95±0.02 1.32±0.05 1.72±0.02 4.12±0.03 3.92±0.05 4.17±0.06

216 4.30±0.02 1.38±0.05 1.78±0.01 2.39±0.06 1.13±0.02 2.98±0.04

240 4.10±0.01 1.42±0.02 1.79±0.06 1.22±0.02 0.57±0.05 1.42±0.03

142

Table-5.98: P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% KNO3 at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Protein (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 6.5±0.2 0.92±0.05 1.28±0.06 21.94±0.03 14.97±0.04 6.82±0.06

48 6.2±0.1 1.49±0.05 1.40±0.03 20.39±0.02 13.41±0.05 9.04±0.01

72 5.9±0.2 1.21±0.01 1.32±0.02 16.32±0.04 12.12±0.01 18.18±0.08

96 5.35±0.03 1.23±0.04 1.35±0.04 15.77±0.04 11.89±0.04 12.40±0.03

120 5.15±0.02 1.23±0.06 1.37±0.02 13.18±0.03 11.92±0.02 9.15±0.04

144 4.85±0.01 1.24±0.02 1.38±0.06 9.56±0.04 7.20±0.04 6.97±0.04

168 4.65±0.01 1.26±0.01 1.39±0.02 6.28±0.07 3.13±0.04 5.95±0.03

192 4.3±0.2 1.32±0.02 1.45±0.01 4.24±0.04 3.96±0.03 3.40±0.05

216 4.2±0.2 1.42±0.06 1.48±0.05 2.98±0.05 1.69±0.01 2.13±0.02

240 4.1±0.1 1.48±0.04 1.58±0.06 1.51±0.03 1.05±0.02 1.24±0.04


sucrose, 5% molasses & 0.4% KNO3 at 30 ± 2 ºC and pH was adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 6.4±0.3 1.00±0.3 1.53±0.02 21.16±0.03 14.19±0.06 6.86±0.04

48 6.1±0.1 1.94±0.04 1.65±0.02 18.54±0.03 13.13±0.06 8.11±0.05

72 5.85±0.02 1.97±0.02 1.76±0.04 15.65±0.05 11.75±0.02 19.14±0.03

96 5.65±0.03 1.12±0.04 1.77±0.07 13.39±0.02 10.12±0.02 11.16±0.01

120 5.15±0.01 1.21±0.04 1.79±0.06 11.89±0.07 8.13±0.04 9.33±0.03

144 4.45±0.02 1.22±0.07 1.84±0.03 8.97±0.02 5.13±0.08 5.98±0.02

168 4.75±0.02 1.28±0.05 1.89±0.07 7.99±0.06 6.15±0.02 4.89±0.07

192 4.05±0.01 1.29±0.01 1.90±0.03 4.82±0.04 3.89±0.02 3.07±0.02

216 4.25±0.03 1.31±0.05 1.88±0.02 2.99±0.01 2.61±0.01 2.13±0.02

240 3.95±0.02 1.34±0.03 1.82±0.06 1.57±0.04 1.03±0.02 1.11±0.05

143

Tables 5.100 to 5.103 shows the results of NH4NO3, when added as nitrogen source in optimized medium for the synthesis of pectinase by A. niger and P.

lilacinum and maximum production of pectinase was achieved 22.40 U/ml and 21.27 U/ml respectively

Table-5.100: A. niger was grown on mineral medium supplemented with 5%

sucrose, 5% molasses and 0.2% NH4NO3 at 30 ± 2 ºC and pH was adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 6.1±0.1 1.09±0.03 1.52±0.04 20.933±0.003 13.74±0.05 6.40±0.04

48 5.4±0.1 1.11±0.03 1.54±0.01 16.98±0.01 11.94±0.02 7.64±0.02

72 5.25±0.01 1.14±0.03 1.59±0.03 14.23±0.03 11.39±0.06 20.35±0.04

96 5.15±0.02 1.16±0.02 1.62±0.02 18.99±0.06 18.21±0.03 11.17±0.04

120 5.05±0.03 1.17±0.05 1.65±0.02 13.22±0.04 12.42±0.05 8.63±0.05

144 4.45±0.02 1.24±0.02 1.67±0.05 12.15±0.04 11.20±0.07 6.91±0.04

168 4.35±0.01 1.26±0.02 1.71±0.03 8.21±0.06 7.65±0.03 5.17±0.04

192 4.65±0.03 1.29±0.06 1.73±0.06 6.22±0.05 5.81±0.01 4.15±0.04

216 4.15±0.01 1.32±0.03 1.79±0.01 4.10±0.04 3.32±0.01 3.32±0.05

240 4.35±0.02 1.34±0.01 1.80±0.07 2.24±0.02 2.10±0.04 1.33±0.03

.

Table-5.101: A. niger was grown on mineral medium supplemented with 5%


Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 5.9±0.2 1.05±0.02 1.49±0.04 21.17±0.06 15.86±0.04 5.23±0.04

48 6.0±0.2 1.21±0.02 1.52±0.02 18.84±0.04 14.74±0.03 8.14±0.01

72 5.4±0.2 1.24±0.03 1.57±0.04 17.18±0.03 14.00±0.2 22.40±0.04

96 5.1±0.1 1.26±0.02 1.59±0.03 14.90±0.02 11.13±0.04 13.73±0.05

120 4.75±0.02 1.28±0.05 1.61±0.03 10.72±0.04 8.24±0.03 8.94±0.01

144 4.25±0.01 1.32±0.04 1.67±0.05 9.92±0.05 7.69±0.01 6.80±0.07

168 4.55±0.02 1.34±0.04 1.69±0.02 5.68±0.05 3.17±0.03 4.39±0.02

192 4.7±0.2 1.42±0.05 1.71±0.01 3.4 ±0.0 4 2.98±0.05 3.13±0.02

216 4.15±0.02 1.43±0.02 1.75±0.03 2.27±0.04 1.83±0.03 2.27±0.01

240 4.2±0.1 1.46±0.02 1.79±0.06 1.07±0.03 0.46±0.03 1.83±0.03

144

Table-5.102: P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% NH4NO3 at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 6.25±0.01 0.99±0.06 1.92±0.02 22.97±0.04 16.01±0.01 5.13±0.05

48 6.15±0.01 1.23±0.02 1.93±0.02 20.82±0.04 15.12±0.06 8.21±0.02

72 6.3±0.1 1.33±0.03 1.95±0.01 16.20±0.03 11.80±0.04 20 .11±0.05

96 6.15±0.02 1.48±0.04 1.97±0.04 15.14±0.04 10.87±0.05 9.44±0.04

120 5.85±0.03 1.59±0.03 1.99±0.03 12.61±0.05 9.87±0.02 9.19±0.05

144 5.25±0.02 1.61±0.04 1.94±0.07 9.38±0.05 7.82±0.05 8.46±0.03

168 4.65±0.01 1.62±0.02 1.94±0.04 6.62±0.05 4.27±0.04 5.39±0.04

192 4.25±0.01 1.75±0.02 1.83±0.02 5.36±0.04 3.89±0.02 3.619±0.008

216 4.4±0.2 1.76±0.01 1.77±0.05 2.97±0.02 2.63±0.02 2.16±0.01

240 4.95±0.03 1.77±0.04 1.57±0.01 1.03±0.03 0.67±0.02 1.78±0.05

Table-5.103: P.lilacinam was grown on mineral medium supplemented with 5%


Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total

Sugar (mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 5.9±0.2 1.07±0.05 1.761±0.004 21.84±0.04 14.12±0.04 6.22±0.02

48 5.7±0.3 1.14±0.03 1.81±0.04 17.55±0.02 13.13±0.06 8.19±0.06

72 5.05±0.02 1.15±0.05 1.85±0.03 14.77±0.05 10.03±0.03 21.27±0.04

96 5.65±0.01 1.20±0.05 1.88±0.02 11.62±0.02 7.18±0.03 13.16±0.05

120 5.5±0.1 1.24±0.01 1.90±0.07 9.13±0.05 6.95±0.03 9.97±0.01

144 5.3±0.1 1.27±0.05 1.71±0.05 8.78±0.05 5.87±0.04 7.87±0.04

168 5.15±0.03 1.31±0.03 1.77±0.05 5.02±0.02 4.92±0.02 5.11±0.05

192 4.85±0.02 1.37±0.04 1.79±0.04 3.91±0.05 2.02±0.01 4.18±0.01

216 4.4±0.1 1.42±0.03 1.79±0.08 1.97±0.04 1.37±0.02 3.19±0.05

240 4.2±0.2 1.44±0.02 1.80±0.01 0.89±0.02 0.50±0.04 1.84±0.04

Previous results in this study show that A. niger produces greater amount

of pectinase than P. lilacinum as shown in tables 5.104 to 5.107. A niger and

P .lilacinum with the adition of (0.2 and 0.4%) peptone as a nitrogen source has

given significant production of pectinase 23.98 U/ml and 22.97 U/ml

145

respectively. The results are in agreement with Neeta et al., (2011) who achieved

maximum production of pectinase from A. niger in Smf system using peptone as

a nitrogen source. Peptone consists of various amino acids that liberate nitrogen

which enhance the growth of fungi (Martin et al., 2004; Margesin et al., 2005).

Table-5.104: A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% peptone at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 7.1±0.1 0.98±0.05 1.55±0.04 20.22±0.08 13.97±0.04 7.80±0.03

48 6.85±0.02 1.07±0.03 1.58±0.03 18.65±0.03 12.89±0.07 8.27±0.02

72 6.3±0.2 1.07±0.04 1.58±0.09 15.76±0.03 11.08±0.03 21.80±0.01

96 6.2±0.2 1.12±0.06 1.59±0.02 12.89±0.03 8.33±0.03 13.73±0.03

120 5.70±0.02 1.16±0.02 1.59±0.06 9.98±0.03 6.60±0.04 9.12±0.01

144 5.25±0.02 1.22±0.02 1.60±0.02 8.88±0.06 5.19±0.05 7.82±0.02

168 5.15±0.03 1.29±0.04 1.59±0.03 5.45±0.05 4.12±0.02 4.78±0.02

192 4.7±0.2 1.41±0.04 1.58±0.06 3.24±0.03 2.99±0.04 3.95±0.03

216 4.15±0.01 1.41±0.01 1.42±0.02 1.69±0.04 1.44±0.02 2.45±0.04

240 4.35±0.03 1.43±0.07 1.42±0.01 1.19±0.01 1.07±0.01 2.17±0.04

Table-5.105: A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.4% peptone at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time Hours


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 7.15±0.02 0.92±0.08 1.43±0.03 21.14±0.03 14.33±0.04 7.76±0.05

48 6.95±0.02 0.917±0.004 1.51±0.05 19.78±0.03 14.13±0.03 11.83±0.05

72 6.55±0.01 0.98±0.03 1.42±0.02 17.92±0.05 13.63±0.05 23.98±0.05

96 6.15±0.01 0.99±0.05 1.47±0.04 14.81±0.05 11.67±0.05 15.66±0.01

120 5.85±0.03 1.13±0.02 1.68±0.03 10.19±0.06 7.90±0.05 9.64±0.03

144 5.15±0.02 1.29±0.04 1.67±0.06 6.72±0.02 5.14±0.01 6.39±0.02

168 5.0±0.2 1.33±0.03 1.68±0.04 5.34±0.03 4.22±0.02 4.74±0.04

192 4.75±0.02 1.34±0.02 1.74±0.04 3.79±0.03 2.69±0.04 3.94±0.06

216 4.7±0.2 1.34±0.03 1.76±0.03 1.98±0.03 1.37±0.04 1.97±0.05

240 4.95±0.02 1.49±0.05 1.71±0.03 1.01±0.01 0.83±0.01 1.78±0.05

146

Table-5.106: P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% peptone at 30 ± 2 ºC and pH was

adjusted at 6.5

Time

hours

Final

pH

Biomass

g/50 ml Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 6.95±0.01 1.09±0.04 1.12±0.04 21.93±0.03 15.17±0.05 6.91±0.04

48 6.15±0.02 1.12±0.04 1.27±0.06 17.32±0.02 12.95±0.02 9.40±0.03

72 5.5±0.1 1.10±0.06 1.32±0.06 14.25±0.02 10.99±0.04 20.57±0.02

96 5.25±0.01 1.12±0.02 1.37±0.04 12.81±0.05 9.11±0.04 11.99±0.03

120 4.85±0.01 1.15±0.04 1.47±0.02 11.98±0.05 8.82±0.05 9.62±0.02

144 4.15±0.02 1.23±0.06 1.47±0.04 9.75±0.03 7.20±0.08 8.40±0.03

168 4.85±0.03 1.24±0.03 1.48±0.02 5.69±0.01 4.22±0.02 5.71±0.06

192 4.75±0.03 1.26±0.01 1.49±0.04 3.41±0.04 3.12±0.03 3.94±0.04

216 4.7±0.2 1.27±0.05 1.49±0.08 2.13±0.07 1.96±0.03 2.36±0.03

240 3.95±0.02 1.28±0.02 1.49±0.02 1.17±0.04 0.53±0.03 1.38±0.06

Table-5.107: P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.4% peptone at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time hours


Broth


Total Sugar (mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

24 6.9±0.2 0.93±0.03 1.37±0.05 20.62±0.05 14.04±0.03 7.20±0.07

48 6.65±0.01 1.03±0.03 1.35±0.01 18.15±0.02 13.94±0.07 8.12±0.02

72 6.55±0.01 1.15±0.04 1.37±0.06 16.05±0.03 12.88±0.03 22.97±0.04

96 6.15±0.02 1.17±0.03 1.40±0.01 13.56±0.05 11.84±0.03 12.86±0.01

120 5.8±0.3 1.18±0.03 1.46±0.01 10.13±0.07 8.95±0.05 7.13±0.03

144 5.6±0.2 1.21±0.01 1.47±0.07 7.88±0.05 6.64±0.04 6.12±0.06

168 5.1±0.1 1.24±0.07 1.50±0.07 4.79±0.07 4.34±0.01 4.82±0.05

192 4.9±0.3 1.26±0.03 1.53±0.04 2.23±0.03 1.94±0.02 2.85±0.03

216 4.7±0.2 1.39±0.03 1.54±0.04 1.36±0.05 1.13±0.07 2.37±0.06

240 5.3±0.2 1.42±0.03 1.52±0.05 0.54±0.04 0.29±0.06 1.11±0.05

Tables 5.108 to 5.111 A. niger and P. lilacinum was grown on 5% sucrose, 5%

molasses and incorporation 0.2% or 0.4% (NH4)2SO4 at 30 ± 2 ºC when pH was

adjusted at 6.5. The maximum production of pectinase 25.14 U/ml and

147

23.13 U/ml respectively was obtained at 72 hours. Loera et al., (1999) and

Scopes (1985) have reported 73 hours to be optimum incubation for maximal

polygalactouranase activity by a diploid construct from two Aspergillus niger

overproducing mutants. Ammonium sulphate appeared to be the most optimal

nitrogen source for pectinase production, which also caused a stabilizing effect

on enzyme. In the literature, ammonium sulphate and potassium phosphate

have been reported to have no significant influence on the production of

pectinase at lower concentrations Hours et al., (1988) but in present study

ammonium sulphate had shown a greater influence on the production of

pectinase after 72 hours with both filamentous fungi used. The present study is

in agreement with Phutela et al., (2005) contrast with , Arijit et al., (2013) reported

that (NH4)2 SO4 showed minimum production of pectinase enzyme by

Streptomyces sp. while Banu et el ., (2010) obtained highest pectinase production

by using ammonium per sulphate as a nitrogen source. Joshi et al., (2006)

reported that ammonium sulphate acted as best nitrogen source for the

production of Pectin methylestrase. Patil and Dayanand (2006 a) also reported

that both ammonium phosphate and ammonium sulphate did influence

production of pectinase positively in both submerged and solid-state conditions.

According to the observation of Phutela et al., (2005) fungi did not produce

pectinase when culture media was devoid of ammonium sulphate. Tariq and

Reyaz (2012) and Reda et al., (2008 ) reported that ammonium sulphate provides

additional nitrogen to the fermentation systems and in result more pectinase was

produced.

148

Table-5.108: A. niger was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% (NH4)2SO4 at 30 ± 2 ºC and pH was

adjusted at 6.5

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 5.6±0.3 1.11±0.04 1.48±0.06 21.44±0.02 14.41±0.04 8.82±0.05

48 5.9±0.1 1.18±0.03 1.53±0.03 19.64±0.01 13.45±0.04 11.74±0.04

72 5.4±0.2 1.32±0.03 1.56±0.03 14.84±0.04 10.79±0.04 23.19±0.04

96 5.35±0.01 1.36±0.03 1.58±0.03 11.37±0.05 8.81±0.01 12.40±0.01

120 4.7±0.1 1.44±0.04 1.60±0.09 9.18±0.03 6.93±0.06 8.79±0.06

144 4.8±0.2 1.25±0.04 1.61±0.01 7.36±0.04 5.95±0.09 7.72±0.02

168 4.6±0.1 1.27±0.02 1.66±0.04 5.79±0.03 4.33±0.03 5.94±0.02

192 4.45±0.02 1.33±0.05 1.69±0.04 2.04±0.03 1.96±0.03 3.40±0.04

216 4.25±0.03 1.36±0.04 1.72±0.04 1.98±0.06 1.68±0.06 2.33±0.03

240 4.1±0.1 1.49±0.05 1.78±0.05 0.54±0.04 0.50±0.06 1.14±0.05

Table-5.109 : A.niger was grown on mineral medium supplemented with 5%

sucrose, 5% molasses and 0.4% (NH4)2SO4 at 30 ± 2 ºC and pH was adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 5.85±0.02 0.93±0.05 1.53±0.03 21.92±0.06 13.73±0.05 7.73±0.03

48 5.55±0.01 1.12±0.03 1.56±0.03 17.93±0.03 13.62±0.02 12.92±0.02

72 5.5±0.1 1.19±0.06 1.58±0.03 15.80±0.07 11.49±0.05 25.14±0.01

96 5.25±0.03 1.23±0.03 1.64±0.02 14.83±0.02 11.17±0.05 14.66±0.05

120 4.85±0.02 1.24±0.03 1.68±0.03 10.59±0.01 8.97±0.01 8.64±0.07

144 4.65±0.03 1.34±0.07 1.68±0.07 6.25±0.03 4.70±0.02 7.74±0.02

168 4.45±0.01 1.43±0.03 1.70±0.01 5.75±0.04 4.24±0.01 5.72±0.06

192 4.35±0.04 1.45±0.03 1.73±0.03 3.21±0.07 3.09±0.04 4.03±0.03

216 4.7±0.2 1.62±0.02 1.79±0.02 1.91±0.05 1.37±0.05 3.37±0.05

240 4.95±0.02 1.63±0.06 1.82±0.02 0.60±0.05 0.31±0.05 1.18±0.05

149

Table-5.110: P. lilacinum was grown on mineral medium supplemented with 5% sucrose, 5% molasses and 0.2% (NH4)2SO4 at 30 ± 2 ºC and pH was

adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 5.8±0.2 1.11±0.04 1.34±0.03 21.37±0.06 14.46±0.04 7.46±0.02

48 5.4±0.3 1.19±0.06 1.37±0.04 19.82±0.05 13.90±0.07 9.98±0.02

72 4.85±0.03 1.25±0.03 1.42±0.05 16.92±0.02 12.14±0.04 21.46±0.06

96 4.25±0.02 1.31±0.01 1.44±0.04 14.28±0.05 11.96±0.03 13.72±0.07

120 5.15±0.02 1.35±0.03 1.49±0.07 12.69±0.01 9.16±0.01 8.72±0.03

144 5.45±0.01 1.39±0.03 1.56±0.03 10.36±0.03 8.12±0.06 8.14±0.01

168 5.75±0.02 1.39±0.01 1.68±0.02 7.24±0.03 6.36±0.05 4.36±0.03

192 5.05±0.01 1.41±0.01 1.71±0.02 5.12±0.02 4.84±0.07 3.11±0.05

216 5.1±0.1 1.42±0.05 1.71±0.01 2.18±0.06 1.74±0.01 2.20±0.03

240 5.3±0.1 1.44±0.04 1.65±0.03 0.53±0.05 0.39±0.06 1.17±0.07


sucrose, 5% molasses and 0.4% (NH4)2SO4 at 30 ± 2 ºC and pH was adjusted at 6.5.

Time

Hours

Final pH Biomass

g/50 ml Broth

Total

Proteins (mg/ml)

Total Sugar

(mg/ml)

Reducing

Sugars (mg/ml)

Pectinase

Activity (U/ml)

24 5.80±0.02 0.91±0.04 1.76±0.03 21.82±0.06 13.82±0.05 7.41±0.08

48 6.15±0.02 1.04±0.03 1.82±0.02 18.75±0.04 12.88±0.07 9.39±0.03

72 5.85±0.01 1.20±0.06 1.91±0.08 14.76±0.03 11.93±0.07 23.13±0.03

96 5.65±0.01 1.21±0.04 1.85±0.05 11.32±0.02 8.54±0.03 12.87±0.05

120 5.50±0.01 1.26±0.04 1.84±0.02 9.83±0.02 6.94±0.01 8.91±0.04

144 5.60±0.03 1.29±0.04 1.75±0.02 7.18±0.03 5.82±0.02 8.36±0.01

168 5.75±0.02 1.34±0.03 1.74±0.06 5.42±0.04 4.94±0.03 6.83±0.06

192 4.95±0.03 1.46±0.03 1.73±0.03 3.93±0.03 3.47±0.05 4.38±0.03

216 4.30±0.02 1.62±0.04 1.72±0.06 2.67±0.05 2.44±0.01 2.59±0.01

240 4.20±0.01 1.64±0.02 1.69±0.02 1.09±0.06 0.73±0.05 1.84±0.04

150

In this study various nitrogen sources with two concentrations

(0.2 and 0.4%) were used to check the effect on the growth and production of

pectinase as results shown in Tables 5.112-5.113. Maximum biomass and

pectinase production 1.19 g/50 ml and 25.14 U/ml respectively recorded, when

A.niger grown on 0.4% ammonium sulphate in comparison to other nitrogen

sources used in this study. The ammonium ion taken up as ammonia, thereby

releasing a proton into the medium and causing a decrease in pH, a proton is taken up

from the medium when nitrate is transported into the cell, and this causes the pH to

increase as reported by (Prior et al., 1992); Torrado et al., (1998) and Gokhale et al., (1992).

Effect of various nitrogen sources were also checked when Penicillium lilacinum grown

on 5% corn steep liquor with addition to sucrose and molasses produced maximum

mycelial biomass and pectinase activity 1.30 g/50 ml and 23.23 U/ml respectively.

Present findings are in agreement with (Gupta et al., 1997) where nitrogen from

inorganic sources activated the production of polygalacturonase. Shastri et al., (1988)

have also showed similar results. The nitrogen source can play an important role in

affecting the pH changes in the substrate during the fermentation. A combination of

these two nitrogen sources can be used to reduce the pH changes during the fermentation

(Prior et al., 1992; Torrado et al., 1998 and Gokhale et al., 1992) also reported the capacity

of urea to prevent the drop in pH during fermentation system. These findings

support the present study also.

During optimizing the nitrogen source in the fermentation experiments, it

is very clear that in the presence of molasses and sucrose, as substrate appeared a

best carbon source. The ammonium sulphate was selected as a best nitrogen

source and Aspergillus niger was selected as best organism, which has produced

higher amount of pectinase in comparison to Penicillium lilacinum on same

optimal conditions as results shown in Fig-5.11

151

Table-5.112 Effect of nitrogen sources on growth and Pectinase production by Aspergillus niger

Nitrogen sources Biomass Pectinase Activity


Corn Steep liquor 0.2 1.27±0.04 21.19±0.06

0.4 1.20±0.03 23.39±0.06

Urea 0.2 1.16±0.01 21.29±0.08

0.4 1.193± 0.003 23.94±0.04

NaNO3 0.2 1.24±0.02 21.11±0.05

0.4 1.09±0.06 22.90±0.05

KNO3 0.2 1.11±0.07 19.22±0.04

0.4 1.17±0.05 20.67±0.03

NH4NO3 0.2 1.14±0.03 20.35±0.05

0.4 1.24±0.04 22.40±0.07

Peptone 0.2 1.07±0.04 21.80±0.01

0.4 1.18±0.06 23.98±0.01

NH4)2SO4 0.2 1.32±0.05 23.19±0.02

0.4 1.19±0.08 25.14 ±0.03

152

Table-5.113 Effect of nitrogen sources on growth and pectinase production by Penicillium lilacinum

Nitrogen sources Biomass Pectinase Activity


Corn steep liquor 0.2 1.30±0.01 21.172±0.006

0.4 1.30±0.06 23.23±0.04

Urea 0.2 1.21±0.01 20.14±0.04

0.4 1.14±0.03 22.16±0.04

NaNO3 0.2 1.21±0.02 20.39±0.03

0.4 1.17±0.01 22.50±0.04

KNO3 0.2 1.21 ±0.02 18.18±0.03

0.4 1.9±0.03 19.14±0.03

NH4NO3 0.2 1.33±0.01 20.11±0.01

0.4 1.15±0.04 21.27±0.05

Peptone 0.2 1.10±0.05 20.57±0.04

0.4 1.15±0.03 22.97±0.01

(NH4)2SO4 0.2 1.25±0.05 21.46±0.05

0.4 1.20± 0.07 23.13±0.01

153

vii- Selection of the organism:

A.niger was selected for further studies on the basis of results of carbon

and nitrogen sources used for the growth and pectinase synthesis through

consecutive experiments. An overview of the results obtained that 5% sucrose,

5% molasses and 0.4% (NH4)2SO4 are the best substrate component for the

production of pectinase by A .niger. The production of the enzymes from agro-

wastes by fungi in submerged fermentation system could not only be cost

effective but it could also offer several process merits. It is suggested that

microorganisms need sugar and nitrogen sources, which are essential for the

growth of microorganism and production of enzymes.

Fig 5.11. Comparison of Pectinase production produced by Aspergillus niger and

Penicillium lilacinum

25.14

23.23

22

22.5

23

23.5

24

24.5

25

25.5

Aspergillus niger Penicillium lilacinum

Pe ctina se Activity U/mL

154

viii- Effect of pH on pectinase production:

Microorganisms have their own individual pH for growth and the production of enzymes

but it is also dependent on the pH of the medium. pH plays a very important and is a

critical role in the synthesis of microbial enzymes.

Table-5.114 reveals the result of pectinase synthesis by A. niger grown in

optimized cultured conditions with different initial pH values. The maximum

production of pectinase 26.87 U/ml was observed at an initial pH 6.0 at 72 hrs

incubation, than its production, was decreased with the increase of pH values.

The concentration of total, reducing sugars and total protein content was

different in different pH values. The pH optimum of 6.0 for pectinase production

by A. niger is in contrast with the reported values of Penicillium sp. As reported

by Martin et al., (2004) and also with A. niger (Jyothi et al., 2005; Díaz-Godínez, et

al., 2001). It is reported that low pH values are favorable for high pectinase

production in Penicillium italicum (Alana et al., 1989). The optimum pH of

mesophilic pectinase has been established to range in-between 4.0-5.5 as reported

by Favela-Torres et al., (2006). The influence of pH on the culture medium may be

directly related with the stability of enzymes (Ueda et al., 1982). Studies with

Pectin Lyase of P. expansum showed that in spite of the optimum activity pH of

Pectin Lyase to be 7.0, the enzyme can be kept stable between the pH 6.5 to 8.0

(Santiago, 1993).

155

Table-5.114 : Effect of pH on Biosynthesis of Pectinase by A. niger grown on

mineral medium supplemented with 5% sucrose, 5% molasses

and 0.4% (NH4)2SO4 at 30 ± 2 ºC for 72 hours.

Initial pH


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

U/ml)

4.0 4.45±0.02 0.84±0.04 1.78±0.05 13.27±0.05 9.12±0.05 13.13±0.03

4.5 4.75±0.01 1.11±0.03 1.75±0.04 12.12±0.02 8.96±0.03 17.94±0.03

5.0 5.9±0.1 1.14±0.03 1.87±0.04 12.99±0.06 9.47±0.02 20.42±0.04

5.5 6.35±0.02 1.59±0.06 1.89±0.04 12.76±0.01 9.52±0.02 23.27±0.06

6.0 6.85±0.02 1.36±0.04 1.94±0.04 13.92±0.02 9.30±0.05 26.87±0.02

6.5 7.6±0.2 1.29±0.04 1.82±0.01 12.92±0.06 9.54±0.03 16.93±0.04

7.0 7.95±0.02 1.26±0.03 1.77±0.04 13.01±0.01 8.98±0.04 11.81±0.04

7.5 8.8±0.1 1.34±0.04 1.73±0.05 13.86±0.03 9.73±0.05 4.96±0.04

8 8.5±0.2 1.22±0.05 1.56±0.04 11.4±0.2 8.90±0.03 4.87±0.04

In the present study, it was noticed that the pH of the culture media

assorted over a broad range on the pH scale. Usually fungi amend the pH of the

medium in which they nurture, due to uptake of the cations or anions in the

medium (Moore-Landecker 1996; Griffin, 1994). Therefore, the different changes

noted in the pH of the culture media may be due to a result of the consumption

of some compounds in the media Juwon and Emmanuel (2012).

The researchers like Uenojo and Pastore, (2007), Cordeiro and Martins,

(2009) investigated that the reason of decrease of pH value is might be due to the

synthesis of galactouronic acid in the medium by the pectinase when acts on

pectin and that may be affects the more pectinase production. The nature of

microorganism and initial pH value for the production of pectinases is significant

because it varies organism to organism as a higher initial optimal pH of 8.5 for

the synthesis of pectinase has been reported by Sharma and Satyanarayana,

(2006). In contrast, to above researchers the optimum initial pH for pectinase

production was found as 7.0 by the thermophilic fungus S. thermophile (Kaur et

al., 2004). According to Young et al., (1996) the genes involved in the production

of certain enzymes in at least some microorganisms may be pH regulated.

156

ix- Effect of Temperature:

Temperature is very important parameter for the growth and other vital activities

of organisms. It is directly connected to the metabolic activities of the

microorganism and it affects the growth and product synthesis by the

microorganisms (Lonsane et al., 1985). Every organism shows its individual optimal

temperature at which it grows well and produces the desired products maximally.

Hence maintenance of optimal temperature is an important and critical factor.

Thermal conditions for the maximum production of pectinase enzyme were studied in

Aspergillus niger . In Table-5.116 enzymes showed maximal activity at 35 °C. The

enzymetic activity was almost similar at 20 °C and 45 °C i.e just below and

above optimal temperature.

The result is in agreement with Said et al., (1991) regarding production of

pectinase by Penicillium frequentans at 30 – 35 °C. The present finding is also

supported by Lonsane et al., (1985), while (Bailey and Pessa , 1990) studied the effect

of temperature on pectinase enzyme production by Aspergillus niger and the optimum

temperature was found to be 30 °C. The results are in contrast with Soni and Bhatia,

(1981) reported 21°C for Fusarium oxysporum. Gummadi and Kumar (2007) and

Nakajima et al., (1999) reported an optimum temperature 30 °C and 37 °C for

pectin lyase and pectin pectate lyase production by A. niger NCIM 548 and

Clostridium butyricum-beijerineki, respectively.

157

Table-5.115: Effect of temperature on biosynthesis of pectinase by A. niger grown

on mineral medium containing 5% molasses 5% sucrose and 0.4% (NH4)2SO4

while pH was adjusted 6.00 for 72 hours.

Initial Tem


Broth

Total Proteins

(mg/ml)

Total Sugar

(mg/ml)

Reducing Sugars

(mg/ml)

Pectinase Activity

(U/ml)

20ºC 6.9±0.2 1.09±0.06 1.94±0.03 22.73±0.03 19.38±0.04 9.84±0.04

25ºC 6.8±0.2 1.11±0.05 2.00±0.1 22.85±0.03 19.12±0.02 17.99±0.02

30ºC 6.1±0.1 1.12±0.02 1.97±0.04 22.63±0.02 19.77±0.02 26.13±0.02

35ºC 6.0±0.3 1.19±0.04 1.99±0.03 21.79±0.04 19.43±0.02 28.25±0.03

40ºC 5.8±0.3 1.02±0.02 1.95±0.03 21.64±0.02 19.36±0.03 18.17±0.02

45ºC 5.7±0.1 1.01±0.01 1.96±0.05 21.58±0.01 19.28±0.02 11.91±0.03

B- Characterization of crude Pectinase Enzyme: The Pectinase produced by

Aspergillus niger when it was grown on 5% molasses, 5% sucrose and 0.4%

(NH4)2SO4 with the initial pH adjusted 6.0 and incubated for 72 hours.The

enzyme was characterized on the basis of time of incubation, substrate

specificity, substrate concentration, enzyme volume, buffer, pH, pH stability,

temperature, thermo stability, activators and inhibitors.

i- Effect of time of incubation on crude Pectinase: The Pectinase activity was

observed at various time periods (10-60 minutes) and result shown in Figure-

5.12, the results indicate that 15 minutes incubation period basis of the highest

activity, because increase and decrease in time reduces the pectinase activity

more or less same results are presented by Akhilesh et al., (2010) who reported

maximum activity of polygalacturonase from M. circinelloides after 20 minutes

and 30 minutes incubation period for highest pectinase activity was reported by

Roosdiana et al., (2013).

The declined activity after 15 minutes is may be due to the presence of other

enzymes in crude sample or the reason behind the decrease in Pectinase activity

might be inactivation of enzyme on prolongs incubation or self digestion or

158

product inhibition. Similar results are reported from earlier studies by (Dahot,

(1992) and Weil et al., (1966)

Figure-5.12: Effect of time of incubation on crude Pectinase

ii- Effect of substrate concentration on crude Pectinase: The effect of substrate

concentration was investigated for the rate of enzymatic reaction of Pectinase by

using pectin with different concentrations ranging from 0.5-4.0%. The rate of

reaction of enzyme directly increases with the increase in substrate concentration

till a certain optimum point is appeared and the substrate saturated with enzyme

present in reaction mixture. The hydrolytic activity of petinase enzymes was

investigated and results are represented in Figure-5.13 shows that 1.5% Pectin

had given optimum pectinase activity while below and above this concentration

activity declines the rate of reaction. The substrate concentratation (1.5 mg /ml)

in this study are in full accordance with NitinKumar and Bhushan, (2010) while

Afifi and Foaad (2002) reported the optimum concentration of substrate for

Pectin lyase as 1.1% citrus pectin. Deshmukh et al., (2012) reported 0.5 substrate

concentration for highest activity of pectinase produced by various strains of

159

Aspergillus. The declination of pectinase activity may be due to alteration in

enzyme and substrate ratio Gillard, (1971) and Price and Stevens (1999).

Figure-5.13: Effect of substrate concentration on crude Pectinase

iii- Effect of enzyme volume on crude Pectinase: The effect of enzyme volume

(0.2-1.4 ml) on the rate of enzyme reaction was studied as shown in Figure-5.14

that an increase in enzyme volume increases the pectinase activity while addition

of above 1.0 ml lowers the activity. The higher pectinase activity was found at 1.0

ml crude enzyme while low pectinase activity was observed when less amount of

enzyme volume was used. Deshmukh et al., (2012) accomplished highest

polygalacturonase activity by using 0.5 ml of crude enzyme isolated from

Aspergillus strains.

0

5

10

15

20

25

30

35

0.5 1 1.5 2 2.5

Pec

tin

ase

Act

ivit

y U

/mL

Substrate concentration %

160

Figure-5.14: Effect of enzyme volume on crude Pectinase

iv- Effect of different buffers on pectinase activity:

Figure-5.15 shows the effect of different buffers 0.1 M (sodium phosphate,

sodium citrate and universal buffer) on Pectinase activity. The 100% relative

activity was determined by using sodium citrate buffer, which was used

throughout the study. The use of sodium citrate buffer in pectinase activity is

reported by Banu et al., (2010) and Akhilesh et al.,(2010)

Figure-5.15: Effect of different buffers on crude Pectinase

0

5

10

15

20

25

30

35

phaosphate citrate universal

Buffers (pH 5.5)

Pec

tinas

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161

v- Effect of pH on crude pectinase

The pectinase activity was checked at various pH values (3.0 to 10.0) and

Figure-5.16 shows that the optimum pH for the crude pectinase was noted 5.0.

The catalytic activity of many enzymes is markedly dependent on pH. pH can exerts its

effect in different ways, on the ionization of groups in the enzyme's active site,

either on the ionization of groups in the substrate or by affecting the conformation of

either the enzyme or the substrate. These effects are influencing to the changes in

kinetic constants, (Dennison, 2003). In the present study Pectinase from A. niger

showed its maximum activity at pH 5.0 which is against the results of Marcia et al.,

(1999) when worked with polygalacturonase the highest activity was found at 6.0.

More or less same results were reported by Banu et al., (2010) that maximum

pectinase activity from P. chrysogenum was found at pH 6.5 using sodium citrate

buffer . The results of this study are in ful agreement with Afifi and Foaad (2002)

who reported that the maximum activity of Pectin lyase was found at pH 5.0 .The

result are also according to the results of Obi and Moneke (1985) and Moharib et

al. (2000) who reported that the pectinase enzyme was highly active at pH 5.0

and 4.5 respectively.

Martos et al.,(2013) reported that polygalcturonase isolated from

Wickerhanomyces anomalus showed maximum activity at pH 4.5-5.0, similar

observations for ploygalacturonase were reported for Rhizopus spp. by Elegado

and Fujio, (1994), for A. niger CH4 Acuña-Argüelles et al., (1995), for Lentinus

edodes by Zheng and Shetty, (2000), for A. awamori and A. japonicas by Jayani et

al.,( 2005). Damásio et al., (2011) reported highest activity of polygacaturonase

from Rhizopus microspores var. rhizopodiformis was recorded at pH 3.5 .

Fungal polygalacturonases from other sources show different optimum pH

conditions like verticillium albo atrum, 6.5 (Wang and Keen, 1970), Ganoderma

lucidum, 5.5 (Kumari and Sirsi. 1971), Pencillum capsulaturn, 4.68 (Gillespie et al.,

1990) and Penicillium frequentans, 4.0-4.7 (Borin et al., 1996). Even though a

marked variation of pH in polygalacturonase was found in different strains, the

162

optimum activity lies in between pH conditions 3.5-6.0. This is the typical

characteristic of fungal polygalacturonases (Rombouts and Pilnik, 1980). This is a

well establish fact that each enzyme has a characteristic pH optimum for its

activity (Lehninger et al. 1992), Wei-Chen et al., (1998) has observed the

maximum pectin layse ctivity at pH 8.0. Favela-Torres et al., (2006) observed that

the optimum pH for exo-Polygalacturonase was higher than the majority of

fungal PGs described, and they are considered as acidic enzymes.The results of

present study are also in accordance to Freitas et al., (2006) who obtained the

maximum activity for exo-PG at pH 5.5 when working with Monascus sp. and

Aspergillus sp. Pectinase. P. viridicatum RFC3 showed an optimum pH 6.0 (Silva et

al., 2007), from Moniliella sp. SB9 at pH 4.5 and Penicillium sp. EGC5 at pH 4.5 -

5.0 (Martin et al., 2004). The optimum activity for PL was pH 5.0, at pH 6.0; PL

activity decreased by 93% but was inactivated in neutral and basic pH. The pH

optima of the previously reported PL have been found to be acidic for Penicillium

canescens (5.5), (Sinitsyna et al., 2007), neutral for Penicillium expansum (Silva et al.,

1993) and basic for Aspergillus flavus (8.0) (Yadav et al., 2008) and Aspergillus

terricola (8.0) (Yadav et al., 2009).

Figure-5.16: Effect of pH on crude Pectinase

163

vi- Effect of pH stability on crude pectinase : Crude enzymatic extract was

diluted (1:1) in different buffers (pH 3- 10 ) incubated at 37 °C and pectinase

activity was checked at various pH values (3.0 to 10.0) Figure-5.17 shows that the

the crude pectinase was stable over a range of 3.0 to 6.0 pH retain more than

70% activity up to pH 8.0.

These results are in accordance with Freitas et al., ( 2006) who observed that exo-

polygalacturonase from Monascus sp. was stable in between pH 4.5 - 6.0, while

that from Aspergillus sp. was stable at pH 4.0..These results are not in agreement

with Silva et al., (2007) who worked with P. viridicatum RFC3 and reported

stability of exo-polygalacturonase in a pH of range 7.0 - 10.0. Marcia et al., (1999)

reported the the stability of in a pH range of 6-8 while Martin et al., (2004)

reported that polygalacturonase was stable at the pH range between 3-8 obtained

from Penicillium sp. and maintained 70% of its initial activity but pectin lyase

was stable in acidic to neutral range of pH (4-8 ) produced from same oraganism.

polygalacturonase from A. sojae ATCC 20235 was stable at pH 5.0 and retained

60 % and 70 % of its activity at pH 3.0 and 7.0 respectively Tari et al., (2008).

polygalacturonase of Penicillium viridicatum RFC3 was observed stable at pH 5.0-

8.0 Silva et al., (2002) and maintaining 80% of its activity at pH 9.0. pectin lyase

was more sensitive to pH variation, presenting maximum stability at pH 3.5 - 4.5

which declined to 80% at pH 5.0. Martin et al., (2004) reported that pectin lyase

produced by Moniliella sp SB9 and Penicillium sp EGC5, and was stable in acidic

to neutral pH (4.0-7.0). However, the results of Yadav et al., (2008 and 2009)

indicated that the stability of pectin lyase was noted in a pH range of 4.0 - 10.0

and 4.0 - 9.0 with A. flavus and A. terricola, respectively. According to Pedrolli

and Carmona, (2010) polygalacturonases of fungal origin are typically stable in

acidic medium, yet, polygalacturonase produced by A. giganteus proved to be

more stable over a neutral and alkaline pH range. It seems that pectinase

produced through A. niger works better in acidic environment.

164

Figure-5.17: Effect of pH stability on crude Pectinase

vii- Effect of temperature on crude Pectinase: The A. niger Pectinase activity was

investigated on different temperatures. The influence of temperature to pectinase

activity was displayed in Figure 5.18. Pectinase activity increased between the

temperature 30 °C and 50 °C and the 40 °C was found optimum temperature.

The decreased activity of the enzyme at temperatures above 40 °C could be due

to protein denaturation at higher temperatures.

Banu et al., (2010) reported the optimum temperature was found to be at 50 °C

when highest activity was recorded for the polygalcturonase obtained from

P. chrysogenum. Alana et al., (1990) also reported the similar results the for pectin

lyase enzyme from P. italicum which showed an increase of activity up to 50 °C.

El-Batal et al., (2013) reported that highest activity of polygalacturonase from P.

citrinum achieved at 40 °C, similar results were shown by Palaniyappan et al.,

(2009) and Arotupin et al., (2012) who reported a decline in the enzyme activity

with a temperature more than 40°C. Arotupin, (2007) investigated that

polygalacturonase produced by A. Flavus, A. fumigatus and A. repens showed

highest activity at 35 °C, 40 °C and 45 °C respectively. According to Arotupin

(1991) who explained these differences in the optimum temperature of fungal

polygalacturonase imply a wide range of temperature, In addition the

0

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40

60

80

100

120

3 4 5 6 7 8 9 10

% R

ela

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mg

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165

environment, sources and differences in the physiological activities of the fungi

may be dependable for this phenomenon.

Temperature °C

Figure-5.18: Effect of temperature on crude Pectinase

166

viii- Effect of temperature stability on crude Pectinase: The activity was

investigated on different temperatures by heating for 10 minutes from 20 -100 °C

with an interval of 10 °C and the remaining activity was checked by adding

substrate according to standard method.

Enzymes are specific to temperature, every enzyme is optimally active and stable

up to a certain temperature and gets denatured at higher temperatures while

crude Pectinase was 100 % stable up to 40 °C and then activity declines slowely,

pectinase and retains more than 30 % activity up to 80 °C as shown in Figure

5.19. These results are in accordance with those obtained by Phutela et al., (2005),

and in contrast with Martin et al., (2004) who reported that polygalactouranase

from Penicillium sp. was stable in temperature lower than 40 °C

polygalactouranase activity of Rhizopus microsporus var. Rhizopodiformis was

stable up to 55 ºC Damásio et al., (2011). An endo- polygalactouranase of A. niger,

produced in solid state fermentation, was stable up to 40 ºC and presented 60 %

of its maximum activity after about 60 min of incubation at 50 ºC Hendges et al.,

(2011).

Temperature °C

Figure-5.19: Effect of temperature stability on crude pectinase

0

20

40

60

80

100

120

20 30 40 50 60 70 80 90 100

% R

ela

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167

ix- Effect of metal ions or compounds on crude pectinase: Effect of various

metal ions and compounds on pectinase activity was investigated by adding the

enzyme with 5 mM of each reagent in 0.1 M Sodium citrate buffer pH 5.5

alongwith enzyme and incubated at optimum temperature for 10 minutes prior

to addition of substrate and their remaining activities were determined.

Figure.5-20 shows the effect of various metal ions and compounds (5mM) on

Pectinase activity produced by A. niger. Among the metal ions tested, the

addition of 5 mM CaCl2 enhanced the activity of pectinase enzyme produced by

A. niger while MnSO4 , AgNO3 , HgNO3 CoCl2 inhibited pectinase enzyme

activity to the level of 40 % to 60 %. There is a significant influence by Ca2+

on the

activity and stability of enzymes (Shevchik et al., 1999). Cabanne and Doneche,

(2002) reported the partial inhibition of polygalacturonase by 1mM of CaCl2 might be

due to the chelating effect of calcium on the substrate (PGA) of the enzyme. Hg had no

effect on Polygalacturonase on Neurospora crassa (Polizeli et al., 1991). ZnSO4 was

an activator of polygalacturonase produced by Rhizopus sp. Elegado and Fujio,

(1994). Afifi and Foaad, (2002) reported that enzyme was inhibited with the

addition of Co++, Mn++, Zn++ and showed complete inhibition with the

addition of Ag++ and Hg++, similar results were produced by Chen et al.,

(1998). The change in electrostatic bonding could change the tertiort structure of

enzyme and may be metal ions change the electrostatic bonding Palmer,

(1991).According to Afifi and Foaad, (2002) reported that it could be due to

participation of sulphydryl groups in the active site of the enzymes. Afifi and

Foaad, (2002) reported that EDTA and L-cysteine did not affect enzyme activity

(Starr and Moran, 1962; Whilaker, 1972; Delgado et al., 1992).

168

Figure-5.20: Effect of metal ions/ compounds on pectinase activity

x- Effect of different concentration of CaCl2 as activator: After investigating that

CaCl2 was best activator for pectinase activity and there fore the effect of

different concentrations CaCl2 were tested and it was observed that increase in

CaCl2 concentration increases Pectinase activity upto 15 mM. As described in

Figure- 5.21.This was also observed that with the increase of CaCl2 concentration

decreased the enzyme activity may be due to the higher amount of activator acts

as inhibitor. However, in some cases the activity was inhibited, in other cases it was

stimulated by CaCl2 Perley and Page, (1971). Some of the polygalacturonases require

Ca+2 whether Ca+2 performs a role in binding and/or catalysis, in maintaining the

conformation of the enzyme, or whether it masks the car boxylic groups of the substrate

is not clear. Polygalacturonase was found to be inhibited by Ca+2 in the cases of,

Neurospora crassa Polizeli et al., (1991) and Rhizopus sp. Elegado and Fujio, (1994)

pectinase and was activated by Ca+2 in Geotrichum candidum (Shastri et al., 1988).

Banu et al., (2010) and Hla et al., (2005) have reported that normal optimal

activity was observed by Clostridium stercorarium at 0.05 mM of CaCl2 and the

enzyme was more or less activated throughout a range of CaCl2 concentrations

0

20

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60

80

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120

140%

of

Acti

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& in

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169

from 0.05 to 0.2 mM but addition of 0.2 mM EDTA inhibited activity to less than

5% of maximum.

Figure-5.21: Effect of different concentrations of CaCl2

xi- Effect of thermostability with and without activator: The thermostability of

pectinase was checked by incubating the crude pectinase with and without

activator (15 mM CaCl2) in 0.1 M Sodium citrate buffer of pH 5.5 at two different

temperatures (60 ºC and 70 ºC) for 60 minutes and remaining activity was

determined under standard assay conditions. Figure-5.24 shows the temperature

profile that crude pectinase retained 81.28% and 74.32% relative activities with

and without activator respectively and lost about 18.72%and 25.68% activity

with and without activator respectively, when heated at 60°C for 60 minutes

respectively as shown in Figure- 5.22.

Similarly in Fig 5.23, The thermostability of pectinase was checked by incubating

the crude pectinase with and without activator (15 mM CaCl2) in 0.1 M Sodium

citrate buffer of pH 5.5 at 70 ºC for 60 minutes and remaining activity was

determined under standard assay conditions. Figure-5.25 shows the temperature

profile that crude pectinase retained 75.32 % and 67.2 % relative activities with

and without activator respectively and lost about 24.28 % and 32.2% activity with

84

86

88

90

92

94

96

98

100

102

0.5 1 15 2 25

% of Re le tive a ctivity

Concentration of CaCl2

mM

170

and without activator respectively, when incubated at 70 °C for 60 minutes

respectively.

Figure-5.22: Effect of themostability at 60 °C on different time periods with and

without activator CaCl2 (15mM) on pectinase activity produced by Aspergillus niger

Figure-5.25: Effect of themostability at 70 °C on different time periods with and without activator CaCl2 (15mM) on pectinase activity produced by

Aspergillus niger

100 97.6493.53

85.278.46 75.34

10096.53

89.56

80.34

71.3467.2

0

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60

80

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10 20 30 40 50 60

Time period (minute)

% o

f R

leti

ve A

ctiv

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with activator without activator

100 99.1 97.3794.11

89.56

81.28

100 98.2395.32

89.23

82.21

74.32

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120

10 20 30 40 50 60

% o

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With Activator without Activator

171

C- Purification of enzyme: In present study pectinase from Aspergillus niger was

purified and characterized. Purification of microbial enzymes is not easy because of the

removal of contaminating substances and also the separation of the enzyme from other

pectinolytic enzymes produced by the microorganisms. Normally one cell or tissue

homogenate contain thousands of different proteins and the purification seems to be

difficult. However in practice four different steps are involved in purification of proteins

and some times a single chromatographic step is enough based on the purpose of

purification.

For structural and functional studies 100% purification is not necessary and

enzymes can be purified several fold but the yield of the enzymes may be very poor.

Industrial enzymes are purified as little as possible i.e. for the removal of

interfering materials. Since additional stages are costly in terms of equipment,

manpower and loss of enzyme activity. As a result, some commercial enzyme

preparations consist especially of concentrated fermentation broth, plus

additives to stabilize the enzyme's activity Wilson and Walker, (2000). It is

important to retain maximum activity of the enzyme during its preparation.

The main objective of the first stage for the recovery of an

extracellular product and the removal of large solid particles as well as

microbial cells usually by centrifugation or filtration. In the next stage, the broth

is fractionated or extracted into major fractions using ultra filtration, reverse

osmosis, adsorption/ion exchange/gel filtration, two phase aqueous

extraction or precipitation. Afterwards, the product-containing fraction is

purified by fractional precipitation. Further more precise chrornatographic

techniques and crystallization are done to obtain a product, which is free from

impurities. Purification of enzyme is very important procedure. Enzyme test while

it is crude does not exhibit isolated action or the presence of a system of multi

enzyme working together to degrade a substrate Pedrolli et al., (2009). It is also

very important line of research that purified enzymes may be characterized as it

differentiates between the enzymic complex components for substrate

172

degradation mechanism, optimum activity conditions and enzyme synthesis

regulation Pedrolli et al., (2009).

i- Removal of microbial cells and other solid matter: Microbial cells and other

insoluble materials are normally separated from the harvested broth by filtration or

centrifugation Stanbury et al., (1997) . Filtation separates particles simply on the basis

of their size. Centrifugation separates on the basis of the particle size and density difference

between the liquid and solid phases Chaplin and Bucke, (1990).

ii- Concentration by precipitation: Enzymes can be concentrated by

precipitation, and this is generally used as the initial step of purification.

Salting out by ammonium sulphate is the best-known method for concentration

and purification of the enzyme. Some enzymes do not survive with

ammonium sulphate precipitation. In such case organic solvents such as ethanol,

propanol and acetone can be used as the alternative. For further purification, the

ammonium sulphate present in the protein precipitate is to be removed. This can be

achieved by dialysis through 10,000 Da cut off membranes (dialysis membrane/bag)

iii- Ammonium sulphate fractionation: The cultured medium of fermentation

was centrifuged at 10,000 rpm for 20 minutes in a refrigerated condition. In this

study enzymatic protein from culture supernatant was precipitated with solvents

such as acetone, ethanol, and methanol with different concentration of

ammonium sulphate (40-80%). The experiments show that more pectinase

activity has been recovered with 60% solid ammonium sulphate precipitation.

The low pectinase enzyme activities were recovered in acetone, ethanol and

methanol may be due to the change of pH or temperature, which denatures the

proteins during the process (Holme and Peck, 1994 and Doi and Nojima, 1971).

Solid ammonium sulphate (GR grade MERCK) was slowly added to the

supernatant of crude enzyme preparation so as to reach 60% saturation.

Addition of ammonium sulphate was carried out with continuous stirring at

4.0 °C, and then it was kept at 4.0 °C for overnight. The precipitated protein was

removed by centrifugation at 10, 000 rpm for 30 minutes at 4.0 °C. The precipitated

173

protein was dissolved in 15 ml Sodium citrate buffer pH 5.0. The protein content

of the fraction was determined by the method of Lowry et al., (1951).

iv- Dialysis: The precipitate obtained after treatment with ammonium sulphate was

dialyzed against Sodium citrate buffer, pH 5.0 for overnight at 4.0°C with

occasional changes of the buffer. Cellulose membrane dialysis tubes was used for

dialysis (Cut off 10,000 Da).

D- Chromatography: Chromatographic techniques are used in the final

stages of purification. For enzyme purification, two types of chromatographic

principles are used.

Gel permeation chromatography: Gel permeation separates molecules on the

basis of pore size. The smaller molecules diffuse into the gel more rapidly than the

larger ones. The most widely used gels include cross-linked dextrans -

sephadex and sephacryl, and cross-linked agarose - sepharose with various pore

sizes Stanbury et al., (1997).

Ion exchange chromatography: Ion exchange can be defined as the reversible

exchange of ions between a liquid and solid phase (ion exchange resin), which is

not achieved by any radical change in the salt structure. Ion exchange materials

are generally water insoluble polymers containing cationic and anionic groups.

Carboxy methyl cellulose is a common cationic exchanger and diethyfaminoethyl

cellulose is a common anionic exchanger.

i- Gel filtration chromatography: The dialyzed sample of pectinase produced

from Aspergillus niger under submerged fermentation was purified by

ammonium sulphate precipitation 10 ml gel filtration chromatography on

sephadex G-100 chromatographic column. The Pectinase was eluted with 0.1M

sodium citrate buffer pH (5.5). Elution pattern is shown in Figure-5-24. Pectinase

activity and protein contents were determined for each fraction. All active

fractions were pooled and assayed for enzyme activity and protein. A recovery

of 9917 U of Pectinase activity with specific activity of 34.55U was obtained. This

gave 2.5 fold purification of the enzyme shown in Table-5.116.

174

Fig.5.24: Gel Chromatography

ii- Ion exchange Chromatography: The pooled and dialyzed fractions (F-3a and

F-3b) were separated on ion exchange DEAE Sephadex A-50 chromatographic

column. Equilibrium and elution shown in Figures 5.25 and were performed first

with 0.1 M Sodium citrate buffer (pH 5.0) to remove unbound proteins and then

with a linear salt gradient from 0.0 to 1.0 N NaCl. Active fractions were pooled

and specific activity of the pooled active fractions was measured as shown in

Table -1.116

175

Fig.5.25: Purifiction of Pectinase (F-3) on ion exchange chromatography

Table-5.116: Purification steps of Pectinase produced by Aspergillus niger

Sample Pectinase Activity Units

Total protein mg

Specific Activity

Purification Fold

% Yield

Culture broth (Crude sample)

13760 995 13.82 1 100

Dialyzed sample 11630 539 21.57 1.56 84.52

G – 100 Column Chromatography

9917 287 34.55 2.5 72.07

Fraction 1 3125 88.2 35.43 2.56 22.71

Fraction 2 2937 71.5 41.0 2.96 21.34

Fraction 3 1750 57.4 30.48 2.19 12.66

Fraction 4 2150 70.3 30.2 2.19 15.44

A – 50 Column Ion exchange

Chromatography

1750 57.4 30.48 2.19 12.66

Fraction 3-a 923 32.2 28.66 2.07 6.70

Fraction 3-b 827 25.2 32.81 2.37 6.0

176

iii- Homogeneity: SDS-polyacrylamide gel electrophoresis (Sambrook and

David 2001) of pectinase revealed multiple protein bands on staining (F-3) while

Fractions F-1, F-2 and F-4 shows single bands when staining with Coomassie

brilliant blue (Figure-5.26). When purified fractions were electrophoresed on

native 10% polyacrylamide gel were observed as different activity bands.

iv- Molecular weight: Electrophoretic mobilities of purified pectinase and

reference proteins on SDS-polyacylamide gel electrophoresis were plotted versus

their molecular weight. The mobility of the purified pectinases; Fraction F-I, F-2,

F-4, F-3a and F-3b corresponded to a molecular weight of 40,000 ± 5000 and of

35,000±3000 Da respectively. Figure-5.26-27. Fractions exhibiting pectinase

activity were pooled and used as purified enzymes indicates that two types of

pectinase are produced by Aspergillus niger. It has been reported by Buga et al.,

(2010) that SDS-PAGE of the active fractions showed two different bands on the

gel with visible molecular weights of 35 KDa and 40 KDa. The presence of

different bands implies the presence of a dimeric protein consisting of both

‘endo and exo’ polygalacturonase activities.

Khairnar et al., (2009) also reported the molecular weight of the pectinase

in his study was found to be 40 KDa. Kester et al., (1996) reported a pectinase

with a molecular weight of 20,000 Da while deVries and Visser (2001) isolated a

pectinase having a range of molecular weight as 35,000 – 80,000 Da. This range is

in agreement with the obvious molecular masses of the pectinase purified in this

study.

177

Figure-5.26: SDS-PAGE (10% Polyacrylamide) of the purified enzymes. Lane 1, low Mw

Marker; Lane 2, Fraction 1; Lane 3, Fraction 2; Lane 4, Fraction 3; Lane 5, Fraction 4; Lane

6, Crude enzyme

Figure-5.27: SDS-PAGE (10% Polyacrylamide) of the purified enzymes. Lane 1, low Mw

Marker; Lane 2, Fraction 3a; Lane 3, Fraction 3b; Lane 4, Crude enzyme

178

E- Characterization of purified Pectinase

i- Effect of Substrate specificity:

Pectins are complex high molecular mass glycosidic macromolecules in

the primary cell wall the major components of the middle lamellae, an adhesive

thin extracellular layer between the walls of adjacent juvenile cells and are

basically responsible for the structural uprightness and consistency of plant

tissues. (Rombouts et al., 1980, Alkorta et al., 1998). There are three major pectic

polyssacharides groups are renowned, to a greater or a lesser extent all contain

D-galacturonic acid. Pedrolli et al., (2009). It is reported by Kabli, (2007) that

pectinase enzyme was treated to check hydrolyzing activity with peels of Mango,

orange, lemon ,grape fruit and with beet pulp.Enzyme activity was exhibited

according to the type of substrate low activity was shown with beet pulp. Singh

and Appu, (2002) also reported that pectinase enzymes preferred their specific

substrate irrespective of the source.

The purified Pectinase enzyme was most active with pure pectin as

shown in Fig -5.28, while crude pectin, Lemon pectin and orange peel given

lower activity as compared to (control) i-e pure pectin. Similar results were

presented by Pedrolli and Carmona, (2010) who described that substrate

specificity for polygalacturonases isolated from Aspergillus giganteus as for citrus

pectin was 100%, polygalactouranic acid 94.9%, citrus pectin 51.9%, citrus

pectin 25.5% and apple pectin 23.9%. The present work is supported by Fahmy et

al., (2008), enzyme from A. niger NRRL3 shown citrus pectins with different

esterification 41 to 97%) Mohamed et al., 2009 reported that pectinase enzyme

isolated from T. harzianum shown citrus pectins with different esterification to

187%. Pectinase from Mucor circinelloides ITCC 6025 hydrolyze citrus pectin 11.0

% and apple pectin 22 % Thakur et al., (2010). More or less similar findings were

reported by Al - Najada et al.,(2012).

179

Figure-5.28: Effect of substrate specificity on pectinase produced by

Aspergillus niger

ii- Effect of substrate concentration on pectinase activity:

Effect of initial reaction rates of pectin hydrolysis by pectinase was estimated

with purified enzyme. Highest pectinase enzyme activity was observed when

1.5% pectin in 0. 1 M sodium citrate buffer pH (5.0) was used as a substrate in the

reaction mixture and incubated for 15 minutes at 37 °C.

Results were shown in Figure-5.29 indicates that significant increase in

pectinase activity was achieved with the increase of substrate concentration.

After the maximum concentration pectinase activity started to declined in

fractions FI, F2 , , F3-b, and F 4 where as the maximum substrate concentration F3-a,

was noted to 1.0 of purified pectinase enzymes. The findings of (Nitinkumar et

180

al., 2010 ) are in agreement with the present study that when 1.5% substrate was

used the enzyme activity was highest.

Fig.5.29: Effect of substrate concentration on Pectinase activity produced by

Aspergillus niger

iii- Effect of pH on pectinase activity produced by Aspergillus niger:

The pectinase activity was investigated at various pH values to check the

optimum pH for the purified enzyme produced by A. niger .The fig-5.30 shows a

graphical representation which exhibits that with the increase in pH, the activity

of the enzyme increased up to an optimum pH. However, further increase in pH

has shown a gradual decrease in pectinase activity in all the fractions like (F1, F2 ,

F3-a, F3-b and F4, ) which was between pH 5.0 and 6.0 and these results are higher

than those generally evident for fungi in the range of 3.0 and 5.0 (Kojima et al.,

1999, Martins et al., 2002, Niture and Pant, 2004, Martin et al., 2004) but the

observations of present study are in full agreement with the results of Pedrolli

et al., (2008) who reported optimal pH for polygalacturonase activity as 5.5–

6.0.In literature fungal pectinase is reported to show maximum activity at a pH

4.0-5.0 ( Jayani et al., 2005, Niture et al., 2008), and mostly more active in acidic or

neutral pH Pedrolli et al., (2009). According to Pedrolli and Carmona, (2010)

0

1

2

3

4

5

6

7

8

0.5 1 1.5 2 2.5 3

Substrate Concentration

Pec

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ase

Act

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y IU

/mL

F-1 F-2 F-3a F-3b F-4

181

Polygalacturonase isolated from A.gigantus shows optimum activity at pH 6.0–

6.5. Kashyap et al., (2001) , Gummadi and Panda (2003), Jyothi et al., (2005), Díaz-

Godínez et al.,(2001) and Tari et al.,(2007) reported that acidic pectinases are

mostly fungal, especially from Aspergillus niger with optimum pH of 4.5-6 . The

pectinase isolated from Kluyveromyces wickerhamii showed optimum pH of 5.0

according to Moyo et al., (2003).

The results of optimum pH of purified pectinase are comparable with

the results of Al-Najada et al., (2012) who has reported that acidic pH optima

was found 4.0 for PGase from F. oxysporum ,while PH optima for PGase I and

PGaseII was found as 4.5 and 6.0 respectively from A. tubingensis. Similar results

were reported by Damasio et al., (2010) for polygalacturonase obtained by P.

variotii pH 4.0, where as Schnitzhofer et al., (2007) reported pH 4.5 to 5.0 for

enzyme isolated from Scleroderma rolfsii. Saad et al., (2007) presented the findings

for pH optima as 4.5 for pectinase from Mucor rouxii NRRL 1894 .

Pectinolytic enzyme shown optimum pH 3.8 from A. niger was reported by

Khairnar et al., (2009), from Mucor circinelloides ITCC 6025 with pH 5.5 Thakur et

al., 2010, by A. tubingensis with pH 4.2 (Gewali et al., 2007) and Aspergillus oryzae

with pH 5.0 , (Riou et al., 1998), Alana et al., (1989) have also reported low pH

values as being favorable for high pectinase production in Penicillium italicum.

The optimum pH of mesophilic pectinases has been established to range

between 4.0-5.5 (Favela-Torres et al., 2006).

Cylindrocarpon destructans pH 5.0 (Sathiyaraj et al., 2011) and typical

characteristic of fungal polygalacturonase according to Rombouts et al., (1980)

182

Fig.5.30: Effect of pH on pectinase activity produced by Aspergillus niger

iv- Effect of pH stability on pectinase activity produced by Aspergillus niger:

The effect of pH stability on Pectinase activity was investigated at various pH

values for the purified enzyme produced by A. niger. The result shown in

Figure-5.31 state that all the Fractions (F1, F2 , F3-a, F3-b and F4, ) are stable up to pH

range 3.0-8.0 . More that 30 % activity was retained when purified Pectinase was

incubated with pH 8.0 The results are in strong accordance to Pedrolli and

Carmona (2010) who isolated a pectionase from A. giganteus which shown

stability over a neutral and alkaline pH range. Yet, fungal pectinase are usually

stable in the acidic range of pH as reported by Devi and Rao (1996), Niture and

Pant, (2004), Jayani et al., (2005), and Dinu et al., (2007).

The results presented by Siddiqui et al., (2012) shown low range of pH stability as

compared to present stuy. He has presented the results of pectinase enzyme

from R. pusillus and the stability of the purified polygalacturonase was recorded

at pH 4.0-5.0. beyond this pH range enzyme stability began to decrease. At pH

8.0 after 4th hour the residual activity was 40.21% of that of the control while no

activity was recorded at pH 9.0 at 4th hour. Martos et al., (2013) reported about a

PG isolated from A. niger which shown highest activity at a pH range between

4.5 to 5.0. At pH 4.0 and 5.5, polygalacturonase activity values were 35 % and 38

0

20

40

60

80

100

120

3 4 5 6 7 8 9 10

pH

% o

f R

elat

ive

Act

ivit

y

F-1 F-2 F-3a F-3b F-4

183

% respectively. The enzyme was stable at 4ºC for 24 h over a pH range between

2.5 to 5.5. At pH 7.5, the residual activity was 54 % (Tari et al., 2008) and A. sojae

ATCC 20235 was stable at pH 5.0 and retained 60 % and 70 % of enzymetic

activity at pH 3.0 and 7.0 respectively

On the basis of above result, it could be predicted and recommended that

A. niger is the best candidate for Pectinase production as compared to other

fungi. The enzyme has a significant future in commercial use especially in food

industries.

Fig.5.31: Effect of pH stability on pectinase activity produced by Aspergillus niger

v- The Effect of temperature on pectinase activity produced by Aspergillus

niger:

The effect of temperature on pectinase activity was investigated at various

temperatures for the purified enzyme produced by A. niger. The result shown in

Figure-5.32 reveals that all the Fractions (F1, F2 , F3-a, F3-b and F4, ) are showing

maximal activity at 40 and 50 °C respectively. These optimal temperature results

were similar to those observed for pectinase enzyme obtained from Streptomyces

erumpens at 50°C ( Karl and Ray, 2011). Dogan and Tari, (2008) presented that

pectinase isolated from Aspergillus sojae (55 °C) shown slightly higher optimum

temperature. The present results are in agreement with the results of Nabi et al.,

0

20

40

60

80

100

120

3 4 5 6 7 8 9 10

pH Stability

% o

f R

elat

ive

Act

ivit

y

F-1 F-2 F-3a F-3b F-4

184

(2003) and Fahmy et al., (2008) for Pectinase enzymes isolated from

Trichoderma harzianum (40 °C) and A. niger NRRL3 (40 °C) respectively. While

Favela-Torres et al., (2006) reported that Pectinase obtained from Streptomyces sp

QG-11-3 have optimal activity at 60 °C. Pectinase from fungi have optimum

activity at 50 °C while yeast pectinase has a wide range of optimum temperature

from 40 °C to 60 °C. Acoording to Pedrolli and Carmona, (2010) purified

polygalacturonase showed highest at 55-60 °C. Pedrolli et al., (2009) reported

that that optimum temperature of PG from fungi was repeatedly recorded at 40-

60 °C. Siddiqui et al., (2012) reported the optimum temperature (55 °C) of the

purified pectinase enzyme produced by R. pusillus. While Martins et al., (2002)

reported high temperature optima 60 °C for the enzyme obtained from

Thermoascus aurantiacus. Kaur et al., (2004) reported little higher temperature

optima for Sporotrichum thermophile apinis at 55 °C . While Thakur et al., (2010)

reported temperature optima for Mucor circinelloides at 42 °C. Andrade et al.,

(2011) reported the high optimum temperature for polygalacturonase enzyme

between 60–70°C which is contrast with the present study.

Fig.5.32: Effect of temperature on pectinase activity produced by

Aspergillus niger

0

20

40

60

80

100

120

20 30 40 50 60 70 80 90 100

Temperature

% o

f R

elat

ive

Act

ivit

y

F-1 F-2 F-3a F-3b F-4

http://www.google.com.pk/url?sa=t&rct=j&q=%20t.%20%20harzianum%20&source=web&cd=2&cad=rja&ved=0CDIQFjAB&url=http%3A%2F%2Fgenome.jgi.doe.gov%2FTriha1%2F&ei=3MVUUZ2LEOuO4gSgtYD4DQ&usg=AFQjCNGSPa7q9pUXSXnwn8BuRn1VDRcQ2Q&bvm=bv.44442042,d.ZWU

185

vi- Effect of temperature stability on pectinase activity produced by

Aspergillus niger :The effect of temperature stability on pectinase activity was

examined at various temperatures for the purified enzyme isolated from A. niger.

The result shown in Figure-5.33 state the graphical presentation of

thermostability of all the Fractions (F-1, F-2 , F-3a, F-3b and F-4,) the enzyme is

maximally stable upto 60 to 70 °C and then decreased as the incubation

temperature was increased. The decrease in temperature stability is probably due

to enzyme denaturation at higher temperatures. More or less same observations

were reported by Pedrolli et al., (2008). Generally Pectinase activity in many

filamentous fungi is stable in a range of 50–60 °C as reported by Kaur et al.,

(2004), Kapoor et al., (2000), Kojima et al., (1999), Silva et al., (2002) and Ortega et

al., (2004). The results having similarity with the results of Siddiqui et al., (2012)

that purified pectinase enzyme from R. pusillus which was found stable at 50 °C

and this study is fully agreed with Martins et al., (2002). The activity of Pectinase

enzymes is depended on thermostability Chaudhri and Suneetha, (2012).

Thermal stability was reported by Damasio et al., (2010) for pectinase enzyme

obtained from P. variotii as 45 to 55 °C. Nabi et al., (2003) reported that pectinase

from T. harzianum was stable upto 60 °C and similar results were presented by

Dogan and Tari, (2008) who reported that thermal stability of pectinase by A.

niger was noted 45 to 55 °C. Kaur et al., (2004) reported temperature stability at

65°C for the pectinase enzyme obtained from Sporotrichum thermophile apinis.

Thakur et al., (2010) reported that the enzyme was stable at 42 °C.

186

Fig.5.33: Effect of thermostability on pectinase activity produced by Aspergillus niger

vii- Effect of activators and inhibitors: The Effect of different metal ions and

compounds at 5mM concentration was tested with purified pectinase activity as

shown in Figure-5.34-5.38. It was observed that 30 to 40% inhibiting effect was

shown on pectinase activity by 1, 10 Phenonthroline in all Fractions (FI, F2,, F3-a,

F3-b and , F4). The Pectinase activity was stimulated in the presence of CaCl2 in all

fractions in the effect of 10-30%, which is contrast to Pedrolli and Carmona (2010)

who reported that no significant effect was shown by Ca2+ on polygalacturonase

activity. ZnSO4 , MnSO4 and Mg SO4 shown higher activity in fractions ( F3-a,

F3-b and F4), while in fractions F1 and F2 ZnSO4 and MnSO4 shown slight

inhibition effect on pectinase activity which is in agreement with Pedrolli and

Carmona (2010) who reported slight inhibition of Zn2+ on prctinase activity Al

Najada et al., (2012 ) reported that Zn 2+ caused inhibition in activity of pectinase

enzyme isolated from F. oxysporum , while CoCl2 , AgNO3, HgNO3, and EDTA

have strong inhibition effect on the pectinase activity in all fractions (FI, F2,, F3-a,

F3-b and F4) which is in partial agreement with Al Najada et al., (2012) who

reported that Hg2+ caused inhibition in pectinase activity isolated from

F. oxysporum , while Co2+ found to enhance the enzyme activity. Pedrolli and

0

20

40

60

80

100

120

20 30 40 50 60 70 80 90 100 100

Temperature Stability

% o

f R

ela

tiv

e A

ctiv

ity

F-1 F-2 F-3a F-3b F-4

187

Carmona, (2010) reported that Co2+ showed an enhancing effect on pectinase

activity but Hg2+ showed total inhibition while EDTA inhibited enzyme

activity.

Zn2+, Pb2+ and Hg2+ caused 12, 39 and 32% inhibition, while Ca2+, Co2+ and

Ni2+ were found to activate the enzyme by 56, 38 and 32%, respect-tively

for F.oxysporum polygalacturonase activity. A. tubingensis PGaseI and PGaseII

were activated by Zn2+, Ca2+, Co2+, where Ni2+ only activated PGaseII. The other

cations Cu2+, Hg2+ and Pb2+ had partially inhibitory effect. In P. viridicatum,

Ca2+ was foundto enhance the stability of polygalacturonase, while Hg2+,

Zn2+ and Cu2+ were strongly inhibited (Gomes et al., 2009). Co2+ and Cu2+ had no

inhibitory effects on the polygalacturonase activity of P. variotii ( Damasio et al.,

2010). Polygalacturonases from M. rouxii (Saad et al., 2007) and A. niger NRRL3

(Fahmy et al., 2008) were partially inhibited by Ca2+, Zn2+, Cu2+, Co2+, Ni2+ and

Hg2+. These results also shown a significant role of the cysteine in the catalysis

and/or substrate binding by the purified pectinase of A. niger and the results

show agreement with the results of Nelson and Cox, (2004), Scopes, (1994) and

Pedrolli and Carmona, (2010) Kaur et al., (2004) , Kapoor et al., (2000), and

Semenova et al., (2003). Kashyap et al., (2000) reported that some pectinases are

dependent on cations like Ca2+. Pedrolli et al., (2008) described that complexes

are formed between anions and cations like Ca2+ or any other which are required

for the activity of the enzyme.

http://www.springerlink.com/content/?Author=Andre+Ricardo+de+Lima+Dam%c3%a1sio

188

Fig.5.34: Effect of activators & inhibitors (F-1)


0

20

40

60

80

100

120

140

Con

trol

Cys

teine

Met

hion

ene

EDTA

1,10

Phe

nont

hrol

ine

ZnSO

4

CaC

l2

MnS

O4

AgNO

3

CoC

l2

MgS

O4

HgN

O3

% o

f A

ctiv

atio

n &

in

hib

itio

n

0

20

40

60

80

100

120

140

Con

trol

Cys

teine

Met

hion

ene

EDTA

1,10

Phe

nont

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ZnSO

4

CaC

l2

MnS

O4

AgN

O3

CoC

l2

MgS

O4

HgN

O3

% o

f A

cti

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& in

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189

Fig.5.36: Effect of activators & inhibitors (F-3a)

Fig.5.37: Effect of activators & inhibitors (F-3b)

0

20

40

60

80

100

120

140

Con

trol

Cys

teine

Met

hion

ene

EDTA

1,10

Phe

nont

hrol

ine

ZnSO

4

CaC

l2

MnS

O4

AgN

O3

CoC

l2

MgS

O4

HgN

O3

% o

f A

cti

vati

on

& in

hib

itio

n

0

20

40

60

80

100

120

140

Con

trol

Cys

teine

Met

hion

ene

EDTA

1,10

Phe

nont

hrol

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ZnSO

4

CaC

l2

MnS

O4

AgNO

3

CoC

l2

MgS

O4

HgN

O3

% o

f A

cti

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& in

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190


CONCLUSION

The major objective of the present study was to extract the important

bioproduct that is extracellular fungal pectinase. From the studies on pectinase

production from Aspergillus niger, it would be appealing to develop this strain

for the production of pectinase enzyme. Pectinase from this strain can be

recommended for the commercial production because of its constitutive and

less catabolically repressive nature, thermostability (upto 50°C), wide range of

pH (4.0-5.5), utilization of agro-industrial waste as well as cost effective

production could be a best opportunity to for industrial use and applications of

this enzyme. However, scale-up studies are needed for the better output for

commercial production. It is also concluded that the pectinase produced by

Aspergillus niger can be used in juice and beverage industry, which save huge

amount of foreign exchange to purchase and import pectinase enzyme.The

0

20

40

60

80

100

120

140

Con

trol

Cys

teine

Met

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ene

EDTA

1,10

Phe

nonthr

oline

ZnSO4

CaC

l2

MnS

O4

AgN

O3

CoC

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O4

HgN

O3

% o

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in

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191

enzyme is found to be thermostable and has wide pH stability and optimum pH

in the acidic range. These factors establish its importance in fruit juice industries.

The acid tolerant property of the polygalacturonase from Aspergillus niger makes

the enzyme an ideal candidate for tissue maceration, juice extraction and

clarification in the fruit and vegetable processing industry.The result revealed

that in comparison to other organisms Aspergillus niger is found best for pectinase

production.The maximum pectinase production was achieved with 5% molasses

along with 5.0% sucrose as carbon source and Ammonium sulphate0.4% as

nitrogen source with initial pH 6.0 and 35 °C temperature at 72 hours.

The crude enzyme characterization proved that enzyme is thermostable

and pH stable. .The enzyme is found to be thermostable and has wide pH

stability and optimum pH in the acidic range. The pectinase enzyme produced

by A. niger was purified by gel chromatography on Sephadex G-100 and Ion

Exchange DEAE A-50. with NaCl gradient concentration 0.0 to 1.0N. The

molecular weight of the purified fractions were found to be 33000 ± 2000 and

38000 ± 2000 Dalton by SDS-PAGE. All fractions show homogeneity on SDS-

PAGE.The purified enzyme characterization proved that enzyme is thermostable

and pH stable, which is suitable for industrial use.

Pectninase from Aspergillus niger could convert pectins from different

sources successfully into simple sugars and thus the enzyme could not only act as

an agent for bioconversion but also could replace the use of highly expensive

commercial pectin in food industry.

192

Further Suggesstions :

The acid tolerant property of the Pectinase from Aspergillus niger makes the

enzyme an ideal candidate and may be used for tissue maceration, juice

extraction and clarification in the fruit and vegetable processing industry.

Pectninase from Aspergillus niger could convert orange peel pectin successfully,

and thus the enzyme could not only act as an agent for bioconversion but also

could be used to replace the use of highly expensive commercial pectin in food

industry.

Present study could open an avenue for the analysis of various other factors,

which can influence the production of pectinase.

The work can be done to analyze active centre, analysis of amino acids and

sequencing of pectinase, which can help to understand its structure.

193

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