j. microb. world…………2013, pp :……. microbiologists society ... · such as modified...

J. Microb. World…………2013, PP :…….

Microbiologists Society

PRODUCTION AND OPTIMIZATION OF AMYLASE FROM GEOBACILLUS

THERMOLEOVORANS AND ITS USE IN DESIZING OF TEXTILE FABRIC

Malik Nagesh and Vijay Vig

Department of Microbiology, V.E.S. College of Arts, Science and Commerce, Chembur,

Mumbai - 400 071. E-mail: [email protected]

ABSTRACT

A thermophilic amylolytic bacterium was isolated from soil. The isolate was identified

by 16S rDNA sequencing as Geobacillus thermoleovorans. The effect of media constituents

such as yeast extract, calcium, glucose, sucrose, tryptone, potassium dihydrogen phosphate,

disodium hydrogen phosphate and magnesium sulphate supplementation of the production

medium on amylase production was studied. Addition of 1 % yeast extract to Starch broth

increased the enzyme production at 24 and 48 hours. Addition of 20 mM calcium chloride

increased the enzyme production at 24 and 48 hours. One percent sucrose showed a little

increase in enzyme production. One percent glucose showed a very strong repression of

enzyme production. In case of tryptone, Potassium dihydrogen phosphate and Magnesium

sulphate, a lesser amylase production was observed at 48 hours as compared to control

whereas Disodium hydrogen phosphate showed decrease in enzyme activity at both 24 hours

and 48 hours. Yeast extract, calcium chloride and sucrose were also added in combinations in

starch broth and the combination of 1 % sucrose and 20 mM calcium chloride were found to

be better than other combinations. Enzymatic desizing of textile fabric was carried out using

amylase and 24 hours enzyme showed 10.04 %, 12.91 % and 8.7 % weight loss at 30°C, 75°C

and 95°C respectively. 48 hours enzyme showed 12.32 %, 13.72 % and 11.33 % weight loss at

30°C, 75°C and 95°C respectively .Hence this enzyme can be used for desizing in textile

industry at all the above temperatures.

KEY WORDS THERMOPHILIC, GEOBACILLUS THERMOLEOVORANS, YEAST

EXTRACT, CALCIUM CHLORIDE, DESIZING

INTRODUCTION

Amylases are enzymes which hydrolyse starch molecules to give diverse products

including dextrins and progressively smaller polymers composed of glucose units (Asgher et

al., 2007). Starch degrading amylolytic enzymes are most important in the biotechnology

industries with huge application in food, fermentation, textile and paper (Mishra and Behera,

2008). Thermophilic and extremely thermophilic microorganisms have gained a great deal of

attention recently. Enzymes from these microorganisms are of special interest since they are

not usually denatured by high temperatures and are even active at elevated temperatures.

Thermostable α-amylases have had many commercial applications for several decades

(Cordeiro et al., 2002).

Textile manufacturing is a major industry. It is based in the conversion of three types

of fibre into yarn, then fabric, then textiles. Environmental awareness in Indian Textile

Industry has increased significantly after the imposition of German ban. With the globalisation

of the textile industry and increase in the awareness towards the ecology the times are

changing very fast for the industry and it has to remain updated continuously. Currently and in

the years to come , the criteria that will be used for judging the new processes and to replace

the old one may be based on the 3 E principle, i.e. Efficiency , Economy and Ecology with a

view to preserve the product quality . This paves the way for bioprocessing in textiles. The

most exciting area of textile research is enzymatic processing. Enzymes have been applied, or

at least tried , in every step of textile wet processing . Among these, many applications have

become well established and routine, while some have not yet been successfully industrialized

due to technical or cost constraints (Anthappan et al., 2007).The implementation of enzymatic

systems in the preparation of cotton is advantageous in terms of decreased water, chemicals,

and energy consumption, reuse of desizing waste baths, milder process conditions, and

environmentally friendly processes (Tzanov et al., 2001).

Sizing of the warp yarn is essential to reduce breakage of the yarn and thus to stop

production on the weaving machine. On the weaving machine, the warp yarns are subjected to

several types of actions i.e. cyclic strain, flexing, abrasion at various loom parts and inter yarn

friction. With sizing the strength of the yarn will improve and the hairiness of yarn will

decrease. Different types of water soluble polymers called textile sizing agents/chemicals

such as modified starch, polyvinyl alcohol (PVA), carboxymethyl cellulose (CMC), acrylates

are used to protect the yarn. The sizing liquor is applied on warp yarn with a warp sizing

machine. After the weaving process the fabric is desized. Desizing is the process of removing

the size material from the warp yarns in woven fabrics.

MATERIALS AND METHODS

Bacterial strain

Geobacillus thermoleovorans was isolated from soil sample from garden of V.E.S.

College campus. The culture was identified by 16S rDNA sequencing.

Enzyme production

The enzyme production was carried out in Sterile starch broth of following

composition g/L: Peptone 10, Sodium chloride 5, Meat Extract 3, Starch 4, Agar 25, Distilled

water (D/W) 1000 ml. The pH of the medium was adjusted to 7.4. The medium was sterilized

by autoclaving at 121°C for 15 min. The microbial isolate was induced for amylase production

by 24 hour incubation at 55°C on starch agar slant. After incubation, the density of bacterial

culture was adjusted to 0.2 at 530 nm by suspending the cells in sterile saline. 1 ml of the

culture suspension was inoculated in to100 ml medium and incubated at 55°C for 48 hour. At

regular intervals (24h) samples were harvested and centrifuged at 3500 rpm for 20 minutes to

obtain a cell free supernatant containing amylase enzyme. This was used for performing

amylase assay (Anthappan et al., 2007).

Enzyme assay

Amylase activity was assayed by measuring the amount of reducing sugar obtained on

the hydrolysis of starch using dinitro salicylic acid (DNSA) method. Reaction mixture

contained 2.5 ml Acetate Buffer (pH 5.0), 2.5 ml starch (1%) and 1 ml NaCl (1%) in test.

Instead of starch, 2.5 ml D/W was added in reagent blank. These mixtures were incubated at

75°C for 10 min. After incubation, 1ml D/W and 0.5 ml crude enzyme were added to test

mixtures and 0.5 ml D/W, instead of enzyme, was added to reagent blank. The reaction

mixtures were then incubated at 75°C for 15 min. Immediately after incubation 1 ml of the

reaction mixture was added to 1 ml of DNSA reagent. The solutions were kept in boiling

water bath for 10 min followed by addition of 5 ml D/W. The absorbance was measured at

530 nm on a colorimeter (Anthappan et al., 2007). The international units (IU) for expressing

an enzyme’s activity is calculated as the micromoles of products released per minute per

milliliter of the reaction mixture (Nigam and Ayyagari, 2007).

Effect of media constituents on amylase production

Effect of media constituents supplementation of sterile 0.4 % starch broth on amylase

production by the isolate was studied by using Yeast extract, Calcium chloride, Glucose,

Sucrose, Tryptone, Disodium hydrogen phosphate, Potassium dihydrogen phosphate and

Magnesium sulphate (Asgher et al ., 2007).The constituents which gave positive effect on

amylase production were also added in different combinations.

Enzymatic Desizing of textile fabric

The crude amylase enzyme obtained was applied in desizing of the grey cotton fabric.

Enzyme application

Starch broth after inoculation of the microbial cells was withdrawn at 24 and 48 hour,

centrifuged to obtain the cell free supernatant and then was applied on the grey cotton fabric

for desizing. The MLR (Material to Liquor Ratio) was kept at 1:30 (2 gram grey cotton fabric:

60 ml liquor) and 15 % broth concentration was used. Also, 0.5 g/L of non-ionic wetting agent

i.e. Tween 80 and 1 g/L of NaCl was added to desizing recipe. Desizing was carried out at

30°C, 75°C and 95°C for 1 hour. Also 2 gram fabric was treated with 60 ml D/W for 1 hour at

30°C and kept as a control (Anthappan et al., 2007).

Application evaluation methods

Conventional method

After desizing, fabric samples were removed and given hot wash and cold wash for 10

minutes. The fabric samples were dried and weight of the fabric was determined. Weight loss

was expressed as a percentage loss in weight of the fabric with respect to the initial dry weight

of the fabric (Anthappan et al., 2007).

Tegewa scale method

The residual starch content was assessed with the Tegewa scale method. After

desizing, fabric samples were given hot wash and cold wash for 10 minutes and dried. Fabric

sample of the size 1 cm x 1 cm was immersed in the Tegewa solution for 1 min. It was then

rinsed with water, dabbed with filter paper and immediately compared with the Tegewa scale

(Anthappan et al., 2007).

RESULTS AND DISCUSSION

The optimum temperature and pH of amylase enzyme was 75°C and 5 respectively.

Effect of media constituents on amylase production

Figure 1 Effect of Yeast extract on amylase production

The amylase synthesis by several microorganisms has been correlated to the presence

or absence of different nitrogen sources and various amino acids in the growth medium.

Organic sources like yeast extract usually have stimulating effects

this case, 1% yeast extract has increased the enzyme production in 24 and 48 hours as

compared to control. 1.5 % and 2.5 % yeast extract has shown increased enzyme production i

24 hours but a very slight increase in enzyme production as compared to control after 48

hours. 2 % yeast extract has shown a very high increase in amylase production in 24 hours but

a decrease after 48 hours. Thus 1% yeast extract was found to be better

concentrations.

Teodoro et al., (2000) studied the cultural conditions for the production of

by Bacillus species. Initially, the organism was grown in the liquid medium and then, in the

liquid medium supplemented with

and peptone (1%) to the liquid medium shortened the lag period and increased both the dry

weight of the cell and the enzyme synthesis. Santos

medium composition on formation of amylase by

extract was found to be important factor in the

thus the influence of this compound on

varying its concentration in the

increased between 2 and 5 g/L yeast extract concentration and then fell very rapi

this point. Asgher et al., (2007) studied the effect of yeast extract on growth and enzyme

production by Bacillus subtilis

0

0.1

0.2

0.3

En

zym

e a

ctiv

ity

IU

concentration of yeast extract %

1 Effect of Yeast extract on amylase production



like yeast extract usually have stimulating effects (Asgher et al


compared to control. 1.5 % and 2.5 % yeast extract has shown increased enzyme production i



a decrease after 48 hours. Thus 1% yeast extract was found to be better as compared to higher

(2000) studied the cultural conditions for the production of

species. Initially, the organism was grown in the liquid medium and then, in the

liquid medium supplemented with yeast extract (0.5 %). The addition of yeast extract (0.5%)


weight of the cell and the enzyme synthesis. Santos et al.,(2003) studied the effect of the

tion on formation of amylase by Bacillus sp. The concentration of yeast

extract was found to be important factor in the α-amylase synthesis by several organisms and

thus the influence of this compound on α-amylase synthesis by Bacillus sp was investigated,

varying its concentration in the medium between 2 and 10 g/L. The activity of the enzyme

increased between 2 and 5 g/L yeast extract concentration and then fell very rapi

(2007) studied the effect of yeast extract on growth and enzyme

JS-2004. Initially, the organism was grown in the liquid

concentration of yeast extract %

Enzyme

activity I.U 24

hoursEnzyme

activity I.U 48

hrs



et al ., 2007). In


compared to control. 1.5 % and 2.5 % yeast extract has shown increased enzyme production in



as compared to higher

(2000) studied the cultural conditions for the production of α-amylase

species. Initially, the organism was grown in the liquid medium and then, in the

yeast extract (0.5 %). The addition of yeast extract (0.5%)


(2003) studied the effect of the

sp. The concentration of yeast

amylase synthesis by several organisms and

sp was investigated,

The activity of the enzyme

increased between 2 and 5 g/L yeast extract concentration and then fell very rapidly beyond

(2007) studied the effect of yeast extract on growth and enzyme

2004. Initially, the organism was grown in the liquid

medium for 24–96 h at pH 7.0 and 40

addition of yeast extract (1%) to the liquid medium increased both the cell dry weight and the

enzyme synthesis at all incubation periods. The result suggests that growth and synthesis of

amylase by B. subtilis JS-2004 is favored by yeast extract

Figure 2 Effect

α-amylase is known to be a calcium metalloenzyme. Enhanced bacterial growth and

enzyme activity may be seen due to increased availability of calcium ions

2007). Calcium chloride was shown to have a

production. 10, 20 and 30 mM CaCl

compared to control. 40 mM CaCl

similar to control at 48 hours. 20 mM CaCl

increased production of enzyme higher than other concentrations but also the enzyme activity

remained constant after 48 hours

Teodoro et al., (2000) studied the cultural conditions for the production of

by Bacillus species. The addition of 10 mM calcium to the liquid medium improved the

growth and amylase production. These results are similar to the findings of Hewitt and

Solomons, (1996) with cultures of

the effect of calcium (10mM) on growth and enzyme production by

The supplementation of liquid culture medium with 10 mM calcium stimulated bacterial

growth and enhanced α-amylase production. The positive effect of calcium was observed at

all time intervals but the maximum enzyme

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Control 10

En

zym

e a

ctiv

ity

I.U

Concentration of Calcium chloride mM

96 h at pH 7.0 and 40°C and then, supplemented with yeast extract (1%).Th

%) to the liquid medium increased both the cell dry weight and the

enzyme synthesis at all incubation periods. The result suggests that growth and synthesis of

2004 is favored by yeast extract.

Effect of calcium chloride on amylase production

amylase is known to be a calcium metalloenzyme. Enhanced bacterial growth and

enzyme activity may be seen due to increased availability of calcium ions (Asgher

Calcium chloride was shown to have a very positive effect on amylase enzyme

production. 10, 20 and 30 mM CaCl2 increased amylase production in 24 and 48 hours as

compared to control. 40 mM CaCl2 shown increase in amylase production in 24 hours but

similar to control at 48 hours. 20 mM CaCl2 were found to be best because not only it


remained constant after 48 hours.

(2000) studied the cultural conditions for the production of

The addition of 10 mM calcium to the liquid medium improved the


with cultures of Bacillus amyloliquefaciens. Asgher et al ., (2007) studied

the effect of calcium (10mM) on growth and enzyme production by Bacillus subtilis


amylase production. The positive effect of calcium was observed at

all time intervals but the maximum enzyme activity was still after 48 h.

20 30 40

Concentration of Calcium chloride mM

Enzyme activity I.U 24 hrs


supplemented with yeast extract (1%).The

%) to the liquid medium increased both the cell dry weight and the

enzyme synthesis at all incubation periods. The result suggests that growth and synthesis of α-

amylase is known to be a calcium metalloenzyme. Enhanced bacterial growth and

(Asgher et al .,

very positive effect on amylase enzyme

increased amylase production in 24 and 48 hours as

shown increase in amylase production in 24 hours but

were found to be best because not only it


(2000) studied the cultural conditions for the production of α-amylase

The addition of 10 mM calcium to the liquid medium improved the


(2007) studied

Bacillus subtilis JS-2004.


amylase production. The positive effect of calcium was observed at



Figure

The synthesis of carbohydrate

Bacillus is subject to catabolic repression by readily metabolizable substrates such as glucose

(Carlos Eduardo de Souza Teodoro

production at 24 and 48 hours. The enzyme activity was 0.072 and 0.078 I.U. at 24 and 48

hours respectively as compared to 0.184 and 0.248 I.U. of control. Thus it was observed that

glucose strongly diminished the synthesis of amylase.

Haseltine et al., (1996) studied the glucose effect and regulation of

synthesis in the hyperthermophilic archaeon

added to the defined minimal medium. The termination of

during growth on starch might re

for α-amylase production. Teodoro

production of α-amylase by Bacillus

to the culture diminished greatly the synthesis of

effect of the medium composition on formation of amylase by

amylolytic system was subject to catabolic repression, the

medium with soluble starch or maltose as a carbon source, at 50

added to cultures of the organism after 30 hours growt

production increased rapidly after 18 hours, with the highest enzyme activity being obtained

after 48 h. On supplementation of the culture with glucose, there was an initial repression of

amylase synthesis. This repression was reversed after 72 h growth, which could

0

0.05

0.1

0.15

0.2

0.25

Control

En

zym

e a

ctiv

ity

I.U

.

Media constituents used

ure 3 Effect of Glucose on amylase

The synthesis of carbohydrate-degrading enzymes in most species of the genus

is subject to catabolic repression by readily metabolizable substrates such as glucose

Carlos Eduardo de Souza Teodoro et al., 2000). 1 % Glucose strongly repressed the



glucose strongly diminished the synthesis of amylase.

(1996) studied the glucose effect and regulation of

synthesis in the hyperthermophilic archaeon Sulfolobus solfataricus. Exogenous glucose was

added to the defined minimal medium. The termination of α-amylase production observed

during growth on starch might reflect the accumulation of glucose as a repressing metabolite

Teodoro et al ., (2000) studied the cultural conditions for the

Bacillus species. It was found that the addition of glucose (0.5%)

to the culture diminished greatly the synthesis of α-amylase. Santos et al., (2003) studied the

effect of the medium composition on formation of amylase by Bacillus sp. To determine if the

amylolytic system was subject to catabolic repression, the Bacillus sp was grown in the liquid

medium with soluble starch or maltose as a carbon source, at 50°C and glucose was then

added to cultures of the organism after 30 hours growth. In the absence of glucose, enzyme



sion was reversed after 72 h growth, which could

Control 1 % Glucose

Media constituents used

Enzyme activity I.U. 24 hrs


degrading enzymes in most species of the genus

is subject to catabolic repression by readily metabolizable substrates such as glucose

. 1 % Glucose strongly repressed the amylase



(1996) studied the glucose effect and regulation of α amylase

Exogenous glucose was

amylase production observed

flect the accumulation of glucose as a repressing metabolite

(2000) studied the cultural conditions for the

It was found that the addition of glucose (0.5%)

(2003) studied the

To determine if the

sp was grown in the liquid

C and glucose was then

h. In the absence of glucose, enzyme



sion was reversed after 72 h growth, which could be correlated



to a depletion of glucose, demonstrating that the amylolytic system of

to catabolic repression. Asgher et a

enzyme production by Bacillus subtilis

production was observed when glucose was added to the fermentation medium. The addition

of 1.0% glucose to the culture medium along with 1% waste potato starch was

repress the growth of B. subtilis JS

Figure 4 Effect of sucrose on amylase production

1 % sucrose increases the production of amylase at 24 and 48 hours.1.5 % and 2 %

sucrose decreases the enzyme production at 24

production remaining same as that of control at 48 hours. 2.5 % sucrose was found to have a

high negative effect on amylase production as it decreased amylase synthesis at both 24 and

48 hours as compared to control. Thus only 1 % sucrose was found to have a positive effect on

amylase synthesis.

0

0.05

0.1

0.15

0.2

0.25

0.3

control 1

En

zym

e a

ctiv

ity

I.U

.

Concentration of sucrose %

to a depletion of glucose, demonstrating that the amylolytic system of Bacillus

et al., (2007) studied the effect of glucose (1 %) on growth and

Bacillus subtilis JS-2004. A decrease in cell growth and enzyme


of 1.0% glucose to the culture medium along with 1% waste potato starch was

JS-2004 and synthesis of α-amylase.

Effect of sucrose on amylase production


sucrose decreases the enzyme production at 24 hours as compared to control while amylase



ol. Thus only 1 % sucrose was found to have a positive effect on

1.5 2 2.5

Concentration of sucrose %



Bacillus sp was subject

) studied the effect of glucose (1 %) on growth and

. A decrease in cell growth and enzyme


of 1.0% glucose to the culture medium along with 1% waste potato starch was found to


hours as compared to control while amylase



ol. Thus only 1 % sucrose was found to have a positive effect on



Figure 5 Effect of tryptone on amylase production

Tryptone is the assortment of peptides formed by the digestion of casein by the

protease trypsin. It provides a source

was found to have no effect at 24 hours while a lesser amylase production was observed at 48

hours as compared to control.

Figure 6 Effect of Disodium hydrogen phosphate (Na

Disodium hydrogen phosphate (Na

hours. The enzyme activity was 0.146 and 0.198 I.U. at 24 and 48 hours respectively as

compared to 0.184 and 0.248 I.U. of control.

0

0.05

0.1

0.15

0.2

0.25

Control

En

zym

e a

ctiv

ity

I.U

.

Media constituent used

0

0.05

0.1

0.15

0.2

0.25

control

En

zym

e a

ctiv

ity

I.U


Effect of tryptone on amylase production


protease trypsin. It provides a source of amino acids for the growing bacteria .0.2 % tryptone


Disodium hydrogen phosphate (Na2HPO4) on amylase produc

Disodium hydrogen phosphate (Na2HPO4) repressed amylase production at 24 and 48


compared to 0.184 and 0.248 I.U. of control.

0.2 % Tryptone



Enzyme activity I.u. 48 hrs

0.25 % Na2HPO4



Enzyme activity I.U. 48 Hrs


of amino acids for the growing bacteria .0.2 % tryptone


on amylase production

) repressed amylase production at 24 and 48



Enzyme activity I.u. 48 hrs



Figure 7 Effect of Potassium

KH2PO4 was found to have no effect at 24 hours while a lesser amylase production

was observed at 48 hours as compared to control. The enzyme activity was 0.208 I.U. at 48

hours as compared to 0.248 I.U. of control.

Figure 8 Effect of Magnesium sulphate (MgSO

MgSO4 .7H2O was also found to have no effect at 24

production was observed at 48 hours as compared to control. The enzyme activity was 0.214

I.U. at 48 hours as compared to 0.248 I.U. of control.

1% Yeast extract, 20 mm Calcium chloride and 1% sucrose

positive effect on amylase production. Therefore

0

0.05

0.1

0.15

0.2

0.25

Control

En

zym

e a

ctiv

ity

I.U


0

0.1

0.2

0.3

Control

En

zym

e a

ctiv

ity

I.U


Potassium dihydrogen phosphate (KH2PO4) on amylase production

was found to have no effect at 24 hours while a lesser amylase production


hours as compared to 0.248 I.U. of control.

Magnesium sulphate (MgSO4 .7H2O) on amylase production

O was also found to have no effect at 24 hours while a lesser amylase


I.U. at 48 hours as compared to 0.248 I.U. of control.

1% Yeast extract, 20 mm Calcium chloride and 1% sucrose were found to have

production. Therefore their effect was also studied in combinations.

0.1 % KH2PO4




Control 0.005 % MgSO4

.7H2O





was found to have no effect at 24 hours while a lesser amylase production



hours while a lesser amylase


were found to have

their effect was also studied in combinations.





Figure 9 Effect of Yeast Extract, Calcium chloride and Sucrose on amylase

Starch broth when supplemented with yeast extract, CaCl

extract and CaCl2 the amylase production increased at 24 hours but almost remained same as

that of control at 48 hours. When yeast extract an

increase in amylase synthesis was observed at 24 and 48

to be best among the combinations. The enzyme activity was 0.312 and 0.340 I.U. at 24 and

48 hours respectively as compared to 0.184 and 0.248 I.U.


Enzymatic desizing of grey cotton fabric was carried out by applying the crude

amylase enzyme on fabric at varying temperatures for 60 minutes using 15 % broth

concentration.

Table 1 Desizing of textile fabric at different temperatures

Sample 30°C

Tegewa

rating

Weight loss

%

24 hour

Enzyme

5 10.04

48 hour

Enzyme

5 12.32

D/W 1 0.01

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

control Yeast

extract, CaCl2

and Sucrose

En

zym

e a

ctiv

ity

I.U

.

Effect of Yeast Extract, Calcium chloride and Sucrose on amylase production in

combinations

Starch broth when supplemented with yeast extract, CaCl2 and Sucrose or only yeast

the amylase production increased at 24 hours but almost remained same as

When yeast extract and sucrose was added to starch broth, a high

increase in amylase synthesis was observed at 24 and 48 hours. Sucrose and CaCl


red to 0.184 and 0.248 I.U. of control.




Desizing of textile fabric at different temperatures

30°C 75°C

Weight loss

%

Tegewa

rating

Weight loss

%

Tegewa

rating

10.04 5 12.91 5

12.32 5 13.72 5

0.01 - - -

extract, CaCl2

and Sucrose

Yeast extract

and CaCl2

Yeast extract

and Sucrose

CaCl2 and

Sucrose

Combination used

production in

and Sucrose or only yeast

the amylase production increased at 24 hours but almost remained same as

d sucrose was added to starch broth, a high

and CaCl2 were found




95°C

Tegewa

rating

Weight

loss %

8.7

11.33

-

Enzyme activity

I.U. 24 hrs

Enzyme activity

I.U. 48 hrs

Figure 10 Tegewa rating at 75°C by24 hr enzyme

Figure 11 Tegewa rating at 75°C by 48 hr enzyme

Figure 12 Tegewa rating by D/W (Control)

Enzymatic desizing was carried out at 30°C, 75°C and 95°C using 24 and 48 hour

enzyme. A higher weight loss was observed at 75°C which is optimum for amylase enzyme

activity. A negligible weight loss and Tegewa rating of 1 given by sample treated with D/W

(Control) confirm the precise action of the enzyme. Weight loss of 10.04 %, 12.91 % and 8.7

% by 24 hour enzyme and 12.32 %, 13.72 % and 11.33 % by 48 hour enzyme at 30°C, 75°C

and 95°C respectively indicates that amylase is carrying out desizing at all the three

temperatures. Tegewa rating was 5 in all the conditions. Thus it can be concluded that this

amylase enzyme is efficient in desizing of the grey cotton fabric at 30°C, 75°C and 95°C.

The chemical desizing and scouring processes are more time consuming and leave a

negative impact on the environment by changing the pH balance of the water bodies in which

the textile effluent is released , causing a serious alteration in the water-cycle due to the use of

strong acids and alkalis. These processes also affect the fabric strength as compared to the

presently established enzymatic processes since the chemicals used are highly non specific

acting on the fibre backbone , thereby weakening it . On the contrary, enzymes being highly

specific have captured the modern textile industry.

Anthappan et al., (2007) used a wild type bacterial strain isolated from soil

rhizosphere to produce amylase and pectinase in a common production media. Desizing was

carried out at 75°C for 90 min and the results show an increase in weight loss with the

increase in broth incubation time. Also weight loss increases distinctly with the increase in the

concentration of the broth used for treatment. The increase in weight loss is proportional to the

incubation time of the broth. Poor weight loss and Tegewa rating given by sample treated with

distilled water confirm the precise action of enzyme in the treatment liquor. At 0.5 % starch in

broth, Tegewa rating and weight loss was found to be 8 and 14.4 % at 75°C and 5 and 7 % at

95°C respectively. At 95°C the decrease in Tegewa rating was due to the inactivation of the

enzyme at high temperature. This experiment is essential as normally textile industries carry

out this process at 95°C. 15 % broth desizing gives the similar results to that of commercial

enzyme desizing and better results as compared to HCl desizing , in terms of Tegewa rating.

CONCLUSION

Out of the eight media constituents analyzed Yeast extract, CaCl2 and Sucrose

individually increased amylase production when supplemented to starch broth , CaCl2 gave the

maximum yield of amylase. 20 mM CaCl2 alone when supplemented to starch broth increased

the amylase production of Geobacillus thermoleovorans at a high level. The crude amylase of

Geobacillus thermoleovorans was applied on grey cotton fabric for Enzymatic desizing of

fabric. Geobacillus thermoleovorans amylase can be used as desizing agent in textile industry

at 30°C, 75°C and 95°C. Thus this single enzyme can be used for desizing in textile industry

at multiple temperatures instead of using different enzymes at different temperatures.

ACKNOWLEDGEMENTS

The authors are highly grateful to Professor R.V. Adivarekar, Head of the department

of Fibres and Textile Processing, Institute of Chemical Technology, Matunga for his valuable

guidance and suggestions on Enzymatic Desizing and also for providing grey cotton fabric and

Tegewa scale.

REFERENCES

Anthappan P., Dalvi P., Darade N., Kanoongo N. and Adivarekar R. (2007). Amylase and

pectinase from single source for simultaneous desizing and scouring. Indian Journal

of Fibre and Textile Research,32:459-465.

Nigam Arti and Ayyagari Archana (2007). Lab Manual in Biochemistry, Immunology and

Biotechnology. Tata McGraw-Hill Publishing company limited, New Delhi. Page no

181,183 and 187.

Asgher M., Javaid Asad M., Rahman S.U., Legge R.L. (2007). A thermostable α-amylase

from a moderately thermophilic Bacillus subtilis strain for starch processing. Journal

of Food Engineering ,79:950–955.

Cordeiro Carlos Alberto Martins, Meire Lelis Leal Martins, Angelica Barbara Luciano.

(2002). Production and properties of α-amylase from thermophilic Bacillus sp.

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