kuhad 2004 produccion de una pectinasa alcalina en especies de streptoyices

7/25/2019 Kuhad 2004 Produccion de Una Pectinasa Alcalina en Especies de Streptoyices

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Enhanced production of an alkaline pectinase from Streptomyces sp. RCK-SCby whole-cell immobilization and solid-state cultivation

Ramesh Chander Kuhad1,2,*, Mukesh Kapoor1 and Renuka Rustagi1

1Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India2Department of Biotechnology, Kurukshetra University, Kurukshetra 132 119, India

*Author for correspondence: Tel.: +91-124-2391054/11-24107576, Fax: +91-11-26885270,

E-mail: [email protected], [email protected]

Received 15 April 2003; accepted 11 November 2003

Keywords: Immobilization, pectinase, polyurethane foam, solidstate cultivation, Streptomyces sp.

Summary

An alkalophilic Streptomyces sp. RCK-SC, which produced a thermostable alkaline pectinase, was isolated from

soil samples. Pectinase production at 45 C in shaking conditions (200 rev min)1) was optimal (76,000 IU l)1) when

a combination of glucose (0.25% w/v) and citrus pectin (0.25% w/v) was added along with urea (0.25% w/v) in the

basal medium devoid of yeast extract and peptone. All the tested amino acids and vitamins greatly induced

pectinase production and increased the specific productivity of pectinase up to 550%. In an immobilized cell system

containing polyurethane foam (PUF), the pectinase production was enhanced by 32% (101,000 IU l)1) compared

to shake flask cultures. In solid-state cultivation (SSC) conditions, using wheat bran as solid substrate, pectinase

yield of 4857 IU g)1 dry substrate was obtained at substrate-to-moisture ratio of 1:5 after 72 h of incubation. The

partially purified pectinase was optimally active at 60 C and retained 80% of its activity at 50 C after 2 h of

incubation. The half life of pectinase was 3 h at 70 C. Pectinase was stable at alkaline pH ranging from 6.0 to 9.0

for more than 8 h at room temperature retaining more than 50% of its activity.

Introduction

Pectins are high molecular weight acid polysaccharides,

primarily made up of a-(1 fi 4) linked D -galacturonic

acid residues with a small number of rhamnose residues

in the main chain and arabinose, galactose and xylose

on its side chain (Deul & Stutz 1958; Singh et al. 1999;

Kapooret al.2000; Lang & Do renburg 2000). Pectinase

is a generic name for a family of enzymes that catalyse

hydrolysis of the glycosidic bonds in the pectic polymers

(Reid & Richard 2000). Pectinases include polygalactu-ronase (EC 3.2.1.15), pectin esterase (EC 3.1.1.11),

pectin lyase (EC 4.2.2.10) and pectate lyase (EC 4.2.2.2),

classified on the basis of their mode of action (Alkorta

et al. 1998; Hoondal et al. 2002; Kapoor & Kuhad

2002). Environmental and legislative pressures have

made pectinases a plausible solution for the degumming

of bast fibres, such as ramie and sunn hemp (Baracat

1989; Kapooret al. 2001; Kobayashi et al. 2003) and as

a biotechnological alternative in the pulp and paper

industries (Sathyanarayana & Panda 2003) and for

treatment of alkaline pectic waste waters (Tanabe et al.

1988). Many reports are available which describe theproduction and characterization of pectinolytic enzymes

from a variety of microorganisms, such as bacteria

including actinomycetes (Cao et al. 1992; Bruhlmann

et al. 1994; Bruhlmann 1995; Beg et al. 2000a), yeast

(Alkorta et al. 1998; Blanco et al. 1999), and fungi

(Elegado & Fujio 1994; Huang & Mahoney 1999).

Given the potential applications of pectinases and the

need for development of economical methods for

improved enzyme production with an overall aim of

reducing the cost of the industrial process, the use of

solid-state cultivation (SSC) conditions and whole-cell

immobilization can serve as an excellent alternatives for

increasing enzyme yields. In the present paper, we report

increased production of alkaline pectinase from a newlyisolatedStreptomyces sp. RCK-SC.

Materials and methods

Chemicals and statistical analysis

Pectin and D -galacturonic acid were purchased from

Sigma (St Louis, USA). All other media components

and chemicals used were of highest purity grade

available commercially. Wheat bran and polyurethanefoam (PUF) were obtained locally. All the experiments

were performed independently in triplicate and the

results given here are the mean of three values. The

World Journal of Microbiology & Biotechnology 20: 257263, 2004. 257 2004 Kluwer Academic Publishers. Printed in the Netherlands.


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standard deviations in the results of all the experiments

were within 10%.

Microorganism

A thermophilic and negligible cellulase-producingStrep-tomyces sp. RCK-SC was isolated from soil samples

collected at South Campus, New Delhi and was main-

tained on actinomycetes isolation agar containing

(g l)1): sodium caseinate 2.0, L-asparagine 0.1, sodium

propionate 4.0, dipotassium phosphate 0.5, magnesium

sulphate 0.1. Pure cultures were stored at 4 C.

Pectinase production in submerged cultivation (SMC)

Horikoshi medium (Ikura & Horikoshi 1987), containing

(g l)1): glucose 5.0, peptone 5.0, yeast extract 5.0,

KH2PO4 1.0, MgSO47H2O 0.1, pH 8.0 was used as a

basal medium. Pectinase production was optimized inSMC by using different concentrations of different carbon

and nitrogen sources in the basal medium devoid of

glucose, peptone and yeast extract. Each 250 ml Erlen-

meyer flask containing 50 ml production medium was

inoculated with 4% of 24 h old seed culture and incubated

at 45 C under shaking conditions (200 rev min)1) for

24 h. Thereafter, the cell-free supernatant was obtained

by centrifugation at 12,000 g for 20 min at4 C and the

pectinase yield determined. The dry biomass in all the

experiments was estimated by drying the mycelial growth

of organism in hot air oven at 80 C for 48 h.

Submerged cultivation using amino acids and vitamins

To study the effect of additives, stock solutions of amino

acids and their analogues and vitamins were filter

sterilized and were added separately at a final concen-

tration of 0.2% (w/v) in the sterilized production

medium. Pectinase yield in submerged cultures was

determined after 24 h of incubation at 45 C with

shaking (200 rev min)1).

Solid-state cultivation

In SSC, wheat bran was moistened in different propor-

tions (1:1 to 1:5) with mineral salt solution containing

(g l)1): KH2PO4 1.0, NaCl 2.5, MgSO47H2O 0.1,

(NH4)2SO4 1.0, CaCl2 0.1 and soil extract 2 ml (v/v)

(prepared by adding 500 g of garden soil to 1500 ml of

tap water followed by sterilizing the suspension at

121 C, 15 psi for 30 min and filtering the suspension

through muslin cloth followed by filter paper), pH 8.0,

so as to see the effect of different moisture contents on

pectinase production by Streptomyces sp. RCK-SC. All

the flasks were inoculated uniformly with 5% of 24 h

old seed culture and incubated at 45 C. Two samples

(1 g each) of spent solid substrate were withdrawnperiodically after every 24 h up to 5 days and suspended

in 10 ml of TrisHCl buffer (100 mM) pH 8.0, vortexed

for 20 min at room temperature to recover maximum

possible enzyme and centrifuged at 12,000 g for

20 min at 4 C. The supernatant was used to assay

pectinase activity.

Immobilized cell system

The effectiveness of PUF as the inert support matrix for

immobilization ofStreptomycessp. RCK-SC was inves-

tigated as described previously for Bacillussp. MG-cp-2

(Kapooret al.2000) andStreptomycessp. QG-11-3 (Beg

et al. 2000b). PUF particles (1 cm2 each), density

32 kg m)3 and pore size varying between 100 to

500 lm were used. One gram of PUF pieces were placed

separately in 250 ml Erlenmeyer flasks, each one con-

taining 50 ml of optimized production medium. The

medium was autoclaved and inoculated with 4% of 24 h

old seed culture of Streptomyces sp. RCK-SC and

incubated at 45 C with shaking (200 rev min)1). After

every 24 h, the spent broth was replaced with freshmedium for the next cycle and the pectinase yield was

determined in the cell-free supernatant. The process was

continued for 10 days.

Partial purification of pectinase

Five hundred millilitre of cell-free supernatant was

saturated with ammonium sulphate to 90% saturation.

The saturated solution was left overnight at 4 C,

centrifuged at 12,000 g for 20 min at 4 C, dissolved

in minimal amount of 100 mM TrisHCl buffer (pH 8.0)

and dialysed against the same buffer for 24 h at 4 C.The dialysed proteins thus obtained were treated as

partially purified enzymes and used for enzyme charac-

terization.

Analytical procedures

Pectinase activity was quantified by measuring the

release of reducing sugar groups by the dinitrosalicylic

acid method (Miller 1959) at 60 C using 1.0% (w/v)

citrus pectin in 100 mM TrisHCl buffer (pH 8.0). One

unit of pectinase was defined as the amount of enzyme

required to release 1 lmol of reducing groups (D -

galacturonic acid) from the substrate. Protein content

was determined by the Lowry method using bovine

serum albumin (BSA) as the standard.

Results and discussion

Streptomyces sp. RCK-SC was an aerobic, Gram

positive, oxidase positive and catalase positive with

branching filaments forming aerial mycelium. Substrate

mycelium was orangish to brown. The organism grew

well in the pH and temperature range of 5.010.0 and

2545 C, respectively. On the basis of morphological,biochemical and physiological characteristics, the acti-

nomycete isolate was identified as a species of the genus

Streptomyces according to Locci (1989).

258 R.C. Kuhadet al.


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Pectinase production in submerged cultivation

Different carbon and nitrogen sources were employed to

study the effects on growth and production of extracel-

lular pectinase from Streptomyces sp. RCK-SC after

24 h of incubation under shaking (200 rev min

)1

). Outof various carbon sources used, pectin (0.25% w/v)

along with glucose (0.25% w/v) gave optimal produc-

tion of pectinase (72,000 IU l)1) after 24 h of incubation

at 45 C and thereafter, the pectinase production

declined (Table 1). Similar results have been reported

earlier in which a readily utilizable carbon source like

glucose or sucrose, when used in conjunction with either

pectin or polygalacturonic acid, resulted in enhanced

enzyme production (Sakellaris et al. 1988; Kumar &

Palanivelu 1998; Piccoli-valle et al. 2001). None of the

sugars tested in the present study repressed pectinase

production from Streptomyces sp. RCK-SC. In fact

fructose, lactose, sucrose, maltose and arabinose in-creased the specific productivity of pectinase by up to

45% as compared to optimal combination of pectin and

glucose (Table 1). In agreement with our results, Polizeli

et al.(1991) reported that polygalacturonase production

from Neurospora crassa was induced 4-fold using

galactose as compared to pectin. However our results

are in sharp contrast to earlier reports, where fructose,

sucrose and arabinose repressed enzyme production

in Lactobacillus plantarum (Sakellaris et al. 1988)

and Penicillum frequentans (Said et al. 1991). The

overall study indicated that pectinase production from

Streptomyces sp. RCK-SC was constitutive in nature.Moreover, our results also suggested that sugar and

pectin-induced pectinase of Streptomyces sp. RCK-SC

might be the products of different independently con-

trolled genes (Polizeli et al. 1991). However detailed

biochemical and genetic studies are still required to

completely understand the phenomenon involved.

Among the various organic and inorganic nitrogen

sources added to medium containing pectin and glucose

as carbon sources, urea (0.25% w/v) favoured maximum

pectinase production (76,000 IU l)1), followed by pep-

tone, tryptone, casein hydrolysate, NaNO3, KNO3,

(NH4)2SO4, while corn-steep liquor and diammonium

hydrogen phosphate did not support high pectinase

production from Streptomyces sp. RCK-SC (Table 2).Yeast extract and peptone have been reported to

stimulate maximum pectinase production fromKluyver-

omyces lactis NRRL 1137 (Murad & Foda 1992).

Effect of amino acids and vitamins on pectinase

production in SMC

The present study revealed that irrespective of the amino

acids and vitamins tested for their effect on pectinase

production fromStreptomycessp. RCK-SC, the specific

productivity of pectinase was observed to increase up to

550% (Table 3). Runco et al. (2001) reported 210, 149,and 70% higher specific productivity of polygalacturo-

nase from Aspergillus terrus by addition of leucine,

methionine and tyrosine. Similarly, a 4.0-fold increase in

polygalacturonase production fromBacillussp. MG-cp-

2 using D L-serine and folic acid and a 2.78-fold increase

in pectinase production from Streptomycessp. QG-11-3

by D L-norleucine, L -leucine, DL -isoleucine, L-lysine

monohydrochloride and D L-b-phenylalanine have been

reported by Kapoor & Kuhad (2002) and Beg et al.

(2000a), respectively. Moreover, amino acids and their

analogues have also been shown to stimulate the

production of other enzymes, e.g. a-amylase (Zhang

et al. 1993) and xylanase (Beg et al. 2000a).

Pectinase production in SSC

In SSC, using wheat bran as solid substrate, Strepto-

myces sp. RCK-SC gave a maximum pectinase yield of

4857 IU g)1 dry substrate at 1:5 moisture ratio after

48 h of incubation at 45 C (Figure 1). The decrease in

Table 1. Effect of carbon sources on production of pectinase fromStreptomycessp. RCK-SC at 45 C with shaking (200 rev min)1) after 24 h of

incubation.

Carbon source (0.5% w/v) Concentration (% w/v) Pectinase yield (IU l)

1) Biomass (g l)

1) Specific productivity(IU g)1 dry biomass)

Sugars

Glucose 0.5 62,000 4096 3.8 0.13 16,316

Sorbitol 0.5 42,800 2145 3.0 0.12 14,267

Mannitol 0.5 45,000 2163 3.2 0.15 20,333

Lactose 0.5 61,000 3210 3.0 0.16 14,062

Fructose 0.5 60,000 2546 2.4 0.12 25,000

Sucrose 0.5 53,700 1985 2.6 0.14 20,654

Maltose 0.5 50,000 3256 2.4 0.15 20,833

Arabinose 0.5 60,000 2136 3.0 0.13 20,000

Galactose 0.5 40,000 2145 2.4 0.12 16,667

Polysaccharides

Citrus pectin 0.25 53,000 2145 3.4 0.10 15,588

Chitin 0.25 42,000 1569 2.6 0.11 9286

Sugars + Polysaccharides

Citrus pectin + glucose 0.25 each 72,000 2300 4.2 0.21 6250

Enhanced Streptomyces pectinase production 259


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enzyme production at a substrate:moisture ratio of more

then 1:5 could be attributed to the change in wheat bran

particle structure, development of stickiness, reduction

in gas volume and exchange and decreased diffusion

(Lonsane et al. 1985; Lonsane & Ramesh 1990). The

differences in pectinase yield in SSC and SMC condi-

tions may be due to higher oxygen levels in SSC at the

solid-to-air interface which supported better growth and

high enzyme production as compared to high oxygen

demand and slow diffusion of substrate producing local

substrate and product gradients of concentration in

SMC, which resulted in less growth and consequently

less enzyme production in the latter. (Diaz-Godinez

et al. 2001). In the literature cited, a yield of 35 units of

exo-pectinase per gram dry substrate has been achievedin SSF using a strain of Aspergillus niger (Solis-Pereyra

et al. 1996). Diaz-Godinez et al. (2001) reported

7150 IU l)1 from Aspergillus niger in SSF using PUF

Table 2. Effect of nitrogen sources on production of pectinase from Streptomycessp. RCK-SC at 45 C with shaking (200 rev min)1) after 24 h

of incubation.

Nitrogen source (0.5% w/v) Pectinase yield (IU l)1) Biomass (g l)1) Specific productivity

(IU g)1 dry biomass)

Organic

Peptone 66,000 1997 3.8 0.12 17,368Tryptone 65,000 1854 3.6 0.15 18,056

Yeast extract 56,500 1956 3.2 0.15 17,656

Casein hydrolyzate 60,000 2459 3.4 0.10 17,647

Inorganic

NH4NO3 50,000 2232 2.2 0.08 22,727

(NH4)2HPO4 48,000 1689 2.0 0.06 24,000

NaNO3 60,000 3210 2.6 0.09 23,077

KNO3 54,000 2158 2.0 0.07 27,000

(NH4)2SO4 62,000 2564 2.2 0.09 28,182

CON2H4 76,000 3223 4.0 0.15 19,000

Table 3. Effect of additives on production of pectinase from

Streptomyces sp. RCK-SC at 45

C with shaking (200 rev min

)1

) after 24 h ofincubation.

Additives (0.2% w/v) Pectinase yield (IU l)1) Biomass (g l)1) Specific productivity

(IU g)1 dry biomass)

Amino acids

Control 76,000 3223 4.0 0.15 19,000

L -Proline 69,500 2123 1.2 0.06 57,917

L -Tyrosine 68,000 2054 1.6 0.10 42,500

L -Cysteine 66,000 3123 2.0 0.13 33,000

3-(3-4-dihydroxyphenyl)- D L -alanine 89,000 4568 2.0 0.12 44,500

D L -Aspartic acid 64,500 1698 1.4 0.09 46,071

L -Cystine monohydrochloride 66,700 1546 1.0 0.05 66,700

L -Glutamic acid 68,900 2178 2.0 0.10 34,450

L -Histidine monohydrochloride 87,000 4689 1.4 0.05 62,143

D L -b-Phenylalanine 85,000 4234 0.8 0.002 1,06,250

L -Lysine monohydrochloride 86,000 4123 1.8 0.04 47,778

D L -Threonine 67,200 1546 1.6 0.05 42,000

Vitamins

Ascorbic acid 83,300 4223 1.6 0.06 52,062

Riboflavin 1,27,000 6987 2.6 0.15 48,846

Biotin 1,36,000 7564 3.4 0.12 40,000

Thiamine 67,800 2150 1.8 0.03 37,667

Nicotinic acid 1,05,000 4500 1.6 0.05 65,625

Wheatbran-to-moisture ratio

1:1 1:1.5 1:2 1:2.5 1:3 1:3.5 1:4 1:4.5 1:5 1:5.5

Pectinaseyield(IUg-1drysubstrate)

1000

2000

3000

4000

5000

Figure 1. Effect of wheat bran-to-moisture ratio on the levels of

pectinase production fromStreptomycessp. RCK-SC in SSC at 45 C

after 72 h of incubation.



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as inert support matrix. More recently, Thermoascus

auranticus has been found to produce 43 and

40,180 U g)1 of polygalacturonase and pectin lyase,

respectively when grown on orange bagasse, sugar cane

bagasse and wheat bran as carbon sources under SSF

(Martinset al. 2002).

Pectinase production in immobilized cell systems

Immobilization studies using PUF as support material

for Streptomyces sp. RCK-SC mycelium, revealed that

pectinase production was enhanced by 32%

(1,01,000 IU l)1) as compared to SMC (Figure 2).

Similarly, Kapoor et al. (2000) reported a 1.5-fold

increase in polygalacturonase production from Bacillus

sp. MG-cp-2 by employing PUF as the inert support

matrix.

Characterization of pectinase

The (NH4)2SO4precipitation procedure resulted in 90%

enzyme recovery with a purification of 3.0-fold. The

pectinase was optimally active at pH 8.0 and retained

more than 50% of its activity in the pH range of 6.09.0

after 8 h of incubation at room temperature (Figure 3).The pectinase from Streptomyces sp. RCK-SC was

found to be maximally active at 60 C. Temperature

kinetics of pectinase suggested that the enzyme activity

rose steadily up to 60 C and thereafter it sharply

declined (Figure 4). The enzyme is thermostable and

retains 80, 68 and 65% of its activity at 50, 60 and

70 C, respectively after 2 h of incubation. The half life

of pectinase was 3 h at 70 C (Figure 5). Pectinases with

alkaline pH optima have been reported from Bacillussp.

NT-2, NT-6, NT-33 and NT-82 (Cao et al. 1992),

Bacillus sp. MG-cp-2 (Kapoor et al. 2000), andBacillus

sp. strain KSM-P15 (Kobayashi et al. 1999). Tempera-

ture optima of 60 C have been reported for pectinases

from Bacillus sp. MG-cp-2 (Kapoor et al. 2000) and

Streptomyces sp. QG-11-3 (Beg et al. 2000b), whereas

lower temperature optima of 45 and 50 C have been

reported for pectinases from Sclerotinia sclerotiorum

(Riouet al. 1992) andSaccharomyces cerevisiae(Blanco

et al. 1994), respectively.

Conclusions

Streptomyces sp. RCK-SC has been found to produce

an alkaline pectinase. Our study reveals that pectinase

production was not a direct function of cell growth and

was induced by various additives, exemplified by a

massive increase in the specific productivity of pectinase.

Time (Days)

0 2 4 6 8 10

Pectinaseyie

ld(IUmL-1)

65

70

75

80

85

90

95

100

105

Figure 2. Course of pectinase production fromStreptomycessp. RCK-

SC immobilized on polyurethane foam (PUF).

pH

2 4 6 8 10

%r

esidualpecti

naseactivity

30

40

50

60

70

80

90

100

Figure 3. Effect of pH (d) and pH stability (s) on pectinase activity

assayed at 60 C for 10 min. The pectinase was assayed in the pH

range 3.010.0 using different buffers (citratephosphate, pH 3.07.2;

phosphate buffer, pH 6.07.5; TrisHCl, pH 7.29.2; carbonatebicarbonate, pH 8.510.0; and glycineNaOH, pH 8.510.0). pH

stability was determined by incubating the enzyme with an equal

amount of buffer for 8 h at room temperature and thereafter residual

activity was determined under standard assay conditions. 100%

pectinase activity was equivalent to 150 IU ml)1.

Temperature (oC)

35 40 45 50 55 60 65 70

%r

esidualpectinaseactivity

75

80

85

90

95

100

Figure 4. Effect of assay temperature on activity of pectinase. The

optimum assay temperature for pectinase was determined by incubat-

ing the assay mixture at pH 8.0 at different temperatures between 40

and 70C. The pectinase activity of 100% was equivalent to

150 IU ml)1.



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The enhanced production of pectinase from Streptomy-

ces sp. RCK-SC using immobilized cell system and

solid-state cultivation might provide a significant impe-

tus to improve economic feasibility and commercial

viability of pectinase for use in degumming of bast fibres

and treatment of alkaline pectic wastewater.

Acknowledgments

The authors thank the Department of Biotechnology,Ministry of Science and Technology and Ministry of

Environment and Forest (MOEF) for financial assis-

tance. The senior research fellowship from the Ministry

of Environment and Forest (MOEF) to M.K. is

gratefully acknowledged. We wish to thank Dr Rani

Gupta for her suggestions during the preparation of the

manuscript.

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Time (min)

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%r

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40

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90

100

Figure 5. Thermostability profile of pectinase. Thermostability was

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150 IU ml)

1.



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kuhad 2004 produccion de una pectinasa alcalina en especies de streptoyices

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