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Asian Journal of Biochemical and Pharmaceutical Research Issue 2 (Vol. 1) 2011 ISSN: 2231-2560

Research Article

329

Asian Journal of Biochemical and Pharmaceutical Research

Screening of Pectinase Producing Microorganisms from Agricultural Waste Dump

Soil

Janani, L. Karthik, Gaurav Kumar, K.V. Bhaskara Rao*

Molecular and Microbiology Research Laboratory, Division of Environmental Biotechnology, School of Bio Sciences and Technology, VIT

University, Vellore 632 014, Tamilnadu, India

Received: 31 March 2011; Revised: 18 April 2011; Accepted: 27 April. 2011

Abstract: The aim of this present study was isolation and Screening of Pectinase producing bacteria from Agricultural waste dump soils in Vellore, Tamilnadu, South India. Total ten bacterial strains were isolated from these soils and only 3 strains were positive in Pectinase depolymerization assay plates. The extracellular pectinase was partially purified by ammonium sulphate precipitation and dialysis. These 3 strains were identified as Bacillus sp. and they produced very high levels pectinase by submerged and semi-solid fermentation. Maximum enzyme production was obtained in the medium containing wheat bran as substrate compare to rice bran. The effect of different temperature shows the optimum temperature for enzyme production is 30C. Key words: Bacillus sp., Pectinase, submerged fermentation, semi-solid fermentation, dialysis

INTRODUCTION:

Pectin is a polymeric material having carbohydrate group esterifies with methanol. It is an important component of plant cell wall. It is present in highest concentration in the middle lamella, where it acts as a cementing substance between adjacent cells. Plant pathogens attack target cells by producing number of cell degrading enzyme which facilitates the entry and expansion of pathogen in the host tissue [1]. The history of pectinases began with an understanding the structure of pectins substances and the mechanism by which pectolytic enzymes degrade pectic substances. Later the microbial production of pectinases became prominent for many decades. Many microorganisms viz.., bacteria, yeast, fungi could produce pectinases [2]. Evidence showed that pectinases are inducible and they can produce from different carbon sources. In the course of time, numerous reports have appeared on the optimization of fermentation and microbiological parameters and different fermentation strategies for the production of pectinases [3]. With the advent of molecular biology, vigorous research has been carried out on cloning and expression of pectinase genes in various hosts. Apergillus nigerpectinases are most widely used in industries because this strain posses GRAS (Generally Regarded As Safe) status so that metabolites produced by this strain can be safely used. This fungal strain produces various pectinases including polymethylgalacturonase (PMG), polygalacturonase (PG), and pectin esterase (PE). Apple and citrus fruits are the main source of commercial pectin at present. Pectin, a major constituent of cereals, vegetables, fruits; fibers are complex, high molecular weight heterogeneous and acidic structural polysaccharide [4]. D-galacturonic acid is one of the major

Asian Journal of Biochemical and Pharmaceutical Research Issue 2 (Vol. 1) 2011

330

components of pectin [5]. The present study was designed to isolate and purify the pectinase from bacteria and also identify the potential pectinase producing strains through conventional methods.

MATERIALS AND METHODS:

Sample Collection

Agriculture and Vegetable waste dump soil samples were collected from Sholingur, Vellore district, Tamil Nadu India. Soil samples are taken with help of sterile spatula, in sterile plastic bags the samples were brought to Molecular and Microbiology laboratory for further processing.

Isolation of Microorganisms

One gram of soil samples from each collection site were pooled and homogenized in sterile distilled water and 10-fold serial dilutions were prepared. One ml aliquots from each dilution was inoculated by spread plate method on to the sterile petriplates containing yeast extract pectin (YEP) medium with pH 7.2 containing pectin 2.5, and yeast extract 5.0 at 37C. Pure cultures were sub cultured onto slant media and maintained for identification and enzyme studies.

Screening Of Pectinase Producing Microorganisms:

Plate Assay of Depolimerized Pectin

The YEP medium was used for isolation of cultures supplemented with 2% agar. Pure culture was inoculated by puncture in the medium and incubated for 48hrs at 30C. After incubation, iodine-potassium iodide solution was added to detect the clearance zone.

Pectinase Assay

Pectinase activity was assayed by the colori-metric method of Miller (6). Briefly, 0.5ml of cell free supernatant was incubated with 0.5ml of pectin in 0.1M acetate buffer with pH 6.0 and the reaction mixture was incubated at 40C for 10 minutes in static condition. After adding 1ml of DNS reagent, the mixture was boiled for 5 min at 90C. The reaction was stopped by adding 1ml of Rochelles salt. Then the mixture was diluted by adding 2ml of de-ionized water. The absorbance was measured Spectrophotometrically at 595 nm. A standard graph was generated using standard glucose solution. One unit of Pectinase activity was defined as the amount of enzyme which liberated 1m glucose per min.

Production of pectinase enzyme on solid-state (SSF) and submerged (SmF) fermentationSubmerged Fermentation

The YEP medium was inoculated with a suspension containing 106 cells/ml. Cultures were grown in 250 ml Erlenmeyer flasks with 50ml of medium in a rotary shaker (150 rpm) at 30C. After


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48hrs, the biomass was separated by centrifugation at 7000 x g for 10 minutes and the supernatant was used to evaluate the Pectinase activity.

Semi-solid Fermentation

The semi-solid fermentation was done using 250 ml Erlenmeyer flask contain 10g wheat bran mixed with appropriate amount of mineral salt solution contain 1% KH2PO4, 5% Nacl , 0.1% MgSO4.7H20, 0.1% Cacl2 soil extract -1 ml was inoculated with 12 hr old culture. It was incubated at 30C for 48hrs. Then 1g of sample suspended in 10ml of glycine-sodium hydroxide buffer (pH 10.0). The sample was vortexed thoroughly and centrifuged 10,000g for 30 min at 4C. Then the supernatant was used to evaluate the Pectinase activity (7).

Partial Purification of Enzyme

The partial purification of the protease was carried out by using standard protocol .The packed cells were suspended in distilled water and this was inoculated into 5ml of broth and incubate for 24hrs at 37C. After 24hrs, the broth was centrifuged at 10,000 rpm for 15 min and cell free supernatant was used for pectinase assay and purification. This supernatant was partially purified using the following two sub-sequential steps [8].

Ammonium Sulphate Precipitation

The supernatant was brought to 60% saturation by mixing ammonium sulphate (pH 7.0) slowly with gentle agitation and allowed to stand for 24hrs at 4C in the cold room. After the equilibration, the precipitate was removed by centrifugation (10,000 rpm at 4C for 20 min). The precipitate obtained was dissolved in 10ml of 0.5ml Phosphate Buffer (7.0).

Desalting by dialysis

The precipitate was desalted by dialysis following the standard protocol, the 10 c. m pretreated dialysis bag was taken and activated by rinsing in double distilled water. One end of the dialysis bag was tightly tied and the precipitate recovered was taken inside the bag. The other end of the dialysis bag was tightly tied to prevent the leakage. After that, dialysis bag was suspended in a beaker containing phosphate buffer (pH 7.0).

Effect of different temperature

For optimization, production of Pectinase was studied using the nutrient medium, under the following condition such as 30C and 37C, pH 7.2. The flasks were incubated for 24-72 hrs and the cells were removed by cold centrifugation at 7000 rpm for 10 min. The cell free supernatant was analyzed for Pectinase activity.

Effect of different carbon and nitrogen source


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To test the effect of nitrogen and carbon source on the Pectinase production, the liquid and solid media was supplemented with various nitrogen and carbon sources such as, pectin, wheat bran, rice bran at 1% concentration. The flasks were incubated for 24-72hrs and cell free supernatant was analyzed for Pectinase activity.

Identification of Pectinolytic Bacteria:Microscopic observation

Gram staining was performed to check the morphology of the cells and spore chain morphology was identified by spore staining technique.

Biochemical characterization

The pectinolytic bacteria isolates were biochemically characterized by Catalase Test, Oxidase Test, Urease test, Mannitol motility test, TSI test, SIM agar deep tube, Starch hydrolysis test Nitrate reduction test, IMVIC test and Carbohydrate Fermentation test.

RESULTS AND DISCUSSION:

The ten bacterial strains were able to grown on medium containing Pectin as a sole carbon source were isolated. These strains were tested for Pectin hydrolysis by plate assay, at pH 7.2. The strains were classified as very good producers of Pecin depolymerizing enzymes when presented clear haloes around colonies of at least 15 mm, good producers when the haloes were of at least 10 mm, weak producers when halos were at least 5 mm and poor producers when no pectinolytic activity and no pectinolytic activity and no clear lysis zones were observed.

From the 10 bacterial strains three strains were able to produce high polygalacturonase activity. Based on the morphology and biochemical characterization the strains were identified as Bacillus sp (SH1), Bacillus sp (SC1) and Bacillus sp. (ST1) (Table 1). These three strains are able to degrade pectin by producing pectinase enzyme. The most common source of commercial pectinases is the filamentous fungus Aspergillus sp [9]. That produces a complex pectinolytic enzyme, the de-esterifying chain-splitting enzymes. They are also obtained from tomatoes and oranges. Pectinase world-wide consumption is above 7106 tons per year [10].

Pectinase production was estimated by submerged and semi solid fermentation and it was observed that Polygalacturonase production by semi solid fermentation was higher than that of Polygalacturonase production by submerged fermentation which was already reported [11].The isolated Bacillus strains were cultivated for polygalacturonase production using submerged fermentation and semi solid fermentation. The submerged fermentation was carried out at different temperature such as 30C and 37C (Fig 1 & 2). The semi solid fermentation was carried out by using wheat bran and rice bran as a substrate. The specific polygalacturonase activity was calculated at 30C for 24 hour culture of 3 potential strains such as Bacillus sp (SH1) (886 U/ml), Bacillus sp (ST1) (908 U/ml) and Bacillus sp (SC1) (988 U/ml). The maximum yield obtained from Bacillus sp (SC1). The


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specific polygalacturonase activity was calculated at 37C for 24 hour culture of 3 potential strains such as Bacillus sp (SH1) (813U/ml), Bacillus sp (ST1) (872 U/ml) and Bacillus sp (SC1) (936U/ml). In solid state fermentation, the medium with wheat bran as a substrate showed maximum yield of pectinase. The earlier studies also proved that wheat bran as a substrate showed maximum yield of pectinase when compare to other substrates such as rice bran, apple pomace etc [9]. In the present study the Bacillus sp (SC1) produced maximum Pectinase activity by using wheat bran as a substrate (1431 U/ml) and showed maximum yield (1199 U/ml) by using rice bran as a substrate (Fig 3).Bacillus sp (SH1) produced maximum pectinase activity by using wheat bran as a substate (1165 U/ml) and the pectinase activity using rice bran is (940 U/ml) (Fig 4). Whereas, Bacillus sp (ST1) showed maximum pectinase activity using wheat bran is (1116U/ml) and the pectinase activity using rice bran is (980 U/ml) (Fig 5). According to various authors the production of these enzymes on solid substrate, such as agricultural residues, was affected by culture conditions such as moisture, pH and type of bioreactor [3].

Off the two substrates (wheat bran, rice bran) used in the present study, wheat bran produced maximum polygalacturonase yield. Among the three Bacillus strains Bacillus sp (SH1) Produced maximum yield (1165 U/ml) and other Bacillus strains such as Bacillus sp (SC1) and Bacillus sp(ST1) were showed (1116 U/ml) during 24th hour and (1431 U/ml) at 72nd hour respectively.The pectinase was partially purified from submerged fermentation broth by using ammonium precipitation and dialysis method. The total activity of dialysed pectinase was Bacillus sp (SH1) (1162U/ml),Bacillus sp (SC1) (1151 U/ml), and Bacillus sp (ST1) (1206 U/ml) (Fig 6).

CONCLUSIONS:

In the present study, Maximum production of Pectinase was obtained from Bacillus sp (ST1) (1206 U/ml) and another strain Bacillus sp (SC1) produced maximum Pectinase activity by using wheat bran as a substrate (1431 U/ml). These organisms will be further identified by molecular methods like 16srDNA analysis to confirm their novelty. And these strains may be used for commercial production of pectinases and also the enzyme yield may be increased by mutational studies.

ACKNOWLEDGEMENTS:

The authors wish to thank the Management and Staff of VIT University for providing necessary facilities to carry out this study.


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Fig. 1: Stability of PG activity at 30C

Fig. 2: Stability of PG activity at 37C

Fig. 3 Effect of PG activity produced by Bacillus sp (SH1) on different substrate


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Fig. 4 Effect of polygalacturonase activity produced by Bacillus sp (ST1) on different substrate

Fig 5 Effect of polygalacturonase activity produced by Bacillus sp (SC1) on different substrate

Fig 6 Pectinase activity of patially purified enzyme from Bacillus sps


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Table1. Morphology and Biochemical characteristics of the 3 isolates

S.No Test Bacillus sps (SH1) Bacillus sps (ST1) Bacillus sps (SC1)

1. Colony morphology Large, irregular, mucoid Small , mucoid Large, mucoid

2. Gram staining Gram positive rod Gram positive rod Gram positive rod

3. Spore staining Central Sub terminal terminal

4. Hanging drop Motile Motile Motile

5. TSI Alkali slant, acid butt, no

H2S production

Alkali slant, acid

butt, no H2S

production

Alkali slant, acid

butt, no H2S

production

6. Mannitol motility Positive Positive Positive

7 Indole Positive Positive Positive

8 Methyl red Positive Positive Positive

9 voges- proskauer test Negative Positive Negative

10 Citrate Positive Positive Positive

11 Catalase Positive Positive Positive

12 Oxidase Negative Negative Negative

13 Urease Negative Positive Negative

14 Nitrate test Positive Positive Negative

15 H2S production Negative Negative Negative

16 Starch Positive Positive Positive

17 Glucose Positive Positive Positive

18 Sucrose Positive Negative Negative

19 Lactose Negative Negative Negative


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2. S. Yadav, P. K. Yadav, D. Yadav and K. D Singh Yadava., Process Biochemistry., 2009, 44 (1),1.

3. R. A. Bayoumi, H. M. Yassin, M. A. Swelim and E. Z. Abdel-All., Journal of Biotechnology.,

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4. T. Doco, P. Williams, S. Vidal and P. Pellerin., Carbohydrate research., 1997, 297 (2), 181.

5. S. N. Gummadi and T. Panda., Process Biochemistry., 2003, 38, 987.

6. G. L. Miller., Analytical Chemistry., 1959, 31,426.

7. D. R. Kashyap, S. K. Soni and R. Tewari., Bioresource Technology., 2002, 88, 251.

8. B. G. Klug-Santner, W. Schnitzhofer , M. Vrsanska , J. Weber, P. B. Agrawal, V. A. Nierstrasz

and G. M. Guebitz., Journal of Biotechnology., 2006, 121 (3) , 390.

9. L. R. Castilhoa, T. L.M. Alvesa and R. A. Medronhob., Process Biochemistry., 1999, 34 (2),181.

10. A. A. Kamnev, C. M. J. Rodriguez, N. M. Ptitchkina and V. V. Ignatov., Food Hydrocolloids.,

1998, 12, 263.

11. M. M. C. N. Soares, R. Silva and E. Gomes., Revista de Microbiologia., 1999, 30, 299.

*Correspondence Author: Dr. Kokati Venkata Bhaskara Rao, School of Bio Sciences and

Technology VIT University, Vellore 632 014, Tamilnadu, India.

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