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

1) 2011

ISSN: 2231-2560
Research Article

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 niger
pectinases 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
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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, iodinepotassium 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) fermentation
Submerged 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 deesterifying 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 Alkali slant, acid Alkali slant, acid
H2S production

butt,

no

H2S butt,

no

production

production

6.

Mannitol motility

Positive

Positive

Positive

Indole

Positive

Positive

Positive

Methyl red

Positive

Positive

Positive

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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*Correspondence Author: Dr. Kokati Venkata Bhaskara Rao, School of Bio Sciences and
Technology VIT University, Vellore 632 014, Tamilnadu, India.

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