18-12-2012, 02:06 PM
Scholars Research Library
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ABSTRACT
The production of polygalacturonase (PG ase) by a locally isolated aerobic bacterium, Bacillus
sp. in submerged fermentation (SmF) was optimized. The effects of the fermentation parameters
namely initial pH, temperature, cultivation time and nitrogen source on enzyme production were
studied using both pure pectin and citrus wastes as the sole carbon source. Polygalacturonase
from the strain was maximally secreted at 37°C, initial pH 7.0 with 0.15% (w/v) of pure pectin
as sole carbon source. Among citrus wastes, 1% (w/v) orange peel and lemon peel gave the
most promising results at pH 7 and 5 respectively. Tryptone was found to be the best nitrogen
source for enzyme synthesis. Under optimized conditions, highest PG ase production was
achieved at 24th hour, at mid stationary phase of growth of the strain. The enzyme was extremely
thermostable and 75% of the activity was restored even after the exposure of the enzyme protein
at 80°Cfor 90 minutes. The enzyme was 86% stable in a broad range of pH of 4 to 9. The PG ase
activity was found to increase in presence of Mn2+ and also by addition of exogenous thiols like
DTT, Cysteine, GSH indicating the presence of thiol groups at the active site.
INTRODUCTION
Pectin is a family of complex polysaccharides that contain 1,4-linked α-D-galactosyluronic acid
residues [1]. Although it is a natural part of human diet, it does not contribute significantly to
nutrition and goes through the small intestine more or less intact. Enzymes that hydrolyze pectic
polymers are broadly known as pectinases and microbial pectinaes have tremendous potential to
offer mankind [2].Amongst the pectic enzymes, polygalacturonases (EC 3.2.1.15 ) catalyze the
random hydrolysis of 1,4 α-D galacturonic acid linkages in smooth region of pectin [3] and have
attracted the attention of scientists from biotechnology or pharmaceutical industry because they
are protein enzymes relevant to phytopathogens invasion, fruit ripening, and potential
antimicrobial drug targets [4] . Commercially, pectinase has a share of 25% in the global sales of
food enzymes [5] and is added to livestock feed to help the animals for better digestion of the
food and are also sold as nutritional supplements for humans to aid digestion . The pectinase
production from microorganisms has been reported under both submerged and solid state
fermentations [6] . At present almost all the pectinolytic enzymes used for industrial applications
are produced by fungi and there are a few reports of pectinase production by bacterial strains [7].
Hence an extensive research is warranted to isolate a bacterial strain for production of
polygalacturonase within a short period of time and standardization of the conditions for
maximum production of the enzyme in a cost effective way.
Optimization of other Parameters
The concentrations of agro wastes used as sole carbon source were varied to optimize the
substrate concentration of submerged culture of Bacillus sp. The optimum pH was determined
by adjusting the initial pH of the fermentation media at a range from 4.0-9.0. Most favorable
production temperature was studied by incubating the production medium at different
temperatures (7°C, 17°C, 27°C, 37°C, and 47°C). To study the effect of concentration of
optimized carbon source for maximal enzyme production, pure pectin as well as the citrus wastes
were used at different concentrations (0.03% to 3% w/v) in the production media. Similarly, the
effects of various nitrogen sources namely peptone, yeast extract, ammonium chloride, tryptone
and urea 0.09% (w/v) were tested. Each experiment was carried out in triplicate and their values
were averaged. The time course of growth and enzyme production by the strain under optimized
culture conditions was studied by checking the enzyme production kinetics fro 0 to 36 hours at
37°C.
Enzyme characterization
The stability of the enzyme at different pH values was studied by incubating the enzyme (0.5ml)
with buffers presenting pH from 4.0 to 9.0 was kept at 37°C for 120minutes followed by the
estimation of the residual activity. Thermostability kinetics was determined by exposing the
pectinase at 80°C for 0 – 90 minutes in water bath and then estimating the residual activity. The
effect of metal ions and thiol compounds was measured by incubating the enzyme at 37°C for 30
minutes with the additives at a concentration of 10mM followed by the assay of enzyme activity
in usual procedure.