Download 100 Pectin is a complex polysaccharide consisting mainly of

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Pectin is a complex polysaccharide consisting mainly of esterified Dgalacturonic acid resided in a (1-4)-chain. The acid groups along the chain are
largely etherified with methoxy groups in natural product. There can also be
acetyl groups present on the free hydroxyl groups. The galacturonic acids
main chain has the occasional rhamnose group which disrupts chain helix
formation. Pectin is also known to contain other neutral sugars which are
present in side chains. The most common side chain sugars are xylose,
galactose and arabinose.
Pectinases are a group of enzymes that contribute to the breakdown of
pectin which are structural polysaccharide found in primary cell wall and
middle lamella of fruits and vegetables. Pectolysis is one of the most
important processes for plant, as it plays a role in cell elongation and growth
as well as fruit ripening. Microbial pectolysis is important in plant
pathogenesis, symbiosis and decomposition of plant deposits. The main
source of the microorganisms that produce pectinolytic enzymes are yeasts,
bacteria and large varieties of fungi and particularly Aspergillus species.
Endopolygalacturonase
production
was first reported
in 1951
using
Saccharomyces fragilis.
Pectic enzymes have two classes namely pectinesterases and pectin
depolymerases. Pectin esterase has the ability to de-esterify pectin by the
removal of methoxy residues. Pectin depolymerases readily split the main
chain and it was further classified as polygalacturonase (PG) and pectinlyases
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(PL). Thus on the whole pectinases are hydrolytic enzymes, which hydrolyze
the pectin molecules and are readily soluble in water.
Pectin substance consists of pectin and pectic acid. The main chain of
pectin is partially methyl-esterified-1, 4, D-galacturonan. Demetylated pectin is
known as pectic acid or polygalacturonic acid. Pectic substances are
commonly amorphous; with a degree of polymerization of about 200-400
substituents that can be found at the C-2 or C-3 position of the main chain.
Substituent's can be either nonsugar (acetyl) or sugar (D-galactose, D-xylose,
L-arabinose and L-mannose). The degree and type of branching varies
depending upon the source of pectic substance.
Pectolytic enzymes are wide spread in nature and are produced by
Bacteria, Fungi, Yeast, Insects, Nematodes and Protozoa. For example
Bacteria like Bacillus species, Clostridium species, Fungi like Aspergillus
species, Penicillium species, Yeast like Saccharomyces, Candida etc.
Microbial pectolysis is important in plant pathogenesis, symbiosis and
decomposition of plant deposits (Lang and Dornenburg, 2000). Thus by
breaking down pectin polymer for nutritional purposes, microbial pectolytic
enzymes play an important role in nature. The enzymes are inducible i.e.
produced only when needed and they contribute to the natural carbon cycle.
Microbial pectinolytic enzymes are enzymes available to attack plant
polysaccharides. However, pathogenic attack on plant tissue is normally
initiated by pectic enzymes because pectic substances are most readily
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accessible. Other carbohydrates appear sequentially and attack the available
polysaccharides.
Production of pectinase from pectin rich agro waste, viz. lemon peel,
sorghum stem, sunflower heads, fruit peels are used as substrate for
microbes in submerged or solid state fermentation systems as increased level
in the production of pectinases was noticed when the agro wastes were
supplemented with additional carbon and nitrogen sources.
By applying these enzymes on fruit pulp, they degrade pectin thereby
reducing the viscosity and the fruit juice can be handled easily. These
enzymes play an important role in maceration and solubilization of fruit pulps
and also in clarification. The traditional method of clarification of pectin
containing juice involves a number of steps, including centrifugation to remove
suspended solid, enzymatic treatment for depectinization, finding agents such
as bentonite and gelatin to remove haze and finally filtration by the
diatomaceous earth to remove
the finding agents. With membrane
technology, juice can be clarified easily using depectinization followed by ultra
filtration.
Textile processing has benefited greatly in both environmental and
product quality aspects through the use of enzymes. Prior to weaving of yarn
in to fabric, the warp yarns are coated with a sizing agent to lubricate and
protect the yarn from abrasion during weaving.
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The most upcoming application of pectinolytic enzymes is, use in the
degumming of plant fibers such as ramie, sun hemp, jute and flax. The
enzymatic processing result in no damage to the fibers and most importantly
in addition to being energy conservative is environmentally friendly. A high pH
optimum of pectinase from microorganisms is reported to be desirable for
degumming of plant fibers since a high pH not only prevents contamination
but also allows an open fermentation system to be adopted.
Using various carbon source and nitrogen sources as well as natural
products was investigated as inducer for the production of amylases and
pectinase using Aspergillus niger. Wheat bran extract was best for the
production of both amylase and pectinases. High pectinase activities were
also observed when polygalacturonic acid, fructose, mannose, saccharose
and cellobiase were used as stimulators. Optimum pH for the production of
pectinase was 7.0 and temperature was 37°C. Bacillus sps. isolated from soil,
has been found to produce significant amounts of an extracellular pectinase
subsequently characterized as pectinlyase.
The present study is aimed at isolation and identification of the
pectinase producing bacteria by microscopic, biochemical and molecular
characterization and also optimizing the production parameters and homology
modeling of the pectinase enzymes.
Soil samples collected from different sites of Guntur district were
enriched with pectin and moistened with water, were incubated at 37°C for
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one month. One gram of the enriched soil sample was transferred and
enriched again into broth containing 1% of pectin and 1% of peptone as the
source of carbon and nitrogen and incubated at 37°C on a rotatory shaker.
One ml of the enriched broth was serially diluted and was inoculated to
selective broth medium (Vincent’s medium) and incubated for 10 days from
which aliquots of culture was inoculated into a selective agar medium
(Vincent’s medium), which supports selectively the growth of pectinase
producing bacteria and the pure culture was transferred on to nutrient agar
medium and was subcultured at regular intervals on the same medium.
The isolated bacteria were identified based on staining, biochemical
tests and molecular characterization by sequencing the 16S rRNA gene. The
genomic DNA of the isolated bacteria was isolated using single step kit
method and the isolated genomic DNA was tested for purity by gel
electrophoresis and spectrophotometer and the 16S rRNA gene present in the
isolated genomic DNA was amplified using the primers in a thermocycler and
the amplified product was analyzed by gel electrophoresis and was then
subjected to sequencing. The sequenced data were compared with the Data
bank of NCBI, and the phylogenetic tree was constructed. The bacterial
isolates were identified as Bacillus subtilis.
The
influence
of different environmental parameters
like
pH,
temperature, nitrogen sources (inorganic and organic) and natural carbon
sources like dry orange peel and banana peel powders on the pectinase
production was studied. The optimum pH and temperature for the enzyme
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production were pH 7 and 37°C respectively, while the maximum enzyme
production was observed between 72 and 96 hrs. Among the nitrogen
sources, maximum enzyme production was observed with Ammonium
chloride (inorganic N source) and Beef extract (organic N source), between 72
to 96 hrs. Of the two natural carbon sources tested, the banana peel powder
yielded higher enzyme compared to the orange peel powder during the same
period. A repeated experiments, conducted employing the different optimal
conditions (pH 7, temperature 37°C, beef extract and dry banana peel
powder), yielded maximum enzyme production between 72 to 96 hrs.
The results showed that the enzyme production by Bacillus subtilis was
high at pH 7 and temperature 37°C and also Bacillus subtilis yielded higher
enzyme in the presence of banana peel powder than Orange peel powder.
The production of the pectinase enzyme by Bacillus subtilis showed that the
enzyme secretion was high in the presence of organic N source than
inorganic N source and maximum activity was observed with organic N source
i.e. beef extract.
Homology modeling of Polygalacturonase of Bacillus subtilis was
worked out by using different commercial and online softwares. Amino acid
composition of the protein was obtained from Swissprot and the composition
of the protein was obtained by software called Bioedit. The primary and the
secondary structures of the protein were predicted by Protparam, EMBOSS
win and PDBsum softwares and the model of the protein was predicted using
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the Phyre server and the structure of the modelled enzymes were valuated
using PROCHECK software.
The salient features of the present study are:
·
Pectinase producing bacteria were isolated from soil samples.
·
The bacterial isolate was identified as Bacillus subtilis
morphological, biochemical and molecular based techniques.
·
The maximum enzyme activity was noticed after 96 hrs of incubation
by Bacillus subtilis.
·
The bacterial isolate yielded maximum enzyme at pH 7 and 37°C
temperature.
·
The pectinase activity of Bacillus subtilis was higher with banana peel
powder than orange peel powder when used as natural carbon source.
·
Bacillus subtilis exhibited higher pectinase activity in the presence of
organic nitrogen source than inorganic nitrogen source.
·
The unknown isolate was identified by amplifying and sequencing of
the 16S rRNA gene.
·
Homology modeling of the Polygalacturonase of Bacillus subtilis was
carried out since the enzyme structures have not been previously
deposited in the Protein Data Bank (PDB).
by
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