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Sum m ary and Conc l usi on 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 100 Sum m ary and Conc l usi on (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 101 Sum m ary and Conc l usi on 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. 102 Sum m ary and Conc l usi on 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 103 Sum m ary and Conc l usi on 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 104 Sum m ary and Conc l usi on 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 105 Sum m ary and Conc l usi on 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 *************************** 106