PARTIAL PURIFICATION AND CHARACTERIZATION OF XYLANASE PRODUCED FROM Aspergillus niger USING WHEAT BRAN

Genetic diversity in chestnuts of Kashmir valley In present exploration, purification and characterization of xylanase was carried out to find its optimum conditions for maximum functionality. The xylanase (EC 3.2.1.8) synthesized by Aspergillus niger in submerged fermentation was partially purified and characterized for different parameters like temperature, pH & heat stability. The molecular mass determined through SDS-PAGE was found 30 kDa. The specific activity of the enzyme was raised from 41.85 to 613.13 with 48.63% yield just in a two step partial purification comprising ammonium sulphate precipitation and Sephadex gel filteration column chromatography. The partially purified enzyme was found to be optimally active at 60°C and 7.5 pH. Conclusively, for the application of xylanase in food, feed or paper manufacturing processes, it is necessary to consider its optimum pH and temperature. Keywords: Xylanase, enzyme, purification, characterization, SDS-PAGE, wheat bran, Aspergillus niger . INTRODUCTION The agricultural waste materials like wheat bran, ground nut cake, rice bran, rice straw, sugarcane bagasse, cotton leaf scraps, fruit and vegetable wastes if not properly handled cause environmental issues. The proper management of these wastes not only reduces their detrimental impact to environment, but also transforms them into value added products of industrial and commercial potential (Bhosale et al., 2011; Javed et al., 2011). Aspergillus niger when grown on agricultural waste materials produces sufficient activities of xylanase (Haq et al., 2002). Xylanase is an extracellular enzyme which hydrolyses β-1, 4 D-xylosidic linkages of highly polymerized and substituted β-1, 4 linked D-xylobiose, xylotriose and glucuronosyl residues. The enzyme holds potential for the degradation of plant cell wall material (Omar et al., 2008). Microbial xylanases (β-1, 4 D-xylan xylanohydrolase, EC 3.2.1.8) are being used in various industries including food, feed, textile and paper processing (Ahmad et al., 2012). In food and feed, they liberate nutrients by hydrolyzing the non-degradable hemicellulose fibers; thus, make the nutrients available (Leisola et al., 2002; Walk et al., 2011). On account of beneficial role of enzymes, different methodologies are being used for their maximum biosynthesis (Chithra and Muralikrishna, 2008). Sugars like xylose, xylobiose and xylo-oligomers can be prepared by the enzymatic hydrolysis of xylan. The depolymerization action of xylanase results in the conversion of polymeric substances into xylo-oligosaccharides and xylose (Omar et al., 2008). The manipulation of biotechnological techniques has played an important role in the potential utilization of agricultural waste materials (Mohammadi et al., 2006; Okafor et al., 2007). The microbial xylanase synthesis has been studied by several researchers (Suneetha et al., 2011), even then efforts are to make to screen and identify suitable xylanolytic enzymes which are well-suited for the specific purpose (Beg et. al., 2000). Therefore, the present study was designed for the purification of xylanase and its characterization to find the optimum conditions for its maximum functionality. MATERIALS AND METHODS Purification Ammonium sulphate precipitation : The crude enzyme (produced in a previous study: Ahmad et al., 2009) was partially purified from the culture supernatant using ammonium sulphate in a Na-acetate buffer of pH 6.8 (Javed et al., 2009). For the purpose, various ammonium sulphate concentrations, i.e. 30, 40, 50, 60, 70 and 80% were used for the precipitation of enzyme. The respective levels were mixed in 500 mL of crude enzyme filtrate and kept at 4°C for two hours with continuous stirring. The precipitates were collected and analyzed for xylanase activity. The optimum xylanase activity at a specific concentration of ammonium sulphate reflected the best concentration to attain maximum enzyme recovery.