Purification and Characterization of Xylanase Enzyme from Bacillus Substilis Isolated from Forest Soil

Abstract

The xylanase obtained from Bacillus substilis isolated from forest soil was purified and characterized to assess its future in industrial application. The purification process was by ammonium sulphate fractionation followed by dialysis, ion exchange chromatography and gel filtration chromatography. Solid ammonium sulphate salt was used in concentration of 80%.The precipitated protein was recovered by centrifugation and dissolving it in minimum quantity of buffer after which the protein were retrieved by dialysis. Xylanase further purified by DEAE cellulose ion exchange column. The yield and fold purification was 60.47% and 2.45% with specific activity of 7.495 U/mg protein. Column chromatography using Bio-Gel P-60 matrix indicate high purity yield of 53.46%  and fold purification 6.25% with specific activity of 19.08 U/mg protein.The molecular weight was found to be 22Kd ,determined by SDSPAGE .Zymogram analysis of xylanase activity using purified protein resulted in a single band. There was a gradual increase in the enzyme activity from 2% to 3% of substrate concentration. The enzyme showed stability at alkaline pH7 and showed an increase in activity at 50°C.The xylanase activity was strongly inhibited by Hg2+ and Cu2+ and enhanced in presence of Mn2+.