Purification, characterization and mass spectroscopic analysis of thermo-alkalotolerant β-1, 4 endoxylanase from Bacillus sp. and its potential for dye decolorization

Abstract

A Bacillus sp., isolated from sludge and sediments of pulp and paper mill, was found to produce xylanase in a synthetic culture media containing oat spelt xylan (1% w/v) and 10% black liquor as inducers along with 2.5% (w/v) sucrose as additional carbon source. The purified enzyme was highly thermostable with half-life of 10min at 90°C and pH 8. The enzyme was stable over a broad range of pH (pH 6–10) and showed good thermal stability when incubated at 70°C. Chemicals like EDTA, Hg2+, Cu2+ and solvents like glycerol and acetonitrile completely inhibited enzyme activity at high concentration. The molecular weights of the purified enzyme, determined by matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF/MS) analysis was analogous to the results obtained from SDS-PAGE, i.e. 55kDa. Kinetic parameters were determined by using oat spelt xylan as substrate. The KM and Vmax values of the enzyme were 4.4mg/ml and 287U/mg respectively. At high xylan concentrations (>70mg/ml) a substrate inhibition phenomenon of the enzyme was observed. In addition, crude xylanase showed enormous potential for decolorization of various recalcitrant dyes.