ONE-STEP PURIFICATION AND CHARACTERIZATION OF CELLULASE-FREE XYLANASE PRODUCED BY ALKALOPHILIC BACILLUS SUBTILIS ASH

The present study describes the one-step purification and characterization of an extracellular cellulase-free xylanase from a newly isolated alkalophilic and moderately thermophilic strain of Bacillus subtilis ASH. Xylanase was purified to homogeneity by 10.5-fold with similar to 43% recovery using ion-exchange chromatography through CM-Sephadex C-50. The purified enzyme revealed a single band on SDS-PAGE gel with a molecular mass of 23 kDa. It showed an optimum pH at 7.0 and was stable over the pH range 6.0-9.0. The optimum temperature for enzyme activity was 55 degrees C. The purified xylanase did not lose any activity up to 45 degrees C, however, it retained 80% and 51% of its activity after pre-incubation at 55 degrees C and 60 degrees C, respectively. The enzyme obeyed Michaelis-Menton kinetics towards birch wood xylan with apparent K-m 3.33 mg/ml and V-max 100 IU/ml. The enzyme was strongly inhibited by Hg2+ and Cu2+ while enhanced by Co2+ and Mn2+. The purified enzyme could be stored at 4 degrees C for six weeks without any loss of catalytic activity. The faster and economical purification of the cellulase-free xylanase from B. subtilis ASH by one-step procedure together with its appreciable stability at high temperature and alkaline pH makes it potentially effective for industrial applications.