Characterization of an endoxylanase produced by isolated bacillus sp.

Abstract

For the production of xylooligosaccharides, a Bacillus sp. with an endoxylanase activity was isolated from compost. The xylanase production reached upto 5 U/ml after 22 hours of incubation in LB medium at $30^\circ C$. The xylanase was purified by ammonium sulfate precipitation and gel filtration chromatography, The molecular weight of the xylanase was estimated to be 20,400 by SDS-PAGE and the isoelectric point was 10.6. Optimal temperature and pH of the xylanase was $60^\circ C$ and 6.5 respectively. The enzyme was stable at temperatures upto $40^\circ C$ and pH valuse from 4 to 10. The xylanase was completely inhibited by the addition of mercury ion and N-bromosuccinimide indicating possible involvement of tryptophan residue in the active site of the enzyme. Apparent $K_m$ and $V_{\max}$ values for oat spelts xylan was 9.2mg/ml and 1954U/mg protein respectively. For birchwood xylan, the values were 6.3mg/ml and 1009U/mg protein. The predominant products of xylan hydrolysate were xylobiose, xylotriose and xylotetraose, indicating that the enzyme was an endoxylanase. The enzyme was highly specific to xylans as substrate with little activity toward lichenan. No activity was detected on CMC and cellulose. The xylanase hydrolysed upto 32\% of reducing sugars from oat spelts xylan and 27\% from birchwood xylan. Upto 85\% of the enzyme was bound to 50mg/ml of insoluble fraction of oat spelts xylan after incubation at $30^\circ C$ for 30 minutes.