PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE XYLANASE FROM BACILLUS-STEAROTHERMOPHILUS T-6

Bacillus stearothermophilus T-6 produces an extracellular xylanase tha t was shown to optimally bleach pulp at pH 9 and 65-degrees-C. The enz yme was purified and concentrated in a single adsorption step onto a c ation exchanger and is made of a single polypeptide with an apparent M (r) of 43,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Xylanase T-6 is an endoxylanase that completely deg rades xylan to xylose and xylobiose. The pIs of the purified protein w ere 9 and 7 under native and denaturing conditions, respectively. The optimum activity was at pH 6.5; however, 60% of the activity was still retained at pH 10. At 65-degrees-C and pH 7, the enzyme was stable fo r more than 10 h; at 65-degrees-C and pH 9, the half-life of the enzym e was approximately 6 h. Kinetic experiments at 55-degrees-C gave V(ma x) and K(m) values of 288 U/mg and 1.63 mg/ml, respectively. The enzym e had no apparent requirement for cofactors, and its activity was stro ngly inhibited by Zn2+, Cd2+, and Hg2+. Xylan completely protected the protein from inactivation by N-bromosuccinimide. The N-terminal seque nce of the first 45 amino acids of the enzyme showed high homology wit h the N-terminal region of xylanase A from the alkalophilic Bacillus s p. strain C-125.