Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6.
Bacillus stearothermophilus T-6 produces an extracellular xylanase that was shown to optimally bleach pulp at pH 9 and 65 degrees C. The enzyme was purified and concentrated in a single adsorption step onto a cation 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 degrades xylan to xylose and xylobiose. The pIs of the purified protein were 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 for more than 10 h; at 65 degrees C and pH 9, the half-life of the enzyme was approximately 6 h. Kinetic experiments at 55 degrees C gave Vmax and Km values of 288 U/mg and 1.63 mg/ml, respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by Zn2+, Cd2+, and Hg2+. Xylan completely protected the protein from inactivation by N-bromosuccinimide. The N-terminal sequence of the first 45 amino acids of the enzyme showed high homology with the N-terminal region of xylanase A from the alkalophilic Bacillus sp. strain C-125.
Khasin
A
A
Department of Food Engineering and Biotechnology, The Technion, Technion City, Haifa, Israel.
Alchanati
I
I
Shoham
Y
Y
D000595:Amino Acid Sequence; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013696:Temperature; D043325:Xylan Endo-1,3-beta-Xylosidase; D014990:Xylans / Q000378:metabolism*
D008670:Metals; D014990:Xylans; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase