Sunday, April 29, 2007

Cloning, expression and characterization of a glycoside hydrolase family 39 xylosidase from Bacillus halodurans C-125

Kurt Wagschal, Diana Franqui-Espiet, Charles C. Lee, George H. Robertson, and Dominic W.S. Wong. USDA Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710

The gene encoding a glycoside hydrolase family 39 xylosidase (BH1068) from the alkaliphile Bacillus halodurans strain C-125 was cloned with a C-terminal His-tag and the recombinant gene product termed XylBH1068 was expressed in E. coli. Of the artificial substrates tested, XylBH1068 hydrolyzed nitrophenyl derivatives of β-D-xylopyranose, α-L-arabinofuranose, and α-L-arabinopyranose. Deviation from Michaelis-Menten kinetics at higher substrate concentrations indicative of transglycosylation was observed, and kcat and Km values were measured at both low and high substrate concentrations to illuminate the relative propensities to proceed along this alternate reaction pathway. The pH maximum was 6.5, maximal activity was at 47 °C, and thermal instability occurred above 43 °C. XylBH1068 was inactive on arabinan, hydrolyzed xylooligosaccharides, and released only xylose from oat, wheat, rye, beech and birch arabinoxylan, and thus can be classified as a xylosidase with respect to natural substrate specificity. The enzyme was not inhibited by up to 200 mM xylose. The oligomerization state was tetrameric under the size-exclusion chromatography conditions employed.