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

Applied Biochemistry and Biotechnology
Kurt WagschalDominic W S Wong

Abstract

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 BH1068(His)(6) was expressed in Escherichia coli. Of the artificial substrates tested, BH1068(His)(6) hydrolyzed nitrophenyl derivatives of beta-D-xylopyranose, alpha-L-arabinofuranose, and alpha-L-arabinopyranose. Deviation from Michaelis-Menten kinetics at higher substrate concentrations indicative of transglycosylation was observed, and k (cat) and K (m) 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, and under the conditions tested, maximal activity was at 47 degrees C, and thermal instability occurred above 45 degrees C. BH1068(His)(6) 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 con...Continue Reading

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Sep 10, 2005·Applied and Environmental Microbiology·Kurt WagschalDominic W S Wong
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Citations

Feb 15, 2012·The Biochemical Journal·Douglas B JordanKurt Wagschal
Nov 19, 2013·Biotechnology Advances·Stijn LagaertGuido Volckaert
Oct 12, 2012·Applied Biochemistry and Biotechnology·Juliana Moço CorrêaRita de Cássia Garcia Simão
Nov 9, 2019·International Journal of Molecular Sciences·Ali RohmanNi Nyoman Tri Puspaningsih
Jul 13, 2019·New Biotechnology·Atilio TomaziniIgor Polikarpov

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