Cloning, expression and characterization of a glycoside hydrolase family 39 xylosidase from Bacillus halodurans C-125.
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 conditions employed.
Wagschal
Kurt
K
USDA Agricultural Research Service, Western Regional Research Center, Albany, CA 94710, USA. kwagschal@pw.usda.gov
Franqui-Espiet
Diana
D
Lee
Charles C
CC
Robertson
George H
GH
Wong
Dominic W S
DW
D000327:Adsorption; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004800:Enzymes, Immobilized; D011134:Polysaccharides; D011994:Recombinant Proteins; C007916:hemicellulose; D006026:Glycoside Hydrolases; D014995:Xylosidases