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United States Department of Agriculture

Agricultural Research Service

Title: Cloning, Expression and Characterization of a Glycoside Hydrolase Family 39 Xylosidase from Bacillus Halodurans C-125

Authors
item Wagschal, Kurt
item Franqui-Villanueva, Diana
item Lee, Charles
item Robertson, George
item Wong, Dominic

Submitted to: Applied Biochemistry and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 14, 2007
Publication Date: July 20, 2008
Citation: Wagschal, K.C., Franquivillanueva, D.M., Lee, C.C., Robertson, G.H., Wong, D. Cloning, Expression and Characterization of a Glycoside Hydrolase Family 39 Xylosidase from Bacillus Halodurans C-125. Applied Biochemistry and Biotechnology.

Interpretive Summary: The gene encoding a glycoside hydrolase family 39 xylosidase (BH1068) from the alkaliphile Bacillus halodurans strain C-125 was cloned with a tag useful for purification and the recombinant gene product termed XylBH1068 was expressed in E. coli. A series of artificial substrates was used to determine the substrate specificity of the enzyme. This is important since it serves to illuminate the potential usefulness of the enzyme for breaking down biomass for use as a feedstock for chemicals and fuel. Of the artificial substrates tested, XylBH1068 hydrolyzed derivatives of the sugars xylose and arabinose, which are chemically similar but differ in structure. The usefulness of the enzyme under potential biomass utilization process conditions was explored by determining that the pH maximum was 6.5, maximal activity was at 47 °C, and thermal instability occurred above 43 °C. XylBH1068 was also tested on natural substrates, which gives a clearer indication of the reactions the enzyme can be expected to perform in an industrial setting. 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, which is important for bioprocess conditions, and this useful characteristic of the enzyme can potentially be incorporated into future designed enzymes using enzyme engineering tools.

Technical 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 XylBH1068 was expressed in E. coli. Of the artificial substrates tested, XylBH1068 hydrolyzed nitrophenyl derivatives of b-D-xylopyranose, a-L-arabinofuranose, and a-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.

Last Modified: 12/22/2014