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Purification and Characterization of a Glycoside Hydrolase Family 43 β-xylosidase from Geobacillus thermoleovorans IT-08

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Abstract

The gene encoding a glycoside hydrolase family 43 β-xylosidase (GbtXyl43A) from the thermophilic bacterium Geobacillus thermoleovorans strain IT-08 was synthesized and cloned with a C-terminal His-tag into a pET29b expression vector. The recombinant gene product termed GbtXyl43A was expressed in Escherichia coli and purified to apparent homogeneity. Michaelis–Menten kinetic parameters were obtained for the artificial substrates p-nitrophenyl-β-d-xylopyranose (4NPX) and p-nitrophenyl-α-l-arabinofuranose (4NPA), and it was found that the ratio k cat/K m 4NPA/k cat/K m 4NPX was ∼7, indicting greater catalytic efficiency for 4NP hydrolysis from the arabinofuranose aglycon moiety. Substrate inhibition was observed for the substrates 4-methylumbelliferyl xylopyranoside (muX) and the arabinofuranoside cogener (muA), and the ratio k cat/K m muA/k cat/K m muX was ∼5. The enzyme was competitively inhibited by monosaccharides, with an arabinose K i of 6.8 ± 0.62 mM and xylose K i of 76 ± 8.5 mM. The pH maxima was 5.0, and the enzyme was not thermally stable above 54 °C, with a t 1/2 of 35 min at 57.5 °C. GbtXyl43A showed a broad substrate specificity for hydrolysis of xylooligosaccharides up to the highest degree of polymerization tested (xylopentaose), and also released xylose from birch and beechwood arabinoxylan.

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Reference to a company and/or products is for purposes of information and does not imply approval or recommendation of the product to the exclusion of others that may also be suitable. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.

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  1. Western Regional Research Center, USDA Agricultural Research Service, 800 Buchanan Street, Albany, CA, 94710, USA

    Kurt Wagschal, Chamroeun Heng, Charles C. Lee, George H. Robertson, William J. Orts & Dominic W. S. Wong

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  1. Kurt Wagschal
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  2. Chamroeun Heng
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  3. Charles C. Lee
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Correspondence to Kurt Wagschal.

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Wagschal, K., Heng, C., Lee, C.C. et al. Purification and Characterization of a Glycoside Hydrolase Family 43 β-xylosidase from Geobacillus thermoleovorans IT-08. Appl Biochem Biotechnol 155, 1–10 (2009). https://doi.org/10.1007/s12010-008-8362-5

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