Monday, May 5, 2008
6-21

Purification and characterization of a family 43 glycoside hydrolase from Geobacillus thermoleovorans IT-08 with dual function arabinofuranosidase/xylosidase activity

Kurt Wagschal, Chamroeun Heng, Charles C. Lee, George H. Robertson, William J. Orts, and Dominic W.S. Wong. USDA Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710

The gene from the thermophilic bacterium Geobacillus thermoleovorans strain IT-08 encoding a glycoside hydrolase family 43 enzyme based on the amino acid sequence was synthesized, and subsequently cloned with a C-terminal His-tag into a pET29b expression vector. The recombinant gene product termed GbtXA was expressed in E. coli and purified to apparent homogeneity. Michaelis-Menten kinetic parameters were obtained for the artificial substrates p-nitrophenyl-β-D-xylopyranose (kcat 0.2 sec-1 and Km 380 μM) and p-nitrophenyl-α-L-arabinofuranose (kcat 1.2 sec-1 and Km 490 μM), indicting greater specificity for the arabinofuranose moiety. The enzyme exhibited end-product competitive inhibition with both xylose and arabinose. The pH maximum was pH 5.5, and the enzyme was not thermally stable above 53 °C, with a T1/2 on the order of 60 min at 56 °C under the conditions tested. GbtXA showed a broad substrate specificity when tested with natural substrates, and released xylose from beechwood arabinoxylan and arabinose from wheat arabinoxylan. GbtXA can thus be classified as a dual function arabinofuranosidase/xylosidase with respect to both artificial and natural substrate specificity.

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