Biochemical characterization of a novel dual-function arabinofuranosidase/xylosidase isolated from a compost starter mixture.

The gene encoding a glycoside hydrolase family 43 enzyme termed deAX was isolated and subcloned from a culture seeded with a compost starter mixed bacterium population, expressed with a C-terminal His6-tag, and purified to apparent homogeneity. deAX was monomeric in solution and had a broad pH maximum between pH 5.5 and pH 7. A twofold greater k cat/ K m for the p-nitrophenyl derivative of α- l-arabinofuranose versus that for the isomeric substrate β- d-xylopyranose was due to an appreciably lower K m for the arabinofuranosyl substrate. Substrate inhibition was observed for both 4-methylumbelliferryl arabinofuranoside and the xylopyranoside cogener. While no loss of activity was observed over 4 h at 40°C, the observed t 1/2 value rapidly decreased from 630 min at 49°C to 47 min at 53°C. The enzyme exhibited end-product inhibition, with a K i for xylose of 145 mM, 18.5 mM for arabinose, and 750 mM for glucose. Regarding natural substrate specificity, deAX had arabinofuranosidase activity on sugar beet arabinan, 1,5-α- l-arabinobiose, and 1,5-α- l-arabinotriose, and wheat and rye arabinoxylan, while xylosidase activity was detected for the substrates xylobiose, xylotriose, xylotetraose, and arabinoxylan from beech and birch. Thus, deAX can be classified as a dual-function xylosidase/arabinofuranosidase with respect to both artificial and natural substrate specificity. [ABSTRACT FROM AUTHOR]