Biochemical and kinetic analysis of the GH3 family β-xylosidase from Aspergillus awamori X-100

The β-xylosidase from Aspergillus awamori X-100 belonging to the family 3 glycoside hydrolase revealed a distinctive transglycosylating ability to produce xylooligosaccharides with degree of polymerization more than 7. In order to explain this fact, the enzyme has been subjected to the detailed biochemical study. The enzymatic hydrolysis of p-nitrophenyl β-d-xylopyranoside was found to occur with overall retention of substrate anomeric configuration suggesting cleavage of xylosidic bonds through a double-displacement mechanism. Kinetic study with aryl β-xylopyranosides substrates, in which leaving group pKas were in the range of 3.96–10.32, revealed monotonic function of log(kcat) and no correlation of log(kcat/Km) versus pKa values indicating deglycosylation as a rate-limiting step for the enzymatic hydrolysis. The classical bell-shaped pH dependence of kcat/Km indicated two ionizable groups in the β-xylosidase active site with apparent pKa values of 2.2 and 6.4. The kinetic parameters of hydrolysis, Km and kcat, of p-nitrophenyl β-d-1,4-xylooligosaccharides were very close to those for hydrolysis of p-nitrophenyl-β-d-xylopyranoside. Increase of p-nitrophenyl-β-d-xylopyranoside concentration up to 80 mM led to increasing of the reaction velocity resulting in kcatapp=81s-1. Addition of α-methyl d-xylopyranoside to the reaction mixture at high concentration of p-nitrophenyl-β-d-xylopyranoside (50 mM) caused an acceleration of the β-xylosidase-catalyzed reactions and appearance of a new transglycosylation product, α-methyl d-xylopyranosyl-1,4-β-d-xylopyranoside, that was identified by 1H NMR spectroscopy. The kinetic model suggested for the enzymatic reaction was consistent with the results obtained.