Characteristics of transxylosylation by β-xylosidase from Aspergillus awamori K4

The Aspergillus awamori K4 β-xylosidase gene (Xaw1) sequence was deduced by sequencing RT-PCR and PCR products. The ORF was 2,412 bp and the predicted peptide was 804 amino acids long, corresponding to a molecular weight of 87,156 Da. The mature protein was 778 amino acids long with a molecular weight of 84,632 Da. A homology search of the amino acid sequence revealed that it was very similar to the Aspergillus niger β-xylosidase gene with only five amino acid differences. K4 β-xylosidase had the same catalytic mechanism as family 3 β-glucosidases, involving Asp in region A. At an early stage in the reaction with xylobiose and xylotriose, the hydrolysis rate was much lower than the transxylosylation rate, decreasing gradually as the substrate concentration increased, whereas the transxylosylation rate increased greatly. Aspergillus awamori K4 β-xylosidase had broad acceptor specificity toward alcohols, hydroxybenzenealcohols, sugar alcohols and disaccharides. A consensus portion involving the hydroxymethyl group of the acceptor was confirmed in the major transfer products 1(4)-O-β-d-xylosyl erythritol, (2-hydroxyl)-phenyl-methyl-β-d-xylopyranoside, 6S-O-β-d-xylosyl maltitol (S: sorbitol residue) and 6G-O-β-d-xylosyl palatinose (G: glucosyl residue). This might suggest that the methylene in the hydroxymethyl group facilitates base-catalyzed hydroxyl group attack of the anomeric center of the xylosyl-enzyme intermediate.