β-Xylosidase activity, encoded by xlnD, is essential for complete hydrolysis of xylan by Aspergillus niger but not for induction of the xylanolytic enzyme spectrum

Abstract

Two proteins exhibiting β-D-xylosidase activity were identified upon fractionation and purification of a culture filtrate of an arabinoxylan-grown Aspergillus niger. A single band of 110 kDa by SDS/PAGE was obtained in both cases and these were active on xylo-oligosaccharides and on xylan. Partial xlnD cDNA clones were immunochemically. identified and isolated from a λ cDNA expression library. Sequence analysis showed that all cDNA clones correspond to a single gene. A genomic clone was isolated and overexpressed in A. niger and A. nidulans. The xlnD gene has an ORF of 2412 nucleotides, encodes a protein of 804 amino acids and contains a potential signal peptide of 26 amino acids. This results in a mature protein of 778 amino acids with a predicted molecular mass of 85 kDa and an isoelectric point of 4.5. The protein is N-glycosylated and contains 15 potential N-glycosylation sites. Sequence similarity is found with β-D-glucosidases both of bacterial and fungal origin. Both β-xylosidase proteins purified have high activity on the artificial substrate p-nitrophenyl β-D-xylopyranoside (XylNp) and a side activity on p-nitrophenyl α-L-arabinofuranoside and p-nitrophenyl β-D-glucopyranoside. A. niger strains in which the xlnD gene was disrupted accumulate mainly xylobiose and xylotriose when grown on xylan and have no significant β-xylosidase activity in the culture medium, indicating that this gene encodes the major extracellular β-xylosidase.