beta-Xylosidase activity, encoded by xlnD, is essential for complete hydrolysis of xylan by Aspergillus niger but not for induction of the xylanolytic enzyme spectrum.
Two proteins exhibiting beta-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 lambda 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 beta-D-glucosidases both of bacterial and fungal origin. Both beta-xylosidase proteins purified have high activity on the artificial substrate p-nitrophenyl beta-D-xylopyranoside (XylNp) and a side activity on p-nitrophenyl alpha-L-arabinofuranoside and p-nitrophenyl beta-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 beta-xylosidase activity in the culture medium, indicating that this gene encodes the major extracellular beta-xylosidase.
van Peij
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NN
Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, The Netherlands.
Brinkmann
J
J
Vrsanská
M
M
Visser
J
J
de Graaff
L H
LH
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry; D004790:Enzyme Induction; D008969:Molecular Sequence Data; D014990:Xylans / Q000378:metabolism*; D014995:Xylosidases / Q000235:genetics / Q000378:metabolism*
D004271:DNA, Fungal; D014990:Xylans; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase