Purification and properties of an extracellular beta-xylosidase from Aspergillus japonicus and sequence analysis of the encoding gene.
An extracellular protein exhibiting beta-xylosidase activity was purified from the culture filtrate of a filamentous fungus, Aspergillus japonicus strain MU-2, grown on oat spelt xylan. The purified enzyme was a monomeric glycoprotein with an apparent M(r) of 113.2 kDa as estimated by SDS-PAGE. beta-Xylosidase activity was optimal at pH 4.0 and 70 degrees C. The enzyme also showed beta-glucosidase and alpha-l-arabinofuranosidase activities. The genomic DNA and cDNA encoding this protein were cloned and sequenced. Southern blot analysis indicated that the beta-xylosidase gene (xylA) was present as a single copy in the genome. An open reading frame, consisting of 2412 bp, was not interrupted by introns, and it encoded a presumed signal peptide of 17 amino acids and a mature protein of 787 amino acids. The deduced amino acid sequence of the xylA gene product showed a high degree of identity (69%) to the primary structure of the Aspergillus niger beta-xylosidase XlnD that belongs to the glycoside hydrolase family 3. Moreover, the xylA gene was functionally expressed in the yeast Pichia pastoris.
Wakiyama
Motoki
M
Department of Applied Chemistry, Faculty of Engineering, University of Miyazaki, 1-1 Gakuen Kibanadai Nishi, Miyazaki 889-2192, Japan.
Yoshihara
Koji
K
Hayashi
Sachio
S
Ohta
Kazuyoshi
K
D001230:Aspergillus / Q000201:enzymology*; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D016678:Genome; D006026:Glycoside Hydrolases / Q000737:chemistry; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D016366:Open Reading Frames; D010843:Pichia / Q000378:metabolism; D013696:Temperature; D014995:Xylosidases / Q000096:biosynthesis* / Q000737:chemistry
D018076:DNA, Complementary; D006026:Glycoside Hydrolases; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; C019713:alpha-N-arabinofuranosidase