Cell-associated β-xylosidase from Aureobasidium pullulans ATCC 20524 : Purification, properties, and characterization of the encoding gene(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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A cell-associated enzyme exhibiting β-xylosidase activity was purified from the cell extract of the dimorphic fungus, Aureobasidium pullulans strain ATCC 20524, grown on oat spelt xylan. The purified enzyme was homogeneous as judged by SDS-PAGE, which showed an apparent M_r of 88.5kDa. β-Xylosidase activity was optimal at pH 3.5 and 70℃. The enzyme exhibited apparent K_m and V_<max> values of 3.5mM and 263μmol/mg/min, respectively, for p-nitrophenyl-β-D-xylopyranoside. The enzyme also showed some α-L-arabinofuranosidase activity. Southern blot analysis indicated that the β-xylosidase gene (xylI) was present as a single copy in the genome. The genomic DNA and cDNA encoding this protein were cloned and sequenced. The open reading frame of 2415bp was interrupted by two introns of 54 and 52bp, and it encoded a presumed signal peptide of 20 residues and a mature protein of 785 residues with a calculated M_r of 85,045Da and a deduced isoelectric point of 4.57. The protein was N-glycosylated and possessed 16 potential N-glycosylation sites. Two distinct transcription start points of the xylI gene were present at nt -32(A) and -26(A) from the start codon. The xylI cDNA was functionally expressed in the yeast Pichia pastoris. The deduced amino acid sequence of the xylI gene product was 49% identical to the Talaromyces emersonii β-xylosidase Bxl1, which belongs to the glycoside hydrolase family 3.