Cell-associated beta-xylosidase from Aureobasidium pullulans ATCC 20524: Purification, properties, and characterization of the encoding gene.
A cell-associated enzyme exhibiting beta-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 88.5 kDa. beta-Xylosidase activity was optimal at pH 3.5 and 70 degrees C. The enzyme exhibited apparent K(m) and V(max) values of 3.5 mM and 263 micromol/mg/min, respectively, for p-nitrophenyl-beta-d-xylopyranoside. The enzyme also showed some alpha-l-arabinofuranosidase activity. Southern blot analysis indicated that the beta-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 2415 bp was interrupted by two introns of 54 and 52 bp, and it encoded a presumed signal peptide of 20 residues and a mature protein of 785 residues with a calculated M(r) of 85,045 Da 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 beta-xylosidase Bxl1, which belongs to the glycoside hydrolase family 3.
Ohta
Kazuyoshi
K
Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen Kibanadai Nishi, Miyazaki 889-2192, Japan. k.ohta@cc.miyazaki-u.ac.jp
Fujimoto
Hirohisa
H
Fujii
Shinya
S
Wakiyama
Motoki
M
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D004789:Enzyme Activation; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase