title: Cloning and expression of A. oryzae β-glucosidase in Pichia pastoris.
abstract: A β-glucosidase gene (bgl) from Aspergillus oryzae GIF-10 was cloned, sequenced and expressed. Its full-length DNA sequence was 2,903 bp and included three introns. The full-length cDNA sequence contained an open reading frame of 2,586 nucleotides, encoding 862 amino acids with a potential secretion signal. The A. oryzae GIF-10 bgl was functionally expressed in Pichia pastoris. After 7-day induction, protein yield reached 321 mg/mL. Using salicin as the substrate, the specific activity of the purified enzyme reached 215 U/mg. The purified recombinant β-glucosidase was a 110-kDa glycoprotein with optimum catalytic activity at pH 5.0 and 50 °C. The enzyme was stable between 20 and 60 °C, and retained 65% of its activity after being held at 60 °C for 30 min. The recombinant β-glucosidase was relatively stable in a broad range of pHs, from 4.0 to 6.5. It showed broad specific activity, hydrolyzing a range of (1-4)-β-diglycosides and (1-4)-α-diglycosides, and Mn(2+) stimulated its activity significantly.
authors.affiliation: College of Life and Physical Science, Sichuan Agricultural University, Yaan, 625014, China, 3530279123456789@163.com.
authors.forename: Zizhong
authors.initials: Z
authors.lastname: Tang
authors.forename: Shan
authors.initials: S
authors.lastname: Liu
authors.forename: Haijun
authors.initials: H
authors.lastname: Jing
authors.forename: Rong
authors.initials: R
authors.lastname: Sun
authors.forename: Moyang
authors.initials: M
authors.lastname: Liu
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.forename: Qi
authors.initials: Q
authors.lastname: Wu
authors.forename: Xueyi
authors.initials: X
authors.lastname: Han
mesh_terms: D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001592:Benzyl Alcohols / Q000378:metabolism; D003001:Cloning, Molecular; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010843:Pichia; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001592:Benzyl Alcohols; D017931:DNA Primers; D018076:DNA, Complementary; D005960:Glucosides; C005696:salicin; D001617:beta-Glucosidase