Cloning and expression of A. oryzae b-glucosidase in Pichia pastoris

A b-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 b- 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 recom- binant b-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)-b-diglycosides and (1-4)-a- diglycosides, and Mn 2? stimulated its activity significantly.