Cloning and expression of the beta-glucosidase gene from Trichoderma longibrachiatum

[Objective] The complete sequence of a β-glucosidase gene from a Trichoderma longibrachiatum strain GM2 previously isolated in the laboratory,namely bglI,was amplified and expressed.[Methods] The gene of bgllI was amplified via homologous cloning.The bglI sequence corresponding to the mature peptide was subcloned into plasmids pET-32a(+) and pPICZα-B,respectively.[Results] Sequencing results showed that the bgl? gene was 2 369 bp in size encoding 744 amino acids,interrupted by two introns.The bgllI protein expressed in E.coli BL21(DE3) existed mostly in inclusion bodies,and there was no detectable β-glucosidase activity in the soluble proteins.The expression vector pPIZα-B-bglI was transformed into Pichia pastoris GS115 by electroporation,and the recombinant protein with the molecular weight around 78 kD,consistent with the expected protein size,was secreted.Under the fermentation conditions of 9% initial inoculum,initial pH of 5.5,30 °C and 1% methanol induction,after shaking for 96 h,the β-glucosidase activity of 60 U/mL was obtained.Enzyme property analyses demonstrated that the optimum pH and the optimum temperature for the recombinant bglI were 5.0 and 70 °C,respectively;furthermore,this bglI exhibited good stability at pH between 3.0 and 10.0 and the temperature range of 40 °C-60 °C.[Conclusion] The gene of bglI was expressed in P.pastoris with β-glucosidase activity.