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

Abstract:

[Objective] The complete sequence of a β-glucosidase gene from a Trichoderma longibrachiatum strain GM2 previously isolated in the laboratory, namely bgl?, was amplified and expressed. [Methods] The gene of bgl? was amplified via homologous cloning. The bgl? 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 bgl? 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 bgl? were 5.0 and 70 °C, respectively; furthermore, this bgl? exhibited good stability at pH between 3.0 and 10.0 and the temperature range of 40 °C?60 °C. [Conclusion] The gene of bgl? was expressed in P. pastoris with β-glucosidase activity.