长梗木霉β-葡萄糖苷酶基因的克隆及表达

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

摘要:

【目的】以实验室筛选获得的一株长梗木霉GM2 (Trichoderma longibrachiatum)为材料, 克隆出其?β-葡萄糖苷酶(β-Glucosidase)基因bgl?并在大肠杆菌和酵母中进行表达。【方法】利用同源克隆扩增出其β-葡萄糖苷酶基因bgl?全长序列, 分别亚克隆到质粒pET-32a(+)和pPICZα-B中, 构建其原核表达载体pET32a(+)-bglI和真核表达载体pPICZα-B-bgl?。【结果】bgl?基因序列全长2 369 bp, 含两个内含子, 编码744个氨基酸。在大肠杆菌BL21(DE3)中表达bgl?, 重组蛋白以包涵体形式存在, 上清液中没有β-葡萄糖苷酶的酶活。将载体pPICZα-B-bgl?电转化入毕赤酵母GS115, 得到78 kD左右重组蛋白, 与预测大小相符。按9%接种量接入50 mL YP培养基(初始pH 5.5), 30 °C振荡培养96 h, 添加终浓度1%的甲醇诱导后β-葡萄糖苷酶酶活达60 U/mL。重组酶bgl?催化水杨苷水解反应的最适pH为5.0, 最适温度为70 °C; 另外, 此bgl?在pH 3.0?10.0和40 °C?60 °C范围内具有比较好的稳定性。【结论】长梗木霉GM2的β-葡萄糖苷酶在P. pastoris中获得可溶性表达, 并证明有一定的活性。

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.