黑曲霉β-葡萄糖苷酶基因克隆及在毕赤酵母中分泌表达

Cloning and Secreting Expression of the β-Glucosidase Gene from Aspergillus niger in Pichia pastoris GS115

摘要:

为大规模制备β-葡萄糖苷酶,构建重组酵母工程菌,实现β-葡萄糖苷酶的分泌表达。根据已报道黑曲霉(Aspergillus niger)β-葡萄糖苷酶cDNA序列设计一对引物,采用RT-PCR扩增,获得黑曲霉β-葡萄糖苷酶基因(bgl)。构建了pMD-18T-bgl克隆载体,bgl亚克隆到质粒pPIC9K,构建表达载体pPIC9K-bgl。经BglⅡ线性化后电转入Pichia pastoris GS115,经过MD、YPD/G418平板筛选阳性克隆,获得分泌表达重组P.pastoris GS115的工程菌。用终体积分数1%的甲醇对其进行诱导表达,SDS-PAGE和酶活测定结果表明,重组毕赤酵母分泌了1个相对分子质量约90 000的蛋白质,与该酶基因产物的理论值一致。酶催化的最适温度为50℃,最适pH 5.5,其酶活达到38 U/mL,比已报导重组酶产酶水平有较大程度的提高。

Abstract:

Based on the β-glucosidase coding sequences from GeneBank,a β-glucosidase gene was amplified from Aspergillus niger by using RT-PCR and vector pMD-18T-bgl was constructed.The expression vector pPIC9K-bgl was constructed by subcloning the gene into plasmid pPIC9K,and then transformed into P.pastoris GS115 through electroporation after linearized by BglⅡdigestion.The recombinant P.pastoris G115 were screened in MD and YPD/G418 plate.The activity of the engineered strain reached to 38 U/mL after induction with the final concentration of 1% methanol.SDS-PAGE analysis showed that the recombinant P.pastoris GS115 had an additional protein band of approximately 90 kD,which was not present in the control,and consistent with the theoretical value of the gene product.The crude enzyme catalysis results indicated that the optimum temperature and pH for the activity were about 50 ℃ and 5.5,respectively.