黑曲霉α-葡萄糖苷酶基因的克隆及其在毕赤酵母中的表达

Cloning of the gene encoding α-glucosidase from Aspergillus niger and its expression in Pichia pastoris

【目的】以毕赤酵母(Pichia pastoris)KM71为宿主菌表达黑曲霉(Aspergillus niger)SG136 α-葡萄糖苷酶(α-glucosidase)。【方法】以实验室保藏的A.niger SG136总DNA为模板,根据NCBI数据库中A.niger CBS 513.88 α-葡萄糖苷酶的cDNA序列(aglu)设计引物,通过PCR和重叠延伸PCR(overlap-PCR)方法,扩增得到aglu,将其克隆到载体pMD18-T simple vector,测序结果表明,aglu编码960个氨基酸。与A.niger CBS 513.88α-葡萄糖苷酶相比仅有一个氨基酸的差异。将得到的aglu亚克隆到质粒pPIC9K,构建表达载体pPIC9K-aglu,经BglⅡ线性化后电转化P.pastoris KM71,经过MD、YPD/G418平板筛选表型,PCR方法验证,获得分泌表达重组P.pastoris KM71/pPIC9K-aglu。摇瓶培养中通过添加终浓度为1%的甲醇诱导α-葡萄糖苷酶的分泌。【结果】SDS-PAGE显示表达蛋白的大小亚基分子量分别为98kDa和33kDa,阴性对照中没有出现此条带,非变性电泳检验为一条带。制备的粗酶液的酶学性质表明,转苷反应最适pH为5,最适温度为55℃。在最适pH和温度下,反应24h时低聚异麦芽糖的总含量达到最大为26.0%。【结论】黑曲霉α-葡萄糖苷酶在P.pastoris中获得可溶性表达,并证明有一定的转糖苷活性。

[Objective]α-glucosidase from Aspergillus niger SG136 was expressed in Pichia pastoris. [Methods] cDNA of mature α-glucosidase（aglu） was amplified from the total DNA of A. niger SG136 by PCR and overlap-PCR with primers designed based on the sequence of A. niger CBS 513.88 in NCBI database. The gene was cloned into pMD18-T simple vector. The sequencing result showed that the gene encoded for a protein of 960 amino acids residues,which was 1 amino acid different from that of A. niger CBS 513.88. The expression vector pPIC9K-aglu was constructed by subcloning the gene into plasmid pPIC9K,and then transformed into P. pastoris through electroporation after linearized by BglⅡ digestion. The recombinant P. pastoris KM71/pPIC9K-aglu were screened in MD and YPD/G418 plates,and identified by PCR. In shaking culture condition,methanol was added to a final concentration of 1% to induce the secretion of α-glucosidase. [Results] Electrophoresis analysis of KM71/pPIC9K-aglu culture supernatant showed that there were two major protein bands corresponding to 98 kDa and 33 kDa respectively in SDS-PAGE,and there was only one band in Native PAGE; while in the control experiment of KM71/pPIC9K,there were no visible bands. Transglucosidation reaction from crude enzyme revealed that contents of isomaltooligosaccharides were up to 26.0% under the optimal conditions of pH 5 and 60℃ at 24 h. [Conclusion] A. niger α-glucosidase was expressed in P. pastoris with transglucosidation activity.