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

摘要:

【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 513.88 α-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Bgl Ⅱ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98 kDa和33 kDa，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24 h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。

Abstract:

[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.