曲霉属糖化酶基因的克隆及其在毕赤酵母中的表达

Gene Cloning of Aspergillus Glucoamylase and its Expression in Pichia Pastoris

从黑曲霉、无花果曲霉、米曲霉中提取总 RNA,RT-PCR 分别获得其糖化酶基因,所得序列在 Gen-Bank 数据库中注册,注册号分别为 AY652617、AY642120和 DQ211971.BLAST 分析显示,克隆的曲霉属糖化酶基因与 GenBank 数据库相应序列同源性很高.将这些糖化酶基因与表达载体 pPIC9重组后,电转化进毕赤酵母株 GS115,均获得了分泌表达糖化酶的酵母工程菌株.甲醇诱导72 h 后,糖化酶的分泌量达到最高,分别为(11.6±0.2)、(12.4±0.2)和(10.0±0.2)U/mL.SDS-PAGE 显示,分泌表达的糖化酶分子量均约为80 kDa.

Total RNAs were isolated and the glucoamylase genes were cloned by RT-PCR from Aspergillus niger, Aspergillus ficuum and Aspergillus oryzae, respectively. The obtained sequences were submitted to GenBank with the accession numbers AY652617, AY642120 and DQ211971, respectively. BLAST analysis suggested that glucoamylase gene sequences ob- tained in this work have high identity with corresponding sequences in GenBank. The recombinant Pichia pastoris expression vectors were constructed by fusion these glucoamylase genes into pPIC9 expression vector. The recombinant plasmids were introduced into GSl15 strain by electroporation and the positive recombinant yeasts were obtained. When induced by 0. 5 % methanol for 72 hours, the recombinants secreted highest level of glucoamylase activity into the supernatants, which were up to （11.6±0. 2）, （12. 4±0. 2）and （10. 0±0. 2） U/mL, respectively. SDS-PAGE electrophoresis showed that the molecular weight of the expressed glucoamylase in recombinant strains was around 80 kDa.