枯草杆菌α-淀粉酶基因克隆及其在毕赤酵母中表达的初步研究  

Cloning and Expression of an α-Amylase Gene from Bacillus subtilis

作　　者：柳辉[1] 杨江科[1] 闫云君[1] 

机构地区：[1]华中科技大学生命科学与技术学院,武汉430074

出　　处：《生物技术通报》2007年第5期104-108,112,共6页Biotechnology Bulletin

基　　金：十五863项目(生物柴油关键技术与应用研究;2003AA214061);武汉市重点攻关项目

摘　　要：以B.subtilis XL-15基因组为模板,运用PCR法成功克隆了α-淀粉酶基因,其开放式阅读框(ORF)为1980bp,编码659个氨基酸残基。分别将该基因转入大肠杆菌BL21(DE3)和毕赤酵母GS115中,进行诱导表达。结果表明,大肠杆菌破碎上清液中未检出酶活,SDS-PAGE电泳分析显示表达产物均以无活性包涵体存在;而毕赤酵母在α-Factor及AOX1基因启动子和终止信号的调控下,经高密度培养,表达产物分泌至胞外,发酵液酶活力为4.3U/ml,实现了B.subtilis α-淀粉酶基因的分泌表达。The α-amylase gene was amplified through PCR by using B.subtilis XL-15 genomic DNA as template.Its Open Reading Frame（ORF）was composed of 1980bp,encoding 659 amino acid.The gene was cloned into the vectors of pET-28a（＋）and pPIC9K,and then transformed into Escherichia coli BL21（DE3）and Pichia pastoris GS115,respectively.The results showed that α-amylase activity was not tested in the supernatant of E.coli,and the expressed protein was all present with inclusion body.However,the α-amylase could secreted from the recombinant strain of Pichia pastoris GS115 with α-Factor singal peptide,promoter of AOX1 gene and termination signal of yeast genomic,the measured activity reached 4.3U/ml when the P.pastoris GS115 was fermented with high density liquid media and induced by 1.0%methanl.

关 键 词：Α-淀粉酶 巴斯德毕赤酵母 基因克隆 分泌表达 

分 类 号：Q78[生物学—分子生物学]