黑曲霉酸性α-淀粉酶基因的克隆及其在毕赤酵母中的表达  

Cloning of Acid-stable α-Amylase Gene from Aspergillus niger and Its Expression in Pichia pastoris

作　　者：曾庆梅[1] 魏春燕[1] 靳靖[1] 吴聪[1] 黄博英[1] 

机构地区：[1]合肥工业大学农产品生物化工教育部工程研究中心,安徽合肥230009

出　　处：《食品科学》2011年第17期219-224,共6页Food Science

基　　金：国家自然科学基金项目(30871739;31071556);国家"863"计划项目(2011AA100801)

摘　　要：以黑曲霉Aspergillus niger基因组DNA为模板,PCR扩增出酸性α-淀粉酶的结构基因,将此基因插入载体pPIC9K,重组质粒pPIC9K-asAA转化毕赤酵母SMD1168,并通过G418平板培养基筛选高拷贝转化子。在酵母α-Factor及AOX1基因启动子和终止信号的调控下,酸性α-淀粉酶大量表达并分泌到胞外,该酶的表达受甲醇的严格调控和诱导,摇瓶培养中,2%甲醇诱导培养168h后酶活力达到最大值2838U/mL。SDS-PAGE结果显示该重组酶的分子质量为58kD。该酶的最适反应pH值为4.0,在pH3.0～6.0之间酶活力基本保持稳定,最适反应温度为70℃,在工业生产常用温度50℃条件下,该酶能够长时间保持稳定,并具有较高的酶活力。重组毕赤酵母遗传稳定性良好。The gene of acid-stable α-amylase was amplified by PCR using Aspergillus niger genomic DNA as template,the amplified gene was cloned into the vector of pPIC9K,and the recombinant pPIC9K-asAA was then transformed into Pichia pastoris SMD1168.High copy transformants were screened in G418 plates.Regulated by α-factor,AOX1 gene promoter and termination signal of yeasts,the recombinant amylase was expressed and secreted from the cells.The expression of recombinant amylase was strictly induced by methanol under shaking culture conditions.The amylase reached its maximal activity of 2838 U/mL after induction with 2% methanol for 168 h.SDS-PAGE analysis showed that the molecular weight of recombinant amylase was approximately 58 kD.The recombinant amylase revealed the highest activity at pH 4.0 and 70 ℃.Meanwhile,the amylase was basically stable at pH 3.0-6.0 and could keep its stability for a long time and high activity at 50 ℃.Therefore,recombinant Pichia pastoris has good genetic stability.

关 键 词：黑曲霉 酸性Α-淀粉酶 PPIC9K 毕赤酵母SMD1168 

分 类 号：Q814.4[生物学—生物工程]