黑曲霉糖化酶基因在毕赤酵母中的克隆和表达  

Cloning and Expression of Glucoamylase Genes from Aspergilus niger cDNA Library in Pichia pastrois

作　　者：汤斌[1] 钱鹏[1] 

机构地区：[1]安徽工程大学微生物发酵安徽省工程技术研究中心,安徽芜湖241000

出　　处：《食品与发酵工业》2012年第6期53-56,共4页Food and Fermentation Industries

摘　　要：从高产糖化酶的黑曲霉的cDNA文库中筛选出糖化酶基因,并研究在毕赤酵母中的表达情况。运用RT-PCR从黑曲霉cDNA文库中克隆糖化酶基因的cDNA片段与载体pPIC9K相连,构建重组载体,电转化毕赤酵母GS115,筛选阳性克隆并进行研究。阳性克隆在MM培养基中发酵72 h和1%的甲醇的诱导的情况下,重组毕赤酵母产生的糖化酶酶活最大为15.6 U/mL。测定结果显示,其糖化酶大小为1 908 bp,编码636个氨基酸残基组成的蛋白质。经柱分离纯化其发酵上清液后,用SDS-PAGE电泳方法,测得分子质量大约为80 ku。黑曲霉糖化酶基因在毕赤酵母GS115中成功得到了表达。An expression cDNA library was constructed from high-yielding glucoamylase strains of A.niger and the glucoamylase gene was isolated,then the expression of the gene in Pichia pastoris was studied.The cDNA sequence of glucoamylase from A.niger was obtained by RT-PCR.The cDNA fragment was cloned into the expression vector pPIC9K and the linearized recombinant vector was transformed to Pichia pastrois GS115 by electroporation.The positive clones were analyzed subsequently.The recombined Pichia pastrois were cultured in the MM medium,using 1% methanol to induce the expression of recombinant gene.The results showed that the maximum activity of glucoamylase was 15.6 U/mL after it was fermented for 72 h.Sequence analysis revealed that glucoamylase had 1908 bp,which encodes a putative polypeptide of 636 amino acids.The expressed protein was purified from the fermented supernatant using DEAE column and determined by SDS-PAGE.The result of SDS-PAGE also showed that the molecular weights of the enzyme was 80 kDa.The expression vector of the glucoamylase gene was constructed successfully,and it could express glucoamylase in Pichia pastrois.

关 键 词：黑曲霉 糖化酶 毕赤酵母 表达 

分 类 号：TQ925[轻工技术与工程—发酵工程]