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 - 食品科学.pdfVIP

※生物工程 食品科学 2011, Vol. 32, No. 17 219 - 曾庆梅，魏春燕，靳 靖，吴 聪，黄博英 (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) 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。 黑曲霉；酸性 α- 淀粉酶；pPIC9K ；毕赤酵母SMD1168 Cloning of Acid-stable α-Amylase Gene from Aspergillus niger and Its Expression in Pichia pastoris ZENG Qing-mei ，WEI Chun-yan ，JIN Jing ，WU Cong ，HUANG Bo-ying (Engineering Research Center of Bio-Process, Ministry of Education, Hefei University of Technology, Hefei 230009, China) Abstract ：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. SDSanalysis 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