疏绵状嗜热丝孢菌糖化酶基因(gla)的克隆及在毕赤酵母中的表达  

Cloning of Glucoamylase Gene(gla) from Thermomyces lanuginosus and its Expression in Pichia pastoris

作　　者：徐荣燕[1] 张亚波[1] 谢晨[1] 张超[1] 李多川[1] 

机构地区：[1]山东农业大学植物保护学院环境生物系,泰安201718

出　　处：《农业生物技术学报》2010年第2期362-367,共6页Journal of Agricultural Biotechnology

基　　金：国家高技术研究与发展计划(863)(No.2006AA10Z304;No.2008AA05Z403)资助

摘　　要：本研究以疏绵状嗜热丝孢菌(Thermomyces lanuginosus)cDNA为模板,克隆了糖化酶基因(gla),序列分析表明gla的开放阅读框由1854个核苷酸组成,编码617个氨基酸。根据氨基酸序列推算该酶的分子量为64kD,属于糖苷水解酶第15家族,具有该家族催化保守区的典型特征。PCR扩增gla的成熟蛋白编码基因,构建表达载体,经线性化后电击转化导入巴斯德毕赤酵母(Pichia pastorisGS115),并成功进行了表达。重组酶经摇瓶发酵后酶活可达11.6U/mL,经硫酸铵沉淀、DEAE-SepharoseFastFlow阴离子交换等步骤纯化了该重组蛋白,SDS-PAGE显示该重组蛋白大小约为67kD,比推测的蛋白分子量稍大,可能与该蛋白的糖基化有关。该重组酶的最适反应温度和最适pH值分别为60℃和5.0,该酶具有较高的热稳定性,70℃保温20min后剩余酶活为54%。The glucoamylase gene gla was cloned by PCR from thermophilic fungus Thermomyces lanuginosus.Sequence analysis revealed that gla had an open reading frame of 1 854 bp, which encodes a putative polypeptide of 617 amino acids and the theoretical molecular mass was 64 kD.The glucoamylase fall into glycoside hydrolase family 15.The fragment encoding mature glucoamylase was inserted into Pichia pastoris vector pPIC9K to construct recombinant plasmid pPIC9K/gla.The pPIC9K/gla was then introduced into Pichia pastoris GS115, and the glucoamylase was secreted into the culture medium by the yeast in a functionally active form and the activity reached 11.6 U/mL after fermatation by Erlenmeyer flask.The recombinant glucoamylase purified was a glycoprotein with a size of about 67 kD, and a little bigger than the deduced molecular mass, possibly related to the glycosylation site.The recombinant glucoamylase exhibited optimum catalytic activity at pH 5.0 and 60℃ respectively.It was thermostable at 60℃ and remained 54% of its original activity after 20 min at 70℃.

关 键 词：疏绵状嗜热丝孢菌 糖化酶 克隆 毕赤酵母 性质 

分 类 号：Q786[生物学—分子生物学]