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.pdfVIP

农业生物技术学报，2010 年，第18 卷，第2 期，第362-367 页 Journal of Agricultural Biotechnology, 2010, Vol.18, No.2, 362-367 研究报告 A Letter 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 徐荣燕 张亚波 谢晨 张超 李多川* 山东农业大学植物保护学院环境生物系, 泰安, 201718 * 通讯作者, lidc20@sdau.edu.cn 摘 要 本研究以疏绵状嗜热丝孢菌(Thermomyceslanuginosus) cDNA 为模板，克隆了糖化酶基因(gla)，序列 分析表明 的开放阅读框由1 854 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 gla 为64 kD，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 的成熟蛋白编码基 gla 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(Pichiapastoris GS115)，并成功进行了表达。重 组酶经摇瓶发酵后酶活可达11.6 U/mL，经硫酸铵沉淀、DEAE-Sepharose Fast Flow 阴离子交换等步骤纯化 SDS显示该重组蛋白大小约为67 kD，比推测的蛋白分子量稍大，可能与该蛋白的糖 了该重组蛋白， 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 20 min 后剩余酶活为54%。 关键词 疏绵状嗜热丝孢菌, 糖化酶, 克隆, 毕赤酵母, 性质 Cloning of Glucoamylase Gene (gla) from Thermomyceslanuginosusand its Expression in Pichiapastoris Xu Rongyan Zhang Yabo Xie Chen Zhang Chao Li Duochuan * College of Plant Protection, Department of Environmental Biology, Shandong Agricultural University, Taian, 201718 * Corresponding author, lidc20@sdau.edu.cn DOI: 10.3969/j.issn.1674-7968.2010.02.026 Abstract The glucoamylase gene glawas cloned by PCR from thermophilic fungus Thermomyceslanuginosus. Se- quence analysis revealed that glahad 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 fam- ily 15. The fragment encoding mature glucoamylase was inserted into Pichiapastorisvector pPIC9K to construct re- combinant plasmid pPIC9K/gla. The pPIC9K/gla was then introduced into Pichiapastoris GS115, and the glu- coamylase 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