| 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 |
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| 引用本文： | 徐荣燕,张亚波,谢晨,张超,李多川.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[J].农业生物技术学报,2010,18(2):362-367. |
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| 作者姓名： | 徐荣燕 张亚波 谢晨 张超 李多川 |
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| 作者单位： | 山东农业大学植物保护学院环境生物系,泰安,201718 |
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| 基金项目： | 国家高技术研究与发展计划(863) |
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| 摘 要： | 本研究以疏绵状嗜热丝孢菌(Thermomyces laltltginosus)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 pastoris 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20 min后剩余酶活为54%.
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| 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 Thermomyces lanuginosus and its Expression in Pichia pastoris |
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| Xu Rongyan,Zhang Yabo,Xie Chen,Zhang Chao,Li Duochuan.Cloning of Glucoamylase Gene(gla) from Thermomyces lanuginosus and its Expression in Pichia pastoris[J].Journal of Agricultural Biotechnology,2010,18(2):362-367. |
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| Authors: | Xu Rongyan Zhang Yabo Xie Chen Zhang Chao Li Duochuan |
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| Institution: | Xu Rongyan Zhang Yabo Xie Chen Zhang Chao Li Duochuan College of Plant Protection,Department of Environmental Biology,Sh,ong Agricultural University,Tai'an,201718 |
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| Abstract: | 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 pasto... |
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| Keywords: | Thermomyces lanuginosus Glucoamylase Clone Pichia pastoris Characteristic |
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