| 枯草杆菌蛋白酶E的156和165位突变 |
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| 引用本文： | 陈为东,马建华,朱榴琴.枯草杆菌蛋白酶E的156和165位突变[J].生物化学与生物物理进展,1997,24(4):331-334. |
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| 作者姓名： | 陈为东 马建华 朱榴琴 |
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| 作者单位： | 中国科学院生物物理研究所，北京 100101;中国科学院生物物理研究所，北京 100101;中国科学院生物物理研究所，北京 100101 |
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| 基金项目： | 国家863-103主题组资助. |
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| 摘 要： | 应用定点突变方法，在M222A突变的枯草杆菌蛋白酶E基因上进行E156S和V165I定点突变. 将突变基因插入大肠杆菌-枯草杆菌穿梭质粒pBE-2中，在碱性和中性蛋白酶缺陷型的枯草杆菌DB104中进行表达，得到突变种(M222A，E156S)和(M222A，E156S，V165I)蛋白酶E. 性质测定表明，E156S突变使蛋白酶比活力增加90%，并不影响酶的热稳定性和抗氧化性. 而V165I突变使蛋白酶比活力降低.
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| 关 键 词： | 枯草杆菌蛋白酶E，蛋白质工程，比活力，稳定性 |
| 收稿时间： | 6/4/1996 12:00:00 AM |
| 修稿时间： | 1996/11/28 0:00:00 |
Site 156 and 165 Mutation of Subtilisin E |
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| CHEN Weidong,MA Jianhua and ZHU Liuqin.Site 156 and 165 Mutation of Subtilisin E[J].Progress In Biochemistry and Biophysics,1997,24(4):331-334. |
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| Authors: | CHEN Weidong MA Jianhua and ZHU Liuqin |
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| Institution: | Institute of Biophysics, The Chinese Academy of Sciences, Beijing 100101, China;Institute of Biophysics, The Chinese Academy of Sciences, Beijing 100101, China;Institute of Biophysics, The Chinese Academy of Sciences, Beijing 100101, China |
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| Abstract: | E156S and V165I mutation were introduced into subtilisin E gene by site-directed mutagenesis. The mutated gene fragments were recombined with pBE-2 which is a shuttle vector between E.coli and Bacillus subtilis. The recombinant plasmids were used to transform B.subtilisDB104,a mutant strain deficient in alkaline and neutral protease,then they were expressed. They were (M222A,E156S) and (M222A,E156S,V165I). The property analysis of these enzymes revealed that the Subtilisin E E156S substitution enhenced the hydrolysis K cat/K m by 90% while keeping thermal stability and oxidation-resistance unchanged,however the V165I mutation reduced the K cat/K m value. |
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| Keywords: | subtilisin E protein engineering specific activity stability |
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