| R-2-氯丙酸脱卤酶的定向进化 |
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| 引用本文： | 秦迎春,刘鹏,杨立荣,徐刚,吴坚平.R-2-氯丙酸脱卤酶的定向进化[J].生物加工过程,2013,11(1):65-69. |
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| 作者姓名： | 秦迎春 刘鹏 杨立荣 徐刚 吴坚平 |
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| 作者单位： | 浙江大学化学工程与生物工程学系 |
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| 基金项目： | 国家重点基础研究发展计划(973计划)资助(2011CB710800);国家高技术研究发展计划(863计划)资助(2011AA02A209);国家自然科学基金面上项目资助(21076187) |
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| 摘 要： | 通过易错PCR手段将R-2-氯丙酸脱卤酶定向进化,并使用基于Cl-浓度显色反应的高通量筛选得到有效突变子库,发现突变子DehDIV-G2和DehDIV-E7的酶比活力分别提高25.2%和38.7%。通过SYBYL对酶与底物进行分子对接显示,DehDIV-G2的活化能下降0.961 4 kJ/mol,DehDIV-E7的活化能下降2.549 8 kJ/mol。由于酶和底物R-2-氯丙酸的活化能下降,亲和能力提高,从而提高酶的比活力。
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| 关 键 词： | 2-氯丙酸 脱卤酶 定向进化 分子对接 |
Directed evolution of R-2-CPA dehalogenase |
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| QIN Yingchun,LIU Peng,YANG Lirong,XU Gang,WU Jianping.Directed evolution of R-2-CPA dehalogenase[J].Chinese Journal of Bioprocess Engineering,2013,11(1):65-69. |
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| Authors: | QIN Yingchun LIU Peng YANG Lirong XU Gang WU Jianping |
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| Institution: | (Department of Chemical Engineering and Biological Engineering,Zhejiang University,Hangzhou 310027,China) |
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| Abstract: | By the error-prone PCR and high throughput screening based on the color reaction of chloride ion concentration, a mutant library of R-2-CPA dehalogenase was created and screened. The specific activity of the mutants, called the DehDIV-G2 and DehDIV-E7, increased by 24. 8% and 39.6%, respectively. Molecular docking of the enzymes with substrates was carried out with SYBYL. Docking scoring displayed that DehDIV-G2 decreased the activation energy of 0. 961 4 kJ/mol, and DehDIV-E7 decreased the activation energy of 2. 549 8 kJ/mol. The mutant enzymes decreased the activation energy, and increased the affinity with R-2-CPA,thus the enzyme specific activities increased. |
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| Keywords: | 2-chloropropionic acid dehalogenase directed evolution molecular docking |
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