|本期目录/Table of Contents|

[1]刘艳** 杨玉洁,李孜一,李同彪,等.定点突变提高羰基还原酶ChKRED03的热稳定性[J].应用与环境生物学报,2021,27(03):1-10.
 Received: 00-xx-xx Accepted: 00-xx-xx.CLC Q814.9[J].Chinese Journal of Applied & Environmental Biology,2021,27(03):1-10.
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定点突变提高羰基还原酶ChKRED03的热稳定性()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
27卷
期数:
2021年03期
页码:
1-10
栏目:
出版日期:
2021-06-25

文章信息/Info

Title:
CLC Q814.9
作者:
刘艳1** 杨玉洁1 2 李孜一1 2 李同彪1 2 吴中柳1
1中国科学院成都生物研究所,中国科学院环境与应用微生物重点实验室,环境微生物四川省重点实验室 成都 610041 2中国科学院大学 北京 100049
Author(s):
Received: 2020-xx-xx Accepted: 2020-xx-xx
(21708038) Supported by the National Natural Science Foundation of China (21708038) ** Corresponding author s ( liuyan@cib.ac.cn) Enhancing the thermal stability of ketoreductase ChKRED03 using site-directed mutagenesis LIU Yan 1**, YANG Yujie1, 2, LI Ziyi 1, 2, LI Tongbiao 1, 2 & WU Zhongliu 1 1 CAS Key Laboratory of Environmental and Applied Microbiology & Environmental Microbiology Key Laboratory of Sichuan Province , Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China 2 University of Chinese Academy of Sciences, Beijing 100049, China
关键词:
羰基还原酶生物催化手性醇热稳定性FireProt在线服务器一致性方法
Keywords:
carbonyl reductase biocatalysis chiral alcohol thermostability FireProt web server consensus approach
摘要:
羰基还原酶不对称还原前手性酮生成相应的手性醇,广泛应用于手性药物、农业化学品和液晶材料等结构复杂的化合物的合成。实验室自有羰基还原酶ChKRED03催化3,5-二(三氟甲基)苯乙酮、1-苯基-2-丙烯基-1-酮类、N-叔丁氧羰基-3-哌啶酮(NBPO)等底物具有优异的立体选择性和良好的活力,但是该酶较低的热稳定性不利于其进一步应用。为此,我们采用蛋白质工程的方法提高其热稳定性。本研究利用FireProt在线工具和序列一致性原理预测了该酶热稳定相关氨基酸位点,通过理性选择8个位点,构建了单点突变体并识别了4个有益突变位点,进一步将其整合得到了热稳定性大幅度提高的组合突变体M8314(T183V/N188L/A211P/S224P)。该突变体最适反应温度为40 ℃,比野生型ChKRED03提高了10 ℃,其催化底物NBPO的活性是野生型的116%。M8314在40 ℃的热失活半衰期为52 h,是野生型的200倍。本研究成功提高了羰基还原酶ChKRED03的热稳定性,增加了该酶工业化应用的潜力,所采用的分子进化策略也为其他酶的热稳定性改造提供了思路。(图6 表3 参40)
Abstract:
Carbonyl reductases ( ketoreductases) can catalyze the asymmetric reduction of prochiral ketones to the corresponding chiral alcohols, which are widely used in the synthesis of pharmaceutical intermediates, agricultural chemicals, liquid crystal materials, and other complicated compounds. Ch KRED03, a carbonyl reductase from our laboratory, is capable of catalyzing a broad spectrum of ketones , such as 1-[3,5-bis(trifluoromethyl)phenyl] ethanone, acrylophenone derivatives and N-Boc-piperidin-3-one (NBPO), with excellent stereoselectivity and good activity. However, the further application of ChKRED03 is facing a challenge due to the limitation of low thermostability. To overcome such a limitation, we employed protein engineering to enhance its thermostability. Herein, the FireProt web server and consensus approach were used to predict potential thermostabilizing amino acid substitutions . And then, eight mutation sites were rationally selected to generate single-point mutants. Four beneficial substitutions were experimentally identified and were combined to form a quadruple mutant M8314 (T183V/N188L/A211P/S224P) with substantially enhanced thermostability. The mutant M8314 displayed an optimal temperature of 40 ℃, 10 ℃ higher than that of the wild type, and a half-life of inactivation of 52 h at 40 °C, which is approximately 200-fold of that of the wild type. Moreover, its catalytic activity toward the substrate NBPO was increased to 116% of the wild type . The current work has demonstrated a successful improve ment of the thermostability of carbonyl reductase ChKRED03 to increase its potential for industrial application. The efficient molecule evolution strategy could be adapted to enhancing the thermostability of other enzymes as well

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更新日期/Last Update: 2020-09-10