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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (9): 59-64    DOI: 10.13523/j.cb.20180909
技术与方法     
定点突变提高醇脱氢酶LkTADH催化制备他汀关键手性砌块的酶活 *
陈方,徐刚,杨立荣,吴坚平()
浙江大学化学工程与生物工程学院生物工程研究所 杭州 310027
Enhancing the Activity of LkTADH by Site-Directed Mutagenesis to Prepare Key Chiral Block of Statins
Fang CHEN,Gang XU,Li-rong YANG,Jian-ping WU()
College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
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摘要：

(S)-6-氯-3-羰基-5-羟基己酸叔丁酯[(S)-CHOH]是他汀类药物合成的关键手性中间体。利用醇脱氢酶催化6-氯-3,5-二羰基己酸叔丁酯不对称合成(S)-CHOH是很有潜力的制备路线,目前存在的主要问题是醇脱氢酶催化活性较低。首先对来源于Lactobacillus kefir DSM 20587的醇脱氢酶的四点突变体LkTADH(A94T/F147L/A202L/L199H)进行回复突变,确定了关键位点147和202,并获得比酶活提高1倍的突变体MF147L-A202L。对这两个位点进行饱和突变,获得比酶活比LkTADH提高1.47倍的突变体MF147I-A202L。其比酶活为10.17U/mg,为目前文献报道最高水平。通过动力学分析和分子对接,分析了突变位点对酶活影响的机制,为后续研究奠定了良好的基础。
关键词： 醇脱氢酶;  (S)-6-氯-3-羰基-5-羟基己酸叔丁酯;  不对称合成;  分子改造;  饱和突变    
Abstract:

(S)-tert-butyl-6-chloro-5-hydroxyl-3-oxohexanoate [(S)-CHOH] is the key chiral intermediate of statins. Asymmetric reduction of tert-butyl-6-chloro-3,5- dioxohexanoate (CDOH) to (S)-CHOH catalyzed by alcohol dehydrogenases is a promising method. Nevertheless, the main problems is the low catalytic activity towards CDOH. First an alcohol dehydrogenase LkTADH (A94T/F147L/L199H/A202L) was further studied by reverse mutation and key sites (147,202) had been identified. MF147L-A202L was obtained, which demonstrated 1-fold improvement in specific activity over LkTADH. After applying saturation mutagenesis at these two sites, MF147I-A202L was obtained with 1.47-fold improvement in specific activity over LkTADH. The specific activity reached 10.17U/mg, which is the highest level as reported. Through dynamic analysis and molecular docking, the effect of mutation sites on enzyme activity was further analyzed.
Key words: Alcohol dehydrogenase    (S)-tert-butyl-6-chloro-5-hydroxyl-3-oxohexanoate    Asymmetric synthesis    Molecular modification    Saturated mutation
收稿日期: 2018-03-09 出版日期: 2018-10-12
基金资助: * 国家自然科学基金面上项目(21676240);国家973计划(2011CB710800)
通讯作者: 吴坚平     E-mail: wjp@zju.edu.cn
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陈方
徐刚
杨立荣
吴坚平

引用本文:

陈方,徐刚,杨立荣,吴坚平. 定点突变提高醇脱氢酶LkTADH催化制备他汀关键手性砌块的酶活 *[J]. 中国生物工程杂志, 2018, 38(9): 59-64.

Fang CHEN,Gang XU,Li-rong YANG,Jian-ping WU. Enhancing the Activity of LkTADH by Site-Directed Mutagenesis to Prepare Key Chiral Block of Statins. China Biotechnology, 2018, 38(9): 59-64.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180909    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I9/59

图1  生物不对称合成(S)-CHOH反应路线
ADHs Enzyme Activity(U/L) Relative activity(%) Specific activity(U/mg)
1 LkADH 7 100 0.09
2 LkTADH 203 2 900 4.11
3 MA202L-L199H-A94T 70 1 000 —
4 MA202L-L199H 35 500 —
5 MA202L 28 400 0.75
6 MF147L-A202L-L199H 140 2 000 —
7 MF147L-A202L 832 11 900 8.47
8 MF147L 636 9 100 6.21
9 MF147L-A94T 490 7 000 —
10 MF147L-L199H 126 1 800 —
11 MF147L-A202L-A94T 277 4 000 —
表1  LkTADH回复突变体对CDOH的活力比较
图2  回复突变体的基因电泳图
图3  重组蛋白诱导表达(a)及纯化(b)电泳图
ADHs Km(mmol/L) Kcat(/s) Kcat/Km[mmol/(L·s)] Kis(mmol/L) Kcat(i) (/s)
MF147L-A202L-A94T-L199H 0.99 4.18 47.10 11.27 1.87 1.05
MF147L-A202L 0.98 2.61 40.11 15.36 3.22 1.88
MF147I-A202L 0.94 2.63 45.52 17.23 3.65 1.89
表2  突变酶动力学参数比较
图4  位点L147和位点L202不同突变体对CDOH的活力比较
图5  醇脱氢酶LkTADH的催化活性中心与底物的作用
图6  94位和199位在蛋白质结构LkTADH (a)和MF147I-A202L(b) 中的位置
图7  94位在蛋白质结构LkTADH(a)和MF147I-A202L (b) 中的位置及相互作用情况
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