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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  2022, Vol. 42 Issue (7): 35-44    DOI: 10.13523/j.cb.2203068
技术与方法     
酵母脂肪酶分子进化及催化合成(S)-1,4-苯并二口恶烷*
张琦**(),唐璐瑶**(),何远志,张凯,祝加伟,崔莉***(),冯雁***()
上海交通大学生命科学技术学院 微生物代谢国家重点实验室 上海 200240
Molecular Evolution of Yeast Lipase for Efficient Synthesis of (S)-1,4-Benzodioxane
Qi ZHANG**(),Lu-yao TANG**(),Yuan-zhi HE,Kai ZHANG,Jia-wei ZHU,Li CUI***(),Yan FENG***()
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
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摘要： 目的 1,4-苯并二口恶烷是普罗克生、多沙唑嗪等抗高血压和MCK-242等抗抑郁药物的手性中间体,在医药领域具有广泛应用。目前化学合成具有产量高的优势,然而存在环境污染和生产安全性低等问题。应用脂肪酶进行催化合成提供了1,4-苯并二口恶烷的绿色合成新途径。研究发现天然酶存在对映体选择性低的局限,因此针对南极假丝酵母脂肪酶B进行分子进化,并建立了1,4-苯并二口恶烷催化合成的技术路线。方法 首先,针对南极假丝酵母脂肪酶B活性中心参与底物结合和转化的关键氨基酸残基进行分析,并构建了相互作用位点协同进化的饱和突变库,通过HPLC检测,筛选获得了系列转化效率高和对映选择性强的突变体,最后对影响最佳突变体D223N/A225K催化效率的反应条件进行系统优化。结果 来自D223和A225位点突变体(D223N/A225K和D223G/A225W等)偏向于合成(S)-型产物;而E188和I189位点突变体(E188D/I189M等)表现出偏向于合成(R)-型产物。与野生型相比,最佳突变体D223N/A225K合成(S)-1,4-苯并二口恶烷的ees值从11.9%提升到29.3%。对反应条件系统优化后,突变体D223N/A225K在37℃、正丁醇/磷酸盐缓冲液(20∶80,V/V)两相溶剂中反应50 min后对底物(R,S)-1,4-苯并二口恶烷-2-甲酸甲酯的转化率为(47.5±2.33)%,ees值达(93.9±0.16)%。结论 通过分子改造与条件优化,成功实现对(R,S)-1,4-苯并二口恶烷-2-甲酸甲酯的高效动力学拆分,为蛋白质工程技术创制新酶提供了新例证,也为酶法高效催化合成(S)-1,4-苯并二口恶烷类分子提供了理论和技术基础。
关键词： 南极假丝酵母脂肪酶B;  蛋白质工程;  1,4-苯并二口恶烷;  手性拆分    
Abstract:

Objective: 1,4-Benzodioxane is an important chiral intermediate for antihypertensive (Proroxan and Doxazosin), antidepressant (MCK-242) and other drugs, and it displays a broad spectrum of applications in the pharmaceutical field. Currently, in spite of high-yield advantage of chemical synthesis, there are some problems of environmental pollution and low production safety. Using lipase to catalyze synthesis of 1,4-benzodioxane provides a new pathway of green synthesis of 1,4-benzodioxane. However, natural enzymes face the dilemma of poor enantioselectivity. Therefore, molecular evolution was performed on Candida antarctica lipase B, and a technical route for the catalytic synthesis of 1,4-benzodioxane was established. Methods: Firstly, the key amino acid residues involved in substrate binding and conversion in the active center of Candida antarctica lipase B were analyzed, and saturation mutagenesis libraries on the interaction sites were constructed. Improved mutants with high efficiency and high enantioselectivity were then obtained using HPLC detection. Furthermore, catalytic synthesis conditions of mutant D223N/A225K were systematically optimized. Results: The results indicated that the mutants mainly derived from the pairwise site D223/A225 (such as D223N/A225K and D223G/A225W) were biased towards the synthesis of (S)-isoforms, while most of the mutants derived from the pairwise site E188/I189 (such as E188D/I189M) showed a bias for the synthesis of (R)-isoforms. Compared with WT, the ees value of the best mutant D223N/A225K to synthesize (S)-1,4-benzodioxane was increased from 11.9% to 29.3%. After systematic optimization of the reaction conditions, an ees value of (93.9±0.16)% and a conversion rate of (47.5±2.33)% were achieved using mutant D223N/A225K to catalyze kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate in n-butanol/phosphate buffered saline (20∶80, V/V) biphasic solvent at 37℃ for 50 min. Conclusion: An efficient kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate was successfully achieved by molecular evolution and optimization of conditions, which provides a new example for the creation of new enzymes by protein engineering technology, and also provides a theoretical and technical foundation for the efficient synthesis of (S)-1,4-benzodioxane molecules by enzymatic methods.
Key words: Candida antarctica lipase B    Protein engineering    1,4-Benzodioxane    Chiral resolution
收稿日期: 2022-03-29 出版日期: 2022-08-03
ZTFLH:  Q819  
基金资助: *国家重点研发计划(2020YFA0907700);中国博士后科学基金(2021M692079)
通讯作者: 张琦,唐璐瑶,崔莉,冯雁     E-mail: cuili@sjtu.edu.cn;yfeng2009@sjtu.edu.cn
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引用本文:

张琦,唐璐瑶,何远志,张凯,祝加伟,崔莉,冯雁. 酵母脂肪酶分子进化及催化合成(S)-1,4-苯并二口恶烷*[J]. 中国生物工程杂志, 2022, 42(7): 35-44.

Qi ZHANG,Lu-yao TANG,Yuan-zhi HE,Kai ZHANG,Jia-wei ZHU,Li CUI,Yan FENG. Molecular Evolution of Yeast Lipase for Efficient Synthesis of (S)-1,4-Benzodioxane. China Biotechnology, 2022, 42(7): 35-44.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2203068    或    https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I7/35

图1  CalB突变位点的选择
图2  CalB野生型及突变体的异源表达与纯化
图3  野生型与突变体催化(R,S)-1,4-苯并二口恶烷-2-甲酸甲酯动力学拆分
图4  温度对野生型和突变体催化反应的影响
图5  共溶剂种类对野生型和突变体催化反应的影响
图6  共溶剂百分比对野生型和突变体催化反应的影响
图7  野生型和突变体催化反应的时间进程
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