酵母脂肪酶分子进化及催化合成(S)-1,4-苯并二烷  

Molecular Evolution of Yeast Lipase for Efficient Synthesis of(S)-1,4-Benzodioxane

作　　者：张琦 唐璐瑶 何远志 张凯 祝加伟 崔莉[1] 冯雁[1] ZHANG Qi;TANG Lu-yao;HE Yuan-zhi;ZHANG Kai;ZHU Jia-wei;CUI Li;FENG Yan(State Key Laboratory of Microbial Metabolism,School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]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

出　　处：《中国生物工程杂志》2022年第7期35-44,共10页China Biotechnology

基　　金：国家重点研发计划(2020YFA0907700);中国博士后科学基金(2021M692079)资助项目。

摘　　要：目的:1,4-苯并二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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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烷的ee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烷-2-甲酸甲酯的转化率为(47.5±2.33)%,ee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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烷类分子提供了理论和技术基础。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 D223 N/A225 K were systematically optimized.Results:The results indicated that the mutants mainly derived from the pairwise site D223/A225(such as D223 N/A225 K and D223 G/A225 W)were biased towards the synthesis of(S)-isoforms,while most of the mutants derived from the pairwise site E188/I189(such as E188 D/I189 M)showed a bias for the synthesis of(R)-isoforms.Compared with WT,the eevalue of the best mutant D223 N/A225 K to synthesize(S)-1,4-benzodioxane was increased from 11.9%to 29.3%.After systematic optimization of the reaction conditions,an eevalue of(93.9±0.16)%and a conversion rate of(47.5±2.33)%were achieved using mutant D223 N/A225 K 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 successful

关 键 词：南极假丝酵母脂肪酶B 蛋白质工程 1 4-苯并二烷 手性拆分 

分 类 号：Q819[生物学—生物工程]