醇脱氢酶催化合成(S)-哌啶醇及产物抑制机制  

Biocatalytic Preparation of a(S)-piperidinol by Alcohol Dehydrogenase and Its Product Inhibitory Mechanism

作　　者：吴彦霏 许国超[1,2] 周婕妤 倪晔[1,2] WU Yanfei;XU Guochao;ZHOU Jieyu;NI Ye(School of Biotechnology,Jiangnan University,Wuxi 214122,China;Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]江南大学工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《食品与生物技术学报》2021年第7期73-80,共8页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(21776112);国家“十三五”重点研发计划项目(2018YFA0901700)。

摘　　要：(S)-N-Boc-3-羟基哌啶((S)-NBHP)是治疗套细胞淋巴瘤药物——依鲁替尼的关键手性中间体。利用醇脱氢酶催化N-Boc-3-哌啶酮(NBPO)的不对称还原是最具开发潜力的(S)-NBHP合成方法。对醇脱氢酶库进行筛选发现,来源于多孢克鲁维酵母的KpADH对NBPO具有最佳的催化效果,纯酶活力高达83.9 U/mg,产物对映体过量值为97.0%(S)。将其与葡萄糖脱氢酶偶联,并考察了单水相、两相、离子液体和不同底物浓度对转化反应的影响。结果表明:高浓度产物(S)-NBHP对KpADH存在抑制作用,抑制动力学分析表明属于非竞争性抑制。采用单水相体系可降低(S)-NBHP对KpADH催化反应的抑制,有利于转化效率的提高。初步探究了醇脱氢酶催化NBPO不对称还原反应的抑制现象,对酶法合成(S)-NBHP具有指导意义。(S)-N-Boc-3-hydroxypiperidine((S)-NBHP)is a key chiral intermediate for the synthesis of ibrutinib treating mantle cell lymphoma.Asymmetric reduction of N-Boc-3-piperidone(NBPO)catalyzed by alcohol dehydrogenase is the most promising method for(S)-NBHP synthesis.After screening of the alcohol dehydrogenase library,KpADH from Kluyveromyces polyspora showed excellent catalytic efficiency on NBPO,with specific activity of pure enzyme as high as 83.9 U/mg and the product enantiomeric excess value of 97.0%(S).Coupled cofactor regeneration reaction was established by introducing glucose dehydrogenase,and the effects of monoaquous phase,biphasic phase,ionic liquids and different substrate concentrations on the conversion were explored.The results indicated that high concentration product(S)-NBHP had inhibitory effect on KpADH,and the inhibitory kinetic analysis showed that it belonged to non-competitive inhibition.The inhibition of(S)-NBHP on KpADH catalytic reaction could be reduced and the conversion efficiency could be improved by using single monoaquous phase catalysis.The inhibition of asymmetric reduction of NBPO catalyzed by alcohol dehydrogenase was studied,which could provide guidance for the enzymatic preparation of(S)-NBHP.

关 键 词：(S)-N-Boc-3-羟基哌啶 醇脱氢酶 单水相催化 产物抑制 非竞争性抑制 

分 类 号：Q814[生物学—生物工程]