高等学校化学学报 ›› 2017, Vol. 38 ›› Issue (10): 1772-1777.doi: 10.7503/cjcu20170164

• 有机化学 • 上一篇    下一篇

羰基还原酶CR2重组酶体系不对称合成(S)-N,N-二甲基-3-羟基-3-(2-噻吩)-1-丙胺

孙太强, 李斌, 聂尧(), 王栋(), 徐岩   

  1. 江南大学生物工程学院, 工业生物技术教育部重点实验室, 无锡 214122
  • 收稿日期:2017-03-20 出版日期:2017-10-10 发布日期:2017-09-21
  • 作者简介:联系人简介: 聂 尧, 男, 博士, 教授, 博士生导师, 主要从事酶工程领域的研究. E-mail:ynie@jiangnan.edu.cn;王 栋, 男, 博士, 副教授, 主要从事酶工程领域的研究. E-mail:dwang@jiangnan.edu.cn
  • 基金资助:
    国家自然科学基金(批准号: 21376107, 21336009, 21676120)、 江苏省自然科学基金(批准号: BK20151124)、 高等学校学科创新引智计划(111计划)项目(批准号: 111-2-06)、 江苏省六大人才高峰计划项目(批准号: 2015-NY-007)和江苏高校优势学科建设工程项目资助

Asymmetric Synthesis of (S)-N,N-Dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine by Cell-free System of Carbonyl Reductase CR2

SUN Taiqiang, LI Bin, NIE Yao*(), WANG Dong*(), XU Yan   

  1. School of Biotechnology, Key Laboratory of Industrial Biotechnology of Ministry of Education,Jiangnan University, Wuxi 214122, China
  • Received:2017-03-20 Online:2017-10-10 Published:2017-09-21
  • Contact: NIE Yao,WANG Dong E-mail:ynie@jiangnan.edu.cn;dwang@jiangnan.edu.cn
  • Supported by:
    † Supported by the National Natural Science Foundation of China(Nos.21376107, 21336009, 21676120), the Natural Science Foundation of Jiangsu Province, China(No.BK20151124), the 111 Project(No.111-2-06), the Program for Advanced Talents within Six Industries of Jiangsu Province, China(No.2015-NY-007) and the Priority Academic Program Development of Jiangsu Higher Education Institutions, China

摘要:

构建了羰基还原酶CR2重组酶体系, 并优化了相关的酶促催化反应条件. 通过在催化体系中添加辅酶NADP+(0.1 mmol/L)和辅底物葡萄糖(120 g/L), 在30 ℃ 及pH=8.0的条件下反应4 h, CR2重组酶体系不对称还原N,N-二甲基-3-酮-3-(2-噻吩)-1-丙胺(DKTP, 10 g/L), 合成了高光学纯度(S)-N,N-二甲基-3-羟基-3-(2-噻吩)-1-丙胺[(S)-DHTP, e.e.值>99.9%], 产率为62%. 在酶促催化过程中, 由于辅酶循环生成葡萄糖酸导致反应体系pH值下降而影响催化效率. 通过调控反应体系pH值, (S)-DHTP的产率提高到68%. 不同浓度底物的反应过程表明底物对CR2酶促反应具有抑制作用, 且在10 g/L底物浓度下反应的时空产率可达1.3 g·L-1·h-1.

关键词: 生物催化, 酶, 重组酶体系, 不对称还原, (S)-N, N-二甲基-3-羟基-3-(2-噻吩)-1-丙胺

Abstract:

(S)-N,N-Dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine[(S)-DHTP] is an important intermediate for the production of (S)-duloxetine, which is a new and effective antidepressant drug. Since the efficiency of biosynthesis of (S)-DHTP is yet limited, development of suitable biocatalytic system is necessary for (S)-DHTP production from asymmetric reduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine(DKTP). In this study, the cell-free biocatalytic system involving recombinant carbonyl reductase CR2 was constructed and the corresponding reaction conditions were optimized. (S)-DHTP with high optical purity(e.e. value>99.9%) and the yield of 61.70% was obtained from DKTP(10 g/L) at pH=8.0[0.1 mol/L triethanolamine(TEA) buffer] and 30 ℃ after reaction for 6 h, by employing glucose(120 g/L) as the co-substrate and coenzyme NADP+(0.1 mmol/L) to drive the cofactor regeneration system. During the reaction process, the pH value decreased significantly due to the formation of gluconic acid from glucose for cofactor recycling. Then the yield of (S)-DHTP was increased to 67.73% by controlling the reaction pH. Substrate inhibition was observed when investigating the conversion of DKTP under different concentrations, and the space time yield of 1.33 g·L-1·h-1 was achieved for DKTP(10 g/L).

Key words: Biocatalysis, Enzyme, Crude enzyme system, Asymmetric reduction, (S)-N, N-Dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamin

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