摘要
以具有高选择性的羰基还原酶(CR2)与可实现辅酶原位循环的葡萄糖脱氢酶(GDH)通过柔性连接肽得到的融合酶CR2-GDH为生物催化剂,催化N,N-二甲基-3-酮-3-(2-噻吩)-1-丙胺(DKTP)不对称还原合成手性药物度洛西汀重要中间体(S)-N,N-二甲基-3-羟基-3-(2-噻吩)-1-丙胺((S)-DHTP)。通过过程调控反应体系的pH,并进一步优化反应转速、底物同辅底物比例、温度及底物浓度,确定单批次反应条件:底物20 g/L、葡萄糖40 g/L、NADP^(+)0.1 mmol/L及酶活7 U,恒定pH 8.4、45℃、150 r/min反应12 h,产物产率达到97.70%,对映体过量值(e.e.)≥99.9%。通过分批补料催化30 g/L底物的反应,产率达92.96%,进一步利用反应器放大体系至100 mL,实现40 g/L底物的转化反应,产率达到85.05%。
We used fusion enzyme CR2-GDH involving a highly selective carbonyl reductase and a coenzyme-regenerating glucose dehydrogenase for asymmetric reduction of(S)-N,N-dimethyl-3-keto-3-(2-thiophene)-1-propanamine(DKTP),the prochiral compound to synthesize(S)-N,N-dimethyl-3-hydroxy-3-(2-thiophene)-1-propanamine((S)-DHTP),the chiral precursor of the important drug duloxetine.In addition to pH control during the reaction,we further optimized the catalytic conditions including rotation speed,ratio of substrate to co-substrate,temperature and substrate concentration.The one-pot reaction system was constructed to comprise substrate DKTP 20 g/L,glucose 40 g/L,NADP^(+)0.1 mmol/L and 7 U enzyme,under the optimized conditions including pH 8.4,45℃,150 r/min for 12 h.Finally,the yield of(S)-DHTP reached 97.70%,with the enantiomeric excess value(e.e.)≥99.9%.By fed-batch strategy,the yield reached 92.96%when the substrate concentration was accumulated to 30 g/L.Through continuous process control for 40 g/L DKTP in reactor system,single enantiomer product(S)-DHTP was obtained and the yield reached 85.05%.
作者
邓陈琪
聂尧
徐岩
DENG Chenqi;NIE Yao;XU Yan(Key Laboratory of Industrial Biotechnology of the Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China)
出处
《生物加工过程》
CAS
2021年第2期178-184,198,共8页
Chinese Journal of Bioprocess Engineering
基金
国家自然科学基金(21336009、21676120)
江苏省六大人才高峰计划(2015-NY-007)
江苏高校优势学科建设工程。
作者简介
邓陈琪(1994—),女,湖南岳阳人,硕士研究生,研究方向:生物化工;联系人:聂尧,教授,E-mail:ynie@jiangnan.edu.cn。
