文献类型：期刊文献

中文题名：构建辅酶自循环系统全细胞催化合成S-2-氨基-1-苯基乙醇

英文题名：Construction of cofactor self-sufficient whole-cell biocatalyst system for producing S-2-amino-1-phenylethanol

作者：赵有玺[1,2];刘松[2];张显[2];徐美娟[2];杨套伟[2];饶志明[2]

第一作者：赵有玺

机构：[1]北京联合大学生物化学工程学院,北京100023;[2]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

第一机构：北京联合大学生物化学工程学院

年份：2020

卷号：26

期号：1

起止页码：152-159

中文期刊名：应用与环境生物学报

外文期刊名：Chinese Journal of Applied and Environmental Biology

收录：CSTPCD;;Scopus;北大核心:【北大核心2017】；CSCD:【CSCD2019_2020】；

基金：国家自然科学基金项目(31870066,31570085);生物质废弃物资源化利用北京市重点实验室开放课题(ZK80201501);北京联合大学人才强校优选计划项目(BPHR2017CZ05)资助。

语种：中文

中文关键词：S-2-氨基-1-苯基乙醇;苯基-1,2-乙二醇;ω-转氨酶;辅酶自循环系统;全细胞生物催化

外文关键词：S-2-amino-1-phenylethanol;phenyl-1,2-ethanediol;ω-transaminase;cofactor self-sufficient;whole-cell biocatalyst

摘要：S-2-氨基-1-苯基乙醇是一种重要的医药中间体,广泛应用于神经递质及抗病毒剂等多种具有生理活性药物的合成.目前其合成主要通过化学手段,虽然工艺成熟,但反应条件剧烈,且会生成许多副产物.基于新鉴定的铜绿假单胞菌来源的ω-转氨酶,设计了一个结合新金色分枝杆菌来源的醇脱氢酶的级联酶催化体系,从而将较为廉价的苯基-1,2-乙二醇一步催化合成具有高附加值的S-2-氨基-1-苯基乙醇.为了解决级联催化过程中辅酶再生和氨基供体再生问题,在该级联体系中引入大肠杆菌来源的谷氨酸脱氢酶,构建一个封闭的氧化还原自循环系统,从而实现辅酶(NADP+)和辅底物(L-Glu)的同时再生.最后将这3个酶串联表达在pETDuet和pACYCDuet质粒上,并转入大肠杆菌中,获得了重组菌Escherichia coli BL21(MPG).通过重组菌全细胞转化,结果显示在100 mL、pH 8.0的NH4Cl溶液中,37℃条件下反应10 h后,OD600=30的该重组菌可一步催化50 mmol/L苯基-1,2-乙二醇生成光学纯的S-2-氨基-1-苯基乙醇,转化率达到99.6%,产物的ee值>99%.本研究提供了一种高效经济催化合成S-2-氨基-1-苯基乙醇的方法,不需要受到辅因子供应不足的限制以及副产物的积累的影响.
In the synthesis of various physiologically active drugs, S-2-amino-1-phenylethanol is widely used as an important pharmaceutical intermediate. Presently, it is synthesized mainly through chemical methods. Although the process is mature, the reaction conditions are severe as several by-products are formed. Based on the newly identified ω-transaminase derived from Pseudomonas aeruginosa PAK, a catalyzed enzyme catalytic system combining alcohol dehydrogenase derived from Mycobacterium neoaurum was designed that synthesized S-2-amino-1-phenylethanol by one-step biocatalysis of phenyl-1,2-ethanediol. By introducing GluDH, a closed cofactor self-sufficient system was constructed that could realize the simultaneous regeneration of coenzyme(NADP+) and cosubstrate(L-Glu). The whole-cell transformation of recombinant Escherichia coli BL21(MPG) showed that in a 100 mL NH4Cl reaction system after a 10 h reaction in pH 8.0 at 37℃. The recombinant strain with OD600=30 could catalyze 50 mmol/L phenyl-1,2-ethanediol to produce optically pure S-2-amino-1-phenylethanol with a conversion of 99.6% and ee > 99%. This study provides an effective method for catalyzing the synthesis of optically pure S-2-amino-1-phenylethanol from cheap substrates without cofactor supply and accumulation of by-products.