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[1]赵有玺,刘松 张显 徐美娟 杨套伟 饶志明**.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*[J].应用与环境生物学报,2020,26(01):1-12.[doi:10.19675/j.cnki.1006-687x.2019.04029]
 ZHAO Youxi,LIU Song,ZHANG Xian,et al.Construction of cofactor self- sufficient whole -cell biocatalyst system for production of S-2-Amino-1-phenylethanol*[J].Chinese Journal of Applied & Environmental Biology,2020,26(01):1-12.[doi:10.19675/j.cnki.1006-687x.2019.04029]
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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*()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
26卷
期数:
2020年01期
页码:
1-12
栏目:
研究论文
出版日期:
2020-01-25

文章信息/Info

Title:
Construction of cofactor self- sufficient whole -cell biocatalyst system for production of 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**
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
Author(s):
ZHAO Youxi1;?2;? LIU Song2;? ZHANG Xian2;? XU Meijuan2;? YANG Taowei2 & RAO Zhiming2**
1 Biochemical Engineering College, Beijing Union University, Beijing 100023, China
2 Ministry of Education Key Laboratory of Industrial Biotechnology, School of Biotechnology, Jiangnan University, Wuxi 214122, China
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
Keywords:
S-2-Amino-1-phenylethanol;? phenyl-1;?2-ethanediol;? -transaminase;? cofactor self-sufficient ;? whole-cell biocatalyst
DOI:
10.19675/j.cnki.1006-687x.2019.04029
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 nm = 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。
Abstract:
S-2-Amino-1-phenylethanol is an important pharmaceutical intermediate and is widely used in the synthesis of various physiologically active drugs. At present, its synthesis is mainly through chemical methods , although the process is mature, the reaction conditions are severe and many 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, which synthesized S-2-Amino-1-phenylethanol by one-step biocatalysis of phenyl-1,2-ethanediol. For cofactor regeneration and amino donor source, an interesting point was involved as that a cofactor self-sufficient system was designed by expression of GluDH. By introducing GluDH, a closed cofactor self- sufficient system was constructed, which could realize the simult aneous regeneration of coenzyme (NADP+) and cosubstrate ( L-Glu). The whole-cell transformation of recombinant Escherichia coli BL21 (MPG) showed that in 100 mL NH 4Cl reaction system, after 10 h reaction at pH 8.0 and 37 °C, the recombinant strain with OD600 nm = 30 could catalyze 50 mmol/L phenyl-1,2-ethanediol produces optically pure S-2-amino-1-phenylethanol with a conversion of 99.6%, ee > 99%. This work provides an effective method for catalyzing the synthesis of optically pure S-2-Amino-1-phenylethano from cheap substrates without cofactor supply of and no accumulation of by-products.

备注/Memo

备注/Memo:
收稿日期 Received: 2019-04-15 接受日期 Accepted: 2019-05-23
*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)和北京联合大学人才强校优选计划项目(BPHR2017CZ05)资助
**通讯作者 Corresponding author (E-mail: raozhm@jiangnan.edu.cn)
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更新日期/Last Update: 2019-06-19