随机-半理性组合突变改造ω-转氨酶催化合成(R)-1-(1-萘基)乙胺  

Engineering ω-transaminase by random mutagenesis and semi-rational design for the synthesis of(R)-(+)-1-(1-naphthyl)ethylamine

作　　者：曹旭东 韩瑞枝[1,2] 方红辉 倪晔[1,2] Xudong Cao;Ruizhi Han;Honghui Fang;Ye Ni(School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China;Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,Jiangsu,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：《生物工程学报》2020年第9期1828-1837,共10页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(Nos.31871738,21776112);国家重点研发计划(No.2018YFA0901700);中国博士后基金(No.2017M621631)资助。

摘　　要：(R)-1-(1-萘基)乙胺是合成拟钙剂药物盐酸西那卡塞的关键手性中间体,利用ω-转氨酶不对称还原1-萘乙酮合成(R)-1-(1-萘基)乙胺具有较好的应用前景。文中针对节杆菌属Arthrobacter sp.来源的ω-转氨酶,采用随机突变和半理性设计相结合的策略,获得了催化效率和热稳定性提高的突变酶F225M、C281I和F225M/C281I。与WT相比,双突变体F225M/C281I的kcat提高85%,Km下降56%,催化效率kcat/Km提高3.42倍。此外,F225M/C281I催化10mmol/L1-萘乙酮反应24h的转化率提高了22%。分子对接和分子动力学模拟结果表明,F225M/C281I相比于WT增加了与底物1-萘乙酮之间的Pi-Pi相互作用力,导致其催化效率的提高;而且突变酶F225M/C281I的134–139位点残基的均方根波动(RMSF)相比WT明显降低,与半衰期的略微提高相关。(R)-(+)-1-(1-naphthyl)ethylamine is a key chiral intermediate for the synthesis of calcimimetic drug cinacalcet hydrochloride. ω-Transaminase has been considered to be potential for producing(R)-(+)-1-(1-naphthyl)ethylamine by asymmetric reduction of 1-acetonaphthone. Here, ω-transaminase from Arthrobacter sp. was engineered by combinatorial strategies of random mutagenesis and semi-rational design. Variants F225M, C281I, F225M/C281I with improved catalytic efficiency and thermostability were obtained. Compared with WT, variant F225M/C281I showed 85% increased kcat, 56% decreased Km and 3.42-fold kcat/Km. Furthermore, 22% higher conversion rate was achieved by F225M/C281I at 10 mmol/L 1-acetonaphthone after 24 h. Based on molecular docking and molecular dynamics simulation, improved catalytic efficiency of F225M/C281I could be attributed to its increased Pi-Pi T-shaped interaction with substrate 1-acetonaphthone. Additionally, a slightly higher half-life of F225M/C281I was validated by its lower root-mean-square fluctuation(RMSF) value of loop 134–139 compared with WT.

关 键 词：ω-转氨酶 随机突变 半理性设计 1-萘乙酮 (R)-1-(1-萘基)乙胺 

分 类 号：TQ246.31[化学工程—有机化工]