分子改造甲烷八叠球菌醇脱氢酶催化合成4-羟基-2-丁酮  

Molecular modification of Methanosarcina thermophila alcohol dehydrogenase for catalytic synthesis of 4-hydroxy-2-butanone

作　　者：张笛 张恒维 张迪 杨套伟[1] 饶志明[1] ZHANG Di;ZHANG Hengwei;ZHANG Di;YANG Taowei;RAO Zhiming(Key Laboratory of Industrial Biotechnology of Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[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

出　　处：《应用与环境生物学报》2024年第5期990-998,共9页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(32370040,32100055);江苏省自然科学基金项目(BK20221537);江苏高校优势学科建设工程项目;江苏高校品牌专业建设工程项目资助。

摘　　要：4-羟基-2-丁酮(4H2B)是一种重要的药物中间体,可以用于合成抗HIV药物度鲁特韦重要手性中间体(R)-3-氨基丁醇,但4H2B价格昂贵,本研究旨在通过酶催化反应,将廉价的1,3-丁二醇(1,3-BDO)催化合成4H2B,以便用于后续开发合成4H2B的衍生物.首先克隆表征了一种甲烷八叠球菌(Methanosarcina thermophila)来源的醇脱氢酶(MeADH),研究发现,其可用于催化1,3-BDO合成4H2B.MeADH最适催化温度为60℃,最适催化pH为10.0.经酶学性质分析发现,MeADH在最适反应温度60℃下孵育48 h仅剩7.51%左右残余酶活力,说明其热稳定性不佳.基于计算机辅助设计选择了15个突变位点,筛选得到一个双突变MeADHE246P/T296P,在60℃条件下孵育48 h,其残余酶活为初始酶活的40.27%左右,比原始酶提高了5.36倍.此外,MeADH催化反应过程中需要辅酶NADP^(+),通过引入鼠李糖乳杆菌(Lactobacillus rhamnosus)来源的NADH氧化酶(LrNox)突变体LrNoxL179S构建了NADP^(+)辅酶循环体系,并将其应用到4H2B的生物合成中.本研究通过优化反应条件和底物流加实验,4H2B合成量最高达到2.2 g/L左右,为后续进行高效生物合成4-羟基-2-丁酮及其衍生物的研究奠定了基础.(图10表3参29)4-Hydroxy-2-butanone(4H2B)is an important pharmaceutical intermediate that can be used to synthesize the chiral intermediate(R)-3-aminobutyl of the anti-HIV drug dulutvir.However,4H2B is costly.In this study,we synthesized 4H2B from inexpensive 1,3-butanediol via enzymatic catalysis and developed and synthesized 4H2B derivatives.Alcohol dehydrogenase(MeADH)from Methanosarcina thermophila was cloned and characterized.This enzyme catalyzed the synthesis of 4H2B from 1,3-butanediol.The optimum catalytic temperature of MeADH was 60℃,and the optimum pH was 10.0.Analysis of its enzymatic properties showed that only approximately 7.51% of residual enzyme activity remained after incubation for 48 h at the optimum reaction temperature,indicating low thermal stability.To improve thermostability,15 mutation sites were selected using computer-aided design,and a double mutant MeADHE246P/T296P with higher stability was obtained.After incubation at 60℃ for 48 h,the residual enzyme activity of the mutant was approximately 40.27% of the initial enzyme activity,which was 5.36-fold higher than that of the original enzyme.In addition,the coenzyme NADP^(+)is required for catalytic activity of MeADH.We constructed an NADP^(+)coenzyme cycle system using the NADH oxidase(LrNox)mutant LrNoxL179S from Lactobacillus rhamnosus for biosynthesis of 4H2B.By optimizing the reaction conditions and experiment of underflow feeding,the maximum levels of 4H2B reached around 2.2 g/L,providing a reference for subsequent research of the efficient biosynthesis of 4H2B and its derivatives.

关 键 词：4-羟基-2-丁酮 醇脱氢酶 辅酶循环再生 热稳定性 

分 类 号：TQ224.6[化学工程—有机化工]