假单胞菌整细胞催化2-取代四氢喹啉类衍生物的不对称氧化拆分研究

发布时间：2018-02-01 06:53

本文关键词： 生物催化不对称氧化拆分单胺氧化酶高立体选择性 Pseudomonas monteilii　出处：《遵义医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的:筛选和获得高活性和高选择性的单胺氧化酶产生菌株,用于催化2-取代四氢喹啉类衍生物的不对称氧化拆分研究,进一步了解菌株内单胺氧化酶的酶学性质,构建单胺氧化酶重组菌。方法:选择2-甲基-1,2,3,4-四氢喹啉作为模板底物,采用悬浮整细胞催化法,筛选高活性的生物催化剂,优化反应条件,建立2-取代四氢喹啉类衍生物较优的反应体系,合成具有高光学活性的2-取代四氢喹啉衍生物;构建来源于Pseudomonas monteilii ZMU-T01菌株的单胺氧化酶重组菌。结果:1.通过筛选发现Pseudomonas monteilii ZMU-T01菌株整细胞对模版底物2-甲基-1,2,3,4-四氢喹啉具有较好的催化活性,在底物浓度为4 m M,细胞浓度为50 g cdw/L,p H为8.0,5 m L的反应体系下,底物的转化率为47%,其ee值达到90%。在此基础上,扩展了8个类似结构的底物,获得手性2-取代四氢喹啉的ee值为89-99%。2.通过控制变量法对生物催化反应体系进行优化,最终确定反应的较优条件为:底物浓度为2 m M,细胞浓度为50 g cdw/L,缓冲溶液为PBS(p H=8.0,50 m M),反应体积为5 m L,反应温度为30 oC,转速为250 rpm,反应时间为24 h。3.在较优反应体系下,对2-取代四氢喹啉类底物进行了普适性考察,当苯环上取代基为吸电子基团时,Pseudomonas monteilii ZMU-T01整细胞对其无催化活性,当2位取代基为供电子基团时,整细胞对其的催化活性较好,共合成得到了8个高光学活性的化合物,所有产物结构经NMR分析,手性HPLC,旋光和HRMS分析确认和指证。4.以Pseudomonas monteilii ZMU-T01整细胞基因为模板,通过分子生物学技术进行序列分析,设计了2段基因,成功获得1个含有单胺氧化酶生物催化活性的基因工程菌。结论:1.筛选获得Pseudomonas monteilii ZMU-T01菌株整细胞能有效的催化2-取代四氢喹啉类衍生物的不对称氧化拆分反应,合成具有高光学活性的2-取代四氢喹啉类衍生物,并具有较好的底物适应范围;为实现手性2-取代四氢喹啉类衍生物的绿色合成提供了一种有效的方法。2.对Pseudomonas monteilii ZMU-T01的基因组进行分析,设计2条基因序列,克隆了1个具有单胺氧化酶活性的基因工程菌,为后续的研究建立了一定的基础。
[Abstract]:Objective: to screen and obtain highly active and selective monoamine oxidase producing strains for catalytic asymmetric oxidative resolution of 2-substituted tetrahydroquinoline derivatives. To further understand the enzymatic properties of monoamine oxidase in the strain, a recombinant strain of monoamine oxidase was constructed. Methods: the template substrate was 2-methyl-1-trimethylamine 4-tetrahydroquinoline and suspension whole cell catalysis method was used. Screening high activity biocatalysts, optimizing reaction conditions, establishing a better reaction system of 2-substituted tetrahydroquinoline derivatives and synthesizing 2-substituted tetrahydroquinoline derivatives with high optical activity. The recombinant strain of monoamine oxidase from Pseudomonas monteilii ZMU-T01 was constructed. Results: 1. Pseudomonas was found by screening. The whole cell pair of monteilii ZMU-T01 strain was 2-methyl-1. In the reaction system with substrate concentration of 4 mm and cell concentration of 50 g cdw / L ~ (-1) H = 8.0 渭 m / L, the catalytic activity of 2C _ (3) C _ (3) O _ (4) -tetrahydroquinoline was better. The conversion rate of the substrate was 47 and the ee value of the substrate reached 90. On this basis, eight substrates of similar structure were expanded. The ee value of chiral 2-substituted tetrahydroquinoline was 89-99.2.The biocatalytic reaction system was optimized by controlling variables. The optimal reaction conditions were determined as follows: substrate concentration was 2 mm, cell concentration was 50 g / L, and buffer solution was PBS(p Hau 8.0 渭 m 路L ~ (-1). The volume of the reaction was 5 mL, the reaction temperature was 30 OC, the rotational speed was 250 rpm, and the reaction time was 24 h 路3. The general properties of 2-substituted tetrahydroquinoline substrates were investigated when the substituents on benzene rings were electron-absorbing groups. The whole cell of Pseudomonas monteilii ZMU-T01 had no catalytic activity. When the two substituents were the electron donor group, the whole cell had better catalytic activity. Eight compounds with high optical activity were synthesized. All the products were characterized by NMR and chiral HPLC. The whole cell base of Pseudomonas monteilii ZMU-T01 was used as template. Two genes were designed by molecular biological technique. A genetically engineered strain containing monoamine oxidase biocatalytic activity was successfully obtained. Conclusion: 1. Pseudomonas monteilii was obtained by screening. The whole cell of ZMU-T01 can effectively catalyze the asymmetric oxidative resolution of 2-substituted tetrahydroquinoline derivatives. The 2-substituted tetrahydroquinoline derivatives with high optical activity have been synthesized and have a good substrate adaptation range. This paper provides an effective method for the green synthesis of chiral 2-substituted tetrahydroquinoline derivatives. 2. for Pseudomonas monteilii. The genome of ZMU-T01 was analyzed. Two gene sequences were designed and a genetically engineered strain with monoamine oxidase activity was cloned.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O621.251

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