本文關(guān)鍵詞： 生物催化 不對稱氧化拆分 單胺氧化酶 高立體選擇性 Pseudomonas monteilii　出處：《遵義醫(yī)學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:篩選和獲得高活性和高選擇性的單胺氧化酶產(chǎn)生菌株,用于催化2-取代四氫喹啉類衍生物的不對稱氧化拆分研究,進一步了解菌株內(nèi)單胺氧化酶的酶學(xué)性質(zhì),構(gòu)建單胺氧化酶重組菌。方法:選擇2-甲基-1,2,3,4-四氫喹啉作為模板底物,采用懸浮整細胞催化法,篩選高活性的生物催化劑,優(yōu)化反應(yīng)條件,建立2-取代四氫喹啉類衍生物較優(yōu)的反應(yīng)體系,合成具有高光學(xué)活性的2-取代四氫喹啉衍生物;構(gòu)建來源于Pseudomonas monteilii ZMU-T01菌株的單胺氧化酶重組菌。結(jié)果:1.通過篩選發(fā)現(xiàn)Pseudomonas monteilii ZMU-T01菌株整細胞對模版底物2-甲基-1,2,3,4-四氫喹啉具有較好的催化活性,在底物濃度為4 m M,細胞濃度為50 g cdw/L,p H為8.0,5 m L的反應(yīng)體系下,底物的轉(zhuǎn)化率為47%,其ee值達到90%。在此基礎(chǔ)上,擴展了8個類似結(jié)構(gòu)的底物,獲得手性2-取代四氫喹啉的ee值為89-99%。2.通過控制變量法對生物催化反應(yīng)體系進行優(yōu)化,最終確定反應(yīng)的較優(yōu)條件為:底物濃度為2 m M,細胞濃度為50 g cdw/L,緩沖溶液為PBS(p H=8.0,50 m M),反應(yīng)體積為5 m L,反應(yīng)溫度為30 oC,轉(zhuǎn)速為250 rpm,反應(yīng)時間為24 h。3.在較優(yōu)反應(yīng)體系下,對2-取代四氫喹啉類底物進行了普適性考察,當苯環(huán)上取代基為吸電子基團時,Pseudomonas monteilii ZMU-T01整細胞對其無催化活性,當2位取代基為供電子基團時,整細胞對其的催化活性較好,共合成得到了8個高光學(xué)活性的化合物,所有產(chǎn)物結(jié)構(gòu)經(jīng)NMR分析,手性HPLC,旋光和HRMS分析確認和指證。4.以Pseudomonas monteilii ZMU-T01整細胞基因為模板,通過分子生物學(xué)技術(shù)進行序列分析,設(shè)計了2段基因,成功獲得1個含有單胺氧化酶生物催化活性的基因工程菌。結(jié)論:1.篩選獲得Pseudomonas monteilii ZMU-T01菌株整細胞能有效的催化2-取代四氫喹啉類衍生物的不對稱氧化拆分反應(yīng),合成具有高光學(xué)活性的2-取代四氫喹啉類衍生物,并具有較好的底物適應(yīng)范圍;為實現(xiàn)手性2-取代四氫喹啉類衍生物的綠色合成提供了一種有效的方法。2.對Pseudomonas monteilii ZMU-T01的基因組進行分析,設(shè)計2條基因序列,克隆了1個具有單胺氧化酶活性的基因工程菌,為后續(xù)的研究建立了一定的基礎(chǔ)。
[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.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：O621.251

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