卡门柏青霉菌脂肪酶PCL催化烯烃环氧化的机理研究

发布时间：2018-05-24 06:51

本文选题：脂肪酶 + 工业应用　；参考：《华南理工大学》2016年硕士论文

【摘要】：脂肪酶是一类可以催化脂类水解反应的酶,由于具有较好的热稳定性和有机溶剂耐受性,因此在食品加工、制药、以及化工领域中都有广泛的应用。脂肪酶可以催化烯烃的环氧化反应,近年来利用脂肪酶催化合成各种环氧化物已成为研究热点,但脂肪酶催化烯烃环氧化反应的机理至今还未阐述清楚。本论文选取来自卡门柏青霉菌的脂肪酶PCL作为研究对象,通过生物化学实验、蛋白质结构研究以及分子对接研究,对PCL催化烯烃环氧化反应的机理进行了探讨。主要研究结果如下:1.脂肪酶PCL及其突变体的克隆表达与纯化:合成了脂肪酶PCL的全长基因,构建了活性中心催化三联体的突变体S145A和H259A,在毕赤酵母中实现高效重组表达,并通过阴离子交换层析纯化,获得纯度较高的蛋白。2.脂肪酶PCL及其突变体催化烯烃环氧化反应的研究:PCL对油酸和1-十八烯都具有较高的环氧化活力;与PCL相比,S145A对两种底物的活力都较低,但仍保留一定的环氧化活力;H259A完全没有环氧化活力。通过电喷雾电离质谱分析环氧化反应过程中生成的物质,发现PCL和S145A催化烯烃的环氧化反应都是酶法-化学过程,首先酶催化羧酸的过氧水解反应产生过氧酸,过氧酸释放后再自发把烯烃环氧化。研究发现pH值对PCL的环氧化活力影响不大,而S145A的活力对pH值十分敏感;与PCL相比,S145A的环氧化活力对过氧化氢的浓度更具有依赖性。3.脂肪酶PCL的结构生物学研究:通过蛋白质结晶实验,获得高分辨率的PCL蛋白质结构,并上传蛋白质结构数据库(PDB ID:5CH8)。通过计算机模拟对PCL与底物进行分子对接,得到脂肪酶PCL-过氧化氢-辛酸复合结构模型。4.脂肪酶PCL催化烯烃环氧化反应机理的研究:综合分析生化实验结果和结构模型,提出脂肪酶催化产生过氧酸的反应由两种机理协同作用,一个是经典的通过活性中心丝氨酸Ser145形成酰酶复合物的机理,另一个是本研究提出和证明的、由活性中心组氨酸His259主导的机理。His259是PCL发挥环氧化活力的关键氨基酸,在两种反应机理中分别夺取Ser145或过氧化氢的质子产生反应。Ser145虽然不是酶分子发挥活力的必要氨基酸,但可以显著地提高脂肪酶PCL的环氧化活力。本论文通过生化实验、结构生物学研究和分子对接研究,提出了在分子水平上卡门柏青霉菌脂肪酶PCL催化烯烃环氧化反应的机理,对今后改造、筛选具有应用前景的环氧化工业用酶具有重要意义。
[Abstract]:Lipase is a kind of enzyme which can catalyze the hydrolysis of lipids. Because of its good thermal stability and solvent tolerance, lipase has been widely used in food processing, pharmaceutical industry and chemical industry. Lipase can catalyze the epoxidation of olefin. In recent years, the synthesis of various epoxides catalyzed by lipase has become a hot topic, but the mechanism of lipase-catalyzed epoxidation of olefin has not been elucidated. In this paper, the lipase PCL from Penicillium Carmensi was selected as the research object. The mechanism of the epoxidation of olefins catalyzed by PCL was studied by biochemical experiments, protein structure studies and molecular docking studies. The main results are as follows: 1. Cloning, expression and purification of lipase PCL and its mutants: the full-length lipase PCL gene was synthesized, and the active center catalytic triad mutants S145A and H259A were constructed, which were highly expressed in Pichia pastoris. The purified protein was purified by anion exchange chromatography. Studies on the Epoxidation of olefin catalyzed by Lipase PCL and its Mutants; the epoxidation activity of PCL to oleic acid and 1-octadecene is higher than that of PCL, and the activity of S145A to both substrates is lower than that of PCL. However, there is no epoxidation activity at all in H 259A. By electrospray ionization mass spectrometry, it was found that the epoxidation of olefin catalyzed by PCL and S145A was an enzymatic chemical process. Firstly, peroxy hydrolysis of carboxylic acid was catalyzed by enzyme to produce peroxic acid. Peroxic acid released and then spontaneously epoxidation of olefins. It was found that pH value had little effect on the epoxidation activity of PCL, while the activity of S145A was sensitive to pH value, and that the epoxidation activity of S145A was more dependent on the concentration of hydrogen peroxide than that of PCL. Studies on structural biology of lipase PCL: high resolution PCL protein structure was obtained by protein crystallization experiment, and the protein structure database was uploaded. The compound structure model of lipase PCL- hydrogen peroxide-octanoic acid was obtained by molecular docking of PCL with substrate by computer simulation. Studies on the Mechanism of Lipase PCL catalyzed Epoxidation of Olefins: the results of biochemical experiments and the structural model were comprehensively analyzed, and the synergistic effect of the two mechanisms for lipase catalyzed peroxic acid production was put forward. One is the classical mechanism for the formation of acylase complexes through the active center serine Ser145, the other is the mechanism proposed and proved in this study. His259 is the key amino acid of PCL to exert its epoxidation activity, which is dominated by the active central histidine His259. The proton production reaction of Ser145 or hydrogen peroxide. Ser145 is not a necessary amino acid for enzyme molecule to exert its activity, but it can significantly increase the epoxidation activity of lipase PCL. In this paper, the mechanism of olefin epoxidation catalyzed by penicillium Carmenella lipase PCL at molecular level was proposed through biochemical experiments, structural biology studies and molecular docking studies. It is of great significance to screen the enzymes used in epoxide industry.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O621.251

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