酮泛解酸内酯还原酶的克隆表达及其在高效不对称合成D-泛解酸内酯中的应用

发布时间：2018-06-04 18:13

本文选题：酮泛解酸内酯还原酶 + 酿酒酵母　；参考：《浙江工业大学》2017年硕士论文

【摘要】：D-泛酸,俗称维生素B5,可以维持头发,血液和皮肤的健康,同时它可以作为辅酶A的前体。目前,主要以D-泛解酸内酯和β-丙氨酸为原料通过化学法合成D-泛酸。前体D-泛解酸内酯主要通过化学法合成和水解酶动力学拆分获得,但化学法步骤繁琐,污染环境,而水解酶拆分过程中需要化学外消旋化和酸化内酯化过程,增大了额外的生产成本。因此,开发全新的D-泛解酸内酯合成工艺具有非常大的意义。本文以酿酒酵母基因组为模板,成功克隆出酮泛解酸内酯还原酶基因SceCPR1,并成功构建了SceCPR1与葡萄糖脱氢酶EsGDH偶联的辅酶循环再生系统,即“一菌双酶”体系,用于高效不对称还原酮泛解酸内酯获得D-泛解酸内酯;SceCPR1和EsGDH蛋白分子大小分别为35 kDa和27 kDa。通过诱导条件优化,最终单位湿菌体中SceCPR1和Es GDH的酶活分别为1179.2 U/g和442.8 U/g。SceCPR1酶学性质表征发现,其最适的pH为5.5,温度45℃,并且酶在45℃下容易变性,但当添加5 mM的NADPH可以保证其温度稳定性;其次,SceCPR1不属于金属离子依赖型还原酶,但是5mM的Fe3+以及大多数有机溶剂对酶活力有抑制作用;底物谱研究发现该酶对于酮泛解酸内酯具有最高的活力,而对于酮泛解酸以及D-或L-泛解酸内酯都无活力;在低底物浓度条件下,测定了SceCPR1的动力学参数,SceCPR1对酮泛解酸内酯和NADPH的Km值分别为0.164 mM和0.029 mM,反应的Vmax分别为131.03 U/mg和137.81U/mg。以BL21(DE3)/pACYCDuet 1-SceCPR1/EsGDH的冻干细胞作为生物催化剂,优化全细胞催化条件,确定最适反应pH为5.5,温度35℃,生物催化剂添加量为0.03 g/mL,辅底物葡萄糖与底物配比为1.5:1.0,搅拌速度400 rpm;通过对底物水解的研究,发现底物自发水解是影响产物得率的关键因素。因此,在最适催化条件下,将酮泛解酸内酯和葡萄糖溶解于p H 2.5的溶液中,采用持续流加补料的方法,最终产物浓度达到475 mM,得率95%,产物光学纯度e.e.p≥99.9%。
[Abstract]:D- pantothenic acid, commonly known as vitamin B5, can maintain the health of hair, blood and skin. At the same time it can be used as a precursor of coenzyme A. Currently, D- pantothenic acid is synthesized by chemical method mainly by D- pantol and beta alanine. Precursor D- pan lactate is obtained by chemical synthesis and hydrolase kinetic resolution, but chemical process steps The process of chemical racemization and acidification in the process of hydrolysis of hydrolase requires the process of esterification of chemical racemization and acidification, which increases the extra cost of production. Therefore, it is very important to develop a new synthesis process of D- flooding acid lactone. This paper successfully cloned the ketone acid lactone reductase gene SceCPR1 by using the Saccharomyces cerevisiae genome as a template. SceCPR1 and glucose dehydrogenase EsGDH coupled coenzyme regeneration system was successfully constructed, that is, "one bacteria double enzyme" system, which is used for high efficiency unsymmetrical reducing ketone acid lactone to obtain D- flooding acid lactone. The molecular size of SceCPR1 and EsGDH protein molecules are 35 kDa and 27 kDa., respectively, by optimization of the inducement conditions, and finally the SceCPR1 in the unit wet mycelium and in the final unit. The enzyme activity of Es GDH was 1179.2 U/g and 442.8 U/g.SceCPR1 characterization respectively. The optimum pH was 5.5, the temperature was 45, and the enzyme was easily denatured at 45, but when the 5 mM NADPH was added to ensure its temperature stability; secondly, SceCPR1 did not belong to the metal ion dependent reductase, but 5mM Fe3+ and most organic solvents. The enzyme activity was inhibited by the enzyme, and the enzyme was found to have the highest activity for the ketone acid lactone, while the ketone flooding acid and D- or L- pan acid lactone were not active, and the kinetic parameters of SceCPR1 were measured under the low substrate concentration. The Km values of SceCPR1 to ketone and NADPH were 0.164 mM and 0.029 mM, respectively. The reaction Vmax is 131.03 U/mg and 137.81U/mg. using BL21 (DE3) /pACYCDuet 1-SceCPR1/EsGDH lyophilized cells as biocatalysts to optimize the whole cell catalytic conditions. The optimum reaction pH is 5.5, the temperature is 35, the addition of biocatalysts is 0.03 g/mL, the auxiliary substrate grape sugar and the substrate ratio is 1.5:1.0, and the stirring speed is 400 rpm; through the The study of substrate hydrolysis found that the spontaneous hydrolysis of the substrate was the key factor affecting the yield of the products. Therefore, under the optimum conditions, the solution of ketoacid lactone and glucose dissolved in P H 2.5 solution under the optimum catalytic condition, the final product concentration reached 475 mM, the yield was 95%, the optical purity of the product was E.E.P > 99.9%..
【学位授予单位】：浙江工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q55;Q78;O621.3

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