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 B 5, maintains healthy hair, blood and skin, and it acts as a precursor to coenzyme A. At present, D-pantothenic acid was synthesized by chemical method from D-panactone and 尾-alanine. The precursor D-panlytic acid lactone was mainly synthesized by chemical synthesis and kinetic resolution of hydrolase, but the steps of chemical method were tedious and polluted the environment, and the hydrolase resolution process needed chemical racemization and acidizing internal esterification. Additional production costs are increased. Therefore, it is of great significance to develop a new synthesis process of D-panalactone. Using Saccharomyces cerevisiae genome as template, the gene SceCPR1 was cloned successfully, and the system of coenzyme cycle regeneration coupled with glucose dehydrogenase (EsGDH) was successfully constructed, that is, "one bacterium double enzyme" system. The molecular sizes of SceCPR1 and EsGDH protein were 35 kDa and 27 kDa, respectively. The enzyme activity of SceCPR1 and es GDH was 1179.2 U / g and 442.8 U/g.SceCPR1, respectively. The optimum pH was 5.5, the temperature was 45 鈩，

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