光合细菌辅酶再生系统的研究及其在生物催化中的应用
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摘要
近年来,用手性技术不对称催化合成手性化合物及其手性中间体已经引起了学术界和企业界的重视,成为研究开发的热点。利用生物不对称催化具有反应条件温和、转化率高及立体选择性好等优点,已经成了诸多手性合成方法的首选。氧化还原酶是在生物催化手性合成中起着重要应用的一类酶。大部分氧化还原酶的催化反应需要辅酶NADPH作为还原剂参与,由于氧化还原酶应用广泛而NADPH价格昂贵,因此NADPH的再生在生物催化中起着重要的作用。另一方面,辅酶NADPH的再生能简化产物的分离,促使反应向正反应方向移动。现已有的NADPH再生方法包括酶法,电化学以及光化学方法等等。本论文是在前期以整体细胞不对称生物催化的基础上研究NADPH的再生,主要是以光合细菌作为实验材料,通过提取菌绿素、载色体,分离纯化氢化酶、氧化还原酶,构建了四种辅酶再生体系,以期实现在生物不对称催化中能够进行NADPH的再生,从而为辅酶依赖性的生物催化体系提供理论基础和方法指导。
其主要内容如下：
(1)从类球红杆菌中提取出的菌绿素作为光敏剂光解水从而实现辅酶NADPH的再生,通过对再生体系所必需的条件实验,分析了利用菌绿素进行辅酶再生的机理,并且考察了辅酶再生的各个因素,得出再生NADPH的最佳条件为：pH 7.0,温度为35℃,NADP+的初始浓度为50μmol/L,反应的最佳时间12 h,电子供体和氢供体的浓度分别为10 mmol/L和10 mmol/L
(2)从类球红杆菌其提取出的载色体通过光电子传递链实现辅酶NADPH的再生。通过对再生体系所必需的条件实验,分析了利用载色体进行辅酶再生的机理。并且考察了辅酶再生的各个因素,得出再生NADPH的最佳条件为：pH7.0,温度为35℃,NADP+的初始浓度为50μmol/L,反应的最佳时间12h,电子供体和氢供体的浓度分别为10 mmol/L和10 mmol/L。
(3)从类球红杆菌中分离纯化出的氢化酶催化氢气产生电子和氢质子,从而实现辅酶NADPH的再生。通过对再生体系所必需的条件实验,分析了利用氢化酶进行辅酶再生的机理。并且考察了辅酶再生的各个因素,得出再生NADPH的最佳条件为：pH7.0,温度为30℃,NADP+的初始浓度为50μmol/L,反应的最佳时间3h。
(4)从类球红杆菌中分离纯化的氧化还原酶共底物催化,从而实现辅酶NADPH的再生。通过对再生体系所必需的条件实验,分析了利用氧化还原酶进行辅酶再生的机理。并且考察了辅酶再生的各个因素,得出再生NADPH的最佳条件为：pH 7.5,温度为30℃,NADP+的初始浓度为501μmol/L,反应的最佳时间3h,葡萄糖和ATP的浓度分别为4.5g/L和11.5g/L
(5)通过对不同的芳香酮类、脂肪酮化合物为底物进行了酶法还原,检测其反应的还原产率和e.e值,结果表明该酶催化的底物适用范围很广；酶对电负性强的基团在苯乙酮α位取代的衍生物以及五碳脂肪酮的专一性较强；酶催化的立体选择性都在9996以上,具有极高的立体选择性。
Recently, chiral technology has highly attracted attentions from industry and academia, and the asymmetrical synthesis of chiral compounds and their chiral intermediates have become an active field in research and development. Because of mild reaction conditions, high conversion ratio and outstanding stereochemical specificity, biocatalysis has become the most promising and first choice method in the asymmetric synthesis. Redox-enzymes have many applications in chemical synthesis, especially in asymmetric synthesis of chiral compounds. As many redox-enzymes depend on the common NADPH-cofactor and the factor is usually expensive. So it plays an important part in building highly efficient NADPH regeneration system that applied oxidoreductase in the catalytic reaction.On the other hand, the NADPH regeneration also can simplify separates the product and facilitates the enzymatic reaction to the direction of positive response. Various approaches to the regeneration of NADPH have been studied, such as enzymatic, electrochemical and photochemical methods etc. This article is based on our previous study of the experiments on whole-cell asymmetric catalytic reaction and enzyme-catalyzed reaction, and the principal objective of this work is to systematic study the NADPH regeneration from the photosynthetic bacteria in our laboratory preservation. Rhodobacter sphaeroides was selected as an experimental material and four NADPH regeneration systems were established by distilling bacteria chlorophyll, preparing chromatophore, purifying hydrogenase and redox enzyme. We aim to provide a theoretical basis and methodological guidance that is designed to build and optimize the system of NADPH-dependent biocatalysis.
The main points are as follows:
(1)The coenzyme regeneration with bacteria chlorophyll as photosensitizer to photolysis the water from the Rhodobacter sphaeroides. Study on the mechanism that the coenzyme regeneration with bacteria chlorophyll through the conditions necessary for experiments on coenzyme regeneration systems.The optimal conditions for NADPH regeneration were obtained from the one factor experiments. The result showed that the optimal regeneration conditions were pH 7.0, temperature 35℃,NADP+ concentration 50μmol/L,time 12h,electron donor concentration 10mmol/L, hydrogen concentration lOmmol/L.
(2) The coenzyme regeneration with chromatophore by photoelectron transfer chain from the Rhodobacter sphaeroides. Study on the mechanism that the coenzyme regeneration with chromatophore through the conditions necessary for experiments on coenzyme regeneration systems.The optimal conditions for NADPH regeneration were obtained from the one factor experiments. The result showed that the optimal regeneration conditions were pH 7.0, temperature 35℃,NADP+ concentration 50μmol/L,time 12h,electron donor concentration 10mmol/L, hydrogen concentration 10mmol/L.
(3)The coenzyme regeneration with hydrogenase by catalyzed the hydrogen to generate the electron and proton from the Rhodobacter sphaeroides. Study on the mechanism that the coenzyme regeneration with hydrogenase through the conditions necessary for experiments on coenzyme regeneration systems.The optimal conditions for NADPH regeneration were obtained from the one factor experiments. The result showed that the optimal regeneration conditions were pH 7.0, temperature 30℃,NADP+ concentration 50μmol/L,time 3h.
(4) The coenzyme regeneration with redox enzyme by catalyzed the different substrates from the Rhodobacter sphaeroides. Study on the mechanism that the coenzyme regeneration with redox enzyme through the conditions necessary for experiments on coenzyme regeneration systems.The optimal conditions for NADPH regeneration were obtained from the one factor experiments. The result showed that the optimal regeneration conditions were pH 7.5,temperature 30℃,NADP+ concentration 50μmol/L,time 3h, glucose concentration 4.5g/L, ATP concentration 11.5g/L.
(5) The yield and ee value were detected by catalyzed the different aromatic ketones and the different aliphatic ketones. The result showed that the scope of the substrate enzyme-catalyzed is broad. The enzyme had a strong specific that the group had strong electrical negative, and had a high stereoselectivity, more than 99%.
其主要内容如下：
(1)从类球红杆菌中提取出的菌绿素作为光敏剂光解水从而实现辅酶NADPH的再生,通过对再生体系所必需的条件实验,分析了利用菌绿素进行辅酶再生的机理,并且考察了辅酶再生的各个因素,得出再生NADPH的最佳条件为：pH 7.0,温度为35℃,NADP+的初始浓度为50μmol/L,反应的最佳时间12 h,电子供体和氢供体的浓度分别为10 mmol/L和10 mmol/L
(2)从类球红杆菌其提取出的载色体通过光电子传递链实现辅酶NADPH的再生。通过对再生体系所必需的条件实验,分析了利用载色体进行辅酶再生的机理。并且考察了辅酶再生的各个因素,得出再生NADPH的最佳条件为：pH7.0,温度为35℃,NADP+的初始浓度为50μmol/L,反应的最佳时间12h,电子供体和氢供体的浓度分别为10 mmol/L和10 mmol/L。
(3)从类球红杆菌中分离纯化出的氢化酶催化氢气产生电子和氢质子,从而实现辅酶NADPH的再生。通过对再生体系所必需的条件实验,分析了利用氢化酶进行辅酶再生的机理。并且考察了辅酶再生的各个因素,得出再生NADPH的最佳条件为：pH7.0,温度为30℃,NADP+的初始浓度为50μmol/L,反应的最佳时间3h。
(4)从类球红杆菌中分离纯化的氧化还原酶共底物催化,从而实现辅酶NADPH的再生。通过对再生体系所必需的条件实验,分析了利用氧化还原酶进行辅酶再生的机理。并且考察了辅酶再生的各个因素,得出再生NADPH的最佳条件为：pH 7.5,温度为30℃,NADP+的初始浓度为501μmol/L,反应的最佳时间3h,葡萄糖和ATP的浓度分别为4.5g/L和11.5g/L
(5)通过对不同的芳香酮类、脂肪酮化合物为底物进行了酶法还原,检测其反应的还原产率和e.e值,结果表明该酶催化的底物适用范围很广；酶对电负性强的基团在苯乙酮α位取代的衍生物以及五碳脂肪酮的专一性较强；酶催化的立体选择性都在9996以上,具有极高的立体选择性。
Recently, chiral technology has highly attracted attentions from industry and academia, and the asymmetrical synthesis of chiral compounds and their chiral intermediates have become an active field in research and development. Because of mild reaction conditions, high conversion ratio and outstanding stereochemical specificity, biocatalysis has become the most promising and first choice method in the asymmetric synthesis. Redox-enzymes have many applications in chemical synthesis, especially in asymmetric synthesis of chiral compounds. As many redox-enzymes depend on the common NADPH-cofactor and the factor is usually expensive. So it plays an important part in building highly efficient NADPH regeneration system that applied oxidoreductase in the catalytic reaction.On the other hand, the NADPH regeneration also can simplify separates the product and facilitates the enzymatic reaction to the direction of positive response. Various approaches to the regeneration of NADPH have been studied, such as enzymatic, electrochemical and photochemical methods etc. This article is based on our previous study of the experiments on whole-cell asymmetric catalytic reaction and enzyme-catalyzed reaction, and the principal objective of this work is to systematic study the NADPH regeneration from the photosynthetic bacteria in our laboratory preservation. Rhodobacter sphaeroides was selected as an experimental material and four NADPH regeneration systems were established by distilling bacteria chlorophyll, preparing chromatophore, purifying hydrogenase and redox enzyme. We aim to provide a theoretical basis and methodological guidance that is designed to build and optimize the system of NADPH-dependent biocatalysis.
The main points are as follows:
(1)The coenzyme regeneration with bacteria chlorophyll as photosensitizer to photolysis the water from the Rhodobacter sphaeroides. Study on the mechanism that the coenzyme regeneration with bacteria chlorophyll through the conditions necessary for experiments on coenzyme regeneration systems.The optimal conditions for NADPH regeneration were obtained from the one factor experiments. The result showed that the optimal regeneration conditions were pH 7.0, temperature 35℃,NADP+ concentration 50μmol/L,time 12h,electron donor concentration 10mmol/L, hydrogen concentration lOmmol/L.
(2) The coenzyme regeneration with chromatophore by photoelectron transfer chain from the Rhodobacter sphaeroides. Study on the mechanism that the coenzyme regeneration with chromatophore through the conditions necessary for experiments on coenzyme regeneration systems.The optimal conditions for NADPH regeneration were obtained from the one factor experiments. The result showed that the optimal regeneration conditions were pH 7.0, temperature 35℃,NADP+ concentration 50μmol/L,time 12h,electron donor concentration 10mmol/L, hydrogen concentration 10mmol/L.
(3)The coenzyme regeneration with hydrogenase by catalyzed the hydrogen to generate the electron and proton from the Rhodobacter sphaeroides. Study on the mechanism that the coenzyme regeneration with hydrogenase through the conditions necessary for experiments on coenzyme regeneration systems.The optimal conditions for NADPH regeneration were obtained from the one factor experiments. The result showed that the optimal regeneration conditions were pH 7.0, temperature 30℃,NADP+ concentration 50μmol/L,time 3h.
(4) The coenzyme regeneration with redox enzyme by catalyzed the different substrates from the Rhodobacter sphaeroides. Study on the mechanism that the coenzyme regeneration with redox enzyme through the conditions necessary for experiments on coenzyme regeneration systems.The optimal conditions for NADPH regeneration were obtained from the one factor experiments. The result showed that the optimal regeneration conditions were pH 7.5,temperature 30℃,NADP+ concentration 50μmol/L,time 3h, glucose concentration 4.5g/L, ATP concentration 11.5g/L.
(5) The yield and ee value were detected by catalyzed the different aromatic ketones and the different aliphatic ketones. The result showed that the scope of the substrate enzyme-catalyzed is broad. The enzyme had a strong specific that the group had strong electrical negative, and had a high stereoselectivity, more than 99%.
引文
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