Coenzyme Regeneration Systems Of Photosynthetic Bacteria And Their Application On Biocatalysis

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%.