Asymmetric Synthesis Of L-tert Leucine By Enzyme Coupled System/Light-enzyme Coupled System

L-tertiary leucine has a wide range of applications in asymmetric synthesis,the synthesis of chiral auxiliaries and ligand,in addition,L-tertiary leucine is a key intermediate in the synthesis of many drugs,such as Zeneca,Sandoz,Atazanavir etc.Chemical synthesis of L-tertiary leucine had disadvantages of low optical purity,low yield,complicated synthetic steps etc.With the development of synthetic biology,genetic engineering,molecular biology,biological catalytic synthesis of L-tertiary leucine began to grow.The way that use trimethylpyruvic acid as the substrate,leucine dehydrogenase catalytic synthesis of L-tertiary leucine has great industrial prospects.The reaction needs to add a certain amount of coenzyme NADH,but coenzyme NADH is expensive.So it is not conducive to saving the cost of production.Thus,leucine dehydrogenase is usually coupling with formate dehydrogenase for NADH regeneration.Photocatalytic NADH regeneration is a new type of NADH regeneration system,at the same time,the light catalyst has the advantages of clean,cheap etc.And photocatalytic NADH regeneration has been gradually developed in recent years.In this paper,we have expressed and purified six different sources of leucine dehydrogenase and four different sources of formate dehydrogenase.With TMP and ammonium formate as the substrate,according to the enzyme activity,we screened out the highest active enzyme,respectively.Leucine dehydrogenase（Am LeuDH,enzyme activity 7.6 U/mg）and formate dehydrogenase（Op-FDH,enzyme activity11.54 U/mg）were the highest active enzymes.And the activity of two kinds of enzymes in different temperature,pH,and metal ions was studied,and we optimized the catalytic conditions for enzyme.At the same time,we built the double enzyme coupling system,and we studied the substrate concentration ratio,NAD+and ratio of two enzyme activity optimization of double enzyme.The optimal reaction system was:the concentration ratio of TMP and ammonium formate was 1:2,the adding amount of NAD+was 1 mM,the enzyme activity ratio of Am LeuDH and Op-FDH was 1:2.Under the optimum reaction conditions,the substrate conversion rate can reach 95%.This paper preliminary constructed light NADH regeneration coupling leucine dehydrogenase catalytic system.The NADH regeneration system was with C₃N₄ as photocatalyst,different conditions of NADH regeneration efficiency was studied.And we coupled the NADH regeneration system with leucine dehydrogenase,then we explored the different light distance,the amount of enzyme,[Cp*Rh（bpy）（H₂O）]²⁺as well as the NAD+.