Multi-enzymatic Kinetic Resolution Of DL-norvaline Combined With Asymmetric Reduction To Produce L-norvaline

Hypertension is a very common chronic cardiovascular disease,Due to a high-salt,high-fat diet or a stressful lifestyle,the current proportion of hypertensive patients is close to 25% of the total population.At present,people mainly control hypertension through antihypertensive drugs,Perindopril is an effective antihypert ensive drug that is commonly used and is in strong demand.L-Norvaline is a vital intermediate in the chemical synthesis of Perindopril,the conventional chemical synthesis of L-norvaline is expensive and has many disadvantages,such as a complicated process,low enantiomeric excess and negative environmental impacts.Due to the high efficiency,high stereo-selectivity and high enantio-sel ectivity of the enzyme catalysts,enzyme-mediated chiral resolution for production of a single enantiomer has a very wide application prospect.We proposed a strategy for production of L-norvaline through enzyme-mediated kinetic resolution combined with asymmetric reduction of DL-norvaline.Firstly,D-Norvaline was oxidized to the corresponding 2-oxovaleric acid by D-amino acid oxidase from the substrate DL-norvaline.Asymmetric reduction of 2-oxovaleric acid to L-norvaline was carried out by leucine dehydrogenase with concomitant oxidation of NADH to NAD+.A NADH regenera tion system was introduced by overexpressing a formate dehydrogenase.The unwanted H2O2 by-product generated during D-norvaline oxidation was removed by adding catalase.In this paper,the expression hosts of DAAO,LeuDH and FDH were optimized and the results showed that Corynebacterium glutamicum ATCC 13032 was suitable for DAAO expression and E.coli BL21（DE3）was suitable for LeuDH and FDH expression.Subsequently,the properties of DAAO,LeuDH and FDH were studied,the optimum reaction temperature and pH of DAAO were 25°C,8.0 and Mn²⁺ had a positive effect,the Km value for D-norvaline was 3.3 mM·L-1;the optimum reaction temperature and pH of LeuDH were 60°C,9.0 and Mg²⁺ had a positive effect,the Km value for 2-oxovaleric acid was 1.42 mM·L-1;the optimum reaction temperature and pH of FDH were 55°C,8.5,Ca²⁺ and Cu²⁺ had a negative effect,the Km value for Sodium formate was 12.3 mM·L-1.Enzyme-mediated kinetic resolution of DL-norvaline combined with asymmetric reduction for L-norvaline production was constructed and the parameters were optimized.The optimum conversion pH and temperature were optimized and the result showed that the optimum temperature and pH were 30°C and 7.5 respectively;the amount of catalase added was optimized and found to be 42.59 U·mL-1;the proportion of DAAO,LeuDH and FDH was optimized and the result demonstrated that 2:1:0.5 was the optimal proportion.In order to prevent the accumulation of 2-oxovaleric acid and increase the L-norvaline production,fed-batch strategy was employed to produce L-norvaline in a 5 L fermentor,under optimal conditions,a total of 54.09 g·L-1 of L-norvaline was achieved,with an enantiomeric excess over 99% and a conversion rate of 96.3%.Subsequently the application of the conversion system was developed and the result showed that other racemic amino acids such as Valine and Arginine can be converted to the corresponding L-amino acids.Compared with chemical synthesis,this method has the advantages of environmental protection and high purity of product.This study provides an effective strategy for production of enantiomerically pure L-norvaline in the pharmaceutical industry.