Molecular Modification Of Amino Acid Dehydrogenas And Synthesis Of D-serine By Multi-enzyme System

D-Serine（D-Ser）plays the role in regulating the function of the human nervous system.In the field of medicine,it can be used as a drug intermediate for the treatment of pulmonary tuberculosis caused by infection to synthesize D-cycloserine.It can also be used as a drug for the treatment of human cocaine dependence,schizophrenia,cerebellar work disorders.In the field of cosmetics,D-Ser and its derivatives can also be used as active substances.At present,the methods of synthesizing D-Ser in industry are mainly divided into chemical method and biological method,which generally have the disadvantages of environmental protection,low yield and low optical purity.Previously,our laboratory has constructed a multi-enzyme cascade route for the synthesis of D-amino acids containing an oxidative deamination module,a reductive amination module and a coenzyme circulation system.The route is coupled with L-amino acid deaminase from Proteus mirabilis,D-amino acid dehydrogenase from Symbiobacterium thermophilum,and Bs FDH from Burkholderia stabilis,which can finally synthesize D-amino acids from cheap and easily available L-amino acids.The product has high optical purity,simple steps and low cost.At present,the laboratory has successfully used this route to synthesize phenylalanine,and the substrate conversion rate and optical purity of80 m M can reach 100%.After investigation,it was found that the yield and optical purity of D-Ser synthesized by this route were very low.In order to construct a multi-enzyme cascade system that can efficiently synthesize D-Ser,this study intends to improve the reaction route.The specific results are as follows:（1）Through catalytic activity detection,the tool enzymes required for each module of the multi-enzyme cascade route were determined,and the catalytic properties of each module were investigated.The optimum temperature of Pmir LAAD was 42°C.Therefore,Pmir LAAD was suitable for catalysis under alkaline conditions and was less affected by p H.The optimum p H of StDAPDH/H227V was p H 9.5,and the catalytic activity was less affected by temperature between 35°C and 50°C.There was no reverse reaction catalyzed by Pmir LAAD and StDAPDH/H227V.The catalytic activity of Bs FDH decreased slightly with the increase of p H in the alkaline environment of p H 7.0-10.5,but the change was not obvious.At 25-50°C,the catalytic activity of Bs FDH increased with the increase of temperature,so its reaction conditions can be compatible with other module tool enzymes.（2）The catalytic activity of StDAPDH/H227V for the intermediate hydroxypyruvate was improved by molecular modification,thereby improving the catalytic efficiency of the entire multi-enzyme cascade route.Four key residues 92D,122D,152M and 253N in the pocket were predicted by molecular docking.Four mutants D92E,D122W,M152S and N253Y with significantly improved activity were obtained by single point saturation mutation,and then iterative mutation was carried out.Finally,a D92E/D122W/M152S mutant was obtained,and the specific activity was increased by 8.11 times.The reason for the increase of enzyme activity may be related to the change of active pocket size and the enhancement of molecular interaction between substrate and key residues.（3）The oxidative deamination module（Pmir LAAD）,the reductive amination module（StDAPDH/H227V/D92E/D122W/M152S）,and the coenzyme cycle module（Bs FDH）were assembled to construct a multi-enzyme cascade system,and its catalytic performance and catalytic efficiency for other amino acids were investigated.In order to maximize the yield of D-Ser produced by the multi-enzyme cascade route,the p H,temperature,and enzyme addition ratio were adjusted and optimized.It was found that the route was 50 m M Tris-HCl buffer,temperature 32°C,sodium formate concentration 100 m M,NADP⁺concentration 2.5m M,NH₄Cl concentration 100 m M,Pmir LAAD whole cells,StDAPDH/H227V mutant pure enzyme and Bs FDH pure enzyme were added at 50 mg·m L^-1,respectively.The maximum catalytic efficiency was achieved at 4 mg·m L^-1and 0.7 mg·m L^-1,which could catalyze the complete conversion of 50 m M L-Ser to D-Ser.Through investigation,it was found that the multi-enzyme cascade route can also catalyze the synthesis of a variety of small molecule D-amino acids,which has great potential in industry.