Gene Mining,Cascade Reaction System Construction Of ?-transaminase And Its Application

?-transaminase is a type of 5'-pyridoxal phosphate-dependent enzyme that can mediate asymmetric reductive amination of aldehydes and ketones or kinetic resolution of chiral amines.It has important research and application value in the synthesis of chiral amines as pharmaceutical intermediates.As an important chiral amine compound,L-2-aminobutyric acid is the chiral precursor of many high-value compounds in the medical field.In this study,by mining a new type of?-transaminase,constructing a cascade reaction system,and optimizing the process,the catalytic conversion of L-2-aminobutyric acid was studied,and the main research results were as follows.(1)Using the amino acid sequence of the?-transaminase derived from Vibrio fluvialis with excellent enzymatic properties as probes,based on the NCBI protein database homology screening strategy,a"pyridoxal phosphate-dependent aminotransferase"(pp TA)derived from Paracoccus pantotrophus was obtained.This enzyme has 83%sequence identity with the?-transaminase derived from V.fluvialis and has all the key residues necessary for the?-transaminase.The enzyme gene was cloned into an E.coli expression system and induced to express.The size of the recombinant protein obtained was 50 KDa.The optimum temperature and p H of the enzyme were 38°C and 9,respectively.The substrate prefers aromatic amines.The K_m for the substrates benzylamine and 2-ketobutyrate were 4.9 m M and 3.3 m M,respectively.(2)Tandem expression of threonine deaminase TD and pp TA:Design a coupling catalytic system that catalyzes the production of L-2-aminobutyric acid from the substrate L-threonine.The verification of coupling catalysis shows that TD can catalyze L-Threonine synthesizes 2-ketobutyric acid,pp TA catalyzes the intermediate product 2-ketobutyric acid to synthesize the final product L-2-aminobutyric acid.During the conversion process,it was found that the reaction involving pp TA was a rate-limiting step,which was inhibited by the substrate side and the product side.(3)Construct a reaction optimization strategy based on the substrate side.The high enzymatic activity of TD catalyzes the conversion of L-Thr and the accumulation of high concentration of 2-OBA causes strong inhibition of pp TA.The ratio of the enzyme activity of TD to pp TA becomes a key factor in the cascade catalytic efficiency.When the ratio of TD to pp TA activity is adjusted to be less than 10:1,the catalytic reaction efficiency of the cascade reaction system reaches the best level.Different strengths of RBS were designed to replace RBS_T7 to assemble into p ETDuet-td-ppta,and the recombinant strain E.coli BL21/p ETDuet-rbs₁-td-ppta with the enzyme activity ratio of TD and pp TA expressed in tandem close to 10:1was screened.Under the catalysis of the recombinant bacterial enzyme solution optimized by RBS,100 m M L-threonine is converted to 21 m M L-2-aminobutyric acid,which is 40%higher than before optimization.(4)Construct a reaction optimization strategy based on the product side.The recombinant strains of E.coli BL21/p ACYCDuet-ahr-gdh and E.coli BL21/p ACYCDuet-ald-fdh were constructed to explore the effect of benzylamine or alanine as amino donors on the release of by-product inhibition.With L-Thr as the starting substrate,different four-enzyme cascade reaction systems were established,and the production of L-ABA under different amino donors was compared.The results show that when benzylamine is used as the amino donor,the amount of catalyzed 100 m M L-Thr to produce L-ABA is increased from 21 m M to 61.7 m M,and when alanine is used as the amino donor,it can catalyze the production of L-ABA from 100 m M L-Thr.The amount of L-ABA increased from 17.8 m M to 69.1 m M.(5)A reaction system based on whole-cell E.coli BL21/p ETDuet-rbs₁-td-ppta and E.coli BL21/p ACYCDuet-ald-fdh catalyzed the production of L-2-aminobutyric acid from L-threonine was established.The results showed that the production rate was 10.5 mmol·(L·h)^-1in the first 4 hours,and 71.6 m M L-2-aminobutyric acid could be produced in one step at 28hours,with a molar yield of 71.6%.