The Soluble Expression Of Transaminase YhxA And Modification Of Its Catalytic Perforamnce

Chiral amine refers to a class of compounds containing a small amino group in the chiral center of small molecule compounds,and is an important intermediate for most medicines and pesticides.For example,the drugs for treating diabetes,sitagliptin,the antibiotic penicillin,and the broad-spectrum contact herbicide glufosinate,all have a chiral amine chemical module.There are two methods for the preparation of chiral amines using aminotransferases,namely the kinetic resolution of racemic amines and the asymmetric synthesis of chiral amines from prochiral ketones.The theoretical yield of chiral amines prepared by asymmetric synthesis can reach 100%,which makes transaminase have a strong application prospect in the production industry.The heterocyclic compound chiral lactam with chiral amine chemistry module is an important and widely biologically active intermediate among the currently approved antibiotics such as penicillin and cephalosporin.The transaminase can catalyze the bioconversion of the aldehyde ester or ketoester,asymmetrically synthesize the corresponding chiral amine,and then obtain an optically active chiral lactam intermediate by spontaneous intramolecular cyclization.Due to the small number of transaminase enzymes that catalyze this reaction,this paper has carried out gene mining on aminotransferases that catalyze the asymmetric synthesis of chiral lactams.Finally,a putative transaminase yhxA?NCBI:CAB12754.2?derived from Bacillus subtilis with a similarity to the template enzyme of 29.4%was screened from the database.The amino acid sequence is 450 aa in length and belongs to the same family of acetylornithine aminotransferases as the template transaminase.The soluble expression of yhxA was subsequently carried out.In order to achieve soluble expression of yhxA,a pET expression system favoring protein NI-NTA affinity chromatography was selected.However,soluble proteins of yhxA were not obtained on pET-21a,pET-28a and pET-28b.To solve this problem,refer to the laboratory's study on the soluble expression of polyamino acid modification to promote the protein,select different lengths of histidine,lysine and arginine to modify the N-terminus and C-terminus of yhxA,and finally a soluble expression of yhxA is achieved.Among them,His6-yhxA is the highest expression of soluble protein;His6-yhxA-Lys15 is the highest soluble protein and the highest proportion of total protein.The preliminary viability assay of the above soluble yhxA expression showed that the highest viability of His6-yhxA-Lys10 was 4.31 U/mg,and the viability of yhxA with higher soluble expression was similar.Subsequently,the asymmetric synthesis of chiral lactam catalyzed by soluble yhxA was carried out,and the reaction effect was not satisfactory.For the problem that the catalytic activity of yhxA was not high,the catalytic performance of yhxA was modified by the rational design of the enzyme.The catalytically active cavity of yhxA was analyzed by homology modeling and molecular docking.It is speculated that the 62-position tryptophan?Trp62?in the large pocket of the substrate binding pocket caused steric hindrance due to its large volume.Therefore,this amino acid site was subjected to a saturation mutation.First,the mutation of His6-yhxA-Lys15?Trp62?was carried out based on the soluble expression of yhxA.The mutants His6-yhxA-Lys15_W62T and His6-yhxA-Lys15_W62V catalyzed the transamination reaction of?-phenethylamine and pyruvate.The yield of the resulting acetophenone was increased to 27.9%and 24.1%,respectively.It was also found that Trp62 may affect the soluble expression of yhxA,and the yhxA?Trp62?expressing all inclusion bodies was saturated,and two soluble mutants yhxA_W62F and yhxA_W62Y were obtained.Among them,yhxA_W62F increased the acetophenone yield to 38.9%,which was 1.95times that of the unmutated His6-yhxA-Lys15.Subsequently,the enzymes yhxA_W62F and yhxA_W62Y were studied with the highest soluble protein expression and the ability to purify His6-yhxA as a reference.The results showed that the optimum temperature of the three was 40°C,and the stability was best at 10°C;the optimum pH was pH 7.0,and the stability was better in the buffer of pH 7.0.From the analysis of the determination results of metal ion stability,the metal ions used in the experiment will reduce the vigor of the three.Finally,yhxA was screened for the substrate.The reaction of racemic-?-phenethylamine and?R?-?-phenethylamine as an amine donor identified that yhxA is?S?-selective?-transaminase;catalyzing the reaction of acetophenone as an amino acceptor,isopropylamine or 1,5-diaminopentane as an amino donor,failing to detect a decrease in acetophenone,yhxA has no catalytic activity on these two amino donors.The substrates identifiable by yhxA include pyruvate and?-phenethylamine by the reaction catalyzed by aminotransferase yhxA.