Study On Biocatalytic Transamination And Reaction Engineering

Optically active lactam compounds are widely present in a variety of biologically active substances.They are important intermediates for the synthesis of complex structures such as polymers.The asymmetric synthesis of amines catalyzed by transaminase has the advantages of simple steps,high yield,high enantioselectivity,and few by-products.Theoretically,the yield of chiral amines prepared by asymmetric synthesis can reach 100%,which makes transaminases have broad prospects in the application of catalyzing the synthesis of such substances.(S)-5-methyl-2-pyrrolidone is a kind of ?-lactam compounds,which are often used as industrial catalysts or pharmaceutical intermediates(such as amino acid peptidase inhibitors)with commercial application value.In this paper,we focus on the asymmetric synthesis of(S)-5-methyl-2-pyrrolidone catalyzed by enzyme,and choose?-transaminase(coTA)as the catalyst.The enzymatic synthesis of(S)-5-methyl-2-pyrrolidone was optimized by modifying the catalytic performance of the enzyme and introducing additives into the reaction system to improve the microenvironment of the reaction system.By means of biomimetic immobilization,the stability of enzyme in the process of catalytic reaction can be improved,and the cost of industrial catalysis can be reduced by repeated use.For the asymmetric synthesis of 5-methyl-2-pyrrolidone catalyzed by ?-transaminase,other potential enzyme sources were screened in the gene library by homologous sequence alignment.Finally,BM-coTA gene chosen from the selected Bacillus megaterium genome were obtained and successfully expressed in E.coli BL21(DE3).When the optimal concentration of IPTG was 0.01 mM and the induction temperature was 16?,the semi-soluble and semi-inclusive protein was successfully obtained,and the enzyme activity was 1.5 U/mg.Then,the catalytic performance of the transaminase for chiral lactam was verified.In the presence of cofactor PLP,the conversion of the product was 8.7%.After 48 h,the conversion of(S)-5-methyl-2-pyrrolidone was increased to 47.7%,and the enantiomeric excess value(e.e.p)was 99.0%.The obtained(S)-5-methyl-2-pyrrolidone had the advantage of high stereoselectivity,and then the conversion of chiral lactam was optimized.The catalytic performance of BM-?TA enzyme was modified,and the interaction between enzyme and ligand was analyzed by molecular docking.Tyrosine(Tyr60)at position 60 was selected and seven mutants were obtained by saturation mutation.The results showed that the activity of BM-?TA(Y60C)was 3.6 U/mg.The conversion of the product was 46.8%,which was almost the same as that of the wild-type catalyst after 36 h.The reaction time of 12 h was shortened,and the high stereoselectivity of the product was not changed.On the other hand,a variety of additives,such as alcohols,polyols,amides and polysaccharides,were introduced into the reaction system.It was found that when 2.5%,v/v formamide was added to the reaction system,the conversion of lactam was greatly affected,and the conversion of product was increased to 65.7%,and the optical purity of product did not decrease.In the process of practical production and application,enzymes usually have the disadvantages of high cost and can not be reused.Therefore,a biomimetic immobilization method is adopted in this paper.This method is fast and convenient,and silicon is used as a non-toxic immobilization carrier,with little damage to the enzymes.The biomimetic silicon immobilization of BM-?TA and TABs directly depends on the presence of a large number of basic amino acids on the transaminases,which induces the silicate precursor to form silica nanoparticles and then embeds the enzyme in it.In this process,there is no need to add other coupling agents.After optimization of the conditions,the immobilization efficiency of the immobilized enzyme BM-?TA-SPs,BM-coTA(Y60C)-SPs and 6His-TABs-SPs were 92.4±2.7%?90.9 ± 2.2%,94.3± 2.8%,and the activity recovery were 134.4±3.5%,81.8±3.2%,102.7 ± 2.3%,respectively.The three immobilized enzymes still have high activity after repeated use for six cycles,which showed that the immobilized method in this study can make the enzyme as a biocatalyst keep its activity and reuse in the actual production.In this paper,we expand the application of enzyme catalyzed transaminase to asymmetric synthesis of chiral lactam,which provides more enzyme sources for biocatalysis of(S)-5-methyl-2-pyrrolidone.By preparing mutants and introducing additives,we optimize the conversion of enzyme catalyzed reaction without changing the high stereoselectivity of the product.Biological biomimetic immobilization was used to improve the reusability of enzyme in industrial catalysis to achieve the purpose of cost saving.