| Sitafloxacin Hydrate,as a broad-spectrum quinolone antibacterial drug with chiral amine structure,has always been a key technical difficulty affecting its production due to the asymmetric synthesis of its five-membered ring key intermediate.Transaminases(TAms;EC2.6.1.X)are widely present in nature and play a very important role in the process of biological cell nitrogen metabolism.Transaminase has many advantages such as high enantioselectivity and regioselectivity,high reaction rate and stability,and environmental friendliness.In summary,the development of an advanced,stable and low-cost transaminase-catalyzed production of sitafloxacin five-membered ring key intermediates is an inevitable requirement of the market,and it is of great significance to promote the domestic market of the drug.In this paper,three Bacillus strains that have been sequenced in the laboratory were enployed,and all transaminase genes were conducted preliminary bioinformatics analysis and classification,and the transaminase genes with specific activity potential were screened out;and the gene was induced,expressed,purified and verified for activity;furthermore,the structure of transaminase and the corresponding catalytic substrate mechanism were explored;for the process of enzymatic asymmetric synthesis of sitafloxacin,the transformation of transaminase and its application in the synthesis of sitafloxacin key intermediates were studied through the integration of modern medicinal chemistry,analytical chemistry,bioinformatics,genetic engineering and other technologies.These results laid a foundation for the fermentation process of transaminase produced by Bacillus and the chiral synthesis of related substrates by using the enzyme.The main results as following:(1)A total of 89 putative aminotransferase genes that may be useful as biocatalysts were identified in the three Bacillus genomes by genome annotation,including ota1-28,otae1-32,and otaf1-29.By domain analysis and classification of transaminase genes in three Bacillus genomes,enzymes encoded by genes ota3,ota8,otae6,otae21,otaf1,otaf8 and otaf26 belong to PLP-dependent enzyme class IV;enzymes encoded by genes ota11,ota20,ota27,otae17,otae22,otae27 and otaf29 belong to glutamine transaminase II;enzymes encoded by genes ota5 and otae24 possess an aminomethyltransferase folate binding domain;the enzyme encoded by ota14 belongs to the aldehyde dehydrogenase superfamily of NAD(P)+-dependent enzymes;the enzyme encoded by otaf4 belongs to the aminotransferase class-V superfamily;and enzymes encoded by the remaining 71 aminotransferase genes belong to aspartate aminotransferase(AAT)superfamily(type I).Furthermore,enzymes encoded by genes otae1 and otaf13 not only have AAT superfamily(type I)characteristics,but also possess an adenylate forming domain,and features of the class I superfamily and condensing enzymes.According to the phylogenetic tree analysis and structure domain classification,transaminase gene ota3,ota8,otae6,otae21,otaf1,otaf8 and otaf26 has the very high homology with the typical ?-TAms from Arthrobacter sp.KNK 168,and has the characteristics of ?-TAms.The active sites of the seven transaminase genes(catalytic sites,substrate-cofactor binding pocket and PLP-binding sites)are highly conserved.Thus,the transaminases expressed by these seven transaminase genes(BPTA-1-7)may have similar biochemical activity to the typical ?-TAms from Arthrobacter sp.KNK 168,but due to low sequence similarity,these enzymes may be novel transaminases that have not yet been explored.(2)A preliminary bioinformatics analysis of the transaminase gene ota3 in the Bacillus altitudinis W3 genome and the typical(R)-selective ?-TAm gene from Arthrobacter sp.KNK168 has revealed that these two genes have high homology and low sequence identity(24.7%).The domain analysis found that the domains of the two enzymes have very high conservation,the result indicated that the enzyme encoded by the gene ota3 may have similar transaminase activity.The protein BPTA-1 and typical(R)-selective ?-TAm have the same half-life in E.coli,yeast and mammalian reticular cells in vitro.Neither protein has a "standard" secretion signal peptide that is transported by the Sec translocator and cleaved by signal peptidase I(Lep).In contrast,the ORF of the gene ota3 covers the entire gene,whereas the typical(R)-selective ?-TAm does not.The instability index(II)of protein BPTA-1 is lower than that of typical(R)-selective ?-TAm,and theoretically,the stability of protein BPTA-1 should be higher.In order to increase the expression level of ota3 in E.coli,the ota3 gene was codon-optimized by making use of E.coli codon preference principle.The molecular weights of the purified proteins were both ?33.4 k Da.Furthermore,enantioselectivity tests with(S)-and(R)-?-phenethylamine revealed its(R)-selectivity.The optimal conditions for catalytic reaction were 45°C and p H 7.0,and BPTA-1 retained stability at 20°C and p H 7.0.(3)Transaminase BPTA-2 and BPTA-1 were compared and analyzed,we found that they have commonalities,for example,the catalytic site is lysine,and they are both homodimers,none of they have the "standard" secretion signal peptide that is transported by the Sec translocator and cleaved by signal peptidase I(Lep).It also revealed the differences between the two enzymes,such as substrate cofactor binding pocket,molecular weight and proportion of secondary structure.We found that this residue V151 may be the key reason for the difference in enzyme activity between BPTA-2 and BPTA-1.The molecular weights of BPTA-2 were both ?40.0 k Da.And the results of CD spectroscopy measurements indicated that BPTA-2 has lower sensitivity to the surrounding environment than BPTA-1 due to its higher ?-helical content.The overall features of the two enzymes were characterized by AFM.And the 2D and 3D AFM imaging results indicated that BPTA-2 and BPTA-1 may exist in nature as aggregations and perform the related transamination reactions in the same form.Compared to BPTA-1,the enzyme BPTA-2 have the same stereoselectivity and higher enzyme activities(2.1207± 0.20 U/mg).The optimal conditions for catalytic reaction were35°C and p H 9.0,and BPTA-2 retained stability at 20°C and p H 7.0.And BPTA-1 was more sensitive to p H and temperature than BPTA-2,so that the catalytic reactions involved have higher requirements on the surrounding environment.(4)Accoding to atomic force microscope imaging experiments,we found that the size of the mutant I215 M single enzyme cluster was larger than that of the wild enzyme BPTA-1(1.32 times the diameter of BPTA-1).Biochemical characteristics and enzyme activity of wild-type and mutant enzymes were initially analyzed.Compared with the wild enzyme,the activity of mutant I215 M,I215F and Y32 L increased by 7.03 times,2.80 times and 9.75 times,respectively;K155A,I215 V and T252 A completely lost enzyme activity.Residues K155 and T252 had a particularly strong influence on catalytic activity.However,with(S)-5-benzyl-5-azaspiro[2.4]heptane-7-amine as the catalytic substrate,the constructed single-point mutants were still unable to catalyze its conversion.(5)Four multi-point mutants(L212M/I215 M,Y32L/S190A/L212M/I215 M,Y32L/Y159F/T252 A and Y32W/Y159F/I215M/T252A)have conversion rates of 77.4%,79.0%,79.0% and 77.1% for(S)-5-benzyl-5-azaspiro[2.4]heptane-7-amine,respectively.Among them,mutants Y32L/Y159F/T252 A and Y32W/Y159F/I215M/T252 A can catalyze conversion of(R)-?-phenethylamine,albeit at an extremely low rate(<8%).In summary,mutants L212M/I215 M and Y32L/S190A/L212M/I215 M are more suitable for industrial production of the antibiotic,sitafloxacin,via an enzymatic approach.This work not only laid the experimental foundation and potential for the biological enzyme production of the sitafloxacin hydrate intermediate,it also provided insights into the structure and function relationship between transaminase BPTA-1 activity and substrate selectivity.In addition,this method establishes a theoretical and experimental basis for the rapid screening of chiral amine drugs for enzymatic production. |
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