| Biocatalysis, thus applying enzymes for organic synthetic transformations, has become a common applied ’green technology’ for selected asymmetric transformations as well as the excellent stereo-, regio-and chemoselectivity of the biocatalysts exceeding many other methodologies. Furthermore, during the last decade the advancements in genomics and protein engineering allow to design enzymes which fulfil the requirements for industrial scale. Focusing on the past years, enzymatic transformations have been paid more attention, whereby it can easily be noted from the examples published recently. In this study, some significant chiral compounds were synthesized with Escherichia coli y-glutamyltranspeptidase, Escherichia coli aspartate aminotransferase, Bacillus sphaericus leucine dehydrogenase and candida boidinii formate dehydrogenase, these enzymes were used to catalyze the natural and unnatural substrates to further investigate the catalytic mechanism and promote biocatalysis of some chiral compounds.In order to investigate the catalytic mechanism of Escherichia coli γ-glutamyltranspeptidase, ten para-and meta-substituted y-glutamyl anilides were chemically prepared and employed as substrates to synthesize L-theanine to assay the activity of y-glutamyltranspeptidase. The reaction was optimized for γ-glutamyl-p-nitroanilide. Key factors such as substrate specificity, pH, temperature, and substrate mole ratio were all investigated. Kinetic studies of the acyl transfer reaction were described and Hammett plot was constructed. This study indicated that the rate-limiting acylation reaction of γ-glutamyltranspeptidase can apparently be accelerated by either electron-withdrawing or electron-donating substituents of y-glutamyl anilides. The reaction could be catalyzed by the general acid and carboxy of Asp-433or phenolic hydroxyl Tyr-444may be the acid by autodock simulation for all prepared y-glutamyl anilides.A new method for the synthesis of β-N-(γ-L(+)-glutamyl)phenylhydrazine is presented. This compound was prepared from L-glutamine and phenylhydrazine through the transpeptidation reaction of Escherichia coli y-glutamyltranspeptidase, while phenylhydrazine had been reported as a typical inhibitor of the enzyme. The optimum reaction conditions for the production of β-N-(γ-L(+)-glutamyl) phenylhydrazine were60mM L-glutamine,300mM phenylhydrazine,40U y-glutamyltranspeptidase/ml, and pH9in approx.800ml. After6h at37℃, the product was obtained with a conversion rate of93%(mol/mol). y-Glutamyltranspeptidase was reversibly inhibited only when phenylhydrazine concentration was above300mM.A new method for the synthesis of β-N-(γ-L(+)-glutamyl)-4-carboxyphenylhydrazine, a precursor of agaritine, is presented. This compound was prepared from L-glutamine and4-hydrazinobenzoic acid through the transpeptidation reaction catalyzed by the Escherichia coli y-glutamyltransferase. The optimum reaction conditions for the production of β-N-(γ-L(+)-glutamyl)-4-carboxyphenylhydrazine were50mM L-glutamine,500mM4-hydrazinobenzoic acid and40U γ-glutamyltransferase/mL at pH8and37℃for24h. The product was obtained with a conversion rate of90%(mol/mol). γ-Glutamyltransferase activity was not inhibited by4-hydrazinobenzoic acid at concentrations up to1000mM. This simple and efficient method would facilitate the synthesis of glutamyl phenylhydrazine analogues, including agaritine.Enantiomerically pure chiral amines are highly valuable functionalized molecules with a wide range of applications including intermediates for the synthesis of pharmaceutical and agrochemical active ingredients, resolving agents for the separation of enantiomers via diastereomeric salt formation, and ligands for asymmetric synthesis using either transition metal catalysis or organocatalysis. Compared with chemical synthesis and kinetic resolution, the option of preparing chiral amines using biocatalytic approaches is now viewed as attractive as a result of recent developments in biocatalyst availability, methods for improving biocatalyst stability, and the inherent high selectivity and catalytic activity that can be obtained through enzyme catalysis. Eight α-keto acids were synthesized to investigate the characteristics of these enzymes including aspartate aminotransferase, leucine dehydrogenase. For aspartate aminotransferase, the conversion rates of phenylpyruvate and o-methoxyphenylpyruvate substrates were higher than o-hydroxyphenylpyruvate and p-dimethylamino phenylpyruvate. And4-methyl-2-oxovaleric acid and5-methyl-2-oxohexanoic acid were catalyzed well using aspartate aminotransferase when compared with the two other alkyl a-keto acids among which the highest conversion ratio was about sixty percent of the phenylpyruvate. In addition,3-hydroxyacetophenone was considered as the substrate of aspartate aminotransferase to prepare the intermediate of rivastigmine, however, it was unfortunate that the enzyme activity of3-hydroxyacetophenone was less than those a-keto acids which the conversion rate was not higher than40%even in the present of10%DMSO.Leucine dehydrogenase was successfully applied for the synthesis of L-leucine and L-tertiary leucine. The necessary NADH regeneration was performed with formate dehydrogenase with formate as the ultimate hydrogen source. It has been demonstrated that in-situ coenzyme regeneration is in principle economically feasible. In this study, a structural gene (leudh) encoding leucine dehydrogenase from B. sphaericus IFO3525was cloned into Escherichia coli cells and sequenced. And a formate dehydrogenase gene from candida boidinii was cloned, sequenced and over-expressed in E. coli. The available a-keto acids described above were tested as substrates in the coupled reaction system of leucine dehydrogenase and formate dehydrogenase. Key factors such as pH, temperature, and NAD usage were all investigated. The alkyl a-keto acids all reacted well at pH9、30℃、1mM NAD after24h, of which the conversion rates were all approximately90%. However, the phenyl a-keto acids did not proceed smoothly except for phenylpyruvate.The number of examples of drugs and drug intermediates prepared by biocatalytic approaches has significantly increased over the past years, so that this study only got a start in biocatalysis and implied a further development of suitable enzymes for asymmetric organic synthesis in the future. |
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