Biocatalysis is gentle and efficient catalytic system, has excellent chemical selectivity, regioselectivity and stereoselectivity. Especially in recent years, genomics, proteomics and other biotech rapid development, greatly promoted the basic and applied research biocatalysis.
Currently, the use of biological systems (such as various cells and enzymes) as Catalytic synthesis of chiral compounds has become a hot topic and bioorganic chemistry and biotechnology research in new growth point of organic synthetic chemistry. This paper uses biocatalytic method for preparing a number of important physiological activity of chiral compounds to glutamyl transferase, aspartate aminotransferase, leucine dehydrogenase and a formate dehydrogenase for the study, combined with the characteristics of the enzyme, in basic research on natural substrate, select some non-normal enzyme substrate catalysis research, and through these studies reveal enzyme - substrate catalysis mechanism. In this paper, high efficiency and high selectivity of the synthesis of these chiral compounds provides a new method, and laid the foundation for their further study. To be able to make γ- glutamyl transferase enzymatic synthesis important γ- glutamyl compound, the catalytic mechanism of the enzyme were investigated. Preparation ten γ- glutamyl aniline analogs them as γ- glutamyl donor for the Y- glutamyl amine receptors, enzyme-catalyzed synthesis theanine, theanine was measured by HPLC generation to evaluate the amount of enzyme activity.
Biocatalysis is gentle and efficient catalytic system, has excellent chemical selectivity, regioselectivity and stereoselectivity. Especially in recent years, genomics, proteomics and other biotech rapid development, greatly promoted the basic and applied research biocatalysis. Currently, the use of biological systems (such as various cells and enzymes) as Catalytic synthesis of chiral compounds has become a hot topic and bioorganic chemistry and biotechnology research in new growth point of organic synthetic chemistry. This paper uses biocatalytic method for preparing a number of important physiological activity of chiral compounds to glutamyl transferase , aspartate aminotransferase, leucine dehydrogenase and a formate dehydrogenase for the study, combined with the characteristics of the enzyme, in basic research on natural substrate, select some non-normal enzyme substrate catalysis research, and through these studies reveal enzyme - substrate catalysis mechanism. In this paper, high efficiency and high selectivity of the synthesis of these chiral compounds provides a new method, and laid the foundation for their further study. To be able to make γ- glutamyl transferase enzymatic synthesis important γ- glutamyl compound, the catalytic mechanism of the enzyme were investigated.
Preparation ten γ- glutamyl aniline analogs them as γ- glutamyl donor for the Y- glutamyl amine receptors, enzyme-catalyzed synthesis theanine, theanine was measured by HPLC generation to evaluate the amount of enzyme activity. In L- γ- glutamyl-p- nitroanilide as substrate for the enzyme conversion conditions such as pH, temperature, substrate molar ratio optimization. Under optimum conversion conditions, respectively enzymatic ten γ- glutamyl aniline formed like theanine, which measured the enzymatic conversion reaction kinetic constants, and converted into Hammett equation, combined Hammett curve shows, γ- glutamyl transferase acylation reaction rate-limiting step of the reaction rate can be electron-withdrawing or electron-donating substituents Y- glutamyl aniline analogs accelerated kinetics of this step by acid-catalyzed acylation reaction, by autodock computer simulation , Asp- 433 carboxyl or phenolic hydroxyl Tyr-444 may be the acid proton source, to further determine the acid protons needed through the implementation of site-directed mutagenesis. Using γ- glutamyl transferase enzymatic synthesis β-N - (γ-L ( +) - glutamyl) phenylhydrazine and β-N- (γ-L (+) - glutamyl) Carboxylazo hydrazine.
Synthesis glutamyl PHENYLHYDRAZINE optimal reaction conditions: preparation pH9 conversion solution, substrate concentration 60mM L- glutamine, 300mM phenylhydrazine, adding 40Uγ, - glutamyl transferase enzyme / ml, 37 ℃ reaction 6h, substrate conversion rate of 93%; synthetic precursor mushrooms leucine, glutamyl carboxyl phenylhydrazine optimal reaction conditions: 50mM L- glutamine, 500mM carboxyl phenylhydrazine, 40U γ- glutamyl transferase / ml, pH8,37 ℃ reaction 24h, product conversion rate of 90%. Although phenylhydrazine was reported as a suicide lacrimal peroxidase inhibitor, but only when phenylhydrazine concentration higher than 300mM only reversible inhibition glutamyl transferase, while the carboxyl phenylhydrazine does not appear even in 1000mM suppression. This new glutamyl transferase enzymatic synthesis method will help mushroom acid synthesis and other important glutamyl compound, and further promote their in-depth study . Optically pure chiral amine is a kind of important value of pharmaceutical and fine chemical intermediates, asymmetric synthesis of chiral amines for a deeper study and application have great economic value. At present, the preparation method of chiral amines are chemical syntheses, chemical and enzymatic resolution and enzymatic synthesis, enzymatic synthesis which has more advantages. In this paper, aspartate aminotransferase, combined with leucine dehydrogenase and formate dehydrogenase catalytic synthesis of chiral amines and amino acids.
To better understand the characteristics of these enzymes, chemically synthesized eight α- keto acid, aspartate aminotransferase higher terephthalic sodium pyruvate and sodium pyruvate o methoxybenzene conversion rate, o-hydroxyphenyl pyruvate sodium and sodium pyruvate dimethylaminobenzaldehyde activity is poor; and for alkyl substituted α- keto acid, 5-methyl-2-keto-4-methyl-sodium has keto acid Sodium conversion activity than the other two alkyl substituents keto good, the highest conversion rate of about 60% benzene sodium pyruvate. In addition, the paper also uses between aspartate aminotransferase catalytic synthesis rivastigmine hydroxyacetophenone chiral amine intermediate 3- (1-aminoethyl) phenol, but aspartate aminotransferase catalytic synthesis of this compound capability It is limited, in the case of 10% DMSO was added, the highest conversion rate of not more than 40%. Combined with leucine dehydrogenase and formate dehydrogenase chiral amine compounds can be prepared.
This paper constructs leucine dehydrogenase and formate dehydrogenase gene engineering bacteria E. coli, joint use of these two enzymes, the above α- acid as substrate, effects of reaction conditions, pH, temperature, the amount of coenzyme NAD Effect of activity at pH9,30 ℃, 1mM NAD in optimal conditions, alkyl-substituted α- keto good response, 24h of the conversion rate at about 90%. In addition to benzene sodium pyruvate, the leucine dehydrogenase and a formate dehydrogenase to a benzene ring substituted α- keto acid catalytic activity is very low. This thesis is based on the enzymatic synthesis of chiral compounds , combined with genetic engineering recombinant expression of enzymes for the synthesis of chiral compounds to broaden the idea, has important theoretical significance and application potential
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