Search

L -Theanine (L -Th) is a non-protein amino acid in the tea leaves, has particular health benefits and is widely used as a dietary supplement, pharmaceutical, nutritional and cosmetic ingredient, which can be synthesized by γ-glutamyltranspeptidase (GGT, EC 2.3.2.2) with L -glutamine (L -Gln) and ethylamine as substrate. In this study, the gene of GGT from Bacillus amyloliquefaciens SK11.001 controlled by P43 promoter was cloned into Bacillus subtilis WB800 incubation to obtain a highly active alkaline GGT. Further research on targeting mutations and promoter screening can be conducted to construct more favorable L -Th producing engineered bacteria. The properties of GGT were determined and the results showed that the activity of the enzyme was optimal at pH 10.5 and the optimal temperature was 35 °C. GGT was kept below 40 °C for 1 h, with more than 90% of initial activity, and remained more than 80% of initial activity after 12 h at pH 4.0–11.5. The optimal reaction system was incubated in 50 mM carbonate buffer (pH 10.5) with 200 mM L -Gln, 1600 mM ethylamine hydrochloride and 2 U/mL GGT for 5 h at 35 °C, 200 rpm in a shaker. The conversion rate of L -Th was 95%, which was higher than in B. amyloliquefaciens BH072 (40%). [Display omitted] • An alkaline γ-glutamyltranspeptidase (GGT) from Bacillus amyloliquefaciens SK11.001 was identified. • The enzyme showed its maximal activity at 35 oC and pH 10.5. • The conversion rate of L -Theanine reached 95% with 200 mM L -Glutamine, 1600 mM ethylamine hydrochloride, 2 U/mL GGT for 5 h at 35 °C, 200 rpm in a shaker. [ABSTRACT FROM AUTHOR]

Several γ-glutamyl compounds produced from gamma-glutamyltranspeptidase (GGT) have alluring features for food, pharmaceutical, and biotechnology applications. GGT from Bacillus altitudinis IHB B1644 was cloned, followed by an expression in pET-47b(+) Escherichia coli BL21(DE3). Recombinant GGT (Ba GGT) gene subsisted of 1755 bp encoding protein with 585 amino acids, predicted molecular weight, and theoretical pI of 63.3 kDa, and 4.92, respectively. Ectopic expression resulted in inclusion bodies formation at 37 °C. The protein was solubilized and different strategies like dialysis, rapid dilution, and on-column refolding were tried to get active recombinant protein (Ba GGT 1). To get protein in soluble fraction, GGT was over expressed at low temperature (20 °C), and IPTG concentration (0.025 mM) (Ba GGT 2). The heterodimeric enzyme consisted of molecular weight of 40, and 22 kDa for large and small subunits, respectively. The specific activities for Ba GGT 1 and Ba GGT 2 were 263.9, and 497.45 U mg−1, respectively. Ba GGT 1 and Ba GGT 2 showed optimum temperature, and pH at 37 °C and 9, respectively. K m values of 0.8 and 0.2 mM, and V max of 666.67 and 333.33 U mg−1 of protein, were calculated for Ba GGT 1 and Ba GGT 2 , respectively. The results demonstrate potential of Ba GGT in biosynthesis of γ-glutamyl compounds with industrial application. [Display omitted] • Bacillus altitudinis IHBB1644 studied for γ-glutamyltranspeptidase(GGT) expression. • Expression was performed at 37 °C,0.5 mM (BaGGT1) IPTG and 20 °C, 0.025 mM (BaGGT2). • Optimum GGT activity was observed at 37 °C and pH 9. • Difference in kinetic parameters was observed for BaGGT1 and BaGGT2. • BaGGT could be a potential candidate for industrial GGT production. [ABSTRACT FROM AUTHOR]

Abstract: Gamma-glutamyltranspeptidases (γ-GTs) catalyze the transfer of the gamma-glutamyl moiety of glutathione and related gamma-glutamyl amides to water (hydrolysis) or to amino acids and peptides (transpeptidation) and play a key role in glutathione metabolism. Recently, γ-GTs have been considered attractive pharmaceutical targets for cancer and useful tools to produce γ-glutamyl compounds. To find out γ-GTs with special properties we have chosen microorganisms belonging to Geobacillus species which are source of several thermostable enzymes of potential interest for biotechnology. γ-GT from Geobacillus thermodenitrificans (GthGT) was cloned, expressed in Escherichia coli, purified to homogeneity and characterized. The enzyme, synthesized as a precursor homotetrameric protein of 61-kDa per subunit, undergoes an internal post-translational cleavage of the 61 kDa monomer into 40- and 21-kDa shorter subunits, which are then assembled into an active heterotetramer composed of two 40- and two 21-kDa subunits. The kinetic characterization of the hydrolysis reaction using l-glutamic acid γ-(4-nitroanilide) as the substrate reveals that the active enzyme has Km 7.6 μM and V max 0.36 μmol min/mg. The optimum pH and temperature for the hydrolysis activity are 7.8 and 52 °C, respectively. GthGT hydrolyses the physiological antioxidant glutathione, suggesting an involvement of the enzyme in the cellular defense mechanism against oxidative stress. Unlike other γ-GTs, the mutation of the highly conserved catalytic nucleophile, Thr353, abolishes the post-translational cleavage of the pro-enzyme, but does not completely block the hydrolytic action. Furthermore, GthGT does not show any transpeptidase activity, suggesting that the enzyme is a specialized γ-glutamyl hydrolase. The GthGT homology-model structure reveals peculiar structural features, which should be responsible for the different functional properties of the enzyme and suggests the structural bases of protein thermostability. [Copyright &y& Elsevier]

Abstract: The purpose of this work was to analyze the effect of diets that contain several oils whose composition in fatty acids were different, on the kinetic parameters of the gamma-glutamyltranspeptidase (GGTP) and the lipoperoxidation of the epididymis because GGTP controls the level of the glutathione that is an molecule that regulates the level of oxidation protecting the maturation and survival of sperm in the lumen of the epididymis. The caput portion of the epididymis was chosen because the epithelium of this segment synthesizes GGTP. Weaned BALB-c mice were fed a commercial or semi-synthetic diet that contained 5% added olein. The mice were maintained on corn oil or fish oil diet for the first 4–8 months of age. The kinetic variables of the GGTP enzyme, analyzed by means of multiple regression analysis using dummy variables, showed that values were similar in olein and corn oil samples, whereas in samples from the fish oil fed group the enzyme behaved as that in animals maintained on commercial diets. Although there were no variations in maximum velocity (V m) of the enzyme, the K m value, was greater (P <0.0001) for the mice fed the olein and corn diets. These groups contained greater percentages of the monounsaturated fatty acids, palmitoleic (16:1 n-7) and oleic acid, 18:1 n-9. Similarly, the amount of lipid peroxidation was also greater in the olein and corn oil groups with respect to commercial and fish groups. The significant increment in K m of GGTP in the olein and corn groups was correlated with greater amount of monounsaturated fatty acids and lipid peroxidation in the epididymis. In conclusion, modifications of dietary lipid sources differentially modulated the epididymis tissue fatty acid profile, lipid peroxidation amounts, and the K m of GGTP. These effects may alter the metabolism of the natural substrate of GGTP, glutathione, a tripeptide with a powerful antioxidant activity, which is necessary in maintaining the oxidative state of the sperm microenvironment, thereby favoring maturation of the male gametes. [Copyright &y& Elsevier]

Abstract: Both elevated liver enzymes and a family history of diabetes mellitus (FHDM) are independent risk factors for type 2 diabetes. This study evaluates the epidemiological association between elevated liver enzymes and FHDM. Subjects included 3512 women workers without diabetes, hepatitis, a smoking habit, or a history of alcohol intake. Blood samples and personal data were collected from all subjects. Subjects with FHDM had a higher mean body mass index (BMI: 23.9kg/m2 vs. 23.4kg/m2; p =0.003). Laboratory testing also revealed higher mean fasting plasma glucose (FPG: 5.67mmol/L vs. 5.22mmol/L; p <0.001), asparate aminotransferase (AST: 20.0IU/L vs. 19.2IU/L; p =0.049), alanine aminotransferase (ALT: 18.4IU/L vs. 16.7IU/L; p =0.004), gamma-glutamyltranspeptidase (GGT: 24.1IU/L vs. 20.5IU/L; p <0.001), and triglycerides (TG: 1.09mmol/L vs. 1.00mmol/L; p =0.011) for FHDM subjects, when adjusted for age and BMI. Multiple linear regression analysis revealed that FHDM, age, BMI, FPG, and TG were correlated with GGT (p =0.004 for FHDM; p <0.001 for age, BMI, FPG, and TG). Elevated liver enzymes were associated with FHDM. In particular, elevated GGT was related to FHDM, independent of the other variables. Elevated liver enzymes, probably due to fat deposition in the liver, may play a role in increasing the risk of diabetes in individuals with FHDM. [Copyright &y& Elsevier]