- Enzymatic Synthesis of γ-Glutamylvaline to Improve the Bitter Taste of Valine
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The taste of several bitter amino acids is reduced, sourness produced, and preference increased by γ-glutamylization. An enzymatic method for synthesizing γ-Glu-Val involving bacterial γ-glutamyltranspeptidase (GGT) was developed. The optimum reaction conditions for the synthesis of γ-Glu-Val were 20 mM Gln, 300 mM Val, and 0.04 U/ml GGT, pH 10. After 3-hr incubation at 37 °C, 17.6 mM γ-Glu-Val was obtained, with the yield being 88%. γ-Glu-Val was purified on a Dowex 1 × 8 column and then identified by NMR.
- Suzuki, Hideyuki,Kato, Kenji,Kumagai, Hidehiko
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- Effect of the inserted active-site-covering lid loop on the catalytic activity of a mutant: B. subtilis γ-glutamyltransferase (GGT)
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Γ-Glutamylpeptides are compounds derived from the acylation of an amino acid or a short peptide by the γ-carboxyl carbon of the side chain of glutamic acid. Due to their altered chemico-physical and organoleptic properties, they may be interesting substitutes or precursors of parent compounds used in pharmaceutical, dietetic and cosmetic formulations. Some of them are naturally occurring flavor enhancers or are endowed with biological activities. Enzymatic approaches to the synthesis of γ-glutamyl derivatives based on the use of γ-glutamyltransferases (GGTs, EC 2.3.2.2) have been proposed, which should be able to alleviate the problems connected with the troublesome and low-yielding extraction from natural sources or the non-economical chemical synthesis, which requires protection/deprotection steps. With the aim of overcoming the current limitations in the use of GGTs as biocatalysts, a mutant GGT was investigated. The mutant GGT was obtained by inserting the active-site-covering lid loop of the E. coli GGT onto the structure of B. subtilis GGT. With respect to the wild-type enzyme, the mutant showed a more demanding substrate specificity and a low hydrolase activity. These results represent an attempt to correlate the structural features of a GGT to its different activities. However, the ability of the mutant enzyme to catalyze the subsequent addition of several γ-glutamyl units, inherited by the parent B. subtilis GGT, still represents a limitation to its full application as a biocatalyst for preparative purposes.
- Massone, Michela,Calvio, Cinzia,Rabuffetti, Marco,Speranza, Giovanna,Morelli, Carlo F.
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- PH-Dependent hydrolase, glutaminase, transpeptidase and autotranspeptidase activities of Bacillus subtilis γ-glutamyltransferase
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γ-Glutamyltransferases (γ-GTs) are heterodimeric enzymes that catalyze the transfer of a γ-glutamyl group from a donor species to an acceptor molecule in a transpeptidation reaction through the formation of an intermediate γ-glutamyl enzyme. In our search for a γ-GT from a generally recognized as safe microorganism suitable for the production of γ-glutamyl derivatives with flavor-enhancing properties intended for human use, we cloned and overexpressed the γ-GT from Bacillus subtilis. In this study, we report the behavior of B. subtilis γ-GT in reactions involving glutamine as the donor compound and various acceptor amino acids. The common thread emerging from our results is a strong dependence of the hydrolase, transpeptidase and autotranspeptidase activities of B. subtilis γ-GT on pH, also in relation to the pKa of the acceptor amino acids. Glutamine, commonly referred to as a poor acceptor molecule, undergoes rapid autotranspeptidation at elevated pH, affording oligomeric species, in which up to four γ-glutamyl moieties are linked to a single glutamine. Moreover, we found that d-glutamine is also recognized both as a donor and as an acceptor substrate. Our results prove that the B. subtilis γ-GT-catalyzed transpeptidation reaction is feasible, and the observed activities of γ-GT from B. subtilis could be interpreted in relation to the known ability of the enzyme to process the polymeric material γ-polyglutamic acid.
- Morelli, Carlo F.,Calvio, Cinzia,Biagiotti, Marco,Speranza, Giovanna
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p. 232 - 245
(2014/01/23)
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- Molecular cloning and characterization of γ-Glutamyltranspeptidase from pseudomonas nitroreducens IFO12694
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y-Glutamyltranspeptidase from Pseudomonas nitroreducens IFO12694 (PnGGT) exhibited higher hydro-lytic activity than transfer activity, as compared with other y-glutamyltranspeptidases (GGTs). PnGGT showed little activity towards most of L-amino acids and towards glycyl-glycine, which is often used as a standard y-glutamyl accepter in GGT transfer reactions. The preferred substrates for PnGGT as a y-glutamyl accepter were amines such as methylamine, ethylamine, and isopropylamine.
- Imaoka, Masashi,Yano, Shigekazu,Okumura, Masashi,Hibi, Takao,Wakayama, Mamoru
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experimental part
p. 1936 - 1939
(2011/06/11)
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- Synthesis of γ-Glutamyl Peptides Catalyzed by Transamidase from Bacillus natto
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Crude ammonium sulfate fraction of a cell free extract from Bacillus natto contained an enzyme (or enzymes) which catalyzed the transamidation reaction specific for glutamine.Both L- and D-isomers of glutamine were active as substrate.On incubation of L- or D-glutamine with the enzyme preparation, two peptides consisting of glutamic acid and glutamine were formed.The main component of the peptides was readily isolated by ion-exchange chromatography and identified as γ-glutamylglutamine by paper chromatography and by paper electrophoresis using authentic peptides.The optical configuration of the amino acid residues in the dipeptide was determined by digestion of the acid hydrolyzate with L-glutamic acid decarboxylase, and the result showed that the dipeptide obtained from L-glutamine was a L-L isomer, while the dipeptide from D-glutamine was a D-D isomer.
- Noda, Kosaku,Igata, Keiko,Horikawa, Yoshiko,Fujii, Hisao
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p. 2419 - 2424
(2007/10/02)
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