Molecular Cloning and Characterization of γ-Glutamyltranspeptidase fromPseudomonas nitroreducensIFO12694

Abstract

-Glutamyltranspeptidase from Pseudomonas nitroreducens IFO12694 (PnGGT) exhibited higher hydrolytic activity than transfer activity, as compared with other -glutamyltranspeptidases (GGTs). PnGGT showed little activity towards most of L-amino acids and towards glycyl-glycine, which is often used as a standard -glutamyl accepter in GGT transfer reactions. The preferred substrates for PnGGT as a -glutamyl accepter were amines such as methylamine, ethylamine, and isopropylamine.

Sensitive Enzymatic Method for the Quantification of Theanine, a Principal Umami Component of Commercial Tea Beverages

“…Calibration curve for theanine Enzymatic determination of theanine concentration in tea samples was performed by following the decrease in absorbance of DCIP at 600 synthesis from glutamine and ethylamine effectively by a transfer reaction, it can also strongly hydrolyze theanine to glutamic acid and ethylamine. PnGGT showed higher hydrolytic activity towards γ-glutamyl compounds than transfer activity, as well as little transfer activity towards most Lamino acids typically used as γ-glutamyl acceptors (Imaoka et al, 2010). Since this type of transfer reaction has not been reported for any other GGTs to date, this reaction characteristic is suited for the determination of theanine concentration in tea samples, and PnGGT is the more appropriate enzyme to use as compared with commercially available mammalian GGTs such as bovine GGT (Furukawa et al, 1983).…”

“…Tris (hydroxymethyl) aminomethane (Tris) and polyvinylpolypyrrolidone (PVPP) were from Nacalai Tesque (Kyoto, Japan). PnGGT and PdADH were prepared according to previously described procedures (Imaoka et al, 2010;Takagi et al, 1999). Appropriately purified PnGGT and PdADH had specific activities of 34.4 U/mg and 0.863 U/mg, respectively.…”

Synthetic transport systems in bacteria

“…In order to ensure efficient cargo release in the cytoplasm of E. coli, it was important to ensure (I) efficient expression of GGT in the cytoplasm of E. coli, (II) high hydrolytic and low transpeptidase activity even in the presence of a suitable peptide or amino acid acceptor, and (III) a wide substrate range. As promising candidates we identified the variants EcGGT from E. coli, which is well-characterized [120,124] and for which a protein structure is available [121], and PnGGT from P. nitroreducens, which has a sequence identity to EcGGT of 45%, was reported with a higher hydrolytic than transpeptidase activity and a broad substrate range [118]. First, we investigated the influence of the location of a hexahistidine tag (6xHis-tag) on expression and activity of both variants.…”

“…GGT is mainly responsible for the degradation of glutathione in the periplasmic space, but in addition, it is known to catalyze the hydrolysis of a broad range of other γ-substituted glutamyl compounds [118][119][120]. Binding of a γ-glutamyl compound to the active site of the enzyme leads to the formation of a γ-glutamyl-enzyme intermediate and results in release of an amine cargo.…”

Synthesis of a Bifunctional Fluorescent Polymer for Cell Imaging and Enzyme Detection

“…γ‐Glutamyltranspeptidase (GGT) is considered to be an important marker for liver carcinoma and is widely used in clinical diagnosis, therefore, the development of simple, rapid detection of GGT is very urgent. Besides its natural substrate glutathione, GGT catalyzes the transfer of γ‐glutamyl group from artificial substrates to amino acids and peptides 18–20. In this paper, we used the synthesized conjugated polymer PFPBOH for GGT detection.…”