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c-Glutamyltransferases (c-GTs) are heterodimeric enzymes that catalyze the transfer of a c-glutamyl group from a donor species to an acceptor molecule in a transpeptidation reaction through the formation of an intermediate c-glutamyl enzyme. In our search for a c-GT from a generally recognized as safe microorganism suitable for the production of c-glutamyl derivatives with flavor-enhancing properties intended for human use, we cloned and overexpressed the c-GT from Bacillus subtilis. In this study, we report the behavior of B. subtilis c-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 c-GT on pH, also in relation to the pK a 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 c-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 c-GT-catalyzed transpeptidation reaction is feasible, and the observed activities of c-GT from B. subtilis could be interpreted in relation to the known ability of the enzyme to process the polymeric material c-polyglutamic acid.

A number of N2-alkyl and N2-acyl derivatives of guanosine 5'-phosphate (GMP) have been synthesized and tested for their synergistic effect with monosodium L-glutamate (MSG), the prototypical substance imparting umami taste to savory-based foods. Capacities to enhance the taste intensity of MSG (gamma values) were estimated through subjective comparisons of MSG/nucleotide mixtures in water with appropriate solutions of MSG alone. Assuming beta = gamma[nucleotide]/gamma[IMP], beta values of the N2-substituted GMPs were found in the range 1.2-5.7. Such values appear to be related to the chain length of the substituent in the 2-position of the purine nucleus and dependent on the replacement of a CH 2 group with an S atom and/or with an alpha-CO group. These findings indicate that the exocyclic NHR group of the guanine moiety is actively implicated in the synergism between GMP derivatives and MSG. Theoretical calculations suggest that an anti conformation is probably assumed by ribonucleotide molecules interacting with umami receptors.