Activation and thermal stabilization of a recombinant γ-glutamyltranspeptidase from Bacillus licheniformis ATCC 27811 by monovalent cations

Abstract

The regulation of enzyme activity through complexation with certain metal ions plays an important role in many biological processes. In addition to divalent metals, monovalent cations (MVCs) frequently function as promoters for efficient biocatalysis. Here, we examined the effect of MVCs on the enzymatic catalysis of a recombinant γ-glutamyltranspeptidase (BlrGGT) from Bacillus licheniformis ATCC 27,811 and the application of a metal-activated enzyme to L-theanine synthesis. The transpeptidase activity of BlrGGT was enhanced by Cs⁺ and Na⁺ over a broad range of concentrations with a maximum of 200 mM. The activation was essentially independent of the ionic radius, but K⁺ contributed the least to enhancing the catalytic efficiency. The secondary structure of BlrGGT remained mostly unchanged in the presence of different concentrations of MVCs, but there was a significant change in its tertiary structure under the same conditions. Compared with the control, the half-life (t_1/2) of the Cs⁺-enriched enzyme at 60 and 65 °C was shown to increase from 16.3 and 4.0 min to 74.5 and 14.3 min, respectively. The simultaneous addition of Cs⁺ and Mg²⁺ ions exerted a synergistic effect on the activation of BlrGGT. This was adequately reflected by an improvement in the conversion of substrates to L-theanine by 3.3-15.1% upon the addition of 200 mM MgCl₂ into a reaction mixture comprising the freshly desalted enzyme (25 μg/mL), 250 mM L-glutamine, 600 mM ethylamine, 200 mM each of the MVCs, and 50 mM borate buffer (pH 10.5). Taken together, our results provide interesting insights into the complexation of MVCs with BlrGGT and can therefore be potentially useful to the biocatalytic production of naturally occurring γ-glutamyl compounds. KEY POINTS: • The transpeptidase activity of B. licheniformis γ-glutamyltranspeptidase can be activated by monovalent cations. • The thermal stability of the enzyme was profoundly increased in the presence of 200 mM Cs⁺. • The simultaneous addition of Cs⁺and Mg²⁺ions to the reaction mixture improves L-theanine production.

Keywords: Biocatalytic synthesis; Enzyme activation; Monovalent cations; Thermal stabilization; γ-Glutamyltranspeptidase.