Effects of Ca²⁺ on the activity and thermal stability of γ-glutamyltranspeptidase from Bacillus subtilis

The γ-glutamyltranspeptidase (GGT) can be used to synthesize the new γ-glutamyl compounds by using γ-glutamyl compounds as acyl donor substrate and transfering their γ-glutamyl moieties to acceptor substrate. However the GGT has the defect of having low thermal stability. In order to improve its poor thermal stability, the method of adding Ca²⁺ was adopted in this study, and the effects of added Ca²⁺ on the activity and thermal stability of GGT obtained from the fermentation of Bacillus subtilis were investigated. It was found that the thermal stability of GGT is greatly improved after it was treated with CaCl₂ of 4 mmol · L^-1 at 37°C for 30 min. As the GGT working temperature increases, the effect of Ca²⁺ treatment on increasing the thermal stability of GGT is more obvious. The kinetic constants of acylation reaction using GGT treated with Ca²⁺ were determined as K_m=0.23 mmol · L^-1, V_max=0.015 mmol · L^-1 · min^-1 and the activation energy E_a=10.50 kJ · mol^-1. The above K_m and E_a values are significantly lower than those of acylation reaction using GGT without treated with Ca²⁺. The inactivation energy E_d of inactivation reaction for GGT was also calculated and it was found to be 60.47 kJ · mol^-1, which is much higher than that of the GGT without treated with Ca²⁺ (50.42 kJ · mol^-1). The study comes to the conclusion that the activity and thermal stability of GGT are obviously improved after treating it with Ca²⁺, and the Ca²⁺ could reduce the deactivation of GGT.