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-1at 37℃ 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 Km= 0.23 mmol·L-1, Vmax= 0.015 mmol·L-Lmin-1 and the activation energy Ea =10.50 kJ·mol-1.The above Km and Ea values are significantly lower than those of acylation reaction using GGT without treated with Ca2+. The inactivation energy Ed 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.%γ-谷氨酰转肽酶(γ-glutamyltranspeptidase,GGT)可将γ-谷氨酰基从酰基供体催化转移至相应的受体上,形成新的γ-谷氨酰基化合物.今针对GGT稳定性差的缺陷,采用添加Ca2+的方法对其进行稳定化.研究了Ca2+对枯草芽孢杆菌GGT活性及热稳定性的影响.以4 mmol·L-1 CaCl2于37℃下处理30 min后,GGT的热稳定性得到了显著改善,且随温度升高Ca2+处理的效果更明显;测定了Ca2+处理后GGT酰基化反应动力学常数Km、Vmax和反应活化能Ea,其值分别为0.23 mmol·L-1,0.015 mmol·L-1·min-1和10.50 kJ·mol-1.与未经Ca2+处理时相比,酰基化反应米氏常数和反应活化能显著降低;GGT的失活反应活化能Ed为60.47 kJ·mol-1,与未加Ca2+时(50.42 kJ·mol-1)相比明显提高.表明Ca2+能提高GGT的活性和热稳定性,有效缓解酶的受热失活.
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