Homologous Cloning and Characterization of Bacillus subtilis 168 γ-Glutamyltranspeptidase

Abstract:

Gammaglutamyltranspeptidase gene from Bacillus subtilis 168 was cloned and overexpressed with pMA5 vector in Bacillus subtilis 168 for overproduction of GGT. GGT was secreted in the extracellular space. The overexpressed enzyme was 3568 fold higher in activity than that from the parent strain and exhibited a specific transpeptidase activity of 49.8 U/mg. The molecular mass of two sub-units was estimated to be 42 000 and 22 000 respectively，by SDS-PAGE. The overexpressed GGT was a very versatile enzyme in alkaline pH. Its optimal temperature was 50 ℃ showing a moderately high thermostability. The effect of ions on purified GGT was also examined. Results showed that NH4+ had the highest activation effect on GGT（>45%），other ions such as Ca2+、K+、Mg2+、Li+ and La3+ had a significant activation effect on the enzyme，whereas Cu2+ and Zn4+ recorded the highest deactivation on GGT activity.