Properties and catalytic mechanism of γ-glutamyltranspeptidase from B.subtilis NX-2

Since it can specially catalyze the transfer of γ-glutamyl moiety, the γ-glutamyltranspeptidase (GGT) has important practical value for the synthesis of analogues of γ-glutamyl-containing compounds. In order to obtain high purity GGT, the GGT produced from Bacillus subtilis NX-2 was purified by ammonium sulfate fractional precipitation and then two-step ion exchange chromatography of DEAE Sepharose FF and Source 15Q. The enzymology properties of GGT in the synthesis of γ-D-Gln-L-Trp (SCV-07) were investigated, and the optimal synthesis conditions of pH 10.0, the γ-glutamyl donor (D-Gln) to acceptor (L-Trp) ratio of 5:7, reaction temperature of 40°C and reaction time of 4 h were found, under these optimal conditions, high conversion ratio reached 42%. According to the conversion process curve of γ-D-Gln-L-Trp, the catalytic mechanism of GGT was discussed. It was demonstrated that the GGT can catalyze not only the reaction of transpeptidation, but also the irreversible hydrolysis of the products, which results in the decrease of the yield of the products. The Michaelis constant of transpeptidation reaction and its maximum reaction rate were determined as Km=5.08 mmol·L^-1 and r_max=0.034 mmol·(min·L)^-1, respectively, while the Michaelis constant of hydrolysis reaction and its maximum reaction rate were measured respectively as Km'=2.267 mmol·L^-1 and r_max'=0.012 mmol·(min·L)^-1.