Properties and catalytic mechanism of -glutamyltranspeptidase from NX-2

Abstract

Since ?-glutamyltranspeptidase (GGT) especially catalyses the transfer of the ?-glutamyl moiety to a variety of amino acids and short peptides, GGT has important practical value for enzymatic synthesis of ?-glutamyl compounds. In this paper, the GGT produced from Bacillus subtilis NX-2 was purified by a combination of ammonium sulfate fractionation and ion exchange chromatography, and the properties of purified GGT were investigated. At the conditions of pH 10.0, D-glutamine (D-Gln)/L-tryptophan (L-Trp) with a molar ratio of 5 : 7, a temperature 40°C and a reaction time of 4 h, a higher conversion rate of 42% was obtained. According to the time course, the catalytic mechanism of enzymatic synthesis of ?-D-glutamyl-L-tryptophan (?-D-Gln-L-Trp) 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 affinity parameter of GGT to D-Gln (Km) was 5.08 mmol·L^-1 and the maximum reaction rate of transpeptidation (r_max) was determined as 0.034 mmol·min^-1·L^-1, while the affinity parameter of GGT to ?-D-Gln-L-Trp (K’_m) was 2.267 mmol·L^-1, and the maximum reaction rate of hydrolysis (r’_max) was 0.012 mmol·min^-1·L^-1.

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