Identification and Characterization of a Gamma-Glutamyl Transpeptidase from a Thermo-Alcalophile Strain of Bacillus pumilus

Abstract

A gamma-glutamyltranspeptidase (GGT, E.C. 2.3.2.2) was isolated from a strain (A8) originating from Lake Bogoria (Kenya) and homologous with Bacillus pumilus. This GGT shows an optimal activity at pH 8.9 and 62 degrees C. The enzyme is thermostable up to 43 degrees C. The best reagent among the potential inhibitors was shown to be DON, which is an inhibitor highly specific for GGTs. Gly-Gly-Ala, Gly-Gly-Gly and Gly-Gly were identified as the best acceptors for the transpeptidation reactions catalyzed by the enzyme. The SDS-PAGE study revealed that the enzyme consists of two non-identical subunits (38,000 and 23,000). Only the large subunit was active when the enzyme was dissociated under denaturing conditions. The behavior of the native enzyme suggests that the active site of the large subunit is masked by the small subunit.

Improved catalytic efficiency of a monomeric γ-glutamyl transpeptidase from Bacillus licheniformis in presence of subtilisin

“…S showed similar kinetics as GGT 67 with K m 0.22 mM and V max 12.5 mM min -1 . The K m of heterodimeric GGT 67 was higher as compared to earlier reports from recombinant GGT in E. coli and other bacilli (Lin et al 2006;Moallic et al 2006). Similarly of the three, GGT 30 ?…”

“…1). The molecular masses of the GGT from the other bacterial strains are in range of 60-68 kDa, with 40-48 kDa and 18-38 kDa larger and smaller subunit respectively (Nakayama et al 1984;Abe et al 1997;Suzuki and Kumagai 2002;Moallic et al 2006;Wu et al 2006;Lin et al 2006). However, this is the first report of a monomeric active GGT 30 from B. licheniformis.…”

“…2b). Generally lower temperature optima ranges 37-45°C have been reported for E. coli, B. subtilis TAM-4 and B. pumilus (Suzuki et al 1986;Abe et al 1997;Moallic et al 2006). Among the three GGT 30 was most thermostable at 60°C with t 1/2 of 240 min followed by GGT 30 ?…”

“…2a). Most of the reported GGT showed optima at pH 8 (Suzuki et al 1986;Abe et al 1997;Moallic et al 2006). GGT 30 and GGT 67 were maximally active at 60°C and GGT 30 ?…”

“…Extracellular GGT from some Bacillus strains, viz. B. pumilus, B. subtilis TAM-4 and B. subtilis NX-2, B. subtilis (natto) have been characterized (Ogawa et al 1991;Abe et al 1997;Moallic et al 2006;Wu et al 2006). Further, a truncated GGT from B. licheniformis has been cloned and expressed in E. coli (Lin et al 2006), but native GGT from B. licheniformis has not been described.…”