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Gamma glutamyl transpeptidase from Bacillus pumilus KS12 (GGTBP) was cloned, expressed in pET-28-E. coli expression system as a heterodimeric enzyme with molecular weights of 45 and 20kDa for large and small subunit, respectively. It was purified by nickel affinity chromatography with hydrolytic and transpeptidase activity of 1.82U/mg and 4.35U/mg, respectively. Sequence analysis revealed that GGTBP was most closely related to Bacillus licheniformis GGT and had all the catalytic residues and nucleophiles for autoprocessing recognized from E. coli. It was optimally active at pH 8 and 60°C. It exhibited pH stability from pH 6–9 and high thermostability with t_1/2 of 15min at 70°C. It had K_m, V_max of 0.045mM, 4.35μmol/mg/min, respectively. Decoupling of autoprocessing by co-expressing large and small subunit in pET-Duet1-E. coli expression system yielded active enzyme with transpeptidase activity of 5 …