γ-Glutamyl transpeptidase from Bacillus pumilus KS 12: Decoupling autoprocessing from catalysis and molecular characterization of N-terminal region

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 20 kDa for large and small subunit, respectively. It was purified by nickel affinity chromatography with hydrolytic and transpeptidase activity of 1.82 U/mg and 4.35 U/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 t1/2 of 15 min at 70 °C. It had Km, Vmax of 0.045 mM, 4.35 μmol/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 5.31 U/mg. Though N-terminal truncations of rGGTBP upto 95 aa did not affect autoprocessing of GGT however activity was lost with truncation beyond 63 aa.

► GGT from Bacillus pumilus KS-12 has been cloned and expressed in E. coli as a heterodimeric protein with sub-unit molecular mass of 45 and 20 kDa. ► Co-expression of large and small subunits revealed that autoprocessing can be decoupled from catalytic activity. ► N-terminal truncations of rGGT revealed that 95 aa from the N-terminal did not affect the autoprocessing of the enzyme however, activity was lost with truncation beyond 63 aa suggesting that it is required for the proper folding of the enzyme.