Gamma glutamyl transpeptidase from Bacillus pumilus KS12 (GGTBP) was cloned, expressed in pET-28-E. coil 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 degrees C. It exhibited pH stability from pH 6-9 and high thermostability with t(1/2) of 15 min at 70 degrees C. It had K-m, V-max of 0.045 mM, 4.35 mu mol/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. (C) 2011 Elsevier Inc. All rights reserved.