Gamma-glutamyl transpeptidase from a mesophilic bacterium Bacillus atrophaeus GS-16 (BaGGT) was expressed heterologously in E. coli using pET-51b vector. Maximum production of BaGGT was obtained at 16 degrees C after 16 h of IPTG induction and the protein, in its native conformation, was active as a heterooctamer which was composed of four heterodimeric units combined together. One heterodimeric unit constituted two subunits with molecular masses of 45 kDa and 21 kDa, respectively. The recombinant enzyme was purified by one step His-tag affinity purification protocol with a specific activity of 90 U/mg and 5.2 fold purity. The purified enzyme had a pH optimum of 10.0 and temperature optiinum of 50 degrees C. It exhibited broad pH stability (6.0-12.0) and was thermostable (t(1/2) of 54 min at 50 degrees C). The enzyme was completely inactivated by Pb2+ ions and strongly inhibited in presence of N-bromosuccihimide, azaserine and 6-diazo-5oxo-L-norleucine. Kinetic characterization of BaGGT using GpNA as a donor and glycylglycine as acceptor revealed that it had a K-m of 0.15 mM and 0.37 mM and V-max of 23.09 mu mol/mg/min and 121.95 mu mol/mg/min for hydrolysis and transpeptidation reactions, respectively. BaGGT also displayed broad substrate specificity for various amino acids. It was studied for its prospective use in the synthesis of an immunomodulatory peptide, gamma-D-glutamyl-L-tryptophan. After optimization of various process parameters, a conversion rate of 50%, corresponding to 25 mM product yield, was achieved within 6 h of incubation using 50 mM D-glutamine as donor and 50 mM L-tryptophan as acceptor and 0.3 U/mL of BaGGT in the reaction, performed at pH 10.0 and 37 degrees C. The product was purified to homogeneity using Dowex 1 x 2 column and its purity was confirmed by HPLC and H-1 NMR. (C) 2017 Elsevier Inc. All rights reserved.