title: Heterologous expression of γ-glutamyl transpeptidase from Bacillus atrophaeus GS-16 and its application in the synthesis of γ-d-glutamyl-l-tryptophan, a known immunomodulatory peptide.
abstract: 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°C after 16h 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 45kDa and 21kDa, respectively. The recombinant enzyme was purified by one step His-tag affinity purification protocol with a specific activity of 90U/mg and 5.2 fold purity. The purified enzyme had a pH optimum of 10.0 and temperature optimum of 50°C. It exhibited broad pH stability (6.0-12.0) and was thermostable (t1/2of 54min at 50°C). The enzyme was completely inactivated by Pb2+ions and strongly inhibited in presence of N-bromosuccinimide, 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 Kmof 0.15mM and 0.37mM and Vmaxof 23.09μmol/mg/min and 121.95μmol/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, γ-d-glutamyl-l-tryptophan. After optimization of various process parameters, a conversion rate of 50%, corresponding to 25mM product yield, was achieved within 6h of incubation using 50mM d-glutamine as donor and 50mM l-tryptophan as acceptor and 0.3U/mL of BaGGT in the reaction, performed at pH 10.0 and 37°C. The product was purified to homogeneity using Dowex 1×2 column and its purity was confirmed by HPLC and H1NMR.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
authors.forename: Meenu
authors.initials: M
authors.lastname: Saini
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
authors.forename: Shruti
authors.initials: S
authors.lastname: Bindal
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India. Electronic address: rani.gupta@south.du.ac.in.
authors.forename: Rani
authors.initials: R
authors.lastname: Gupta
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004151:Dipeptides / Q000096:biosynthesis*; D004795:Enzyme Stability; D005798:Genes, Bacterial; D007218:Industrial Microbiology; D007700:Kinetics; D008970:Molecular Weight; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D005723:gamma-Glutamyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004151:Dipeptides; D011994:Recombinant Proteins; C102867:gamma-glutamyltryptophan; D005723:gamma-Glutamyltransferase
keywords: Bacillus atrophaeus; Heterologous expression; Immunomodulator; γ-Glutamyl transpeptidase; γ-d-Glutamyl-l-tryptophan