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Murty, NA, Tiwary, E, Sharma, R, Nair, N and Gupta, R (2012) γ-Glutamyl transpeptidase from Bacillus pumilus KS 12: decoupling autoprocessing from catalysis and molecular characterization of N-terminal region. Enzyme Microb. Technol. 50:159-64
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 t(1/2) of 15 min at 70°C. It had K(m), V(max) 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.
Amino Acid Sequence; Bacillus/classification; Bacillus/enzymology; Bacillus/genetics; Bacillus/growth & development; Biotechnology/methods; Catalysis; Chromatography, Affinity; Cloning, Molecular; Dimerization; Escherichia coli/genetics; Escherichia coli/metabolism; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Molecular Sequence Data; Molecular Weight; Mutagenesis, Site-Directed; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Sequence Alignment; Sequence Analysis, DNA; gamma-Glutamyltransferase/chemistry; gamma-Glutamyltransferase/genetics; gamma-Glutamyltransferase/isolation & purification; gamma-Glutamyltransferase/metabolism
|Gene product||Qualifier||GO Term||Evidence Code||with/from||Aspect||Extension||Notes||Status|
|GO:0036374: glutathione hydrolase activity||
ECO:0000314: direct assay evidence used in manual assertion
Fig. 6: E. coli transformed with GGTBP show gamma-glutamyltransferase activity using many different amino acids acceptors.
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