title: Purification and characterization of γ-glutamyltranspeptidase from Bacillus subtilis SK11.004.
abstract: An extracellular γ-glutamyltranspeptidase (GGT) with a specific activity of 683.4 U/mg was purified to homogeneity from a culture filtrate of Bacillus subtilis SK11.004 in three steps and then characterized. The GGT is composed of one large subunit of 40 kDa and one small subunit of 21 kDa that was determined by SDS-PAGE and a molecular mass of 62 kDa that was determined by gel-filtration chromatography. The purified GGT had an optimal pH and temperature of 10 and 37 °C, respectively, and it was stable at pH 4.0-11.0 or <50 °C. The enzyme exhibited the highest affinity to imino acids (L-Pro) and then decreasing affinities for aromatic amino acids, ethylamine and basic amino acids. The K(m) values of hydrolysis and of transpeptidation for L-Gln were 3.16 mM and 0.83 mM, respectively, suggesting that the GGT likely synthesizes valuable γ-glutamyl peptides using L-Gln as γ-glutamyl donor. The effects of inhibitors on the enzyme suggested that the tryptophan residues and hydroxy groups of Ser or Thr are essential to enzyme activity. Based on the biochemical characteristics of the enzyme and lack of homology to previously identified proteins, it can be concluded that the GGT from B. subtilis SK11.004 is a novel enzyme.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 214122 Wuxi, Jiangsu, People's Republic of China.
authors.forename: Yuying
authors.initials: Y
authors.lastname: Shuai
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
authors.forename: Wanmeng
authors.initials: W
authors.lastname: Mu
authors.forename: Bo
authors.initials: B
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D005723:gamma-Glutamyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005723:gamma-Glutamyltransferase