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.

Rapid Purification and Characterization of γ‐Glutamyl‐Transpeptidase from Shiitake Mushroom (Lentinus Edodes)

“…Summary of the purification of GGT is given in Table 1. In a previous work (Shuai and others 2011), the final purification fold and recovery of GGT from Bacillus subtilis were 28.99 and 34.57%, respectively. Another work about the purification of tomato GGT showed a purification of 675‐fold and recovery of 4.1% (Martin and Slovin 2000).…”

Properties of a novel polydatin‐β‐d‐glucosidase from Aspergillus nigerSK34.002 and its application in enzymatic preparation of resveratrol

“…The pure enzyme was identified by MALDI‐TOF/TOF‐MS (UltrafleXtreme, Bruker Daltonics, Bremen, Germany) according to Shuai et al …”

pH‐Dependent hydrolase, glutaminase, transpeptidase and autotranspeptidase activities of Bacillus subtilis γ‐glutamyltransferase

“…The low propensity of serine and alanine to act as acceptor substrates in c-GT-catalyzed transpeptidation reactions has already been established for B. subtilis c-GT [4] and several related enzymes [17,19,28], but this cannot explain the lack of the autotranspeptidation reaction involving glutamine. Such a behavior could be indicative of an inhibitory activity of a component of the reaction mixture towards the enzyme.…”

“…In this study, the donor and the acceptor substrates were used in the enzyme-catalyzed reactions in equimolar amounts, usually at concentrations of 100 mM. This approach gave us the opportunity to gain some insights into the enzyme activity, and to find new and unexpected behaviors with respect to those previously reported [16][17][18][19]40].…”

“…Among the bacterial c-GTs, those from Bacillus species have attracted interest [16][17][18][19], owing to some peculiar features.…”