Gamma Glutamyl Transpeptidase from Brevibacillus Brevis: Production, Biochemical Characterization and Expression in E. Coli

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environment to form cysteine and glycine molecules (Meister and Tate, 1976).

Once glutathione is cleaved by γ -GT, the constituent moieties can be actively transported into the cell.. The cysteine molecule can be used by the cell in the de novo synthesis of glutathione or by the cell for protein or DNA synthesis. The γ-glutamyl-cysteine molecule can either enter the cycle to manufacture glutathione with the aid of the enzyme glutathione synthetase or be cleaved by y-glutamyl cyclotransferase to form cysteine and 5- oxo-proline .De novo synthesis of glutathione occurs through the action of the enzyme y-glutamylcysteine synthetase (γ -GC synthetase) and amino acids glutamic acid, cysteine and glycine.

An overview on the biochemical properties of γ-glutamyl transpeptidase

1. Temperature Optima

The optimal temperature of GGT activity has been seen to vary considerably with respect to organism under consideration. The optimal temperature of GGT activity for E. Coli Novablue has been observed at 37˚C (Yao et al., 2006) which was similar to that of Bacillus licheniformis (Lin et al., 2006) and Bacillus subtilisNX-2 (Wang et al., 2008). However, optimal temperature was as low as 20˚C for E. Coli K-12 (Suzuki et al., 1986) and as high 62˚C for Bacillus pumulisA8 (Moallic et al., 2006). The temperature stability was reported in case of E.coli K-12 and the enzyme was found to retain 84% activity at 45˚C for 15 min and lost 92% activity on incubation at 50˚C (Suzuki et al., 1986).

2. pH optima

GGTs are known to be alkalophilic enzymes thus are likely to have pH optima above pH 7.0. Unlike temperature, there was not much difference between the optimal pH of the enzyme with the pH optima lying within the range of pH 6-8 for all reported microbial GGTs. Strong activity of the enzyme was also noted at pH 9. Exception to this, however, was reported by (Wang et al. 2006) who have reported maximum activity of GGT from B .subtilis NX-2 at pH 10.

3. Effect of Metal ions

The activator or inhibitory effects of mono and divalent cations on GGT activity has also been studied. In case of B. pumilus Li+, K+, Ca2+, Mn2+ and Co2+ had no significant effect whereas Zn2+ and Cu2+ slightly inhibited the activity by about 30 % (Moallic et al., 2006). Lin et al. 2006reported no significant effect of Co2+ on enzyme activity, inhibitory effect in presence of Hg2+, Zn2+, Pb2+ and Ni2+ and strong activation in presence of chloride salts of Mg2+, K+ and Na+ in B. licheniformis.

4. Effect of Inhibitors

The effect of different inhibitors has been tested on γ-glutamyl transpeptidase purified from different sources. In case of Bacillus pumilus A8 partial inhibition by chelating agents such as EDTA and EGTA have been observed but not by 1,10 phenanthroline which is known as specific inhibitor of metallopeptidase. Phenylmethylsulfonyl fluoride (PMSF) which specifically inhibits the enzyme containing a serine residue at the catalytic site produced moderate inhibition of the activity. These effects can be compared to those described by Suzuki et al (1986) for E.coli GGT and Inoue et al, (1978) for rat kidney GGT. Specific inhibitors of GGT include 6-diazo-5-oxo-L-norleucine (DON), azaserine and L-serine in presence of borate buffer. These inactivate the enzyme by acting as γ- glutamyl analogs reacting specifically and stoichiometrically to the γ- glutamyl site of the enzyme (Tate & Meister, 1976). These could mimic the transition state of the reaction and thus totally inhibiting the enzyme activity.

Apart from its physiological importance, GGT has also been recognized as one of the important enzymes with a wide range of industrially viable applications.

• Improvement of taste on γ-glutamylation- Amino acids such as phenylalanine, valine, leucine and histidine are added to nutritional supplements at high concentrations. However, these L-amino acids taste bitter. It was found that when a bitter amino acid is γ-glutamylated, its bitterness is reduced dramatically and the sourness is increased (Suzuki et al, 2002). The sourness gives a refreshing lemon-like taste, so preferences for γ-glutamyl amino acids have increased.

• Increase in solubility with γ-glutamylation- Some amino acids and peptides having low solubility in water become more soluble following γ-glutamylation. Solubility in water is critical to the use of a compound for medication, therefore γ-glutamylation is an effective tool to increase the solubility of certain compounds used as drugs.

• Increase in stability of compounds in serum by γ-glutamylation- γ-glutamylation increases the stability of compounds in the bloodstream. γ-glutamyl linkage cannot be cleaved by normal peptidases in serum, the half lives of the compound becomes much longer with γ-glutamylation (Hara et al, 1992) and the γ-glutamyl linkage is first cleaved in organs which express GGT. Therefore, some γ-glutamyl compounds may possibly be used as pro-drugs specific for the organs that express GGT.

• Increase in stability of glutamine by γ-glutamylation- Glutamine is a known to be an important amino acid that protects the intestinal tract (Thomas et al, 2005) and liver (Bergamini et al, 1995) and increases immune competence. It has also been reported that the degradation of protein from muscle by exercise was decreased by the ingestion of glutamine. However, glutamine is highly unstable in aqueous solution and is converted into pyroglutamic acid. It has been found out that γ-glutamylation can stabilize glutamine in aqueous solutions and also tastes better than glutamine.

• γ-glutamyl compounds produced by the enzymatic method using bacterial GGT-

1. γ-L-glutamyl-L-DOPA- γ-L-glutamyl-L-DOPA was produced in high yields by bacterial GGT (Kumagai et al, 1998). After its administration to mice, it was observed that amount of dopamine in the brain increases markedly (Ichinose et al, 1987). Hence, role of γ-L-glutamyl-L-DOPA as a pro-drug for Parkinson’s disease is promising.

2. γ-glutamyltaurine- The discovery of this dipeptide in the parathyroid in 1980 and later in brain of mammals gave rise to studies on intrinsic and synthetic taurine peptides. GGT has been suggested to be responsible for its synthesis in vivo (Bittner et al, 2005). It is potent anti epileptic agent when injected directly into the kindlind site; it prevents electroconvulsive shock induced amnesia.

3. γ-D-glutamyl-L-tryptophan- It stimulates the differentiation of T-lymphocytes and specific immune response and enhances interleukin