Bacterial Gamma-Glutamyl Transpeptidase, an Emerging Biocatalyst: Insights Into Structure-Function Relationship and Its Biotechnological Applications.

Gamma-glutamyl transpeptidase (GGT) enzyme is ubiquitously present in all life forms and plays a variety of roles in diverse organisms. Higher eukaryotes mainly utilize GGT for glutathione degradation, and mammalian GGTs have implications in many physiological disorders also. GGTs from unicellular prokaryotes serve different physiological functions in Gram-positive and Gram-negative bacteria. In the present review, the physiological significance of bacterial GGTs has been discussed categorizing GGTs from Gram-negative bacteria like Escherichia coli as glutathione degraders and from pathogenic species like Helicobacter pylori as virulence factors. Gram-positive bacilli, however, are considered separately as poly-γ-glutamic acid (PGA) degraders. The structure-function relationship of the GGT is also discussed mainly focusing on the crystallization of bacterial GGTs along with functional characterization of conserved regions by site-directed mutagenesis that unravels molecular aspects of autoprocessing and catalysis. Only a few crystal structures have been deciphered so far. Further, different reports on heterologous expression of bacterial GGTs in E. coli and Bacillus subtilis as hosts have been presented in a table pointing toward the lack of fermentation studies for large-scale production. Physicochemical properties of bacterial GGTs have also been described, followed by a detailed discussion on various applications of bacterial GGTs in different biotechnological sectors. This review emphasizes the potential of bacterial GGTs as an industrial biocatalyst relevant to the current switch toward green chemistry.

High level extracellular production of recombinant γ-glutamyl transpeptidase from Bacillus licheniformis in Escherichia coli fed-batch culture.

Increasing demand of microbial γ-glutamyl transpeptidase (GGT) in food and pharmaceutical sectors raised the need for process development for high level production of the enzyme. In this respect, GGT from Bacillus licheniformis ER15 (SBLGGT) was cloned along with its native secretion signal and expressed in E. coli using different expression vectors. Native signal of the enzyme assistedits extracellular translocationin E. coli.Maximum enzyme expression was shown by construct pET51b-sblggt,in comparison to other clones, in E. coli. Shake-flask cultivation and expression using Luria-Bertani (LB) medium resulted in 2800 U/l enzyme titers in 48 h which was furtherenhancedto 4.3-fold after optimizing various cultivation conditions viz. inducer concentration, agitation, medium and induction optical density. High cell density cultivation using fed-batch fermentation strategy resulted in 20-fold increase over shake flask studies to a level of 61250 U/l. After 24 h,the specific product yield was 2355 U/g dry cell weight (DCW)with volumetric productivity of 2552 U/l/h. Of the total enzyme expressed,40% was translocated extracellularly during high cell density fed-batch fermentation resulting in an enzyme activity of 24500 U/l in the extracellular medium after 24 h. This is the highest reported enzyme titers of bacterial GGT enzyme in E. coli expression system. Thus, the current study provides a cost-effective method for the over-expression and preparation of bacterial GGT enzyme for its industrial applications.

Evolving transpeptidase and hydrolytic variants of γ-glutamyl transpeptidase from Bacillus licheniformis by targeted mutations of conserved residue Arg109 and their biotechnological relevance.

γ-Glutamyl transpeptidase (GGT) catalyzes the transfer of the γ-glutamyl moiety from donor compounds such as l-glutamine (Gln) and glutathione (GSH) to an acceptor. During the biosynthesis of various γ-glutamyl-containing compounds using GGT enzyme, auto-transpeptidation reaction leads to the formation of unwanted byproducts. Therefore, in order to alter the auto-transpeptidase activity of the GGT enzyme, the binding affinity of Gln should be modified. Structural studies of the Bacillus licheniformis GGT (BlGT) complexed with the glutamic acid has shown that glutamic acid has strong ionic interactions through its α-carboxlic group with the guanidine moiety of Arg109. This interaction appears to be an important contributor for the binding affinity of Gln. In view of this, six mutants of Bacillus licheniformis ER15 GGT (BlGGT) viz. Arg109Lys, Arg109Ser, Arg109Met, Arg109Leu, Arg109Glu and Arg109Phe were prepared. As seen from the structure of BlGT, the mutation of Arg109 to Lys109 may reduce the affinity for Gln to some extent, whereas the other mutations are expected to lower the affinity much more. Biophysical characterization and functional studies revealed that Arg109Lys mutant has increased transpeptidation activity and catalytic efficiency than the other mutants. The Arg109Lys mutant showed high conversion rates for l-theanine synthesis as well. Moreover, the Arg109Met mutant showed increased hydrolytic activity as it completely altered the binding of Gln at the active site. Also, the salt stability of the enzyme was significantly improved on replacing Arg109 by Met109 which is required for hydrolytic applications of GGTs in food industries.

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.

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 (t1/2 of 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 Km of 0.15mM and 0.37mM and Vmax of 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 H1 NMR.

Hyperproduction of γ-glutamyl transpeptidase from Bacillus licheniformis ER15 in the presence of high salt concentration.

BACKGROUND: Microbial γ-glutamyl transpeptidases (GGTs) have been exploited in biotechnological, pharmaceutical, and food sectors for the synthesis of various γ-glutamyl compounds. But, till date, no bacterial GGTs are commercially available in the market because of lower levels of production from various sources. In the current study, production of GGT from Bacillus licheniformis ER15 was investigated to achieve high GGT titers. RESULTS: Hyperproduction of GGT from B. licheniformis ER15 was achieved with 6.4-fold enhancement (7921.2 ± 198.7 U/L) by optimization of culture medium following one-variable-at-a-time strategy and statistical approaches. Medium consisting of Na2HPO4: 0.32% (w/v); KH2PO4: 0.15% (w/v); starch: 0.1% (w/v); soybean meal: 0.5% (w/v); NaCl: 4.0% (w/v), and MgCl2: 5 mM was found to be optimal for maximum GGT titers. Maximum GGT titers were obtained, in the optimized medium at 37°C and 200 rpm, after 40 h. It was noteworthy that GGT production was a linear function of sodium chloride concentration, as observed during response surface methodology. While investigating the role of NaCl on GGT production, it was found that NaCl drastically decreased subtilisin concentration and indirectly increasing GGT recovery. CONCLUSION: B. licheniformis ER15 is proved to be a potential candidate for large-scale production of GGT enzyme and its commercialization.

Thermo- and salt-tolerant chitosan cross-linked γ-glutamyl transpeptidase from Bacillus licheniformis ER15.

Gamma-glutamyl transpeptidase enzyme, from Bacillus licheniformis ER15 (BLGGT), was produced extracellularly using a complex medium with high enzyme titers. Enzyme was concentrated and purified using ultra-filtration and ion exchange chromatography, respectively, with a purification fold of 4.6 and 50.11% yield. Enzyme was covalently immobilized onto chitosan microspheres (CMS). Immobilization was standardized with respect to pH, enzyme load and time. Immobilization efficiency of 11.9U/mg dry weight of microsphere was obtained in Tris-HCl buffer (pH 9.0) at 18°C in 4h. Immobilized enzyme (CMS-GGT) exhibited improved thermal stability (t1/2 of 70.7min at 60°C), activity in a broader pH range and improved salt stability in 18% (3M) sodium chloride solution as compared to free enzyme. Both free and immobilized enzymes specifically converted glutamine to glutamic acid in a mixture of amino acids. CMS-GGT had a better shelf life and high recyclability retaining 90% catalytic efficiency upto 10 reaction cycles. For long-term storage, CMS-GGT can be disinfected using either sodium azide or sodium hypochlorite solution without affecting enzyme activity. Thus, the present study provides an easy and efficient method for GGT enzyme immobilization that results in an improved and robust enzyme preparation.

L-theanine synthesis using γ-glutamyl transpeptidase from Bacillus licheniformis ER-15.

Recombinant γ-glutamyl transpeptidase (rBLGGT) from Bacillus licheniformis ER-15 was purified to homogeneity by ion-exchange chromatography. Molecular masses of large and small subunits were 42 and 22 kDa, respectively. The enzyme was optimally active at pH 9.0 and 60 °C and was alkali stable. K(m) and V(max) for γ-glutamyl-p-nitroanilide hydrochloride were 45 µM and 0.34 mM/min, respectively. L-Theanine synthesis was standardized using a one variable at a time approach followed by response surface methodology, which resulted in approximately 85-87% conversion of L-glutamine to L-theanine within 4 h. The standardized reaction contained 80 mM L-glutamine, 600 mM ethylamine, and 1.0 U/mL rBLGGTin 50 mM Tris-Cl (pH 9.0) at 37 °C. Similar conversions were also obtained with the enzyme immobilized in calcium alginate. Using immobilized enzyme, 35.2 g of L-theanine was obtained in three cycles of 1 L each. The product was purified by Dowex 50W X 8 hydrogen form resin and was confirmed by HPLC and proton NMR spectroscopy.