Purification And Characterization Of γ-Glutamyltranspeptidase And Itsapplication For The Syntheses Of Theanine

Theanine, a unique amino acid found almost exclusively in tea plants, has various favorable physiological and pharmacological functions for human beings without any toxic or side effect. In addition, it has diverse applications in food and medicine areas. Therefore, a study on biological synthesis of theanine has important theoretical and practical significances. Gamma-glutamyltranspeptidase （GGT, EC 2.3.2.2） is considered to be the most effective enzyme for the production of theanine. GGT is the key enzyme in glutathione metabolism, and can catalyze the transfer of theγ-glutamyl group. It can catalyse L-glutamine and ethylamine to theanine （γ-glutamyl-ethylamine）. GGT’s application in catalysis has become a research focus.In this study, strain SK 11.004 isolated from fermented shrimp paste through two steps was demonstrated as an effective strain for theanine production. It was identified as Bacillus subtilis based on its physiological and biochemical properties, as well as its 16S rDNA sequence analysis, and named as Bacillus subtilis SK11.004. The 16S rDNA sequence data from B. subtilis SK11.004 was submitted to the GenBank databases under accession no. FJ437210. GGT production was synchronous with the growth of B. subtilis SK11.004, and the highest enzyme activity （2.5 U/mL） in the culture was achieved after 16h.The goal of developing an effective method for the production of theanine was achieved using the GGT from B. subtilis SK11.004. Firstly, the optimum conditions for the synthesis of theanine were determined. In the optimized conditions, the side reaction was inhibited effectively, and the conversion rate of L-Gln to theanine reached 94%. Secondly, the technology of decoloration and purification of theanine was established. The theanine with 85% purity was obtained through activated carbon decoloration, 001×7 strong acidic resin （H⁺ form）, 201×7 strong basic resin （Cl^- form）, vacuum concentration and freeze-drying, with the recovery rate of theanine as 80.4%. Finally, the crystallization process of theanine was also studied. After crystallization, the theanine with purity of 97% was obtained with a yield of 48%. The crystals appeared as compact layered-structure with tetra-rectangle under scanning electron microscopy.Two GGTs （GGT-1 and GGT-2） were purified to electrophoretical homogeneity by ultrafiltration, hydrophobic interaction and gel filtration chromatography, and exhibited the special activity of 684.9 and193 U/mg, respectively. Based on SDS-PAGE and gel filtration analysis, it was confirmed that GGT-1 was composed of one large subunit of 40 kDa and one small subunit of 21 kDa, with the molecular mass of 62 kDa, GGT-2 was showed as a monomer, with the molecular mass of 58 kDa.The properties of GGT-1 were investigated. The enzyme showed maximal activity in hydrolase reaction at pH 8, while in transpeptidase activity at pH 10. It exhibited strong stability in pH 6-12.The isoelectric point of GGT-1 was determined to be 7.8, suggesting that the enzyme had higher activity when it was zwitterion and anion. The enzyme showed maximal activity at 37 oC, and was highly stable below 50°C. Al³⁺ and Mg²⁺ stimulated the enzyme activity of GGT-1, whereas Cu²⁺, Zn²⁺, Fe²⁺and Fe³⁺ caused its inhibition. The acid radicals SO₄^2-, Cl^-, and NO₃^- did not influence enzyme activity.The enzyme exhibited the highest affinity to imino acids （L-Pro） and then decreasing affinities for aromatic amino acids, ethylamine and basic amino acids. GGT-1 has low K_m values forγ-glutamyl donors, but high ones for acceptors, and low affinity forγ-GpNA, but high for L-Gln. 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-1 likely synthesizes valuableγ-glutamyl peptides using L-Gln asγ-glutamyl donor.Chemical modifiers PMSF and NBS showed strong inhibition of the GGT-1 enzyme activity, indicating the tryptophan residues and hydroxy groups of Ser or Thr are essential to enzyme activity. The addition of excess substrate did not reverse the inhibition process of NBS, it was suggested that the tryptophan residues were not located in the substrate binding site of GGT-1, but it was necessary group which maintain the structure of the enzyme. However, the addition of excess substrate reduced the inhibition effect of PMSF, revealing that the hydroxy groups were located in the substrate binding site of GGT-1.Circular dichroism spectra reflected the conformational changes of GGT-1. CD of the native enzyme showed that ratio ofα-Helix,β-sheet, turn and random coils structure were 34.4%, 11.7%, 22.2% and 31.7%. The modified GGT-1 by PMSF resulted in the decrease ofα-helix contente and increase of random coils content, showing that the secondary structure of the enzyme was destroyed to some extent. However, when GGT-1 was modified by NBS, there was only 6.8%α-Helix left, with 93.2% random coils. It was confirmed that the tryptophan residues were very important in maintaining the structure of the enzyme.The MALDI-TOF-TOF MS data were used to query the Mascot proteomics database, resulting in one significant match, GGT [Bacillus amyloliquefaciens FZB42]. After in-gel digestion, we obtained a Mascot score of 84 with a sequence coverage of 18%. The N-terminal amino acid sequence of the first 8 residues of heavy subunit was determined as FSYDTYKQ, which almost fully corresponded to residues 35-43 of B. amyloliquefaciens FZB42 GGT. The result confirmed the accuracy of identification by MALDI-TOF-TOF MS. A comparison with other existing GGT enzymes proved the observed N-terminal amino acid sequence to be different.