Molecular Modification Of γ-glutamyl-transpeptidase And Its Application

γ-glutamyltranspeptidase（GGT）synthesizes theanine and bitter amino acid derivatives which are widely used in food and medicine by transferring theγ-glutamyl group of donor substrate L-Gln to acceptor substrate.In this study,the expression element and molecular structure of GGT were optimized in order to improve GGT synthesis and enzyme activity,while the conversion conditions of theanine andγ-Glu-Phe catalyzed by recombinant GGT were further optimized to improve the yield of target product.The results obtained from this study are as follows:（1）The GGT gene from Bacillus pumilus ML413 was successfully cloned and expressed as an active enzyme in Bacillus subtilis 168.SDS-PAGE analysis showed that GGT had a molecular weight of 38 k Da and 23 k Da.The enzymatic properties of GGT were then studied and results showed that the optimum catalytic temperature and p H of GGT were 40°C and10.0 respectively,with the GGT enzyme displaying significant enzyme activity in a broad range of temperature and p H.（2）GGT-catalyzed transpeptide reaction is often accompanied by NH₄⁺generation,which NH₄⁺reacts with o-phthalaldehyde,trichloroacetic acid,and dimethyl sulfoxide forming blue compounds.In this study,to characterize the enzyme activity in the system,the concentration of NH₄⁺released in the GGT catalytic process was measured at 600 nm wavelength.Based on this,a high-throughput screening method was therefore established and applied in the identification of high enzyme activity mutants.（3）In order to improve the expression level of GGT,seven upstream bases of promoter Hpa II-10 region were initially mutated iteratively by error prone PCR thus constructing the mutant library.Using the established screening method,the positive promoter EF3Hpa II was obtained,which increased the expression level of GGT by 63%compared with the original strain.The study further explored RBS library design and screening using RBS calculator in order to obtain RBS more suitable for EF3Hpa II promoter,thus obtaining the artificial RBS362 by high-throughput screening.Subsequently,RBS362 was combined with EF3Hpa II promoter to regulate the expression of GGT gene,and the resultant expression level of GGT in the recombinant strain was increased to 60.25 U·m L^-1,which was 2.02 times that of the original strain.（4）In order to increase the specific activity of GGT,ggt was randomly mutated by error prone PCR,and the mutant library constructed and this combined with high-throughput screening method,resulted in a forward mutant GGT_T463S with specific enzyme activity of29.66 U·mg^-1.Through homologous modeling,molecular docking and molecular dynamics simulation,the structure of the protein before and after the mutation was analyzed.It was found that the change of amino acid residues at 463 site resulted in widening of the distance between the amino acid residues at position 463 and the threonine residues at 413 site,which possibly made substrates access into the tunnel and the release of products more convenient and easier,hence explaining the higher specific enzyme activity of the mutant GGT_T463Sobserved.（5）The mutant GGT_T463S then catalyzed the synthesis of theanine,with the final yield of theanine up to 88%from the substrate concentration of 0.2 mol·L^-1 L-Gln and 2 mol·L^-1ethylamine,with the enzyme concentration of 1.0 U·m L^-1,the initial reaction temperature of35℃and p H 10.0.The final yield of theanine reached 58.73 g·L^-1,with the final yield of theanine obtained was 60%..（6）A preliminary study on the catalysis of bitter amino acids by mutant GGT_T463S.The optimal conditions for the synthesis ofγ-Glu-Phe were as follows:the concentration of L-Gln was 0.05 mol·L^-1 and 0.2 mol·L^-1 L-Phe,the enzyme concentration was 1.0 U·m L^-1,at the initial reaction temperature of 35℃and p H 10.0.The final yield ofγ-Glu-Phe obtained was 94%.