Molecular Cloning,Mutagenesis And Optimal Induction Conditions Of ?-glutamyltranspeptidase

L-Theanine ?N-ethyl-gamma-glutamine? is a peculiar amino acids and one of the important active ingredient of tea. A large number of studies have manifested that theanine has a number of important medicinal values, such as it can lower blood pressure, anti-tumor and enhance the curative effect of antitumor drug, the role of anti-fatigue enhance immunity, calm and relax, protect brain cells to enhance learning and memory function and a series of medical and health care function, y-glutamyltranspeptidase ??-GGT, EC 2.3.2.2? is a key enzyme in metabolic pathways of organism glutathione, it can not only catalytic gamma pancreatic hydrolysis reaction of acyl compounds generated gamma glutamic acid, but also can be the catalytic pancreatic acyl compounds on pancreatic acyl group transferred to amino acids, peptides and H₂O receptors. It can catalytic synthesis of glutathione and theanine by using y-GGT. This paper aims at the biological preparation of theanine technology, carrying out the engineering construction work, the biological technology used in natural product preparation technology. Through gene cloning constructed recombinant bacteria, with the help of error-prone PCR screening high-yielding y-glutamyltranspeptidase genetic engineering strain, optimize induced expression of y-glutamyltranspeptidase conditions. The main results are as follows:1 ? We amplified the gene coding for y-glutamyltranspeptidase from the genome of E.coli DH-5a by PCR, and the amplified fragment of y-GGT gene was successful cloned into pMD19-T vector, then recombinant plasmid pET-32a-GGT was construted with the expressing vector pET-32a, Subsequently transformed the recombinant plasmid into Escherichia coli BL-21 ?DE3?, obtaining engineering bacteria pET-32a-GGT-BL-21.2 ?With the help of error-prone PCR, we substituted, inserted or deleted specific nucleotide, changed the specific genes of individual amino acid residues of y-GGT gene sequences, transformed y-GGT without directional, contructed ?-GGT with the combinant plasmid and transformed the recombinant plasmid into Escherichia coli BL-21 ?DE3?. We induce the combinant bacteria and screened out to achieve the purpose of improving enzyme activity.3?To abtain expression optimization, using IPTG as inducer to induce recombinant bacteria, we chose six single factors- the initial OD600 of microbe, the inducing time, the concentration of IPTG, the inducing temperature, the rotate speed and the pH value, to study the influence of expressing y-GGT, then we investigated the first four factor as independent variable and the expressing recombinant ?-GGT in the amount of total protein content as response value, using the response surface analysis method to study the variables and their interactions to the expressing of ?-GGT, and determined the optimal induction conditions:the inducing temperature is 23 ?; the initial OD600 of microbe is 0.65; the inducing time is 7.8h and the concentration of IPTG is 0.63 mM. Under the above conditions, the expressing recombinant y-GGT in the amount of total protein content is 72.83%.