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Enzymatic Production of Glutathione, Theanine Through the Transpeptidation Reaction of γ-glutamyltranspeptidase from Escherichia Coli k-12

Author: ZuoJunFeng
Tutor: YinZhiMin
School: Nanjing Normal University
Course: Biochemistry and Molecular Biology
Keywords: γ- glutamyl GGT Recombinant bacteria Catalytic Synthesis Glutathione Theanine Clone Heat shock protein 70 Heat shock protein 90 Transforming growth factor beta activated kinase 1
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 238
Quote: 0
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Abstract


Glutathione (Glutathione) widely exist in a variety of organisms, mainly for the maintenance of normal intracellular redox state vivo free radical scavenger, antioxidant, detoxification, protect cells and other important physiological role has a very wide range of uses in clinical medicine, the food industry, health care products. In recent years, with Glutathione the production methods are a solvent extraction method, fermentation method, enzymatic conversion method and chemical synthesis method. Due to the relatively high cost of separation of extracted directly from the organization, chemical synthesis, separation and purification process is very difficult, the high yield of the enzymatic conversion of synthesis of GSH, subsequent separation and extraction is relatively simple and has drawn greater attention. Theanine (L-Theanine) the study called N-ethyl-of γ-amino-L-glutamine (N-ethyl-gamma-L-glutamine), natural tea in a special amino acid, is the tea has Xianshuang taste of the main component, obtained in 1985 the FDA approved, and synthesized theanine are recognized as GRAS, when in use as a novel food additive does not limit the dosage of theanine, not only soluble in water, but also inhibit bitter substances, improve food flavor, widely used in the food industry. In addition, theanine also have lower blood pressure, enhance the efficacy of anti-cancer drugs, improve immunity, relaxation nervous pharmacological effects. Directly from the tea extract high purity theanine cost is relatively high, theanine synthetic, especially microbial fermentation synthesis method has been a focus for researchers. γ-glutamyl peptide switch enzyme (γ-glutamyltranspeptidase, γ-GGT, EC 2.3.2.2) is a key enzyme in vivo the glutathione metabolic pathway, and is widely distributed in living tissue. This enzyme in E. coli, is transported in its own signal peptide mediated their physiological role in the periplasmic space. It not only can the reaction of hydrolysis of the compound of the catalytic γ-glutamyl γ-glutamic acid is generated, can also be catalytic γ-glutamyl compounds at the γ-glutamyl group is transferred to the amino acids, peptides, H 2 O, etc. receptors. Take advantage of this feature, can catalyze the synthesis of glutathione and theanine. In order to improve the yield and the soluble component of the γ-glutamyl microorganism GGT, this experiment from Escherichia coli K-12, γ-ggt gene fragment was cloned into the prokaryotic expression vector pET28a (), pET32a () and pGEX-4T-1 to construct the recombinant plasmid, and transformed into E. coli BL21 (DE3), as a cheap, non-toxic lactose inducer measured the expression of the target protein characteristics, based on the use of the Enzymatic synthesis of glutathione and theanine. Experimental results show that the recombinant plasmid pGEX-4T-1/γ-ggt transformed into E. coli BL21 (DE3), the lactose induction expression of γ-glutamyl peptide switch soluble and enzyme activity of the enzyme has a higher protein in Optimization on the basis of the reaction conditions, the use of recombinant E. coli gamma-glutamyl peptide switch the maximum generation amount of the enzyme, the theanine can reach 80 to 90g / L, in a still further study of the synthesis of glutathione. It has been reported that the existence of a close link between the inducible HSP70 and apoptosis, apoptosis signal-regulated kinase ASK1 (Apoptosis signal regulating kinase) is a critical kinase in the apoptotic pathway to induce activation of ASK1 through the mitochondrial pathway apoptosis, inducible HSP70 can directly bind effectively inhibit the intracellular activation of ASK1 and caused apoptosis. Inducible HSP70 mRNA or protein levels also can regulate ASK1 role, therefore, this experiment from a human cervical cancer Hela cells to amplify and clone inducible HSP70 gene to the true nuclear carrier pcDNA3.1 (-) in vitro identified by the expression of apoptotic pathway for the follow-up to explore its specific role and mechanism of the foundation. Heat shock proteins play a very important role in the cellular stress involved in the occurrence and development of a variety of diseases, heat shock protein 90 (HSP90) is a class of heat shock protein research in recent years more. At present, the international study of the function of the HSP90 proteins involved in cell mainly in protecting cells, inhibition of apoptosis, and whether they are involved in the regulation of IL-1 related inflammatory signaling pathways is still rarely reported. In mammals, TAK1 has an important role in cell signaling pathways in the innate and acquired immune. The results of our study show that HSP90 can be combined with TAK1 and affect the IL-1 induced the TAK1 ubiquitination and stability, thereby affecting the IL-1-induced activation of the downstream signaling. The coimmunoprecipitation cells show of HSP90 can bind to TAK1, this experiment will be found on the basis of the preliminary work role TAK1 HSP90 molecules reveal the molecular mechanism of the effect of HSP90 TAK1. This experiment was successfully constructed three sections HSP90 deletion mutants, respectively, HSP90 (1-232aa), (233-732aa), (402-732aa) and TAK1 two sections deletion mutants were TAKl (1-299aa), ( 301-579aa), by the body's immune coprecipitation experiments show of HSP90 protein in the 1-401aa is necessary for formation of the HSP90-TAK1 complex. The present study demonstrated that HSP90 interaction with TAK1 functional areas, provided the experimental basis for the study of the inflammatory response signaling networks and molecular mechanism.

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