45 results about "Glutathione synthetase" patented technology

The invention relates to a method for synthesis of glutathione in vitro, comprising the step of: generating ATP (adenosine triphosphate) from ADP (adenosine diphosphate) and polyphosphoric acid compound under the catalysis of polyphosphate kinase; under the catalysis of gamma-glutamylcysteine synthetase and glutathione synthetase, synthesizing glutathione in vitro by glutamic acid, cysteine and glycine with the energy provided by the ATP. Compared with the prior art, the method of the invention can bring enzymes into complete contact with a substrate so as to improve the mass transfer efficiency and the utilization rate of enzymes. And the reaction system of the method is simple and strong in controllability. In addition, the method can solve the problem of great investment of ATP during production and reduce the production cost.

The invention provides a glutathione synthetase-system recombinant plasmid as well as a construction method and application thereof, a recombinant strain for expressing glutathione synthetase systems as well as a construction method and application thereof, and a method for producing glutathione. The glutathione synthetase-system recombinant plasmid comprises a GSH1 sequence, a GSH1 sequence and a suitable vector fragment. The recombinant strain for glutathione synthetase systems is constructed by use of the glutathione synthetase-system recombinant plasmid. The method for producing glutathione comprises the step of fermenting the recombinant strain for glutathione synthetase systems. Glutathione-producing Pichia pastoris can be constructed by use of the coexpression plasmid provided by the invention; the obtained glutathione-producing Pichia pastoris is fermented and cultured; and the yield of glutathione in obtained fermentation broth is significantly higher than the current reported highest level of glutathione fermentation production. The invention has the advantages of improving the utilization rate of raw materials and reducing production cost and energy consumption, and can be used for industrial production.

Disclosed are stable recombinant multi-gene nucleic acid constructs, such as plant binary vectors, comprising (i) a gene encoding gamma-glutamylcysteine synthetase and (ii) a gene encoding glutathione synthetase, plus preferably at least one, preferably two, genes which encode enzymes involved in the redox cycling of glutathione between its reduced and its oxidised forms e.g. glutathione reductase and/or glutathione peroxidase. Preferably the promoters linked to the genes are different and of different strengths, and may optionally be inducible. Also provided are related materials and corresponding methods and uses e.g. in plants to improve oxidative stress tolerance enhance root development, or to increase the post-harvest shelf life of the plant or part thereof.

The invention discloses a preparation method of a genetically engineered bacterium for synthesizing glutathione. The preparation method comprises the following steps: (i) converting a gene for encoding gamma-glutamoyl cysteine synthetase-linker peptide-glutathione synthetase fusion protein into a host bacterium cell, and highly expressing the gene; (ii) converting a gene for encoding gamma-glutamoyl cysteine synthetase-linker peptide-glutathione synthetase into the host bacterium cell, and highly expressing the gene; (iii) knocking off the gamma-glutamyltranspeptidase of the host bacterium cell; (iv) knocking off an inositol-3-phosphatep synthase gene of the host bacterium cell; and completing the steps (i), (ii), (iii) and (iv) in any sequence, thereby obtaining the genetically engineered bacterium for synthesizing glutathione. The invention further relates to the prepared genetically engineered bacterium. The genetically engineered bacterium for synthesizing glutathione, which is disclosed by the invention, is not only high in efficiency in synthesizing glutathione, but also can greatly improve the conversion efficiency of exogenous cysteine, and can be applied to glutathione production.

The invention discloses a pichia-pastoris engineering bacterium for producing glutathione and provides a method for constructing a recombinant bacterium. The method comprises the step of guiding the coding gene of bifunctional glutathione synthetase into an original strain to obtain the recombinant bacterium. Experiments show that the recombinant bacterium prepared by adopting the method can be used for producing the glutathione, ensures the high yield and lays a foundation for the industrial production and the preparation of the glutathione.

The invention discloses a pichia genetic engineering bacteria used for generating the glutathione and a recombinant plasmid used for constructing the pichia genetic engineering bacteria, which is characterized in that: the enzyme system GSH I and GSH II genes synthesized through the exogenous glutathione are respectively put in the promoter such as the CAP promoter, and integrated to the methanol yeast under the regulation of the GAP promoter, so as to obtain the high yield GSH genetic engineering bacteria. The pichia genetic engineering bacteria used for generating the glutathione and a recombinant plasmid used for constructing the pichia genetic engineering bacteria has the advantages that: the pichia genetic engineering bacteria can be carried out high density cultivation, so as to decrease the cost of producing the GSH and be beneficial to large scale industrial production.

The invention discloses a Cr (VI) stress resistant petroleum contaminated soil immobilized bioremediation technology. The method comprises the following steps of: by taking tartary buckwheat shells as a raw material, carrying out hydrothermal carbonization in an ethylenediamine aqueous solution and a ascorbic acid solution, carrying out rotary heating in formic acid and loading nano zero-valent aluminum, so as to gradually improve the reducibility of biochar, thereby realizing the reduction of Cr (VI); and loading glutamylcysteine synthetase and glutathione synthetase to enhance the detoxification capability of microorganisms. The method is very effective for bioremediation of petroleum contaminated soil stressed by heavy metal Cr (VI), and the heavy metal Cr (VI) in the soil is partially removed while petroleum is removed.

The invention provides a bifunctional glutathione synthetase expression cassette derived from lactobacillus. The bifunctional glutathione synthetase expression cassette includes cloning a gshF gene derived from the lactobacillus. The invention further provides a preparation method of the bifunctional glutathione synthetase expression cassette derived from the lactobacillus. The preparation methodcomprises the following steps: designing a primer for amplifying the gshF gene by taking a lactobacillus genome as a template; performing enzyme digestion with Ndel and Xhol; connecting to a polyclonal site of an escherichia coli pET expression vector through DNA ligase in order to construct the expression cassette expressing the lactobacillus gshF. The invention further provides a lactobacillus-containing gshF expression cassette escherichia coli genetically engineered bacterium. The invention further provides application of the lactobacillus-containing gshF expression cassette escherichia coli genetically engineered bacterium to preparation of glutathione. After the bifunctional glutathione synthetase expression cassette is applied to GSH synthesis, the GSH yield is up to 103.1mu m, theescherichia coli GSH yield is increased by 11.5 times or more, and the GSH synthesis ability of the escherichia coli is significantly improved.

The invention provides recombinant escherichia coli for synthesizing glutathione and application of the recombinant escherichia coli. The N end of bifunctional glutathione synthetase GshF and the C end of polyphosphoric acid kinase PPK are connected with SpyCatcher and SpyTag respectively, two modified gene segments are integrated to the same plasmid vector at the same time, and the recombinant escherichia coli is obtained through conversion. After the obtained recombinant escherichia coli is subjected to high-density culture and optimization of the inducible expression condition, the maximum expression of a multi-enzyme compound is obtained, escherichia coli thalli are collected for efficient catalysis of whole cells or assembly of the multi-enzyme compound, centrifugation or membrane separation is directly conducted after the reaction is finished, and the cells or the multi-enzyme compound can be repeatedly used after being simply recycled. According to the process, the production cost of glutathione is effectively reduced, the utilization efficiency of a catalytic system is improved through efficient repeated utilization, the recombinant escherichia coli is simple to operate, and the application prospect is good.

An anticancer composite medicine applied locally is prepared from anticancer nitrosource medicine or its analog, the glutathion synthetase depressant and/or the nitrogen oxide synthetase depresant for destroying the DNA repairing function in cell and lowering the tolerance of tumor cells to nitrosource medicine or its analog, and the biocompatible and biodegradable high-modular polymer as medicinal additive.

The invention relates to a preparation method of a dye leveling agent, and belongs to the technical field of dye additives. Mixed enzymes and acrylamide are mixed for polymerization under the action of an initiator to form polyacrylamide, the enzymes are encapsulated, metal elements in mixed liquid are adsorbed, the enzyme activity is increased by using the metal elements, cross-linking with chitosan and mixed sugar is achieved under the action of an assistant, incomplete cross-linking is achieved under the action of N-methylenebisacrylamide and glutaraldehyde, and an acylating agent is used for acylation to increase active groups. When the dye leveling agent is in use, firstly the mixed enzymes increase surface active groups by partially destroying the surface of a fabric; then the dyeingagent is bonded with the surface of the fabric under the action of glutathione synthetase through an incompletely cross-linked material, and the adoption of fatty alcohol is more conductive to infiltration of raw materials; finally the dyeing agent is adsorbed on the surface of the fabric under the action of the formed polyacrylamide and loaded chitosan and is separated from the surface of the fabric with the increase of the temperature, and thus coloring can be better achieved.

A gene encoding glutathione synthetase from Candida utilis is provided and food containing γ-glutamylcysteine cysteine or cystenylglycine is produced by cultivating Candida utilis modified by means of a gene encoding glutathione synthetase under a suitable condition and mixing the obtained culture or a fraction thereof or the culture or a fraction thereof subjected to heat treatment with a raw material of food or drink to process food or drink.

The invention provides a recombined strain capable of simultaneously compressing glutathione synthetase system and adenosyl methionine synthetase and a method for simultaneously producing glutathione and S-adenosyl methionine at high yield. The recombined strain comprises: fragment capable of exogenously expressing gama-glutamyl cysteine synthetase or glutathione synthetase and fragment capable of exogenously expressing adenosyl methionine synthetase and the strain is capable of expressing glutathione synthetase system and/or adenosyl methionine synthetase, thereby fermenting glutathione and adenosyl methionine. The recombined strain is capable of simultaneously producing glutathione and adenosyl methionine by fermentation at high yield and independently producing glutathione or adenosyl methionine by fermentation.

The present invention provides a method for preparing glutathione (GSH) through an enzyme method. According to the method, two steps of reactions for GSH synthesis are respectively performed in different reaction tanks, and enzymes used in each reaction are separated after each reaction is performed, such that enzyme activities of GSH I and GSH II are utilized to the maximal degree, and mutual inhibition between the two enzymatic reactions are reduced, wherein the two steps of the reactions comprise a gamma-glutamylcysteine generation reaction ??and a GSH generation reaction, and the enzymes used in each reaction comprise gamma-glutamyl cysteine synthetase (GSH-I) and glutathione synthetase (GSH-II). With the method, recycling of GSH I and GSH II is achieved, an ATP regeneration system required by the GSH preparation reaction is adopted, production cost for GSH preparation is reduced, GSH yield is improved, large-scale continuous production is achieved, and economic benefits are significant.

The invention relates to a xanthene fluorescent probe and a preparation method and application thereof. The xanthene fluorescent probe has a chemical structure of a formula (1) which is shown in the description. Compared with the prior art, the xanthene fluorescent probe has the following characteristics that 1, the synthesizing method is simple; 2, the selectivity on GSH (glutathione synthetase)is higher; 3, the response concentration on GSH is millimole level, and the xanthene fluorescent probe is suitable for quantitatively detecting the GSH in cells; 4, the toxicity on cells is small; 5,the fluorescent transmission wavelength of a resultant after functioning with GSH can reach 630nm, and the xanthene fluorescent probe can be used for cell imaging.