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 present invention discloses a method for synthesizing glutathione by enzymatic catalysis, comprising the following steps: S1, mixing Upsilon-glutamylcysteine synthetase liquid and glutathione synthetase liquid to obtain mixed liquid A, mixing the mixed liquid A with acetate kinase to obtain mixed liquid B; S2, adding an immobilized carrier in the mixed liquid B, stirring for immobilizing, and filtering to obtain an immobilized enzyme; S3, blending glutamic acid, L-cysteine, glycine, magnesium sulfate, ATP (adenosine triphosphate) and acetyl phosphate into a reaction liquid, adding the immobilized enzyme, and stirring for reacting; S4, after reacting, filtering the reaction liquid, and extracting and refining filtrate to obtain the glutathione. The method of the invention has high synthetic efficiency, the production content of the reaction liquid is higher than 10 g / L, and substrate conversion rate reaches higher than 90%; by introducing acetate kinase to construct an ATP regeneration and coupling system, ATP charge is greatly reduced and production cost is significantly reduced; the immobilized enzyme is reusable and highly stable.

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 relates to an immobilized glutathione synthetase, preparation thereof and applications thereof. In particular, the immobilized glutathione synthetase having largely improved stability and reactivity is obtained by immobilization of a glutathione synthetase and a carrier. In particular, a long connecting component between the glutathione synthetase and the immobilization carrier can achieve an excellent immobilization effect. The immobilized glutathione synthetase has a long half-life period so that the enzyme productivity per unit is effectively enhanced. The immobilized glutathione synthetase can be used as a GSH biosynthesis system, thus simplifying production processes, increasing the production efficiency and simplifying purification technology.

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 provides novel application of sodium hydrogen sulfide aqueous solution, particularly provides application of sodium hydrogen sulfide aqueous solution in improving heavy metal stress resistance of a plant. The sodium hydrogen sulfide aqueous solution has a concentration of 10-500 mumol / L, preferably 50-100mumol / L. The sodium hydrogen sulfide aqueous solution sprayed on plant left surface can induce high expression level of glutathione synthetase gene 1 (GSH1) and glutathione synthetase gene 2 (GSH2), so as to achieve the effect of synthesizing a large amount of glutathione (GSH). Meanwhile, the sodium hydrogen sulfide aqueous solution can supplement sulfur (S) required by the plant, so as to provide guarantee for heavy metal stress resistance of the plant.

A yeast which harbors a mutant glutathione synthetase having one or both of mutations selected from the group consisting of a mutation to replace a threonine residue at the position 47 with an isoleucine residue and a mutation to replace a glycine residue at the 387-position with an aspartic acid residue and produces γ-glutamylcysteine. The yeast is cultured under suitable conditions, the resultant culture or fractionation product thereof or the resultant culture or fractionation product thereof that had been heat-treated is mixed with food or beverage materials and processed into foods or beverage to produce γ-glutamylcysteine- or cysteine-containing foods or beverage.

Disclosed is an anti-cancer implant which comprises anti-cancer active constituent and medicinal adjuvant, wherein the active anti-cancer constituents mainly include blood vessel inhibitor and anti-cancer medicament, the anti-cancer medicament is selected from glutathione synthetic enzyme inhibitor or nitrogen oxide synthetic enzyme inhibitor or their combination, The medicinal subsidiary materials mainly comprise bio-compactable and degradable macromolecular polymers, which can slowly release the anti-cancer medicament onto tumor partially during the degradation and absorption process, thus the whole body toxicity reaction is reduced appreciably , and the effective medicinal concentration can be sustained to the tumor partially. when locally dispensed on the tumor, the composition can lower down the whole body toxicity reaction of the anti-cancer medicament, selectively increase the tumor local medicinal concentration, and the treatment effect of the non-operative treatment methods such as chemotherapy, medicament and radiation can be improved.

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 discloses a method for producing glutathione by using bifunctional glutathione synthetase contained in actinobacillus pleuropneumonia, actinobacillus succinogenes, bacillus cereus, streptococcus sanguis, streptococcus gordonii, streptococcus uberis and streptococcus thermophilus. The method comprises heterologous expression by using the seven microbes or enzymes of the microbes in other microbes. The glutathione synthesized by using the method has the structure of natural glutathione, and has high response rate and high output and yield of the glutathione.

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 relates to a preparation method of recombinant bacillus subtilis and glutathione, and the glutathione is produced by fermenting a bacillus subtilis gene engineering strain for overexpression of a bifunctional glutathione synthetase gene, so that the glutathione is high in yield, simple and convenient to operate. Meanwhile, the food-grade bacillus subtilis is used as a production strain, so that the method is safer and more reliable, and effective reference is provided for industrial green production of glutathione.

A mutant glutathione synthetase (GSHB) suitable for generating γ-Glu-Val-Gly, and a method for producing γ-Glu-Val-Gly using the same are provided. γ-Glu-Val-Gly is produced by using a mutant GSHB having a mutation at such a position as V7, N13, I14, N15, K17, F22, F95, M165, N199, Y200, P202, I274, T285, and P287.

The invention discloses a method for purifying double-function glutathione synthetase. The method includes the steps of firstly, preparing crude extract containing the double-function glutathione synthetase; secondly, adding divalent metal ions with concentration of 0.001-0.5M into the crude extract to remove sediment so as to obtain supernate; adjusting the pH of the supernate to 4-9, adding one or more of divalent copper ions (Cu<2+>), divalent manganese ions (Mn<2+>) or divalent zinc ions (Zn<2+>) with concentration of 0.005-0.4M, and collecting sediment, namely the double-function glutathione synthetase. The method for purifying the double-function glutathione synthetase has the advantages that the method is simple, easy to operate, short in time consumption and low in cost; the double-function glutathione synthetase (GshF) produced by the method is insoluble in water and reusable, and the method is suitable for large-scale industrial production of GshF.

The present invention relates to a method of producing glutathione, wherein photosynthetic membrane vesicles and enzymes catalyzing glutathione synthesis are combined and glutamate, cysteine and glycine are used as reaction substrates. As enzymes catalyzing glutathione synthesis, γ-glutamylcysteine synthetase and glutathione synthetase may be used together, or bifunctional glutathione synthetase may be used alone. According to the conventional methods, there is a problem in that expensive adenosine triphosphate should be continuously supplied when glutathione is produced. However, according to the present invention, since photosynthetic membrane vesicles are used as a source to regenerate adenosine triphosphate, it is possible to continuously produce glutathione without additionally adding adenosine triphosphate, thereby reducing production costs of glutathione.

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 invention discloses a mutant of difunctional glutathione synthetase. The mutant is an amino acid sequence as shown in SEQ ID NO.3 or a mutant thereof obtained by recombining amino acid sequences as shown in SEQ ID NO.2 and SEQ ID NO.1 and / or carrying out site-directed mutagenesis on a recombinant. The mutant has the advantages that the thermal stability of the mutant is remarkably improved, and the half-life period t1 / 2 is 10-60 times that of wild enzyme. The half-life period t1 / 2 of the optimal mutant is 163.3 times that of the wild enzyme, and the specific enzyme activity is improved by 24% compared with that of the wild enzyme. Compared with the temperature of the wild enzyme, the optimal temperature of the optimal mutant is increased from 45 DEG C to 50 DEG C. Tests such as enzyme catalysis, intracellular self-assembly whole-cell catalysis and fermentation prove that the finally obtained mutant effectively improves the yield and synthesis efficiency of GSH.

The invention discloses a method for immobilized bioremediation of oil polluted soil against Cr(VI) stress. Using tartary buckwheat shells as raw materials, the reducibility of biochar was gradually improved by hydrothermal carbonization in ethylenediamine aqueous solution and ascorbic acid solution, rotating heating in formic acid, and loading nano-sized zero-valent aluminum, in order to achieve the reduction of Cr(Ⅵ). ; Enhance the detoxification ability of microorganisms by loading glutamylcysteine ​​synthetase and glutathione synthase; At the same time, the heavy metal Cr(Ⅵ) in the soil was partially removed.

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 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 canbe used for industrial production.

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.

The invention provides a feed additive for improving the survival rate of large yellow croaker fry. The feed additive comprises the following raw materials in percentages by weight: 35-42% of a tea fertilizer, 10-16% of astaxanthin, 3-7% of glutathione synthetase, 12-17% of glycerin, 2-5% of carboxymethyl dextran and the balance of a carrier, wherein the tea fertilizer is prepared from tea leaves,zinc methionine and sodium alginate. The additive can be directly added into fish feed to enhance immunity, prevent diseases of the fry and improve the survival rate.

The invention relates to the field of polypeptide synthesis, and in particular relates to immobilized fusion enzyme and a method for preparing glutathione by using the immobilized fusion enzyme. According to the invention, a glutathione synthetase gene and a triphosadenine regenerative enzyme gene are respectively connected with a chitin protein domain gene; fusion enzyme is prepared by expression; by utilization of the fact that a chitin carrier can be bound to a chitin protein domain specifically and firmly, glutathione synthesis fusion enzyme and triphosadenine regenerative fusion enzyme are purified and immobilized through one step; the process is simple; and the space density of the immobilized enzyme is high. The immobilized fusion enzyme prepared by the invention is high in enzyme activity; continuous generation of glutathione can be realized; the batch and continuous process route of the immobilized fusion enzyme is adopted in the invention; glutathione up to 31 g / L can be generated; the concentration of a target product is high; no enzyme residue exists in product solution; and the later purification process is greatly simplified.