21 results about "Malate dehydrogenase" patented technology

The invention provides a recombinant pichia kudriavzevii strain and application thereof, pichia kudriavzevii is taken as an original strain, and a reduced TCA pathway is introduced and comprises the following steps: overexpressing fumarate reductase FRD in the strain; weakening or inactivating an ethanol pathway, comprising: knocking out any one or a combination of at least two of encoding genes of ethanol dehydrogenase ADH1, ADH3 or ADH6; enhancing the PPP pathway in the pentose phosphate pathway of the strain, including: overexpressing glucose-6-phosphate dehydrogenase ZWF1 and / or 6-phosphogluconate dehydrogenase GND1; the method for reducing the activity of the malate dehydrogenase MDH2 in the TCA circulation pathway comprises the step of knocking out the coding gene of the malate dehydrogenase MDH2 in the TCA pathway. The pichia kudriavzevii is modified by using genetic engineering and metabolic engineering methods, so that the yield and the yield of succinic acid are increased, and the pichia kudriavzevii has important application prospects in industrial production.

The invention discloses an engineering strain for producing glycine. The engineering strain is obtained by modifying the following steps: (i) respectively introducing an exogenous acetyl coenzyme A synthetase gene acs, an isocitrate lyase gene aceA and an isocitrate dehydrogenase kinase gene aceK; (ii) introducing a glutamic acid glyoxylate transaminase gene cgaT from quinoa; and (iii) respectively knocking out a DNA (Deoxyribose Nucleic Acid) binding transcription inhibition factor gene iclR, a glycine dehydrogenase gene gcvP, an alanine transaminase gene alaA, a malate dehydrogenase gene maeA and a malate synthetase G gene glcB. According to the invention, an engineering strain capable of efficiently synthesizing glycine is constructed by transforming a glycine biosynthesis metabolic pathway, and the strain can produce glycine by fermentation with glucose as a carbon source and has a relatively high conversion rate.

A modified Escherichia coli and the use thereof in the production of L-amino acid by means of fermentation. Specifically, disclosed is the use of pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, branched-chain amino acid transaminase derived from Bacillus subtilis, thiamine phosphate synthase and malate dehydrogenase in the construction of a genetically engineered bacterium for producing L-amino acid. Experiments prove that the recombinant bacterium, which is conducive to the accumulation of L-amino acid, is obtained by means of knocking out the encoding genes of the pyruvate formate lyase, the alcohol dehydrogenase, the branched-chain amino acid transaminase derived from Escherichia coli, the thiamine phosphate synthase and the malate dehydrogenase of Escherichia coli capable of producing L-amino acid, and introducing the encoding gene of the branched-chain amino acid transaminase derived from Bacillus subtilis. The recombinant bacteria can greatly increase the yield of L-amino acid and reduce the costs, and is of great significance to accelerate the industrial progress of L-amino acid.

Provided is a genetically engineered strain of Saccharomyces cerevisiae ST100-Suc3 for producing succinic acid. The Saccharomyces cerevisiae ST100-Suc3 is obtained by overexpressing malate dehydrogenase (ScMDH3R), fumarase (RoFUM) and fumarate reductase (TbFRD) in Saccharomyces cerevisiae PY-EDS8 while knocking out a glycerol-3-phosphate dehydrogenase (GPD1) gene, an alcohol dehydrogenase (ADH1) gene and an aldehyde dehydrogenase (ALD6) gene. The Saccharomyces cerevisiae strain is Saccharomyces cerevisiae PY-EDS8, with the deposit number GDMCC No: 63815. The strain exhibits tolerance to high-concentration succinic acid and can grow in a liquid medium supplemented with 100 g / L of succinic acid (pH=2.68), but produces almost no succinic acid.

The present invention relates to an improved process for the preparation of 2, 4-dihydroxybutyrate from a carbon source via homoserine, comprising a two-step route: a first step of catalytically converting the primary amino group of homoserine to a carbonyl group by an enzyme having homoserine transaminase activity as defined by E.C.2. 6. 1.1 or E.C.2. 6. 1.42 or E.C.2. 6. 1.57 to obtain 2-oxo-4-hydroxybutyrate, and a second step of catalytically converting the primary amino group of homoserine to a carbonyl group by an enzyme having homoserine transaminase activity as defined by E.C.2. 6. 1.1 or E.C.2. 6. 1.57 to obtain 2-oxo-4-hydroxybutyrate. And-a second step of catalytically reducing the obtained 2-oxo-4-hydroxybutyrate (OHB) to 2, 4-dihydroxybutyrate by an enzyme having OHB reductase activity, the enzyme having OHB reductase activity being a lactic dehydrogenase as defined by E.C. 1.1. 1.27 or E.C. 1.1. 1.28, or a malate dehydrogenase as defined by E.C. 1.1. 1.37, E.C. 1.1. 1.82, or E.C. 1.1. 1.299, and-a second step of catalytically reducing the obtained 2-oxo-4-hydroxybutyrate (OHB) to 2, 4-dihydroxybutyrate by an enzyme having OHB reductase activity, the enzyme having OHB reductase activity being a lactic dehydrogenase as defined by E.C. 1.1. 1.28, or a malate dehydrogenase as defined by and wherein at least one enzyme having aldolase activity, as defined by E.C.4.1. 2.28, is inactivated to prevent degradation of OHB.

The application discloses a succinic acid-producing strain of Issatchenkia orientalis, and construction and application thereof, and belongs to the technical field of microorganisms. Through metabolic engineering, the application knocks out a pyruvate decarboxylase coding gene on a genome of the Issatchenkia orientalis and integrates expression of a pyruvate carboxylase, a malate dehydrogenase, a fumarase and a fumarate reductase, knocks out a glycerol dehydrogenase coding gene on the genome and integrates expression of a dicarboxylic acid outer transport protein from a schizosaccharomyces pombe, simultaneously knocks out carboxylic acid inner transport proteins JEN2-1 and JENE2-2 on the genome, and overexpresses the fumarate reductase and / or the malate dehydrogenase. The modified yeast strain significantly improves a succinic acid yield in a fermentation process, and the succinic acid yield reaches 125.77 g / L, and a production intensity is 1.75 g / L / h. The engineering strain has good industrial application potential in high-efficiency succinic acid production.

The invention relates to the technical field of enzyme engineering, in particular to an expression and purification method of cyanobacteria malate dehydrogenase, which comprises the following steps: S1, transforming a constructed recombinant plasmid into an escherichia coli competent cell; s2, carrying out inoculation and fermentation culture on the bacterial liquid obtained in S1, and collecting thalli; s3, adding the thalli into a buffer solution for cell disruption to obtain a supernatant; s4, adding the supernate into resin, and performing chromatography and elution to obtain purified cyanobacteria malate dehydrogenase protein. The invention lays a foundation for the structure and function research of cyanobacteria MDH. Researches show that the cyanobacteria MDH family possibly has a new characteristic of double-coenzyme dependence, and MDH from part of species of cyanobacteria has good heat resistance and thermal stability and has good industrial application potential.

The application belongs to the technical field of biological medicine, and particularly relates to a sulfonamide compound and a preparation method and application thereof. It is found for the first time that malate dehydrogenase is closely related to aging, and inhibiting malate dehydrogenase can achieve a significant anti-aging effect. Based on this, the application provides a sulfonamide compound, the compound has a significant inhibitory effect on malate dehydrogenase, can prolong the life of nematodes, achieves a significant anti-aging effect, and has good safety, thereby providing more safe compounds for anti-aging drugs.

The present invention belongs to the field of biological breeding technology, and specifically relates to the application of soybean malate dehydrogenase in regulating plant stress resistance. The technical problem to be solved by the present invention is: how to improve the stress resistance of soybean, for example, how to improve the salt tolerance of soybean. To solve this technical problem, the present invention provides the application of protein or a substance that regulates gene expression or a substance that regulates the activity or content of the protein in regulating plant salt tolerance, the gene encodes the protein, and the protein may be a GmMDH2 protein; the GmMDH2 protein is a protein whose amino acid sequence is shown in positions 1-352 of SEQ ID No.1. The results of the present invention show that GmMDH2 can significantly improve the salt resistance of transgenic plants, and GmMDH2 can be used as a salt-tolerant gene to cultivate soybean stress-resistant varieties.

The invention discloses a freeze-drying reagent for citric acid determination. The freeze-drying reagent is prepared from a first freeze-drying reagent ball and a second freeze-drying reagent ball which are obtained by freeze-drying liquid drops of a first citric acid determination reagent and a second citric acid determination reagent, the first citric acid determination reagent comprises citrate lyase, lactic dehydrogenase, malate dehydrogenase, a first buffer solution, an additive, a first cryoprotectant and a first surfactant; the second citric acid determination reagent comprises reduced coenzyme, a second surfactant, a second buffer solution and a second cryoprotectant. The biochemical analysis chip comprises a chip body, the chip body is provided with a colorimetric hole and a diluent groove, the first freeze-drying reagent ball and the second freeze-drying reagent ball are contained in the colorimetric hole, and diluent is contained in the diluent groove. The method for measuring the citric acid comprises the following steps: treating a sample, and adding the treated sample into a heated colorimetric hole to react with a freeze-drying reagent ball; testing the absorbance variation and / or the decline rate of the absorbance value of the solution in the colorimetric hole under the specific wavelength; based on this, a citric acid concentration value is determined.

The invention discloses a malate dehydrogenase (MDH) mutant MaMDHK240A with increased enzyme activity and an application of the malate dehydrogenase (MDH) mutant MaMDHK240A. The invention relates to a malate dehydrogenase mutant MaMDHK240A, and the amino acid sequence of the malate dehydrogenase mutant MaMDHK240A is as shown in SEQ ID NO. 3. According to the mutant MaMDHK240A, site-specific mutagenesis is carried out on an amino acid sequence as shown in SEQ ID NO.2, and 240th Lys is mutated into Ala, so that the activity of the malate dehydrogenase is increased, and the enzyme activity of the malate dehydrogenase is increased by 1.87 times. The malate dehydrogenase mutant MaMDHK240A obtained by the invention can be used as a mutant material capable of remarkably increasing the catalytic activity of the malate dehydrogenase, and provides reference for further research on the functions of the malate dehydrogenase.

The invention belongs to the technical field of molecular biology, and particularly relates to yarrowia lipolytica engineering bacteria for producing L-malic acid and application. Glycerin is used as a carbon source, and the metabolic flow of a glyoxylic acid circulation path in the yarrowia lipolytica peroxisome is enhanced to promote the synthesis of L-malic acid. In addition, malic acid transporter SpMAE1 from schizosaccharomyces pombe is further introduced, feedback inhibition is relieved, and product efflux is accelerated. Meanwhile, it is accidentally found that the acid resistance of the Po1g-CEE2 + Sp strain obtained after over-expression of endogenous genes ICLC (isocitrate lyase), MSE (malate synthase), MDHE2 (malate dehydrogenase) and heterologous transporter gene SpMAE1 on the basis of the original strain Po1g delta ku70 is improved to 2.76 from 3.58 compared with that of the original strain, and the enhancement of the acid resistance is beneficial to production of L-malic acid.

The invention relates to the technical field of stichopus japonicus breeding, in particular to a method for losslessly evaluating heat resistance of stichopus japonicus, which comprises the following steps: S1, collecting foot tissues from a living stichopus japonicus sample, and pretreating to obtain enzyme-containing tissue fluid; s2, determining the thermal stability of malate dehydrogenase of the enzyme-containing tissue fluid; and S3, performing covariance analysis on the measured data, and comparing the time change slopes of the hot residual enzyme activity of different stichopus japonicus samples, so that the smaller the slope is, the higher the heat resistance of the stichopus japonicus sample is. According to the technical scheme, the heat resistance of the stichopus japonicus individual can be accurately determined, the death of the living stichopus japonicus of the detected sample is avoided, and the method is suitable for screening the heat resistance in stichopus japonicus breeding.

The invention relates to the technical field of enzyme engineering, and discloses malic dehydrogenase, engineering pichia kudriavzevii containing the malic dehydrogenase and application of the malic dehydrogenase and the engineering pichia kudriavzevii. In the invention, a pichia kudriavzevii engineering strain with NADH (Nicotinamide Adenine Dinucleotide Hydrocarbon) selection pressure after double knockout in an ethanol / glycerol synthetic route is used as a chassis bacterium, and in-vivo directed evolution screening is carried out to obtain various malate dehydrogenase mutants. The malate dehydrogenase mutants have high activity and can be used as novel efficient tool enzymes for preparing malic acid.

The invention discloses a method for obtaining a high-yield epsilon-polylysine streptomyces diastatochromogenes engineering strain S.diastatochromogenes MDH-OGDH by overexpressing a key enzyme malic dehydrogenase gene MDH and an alpha-ketoglutaric acid decarboxylase gene OGDH in a TCA (Trichoderma Carboxygenase) pathway, the epsilon-polylysine is produced by fed-batch fermentation in a 5L fermentation tank, and the yield (g / L) of the epsilon-polylysine is 137.18% of that of a chassis strain S.diastatochromogenes 6 #-7, and the yield (g / L) of the epsilon-polylysine is 37.18% of that of the chassis strain S.diastatochromogenes 6 #-7. The production intensity (g / L.h) of the strain is 133.68% of that of a chassis strain S.diastatochromogenes 6 #-7; the saccharic acid conversion rate of the strain is 116.04% of that of a chassis strain S.diastatochromogenes 6 #-7, the production cost is remarkably reduced, and an excellent strain is provided for industrial production of epsilon-polylysine.

The present application relates to the technical field of enzyme engineering, and discloses a malate dehydrogenase, an engineered Pichia kudriavzevii containing the same, and application of the engineered Pichia kudriavzevii. In the present application, the Pichia kudriavzevii engineering strain with NADH selection pressure after double knockout of ethanol / glycerol synthesis pathway is used as a chassis strain, in vivo directed evolution screening is carried out, and various malate dehydrogenase mutants are obtained. The malate dehydrogenase mutants have high activity and can be used as a novel and efficient tool enzyme for preparing malic acid.

Provided a genetically modified yeast strain for producing succinic acid, which strain has the activity or an enhanced activity of an NADPH-dependent malate dehydrogenase (EC 1.1.1.82), and optionally also has the activity or an enhanced activity of at least one of the following: (i) soluble fumarate reductase (EC 4.2.1.2), (ii) a pyruvate carboxylase (EC 6.4.1.1), (iii) a fumarase (EC 4.2.1.2), and (iv) succinate transport protein; and a preparation method therefor, a method for producing succinic acid using same, and the use thereof.

The invention discloses construction and application of an issatchenkia orientalis strain for producing succinic acid, and belongs to the technical field of microorganisms. Through a metabolic engineering means, a pyruvate decarboxylase coding gene on an issatchenkia orientalis genome is knocked out, and pyruvate carboxylase, malate dehydrogenase, fumarase and fumarate reductase are integrally expressed; a glycerol dehydrogenase coding gene on a genome is knocked out, dicarboxylic acid outer transport protein from schizosaccharomyces pombe is integrally expressed, carboxylic acid inner transport protein JEN2-1 and JENE2-2 on the genome are knocked out at the same time, and fumarate reductase and / or malic acid dehydrogenation are / is overexpressed. The succinic acid yield of the modified yeast strain in the fermentation process is remarkably increased and reaches 125.77 g / L, and the production intensity is 1.75 g / L / h. The engineering strain provided by the invention has good industrial application potential in efficient succinic acid production.

The present invention relates to the construction and application of a genetically engineered strain capable of realizing the co-production of branched-chain amino acids, and can realize the co-production of branched-chain amino acids (BCAA) such as valine, leucine and isoleucine by fermentation. A genetically engineered strain is obtained by a genetic engineering method, wherein the genetically engineered strain is a synthetic pathway of leucine introduced into an Escherichia coli host cell capable of producing isoleucine and / or valine, and isopropylmalate synthase encoding gene leuAm, isopropylmalate dehydrogenase encoding gene leuB, isopropylmalate dehydratase encoding gene leuCD, aromatic amino acid transaminase gene tyrB and leucine efflux protein gene leuE are expressed to release the feedback inhibition of L-leucine. The co-production of two or three branched-chain amino acids is finally realized, and the co-production process has at least 30% improvement in the utilization rate of overall glucose. The genetically engineered strain described in the present invention has a short fermentation cycle, high yield, high conversion rate, and has a high production application value.

The embodiment of the application belongs to the technical field of malate dehydrogenase preparation, and relates to a thermus thermophilus malate dehydrogenase recombinant gene and a preparation method and application thereof, comprising the following steps: adopting a gene sequence of thermus thermophilus malate dehydrogenase, optimizing the gene sequence of the thermus thermophilus malate dehydrogenase by using the synonymous codon preference of escherichia coli, obtaining a thermus thermophilus malate dehydrogenase recombinant gene, constructing a thermus thermophilus malate dehydrogenase recombinant plasmid based on the thermus thermophilus malate dehydrogenase recombinant gene, transforming the thermus thermophilus malate dehydrogenase recombinant plasmid into escherichia coli and culturing, obtaining a bacteria liquid for expressing malate dehydrogenase, purifying the bacteria liquid for expressing malate dehydrogenase, and obtaining malate dehydrogenase. The malate dehydrogenase expressed by the application has high yield, short production cycle and high activity.

The invention provides an optimized Lv-Bip-like protein and an application of the optimized Lv-Bip-like protein in improving the heat resistance of malate dehydrogenase. The method comprises the following steps: cloning an Lv-Bip-like gene from a litopenaeus vannamei body, optimizing the Lv-Bip-like gene, further constructing a recombinant vector by using a gene cloning technology, carrying out induced expression of prokaryotic protein, purifying the prokaryotic protein by using an affinity chromatography method, and carrying out ultrafiltration concentration to obtain the optimized Lv-Bip-like protein. The MDH protein is unstable when encountering heat, and experiments prove that after the optimized rBip-like protein is added under the heating condition, aggregation of the MDH protein can be reduced by optimizing the Lv-Bip-like protein, and then the heat stability of the MDH protein is improved. Therefore, the optimized Lv-Bip-like protein can be applied to feed additives, especially formula improvement of pig feed, and has a good market application prospect.