49 results about "Pyruvic oxidase" patented technology

The invention relates to a method for preparing pyruvic oxidase. The method comprises the steps as follows: (1), cultivation of microbial cells containing the pyruvic oxidase, centrifugal collection of bacterial cells, and cell disruption; (2), preparation of a polyethylene glycol / inorganic salt two-aqueous phase extraction system solution; (3), three times of extraction separation by the aid of a two-aqueous phase extraction system, and obtaining of an inorganic salt solution of the pyruvic oxidase; and (4), flirtation and concentration of an ultrafiltration membrane, freezing and drying of a trapped fluid, and preparation of a pyruvate oxidase product. The method can prepare the pyruvic oxidase continuously in a large scale, the preparation technology of the oxidase is simple and convenient, the production period is short, the operation condition is mild, the recovery rate of the oxidase activity is high, and the production cost of the pyruvic oxidase is lower.

The invention discloses engineering bacteria for producing trans-4-hydroxy-L-proline and a construction method and an application thereof. The construction method of the engineering bacteria provided by the invention includes the steps of A1) and A2): A1) introducing a L-proline-4-hydroxylase gene, a glutamate-5-kinase gene and a glutamate-5-semialdehyde dehydrogenase gene into a receptor cell; and A2) knocking an alpha-ketoglutaricdehydrogenase gene, an isocitratlyase gene or a proline dehydrogenase gene of the receptor cell out, or replacing a pyruvic oxidase gene of the receptor cell with an acetylcoenzyme A synthetase gene; and carrying out a reaction of the recombinant cell and a substrate to obtain the trans-4-hydroxy-L-proline. Experiments prove that the production method of the trans-4-hydroxy-L-proline can be used for production of the trans-4-hydroxy-L-proline.

The invention discloses a pyruvic oxidase, the amino acid sequence of which is (1) shown in SEQ ID NO: 1, or (2) obtained by losing, adding and / or substituting of one or a plurality of amino acids ofthe amino acid sequence in (1), but the pyruvic oxidase function of which is invariable. The invention also discloses a nucleotide sequence encoding the pyruvic oxidase, and comprises the recombinantvector of the nucleotide sequence, the recombinant host cell of the recombinant vector, and the kit of at least one of the pyruvic oxidase, the nucleotide sequence, the recombinant vector and the recombinant host cell. The pyruvic oxidase has good heat resistance, high activity of residual enzyme after repeated freeze thawing and high stability within wide pH value range.

The invention discloses a genetic engineering bacterium for synthesizing pyruvic acid and D-alanine as well as a construction method and application thereof. Recombinant escherichia coli of the invention is obtained by knocking out apyruvate dehydrogenase complex achEF gene, a pyruvate lyase pflB gene, a pyruvate oxidase poxB gene, a phosphoenolpyruvate synthase pps gene and a lactic dehydrogenaseldhA gene in escherichia coli BL21 (DE3), blocking a key pathway of further metabolism of pyruvic acid and knocking out genes btsT and cstA for encoding a pyruvic acid transport protein. For the genetic engineering bacterium disclosed by the invention, L-amino acid deaminase pm1 is utilized to catalyze D, L-alanine, so that L-alanine can be converted to the pyruvic acid and can be separated to obtain D-alanine at the same time, and a new idea and method is provided to chiral separation of the D, L-alanine; and the recombinant escherichia coli capable of efficiently synthesizing the pyruvic acid and the D-alanine is obtained through modification, the yield of the pyruvic acid reaches 32.0g / L, the conversion rate of the L-alanine reaches up to 80 percent, and the separation rate of the D / L-alanine reaches up to 80 percent.

The invention relates to a method for measuring homocysteine by a cystathionine beta-synthase, a cystathionine beta-catabolic enzyme, and coupled pyruvic oxidase. According to the principles: with tris(2-carboxyethyl)phosphorus hydrogen chloride, oxidized homocysteine is converted into free homocysteine; L-cystathionine is generated under catalysis of cystathionine beta-synthetase and alpha-amino-beta-hydroxyrpropionic acid reaction; homocysteine, ammonia and pyruvic acid are generated under the catalysis of cystathionine beta-clastic enzyme on the L-cystathionine; hydrogen peroxide is generated under the catalysis of pyruvic oxidase on the pyruvic acid; and then Trinder reaction color developing is performed. The homocysteine produced during the reaction makes reaction again to form a cyclic reaction. Besides, a homocysteine measurement kit can be made.

The present invention relates to 5í»-nucleotidase activity detecting method and 5í»-nucleotidase diagnosing reagent kit in medical detecting technology. The 5í»-nucleotidase diagnosing reagent kit includes buffering liquid, adenosine monophosphate, pyruvic acid, pyruvic oxidase, peroxidase, stabilizer, and reduction type chromogen combined together. Through mixing the sample and reagent in certain volume ratio to produce enzyme coupling reaction, and detecting in biochemical analyzer the main wavelength absorbency change speed, the activity of 5í»-nucleotidase is calculated. The present invention can obtain the measurement result in biochemical analyzer in high sensitivity, high precision and no contamination of various foreign and internal matters.

The invention discloses an engineering bacterium lacking organic acid production paths and use thereof in co-production of 1,3-propanediol, 2,3-butanediol and ethanol. Lactic dehydrogenase gene of lactic acid synthesis path of Klebsiella, acetate kinase gene and pyruvic oxidase gene in acetic acid synthesis path, and fumarate reductase gene and isocitrate lyase gene of succinic acid synthesis path are inactivated, so that production of organic acids in metabolism of glycerol of the Klebsiella can be reduced, and yield and conversion rate of alcohols such as1,3-propanediol, 2,3-butanediol and ethanol can be improved, and the co-production of the 1,3-propanediol, 2,3-butanediol and ethanol can be achieved. As a significant reduction in the production of the organic acids, the extraction process of the alcohols can be simplified. The production efficiency of production of high value products by microbial conversion of glycerol can be improved, the production cost is reduced, and the engineering bacterium has important application value.

The invention belongs to the technical field of microorganism genetic engineering and in particular relates to an escherichia coli genetically engineered bacterium establishment method for increasingaccumulation of pyruvic acid, and production and application. The genetically engineered bacterium provided by the invention is established by using a method of the following steps: deleting encodinggenes of lactic dehydrogenase, encoding genes of pyruvic oxidase, encoding genes of phosphotransacetylase and encoding genes of acetokinase from wild derived escherichia coli, and establishing an escherichia coli genetically engineered bacterium KLPP with accumulated pyruvic acid. From KLPP, a mutation library is established through Tn5 transposons, a mutation strain with increased pyruvic acid accumulation is screened in a high flux manner, and genes for increasing pyruvic acid accumulation can be found through whole genome sequencing.

The invention provides an enzyme-free pyruvic acid sensing detection method based on H2O2. The method comprises the following steps: adopting a three-electrode system, wherein an electrolyte solutionof the three-electrode system contains H2O2; adding sodium pyruvate with different concentrations into the electrolyte solution, and acquiring a standard curve of current response and sodium pyruvateconcentration by utilizing a chronoamperometry, wherein the working electrode of the three-electrode system is an H2O2 sensitive electrode; and comparing the standard curve according to the current response value generated by the to-be-detected sample solution containing H2O2 and pyruvic acid to obtain the concentration of sodium pyruvate. According to the method, the H2O2 consumption rate is monitored on line in real time by utilizing a Prussian blue modified electrode based on a carbonic acid removing reaction of pyruvic acid-H2O2, so that the concentration of pyruvic acid is quantitativelyobtained. The pyruvic acid concentration detection strategy adopting the method has a wide linear detection range, can effectively avoid the problems of poor repetitive effect and the like caused by poor stability of pyruvate oxidase, and can realize rapid and efficient real-time detection in a complex biological system, and the detection limit can reach 0.5 mM.

The invention provides an anti-interference film and a liver function combined test strip. The anti-interference film is obtained by wetting and treating a substrate with anti-interference treatment fluid. The anti-interference treatment fluid comprises 10-100KU / L of pyruvic oxidase, 100-500KU / L of catalase, 3-30 micronM of cupric salt and 0.1-0.3g / L of erythrocyte antibody. The liver function combined test strip comprises a diffusion film, the anti-interference film, a blood filter film, a reaction film, a color development film and a bottom board, which are stacked up in sequence. The liverfunction combined test strip prepared by the anti-interference film can detect ALT and AST two substances simultaneously and can obtain ALT, AST and ALT / AST three indicators, thereby providing more reliable guidance for clinical diagnosis; and the test strip is simple and fast, adopts smaller matched instruments and is flexible and wide in application.

The invention relates to a method for determining the activity of adenosine deaminase, and also the reagent kit for adenosine deaminase diagnosis. The reagent kit comprises cushioning solution, adenosine, glutacid, deacidized type coenzyme, adenosine triphosphate, pyroracemic acid, phosphoenolpyruvate phosphatase, glutamine synthetase, pyruvic oxidase, phosphoenolpyruvate pyruvate carboxylase, malic dehydrogenase, and stabilizer. By mixing sample and reagent of a predetermiend volumetric ratio, generating coupling reaction between them, subjecting the final reactant to biochemiscal analyser, the main wavelength absorbancy variance ratio (speed) can be detected, and the activity of the adenosine deaminase can thus be measured. The method of the invention can be used to obtain the needed measurement result purely through biochemical analytic instruments, and advantages of the method include higher sensibility, better accuracy, less susceptibility to contamination of internal or external materials, and easy application.

The invention relates to an amino acid diagnosis / measurement reagent (kit) utilizing an enzyme-colorimetry method and an enzyme immunoassay technology, and relates to a method for measuring the concentration of amino acid as well as the composition and the components of the reagent, belonging to the technical field of medical / food / environmental detection and measurement. The reagent (kit) mainly comprises the components of buffer solution, NADH, sodium bicarbonate (carbon dioxide), acetyl phosphate, taurine, amino acid oxidase, pyruvic oxidase, taurine dehydrogenase and a stabilizer. The concentration of amino acid is measured by the steps of mixing a sample with the reagent in a certain volume radio to carry out a series of enzymatic reaction, placing the reactants under an ultraviolet / visible light analyzer, and detecting the reducing degree of absorbance at the main wavelength of 340 nm.

A method for measuring the content of creatinine by use of the creatinine testing reagent kit includes such steps as proportionally mixing said reagents with specimen, enzyme coupling reaction, taking resultant, detecting the variation of primary wave in optical absorbancy by biochemical analyzer and calculating the content of creatinine. Said reagent kit is composed of 9 reagents including buffer liquid, adenosine, pyruvic acid, stabilizer, etc.

A method for measuring the content of creatinine by use of the creatinine testing reagent kit includes such steps as proportionally mixing said reagents with speciment, enzyme coupling reaction, taking the resultant, detesting the variation of primary wave in optical absorbancy by biochemical analyzer, and calculating the content of creatinine. Said reagent kit is composed 11 reagents including buffer liquid, adenosine triphosphate, oxidizing coenzyme, stabilizer, etc.

The invention discloses a test card for detecting glutamic-pyruvic transaminase by a photochemical method and a preparation method of the test card. The test card comprises a test card upper shell, atest card lower shell, a main test hole, a contrast test hole, a sample adding hole, a diffusion film, a first blood filtering film, two second blood filtering films, a first reaction film, a second reaction film and a single-sided adhesive tape, wherein the formula of a first reaction solution comprises L-alanine, pyruvate oxidase, alpha-ketoglutaric acid, peroxidase, thiamine pyrophosphate, magnesium chloride, 4-aminoantipyrine, a color developing agent, a film forming agent, a stabilizer and a buffer solution; the formula of a second reaction solution comprises pyruvate oxidase, peroxidase,thiamine pyrophosphate, magnesium chloride, 4-aminoantipyrine, a color developing agent, a film forming agent, a stabilizer and a buffer solution; by using the test card and the preparation method provided by the invention, endogenous pyruvic acid interference can be avoided, and the detection result is well matched with the hospital inspection result.

The invention relates to a reagent (kit) for diagnosing / determining amino acid by using an enzyme colorimetric method and an enzyme link method, and also discloses a method for determining the concentration of the amino acid, a composition and components of the reagent, belonging to the technical field of medicine / food / environmental test determination. The reagent (kit) comprises the main components: a buffer solution, a coenzyme, sodium bicarbonate (carbon dioxide), an acetylcoenzyme A, aldehyde, an amino acid oxidase, a pyruvic oxidase, an acetaldehyde dehydrogenase and a stabilizing agent. A sample and the reagent are mixed according to a certain volume ratio to generate a series of enzymatic reactions, and reactants are placed under an ultraviolet / visible light analyzer for detecting the degree of absorbance rise at the dominant wavelength of 340 nm, thereby measuring and calculating the concentration of the amino acid.

The invention relates to a reagent (kit) for diagnosing / determining amino acid by using an enzyme colorimetric method and an enzyme link method, and also discloses a method for determining the concentration of the amino acid, a composition and components of the reagent, belonging to the technical field of medicine / food / environmental test determination. The reagent (kit) comprises the main components: a buffer solution, a coenzyme, sodium bicarbonate (carbon dioxide), an acetylcoenzyme A, dihydro-monascus L, S-adenosyl-homocysteine, an amino acid oxidase, a pyruvic oxidase, a Lovastatin 9-ketone synthetase and a stabilizing agent. A sample and the reagent are mixed according to a certain volume ratio to generate a series of enzymatic reactions, and reactants are placed under an ultraviolet / visible light analyzer for detecting the degree of absorbance rise at the dominant wavelength of 340 nm, thereby measuring and calculating the concentration of the amino acid.

The invention relates to a method for determining the activity of adenosine deaminase, and also the reagent kit for adenosine deaminase diagnosis. The reagent kit comprises cushioning solution, adenosine, adenosine triphosphate, glutacid, pyroracemic acid, ethanol, oxidized type coenzyme, glutamine synthetase, pyruvic oxidase, hydrogen peroxidase, aldehyde dehydrogenase, and stabilizer. By mixing sample and reagent of a predetermiend volumetric ratio, generating coupling reaction between them, subjecting the final reactant to biochemiscal analyser, the main wavelength absorbancy variance ratio (speed) can be detected, and the activity of the adenosine deaminase can thus be measured. The method of the invention can be used to obtain the needed measurement result purely through biochemical analytic instruments, and advantages of the method include higher sensibility, better accuracy, less susceptibility to contamination of internal or external materials, and easy application.

The invention relates to a method for determining the activity of adenosine deaminase, and also the reagent kit for adenosine deaminase diagnosis. The reagent kit comprises cushioning solution, adenosine, adenosine triphosphate, glutacid, pyroracemic acid, glutamine synthetase, pyruvic oxidase, peroxydase, deacidized chromogen combination, and stabilizer. By mixing sample and reagent of a predetermiend volumetric ratio, generating coupling reaction between them, subjecting the final reactant to biochemiscal analyser, the main wavelength absorbancy variance ratio (speed) can be detected, and the activity of the adenosine deaminase can thus be measured. The method of the invention can be used to obtain the needed measurement result purely through biochemical analytic instruments, and advantages of the method include higher sensibility, better accuracy, less susceptibility to contamination of internal or external materials, and easy application.

The invention relates to a method for determining the activity of creatinine, and also the reagent kit for creatinine diagnosis. The reagent kit comprises cushioning solution, pyroracemic acid, phosphoenolpyruvate phosphatase, creatinine enzyme, creatinase, urease, phosphoenolpyruvate carboxylase, pyruvic oxidase, peroxydase, stabilizer, and deacidized chromogen combination. By mixing sample and reagent of a predetermiend volumetric ratio, generating coupling reaction between them, subjecting the final reactant to biochemiscal analyser, the main wavelength absorbancy variance ratio (speed) can be detected, and the creatinine content can thus be measured. The method of the invention can be used to obtain the needed measurement result purely through biochemical analytic instruments, and advantages of the method include higher sensibility, better accuracy, less susceptibility to contamination of internal or external materials, and easy application.

The invention discloses an engineering bacterium lacking organic acid production paths and use thereof in co-production of 1,3-propanediol, 2,3-butanediol and ethanol. Lactic dehydrogenase gene of lactic acid synthesis path of Klebsiella, acetate kinase gene and pyruvic oxidase gene in acetic acid synthesis path, and fumarate reductase gene and isocitrate lyase gene of succinic acid synthesis path are inactivated, so that production of organic acids in metabolism of glycerol of the Klebsiella can be reduced, and yield and conversion rate of alcohols such as1,3-propanediol, 2,3-butanediol and ethanol can be improved, and the co-production of the 1,3-propanediol, 2,3-butanediol and ethanol can be achieved. As a significant reduction in the production of the organic acids, the extraction process of the alcohols can be simplified. The production efficiency of production of high value products by microbial conversion of glycerol can be improved, the production cost is reduced, and the engineering bacterium has important application value.

The invention relates to a reagent (kit) for diagnosing / determining amino acid by using an enzyme colorimetric method and an enzyme link method, and also discloses a method for determining the concentration of the amino acid, a composition and components of the reagent, belonging to the technical field of medicine / food / environmental test determination. The reagent (kit) comprises the main components: a buffer solution, a coenzyme, sodium bicarbonate (carbon dioxide), an acetylcoenzyme A, long-chain aliphatic acid, an amino acid oxidase, a pyruvic oxidase, a fatty acid synthetase and a stabilizing agent. A sample and the reagent are mixed according to a certain volume ratio to generate a series of enzymatic reactions, and reactants are placed under an ultraviolet / visible light analyzer for detecting the degree of absorbance rise at the dominant wavelength of 340 nm, thereby measuring and calculating the concentration of the amino acid.

The invention relates to a reagent (kit) for diagnosing / determining amino acid by using an enzyme colorimetric method and an enzyme link method, and also discloses a method for determining the concentration of the amino acid, a composition and components of the reagent, belonging to the technical field of medicine / food / environmental test determination. The reagent (kit) comprises the main components: a buffer solution, a reduced coenzyme, sodium bicarbonate (carbon dioxide), acetylphosphate, an amino acid oxidase, a pyruvic oxidase, an N-methyl alanine dehydrogenase and a stabilizing agent. A sample and the reagent are mixed according to a certain volume ratio to generate a series of enzymatic reactions, and reactants are placed under an ultraviolet / visible light analyzer for detecting the degree of absorbance decrease at the dominant wavelength of 340 nm, thereby measuring and calculating the concentration of the amino acid.

The invention relates to a reagent (kit) for diagnosing / determining amino acid by using an enzyme colorimetric method and an enzyme link method, and also discloses a method for determining the concentration of the amino acid, a composition and components of the reagent, belonging to the technical field of medicine / food / environmental test determination. The reagent (kit) comprises the main components: a buffer solution, a reduced coenzyme, sodium bicarbonate (carbon dioxide), acetylphosphate, an amino acid oxidase, a pyruvic oxidase, an alanine dehydrogenase and a stabilizing agent. A sample and the reagent are mixed according to a certain volume ratio to generate a series of enzymatic reactions, and reactants are placed under an ultraviolet / visible light analyzer for detecting the degree of absorbance decrease at the dominant wavelength of 340 nm, thereby measuring and calculating the concentration of the amino acid.

The present invention relates to 5í»-nucleotidase activity detecting method and 5í»-nucleotidase diagnosing reagent kit in medical detecting technology. The 5í»-nucleotidase diagnosing reagent kit includes buffering liquid, adenosine monophosphate, pyruvic acid, phosphoenolpyruvic acid, reduction type coenzyme, pyruvic oxidase, phosphoenolpyruvate carboxylase, malate dehydrogenase and stabilizer. Through mixing the sample and reagent in certain volume ratio to produce enzyme coupling reaction, and detecting in biochemical analyzer the main wavelength absorbency change speed, the activity of 5í»-nucleotidase is calculated. The present invention can obtain the measurement result in biochemical analyzer in high sensitivity, high precision and no contamination of various foreign and internal matters.

The invention relates to a monoamine oxidase diagnosis kit using the technology of an enzyme colorimetric method and an ELISA method, a method for determining monoamine oxidase activity concentration and composition and components of a reagent, and belongs to the technical field of medical test and determination. The kit comprises the following main components: buffer solution, reduced coenzyme, amines, sodium bicarbonate (carbon dioxide), acetyl phosphate, pyruvic oxidase, alanine dehydrogenase and a stabilizer. The method comprises the following steps: mixing a sample and the reagent according to a certain volume ratio to perform a series of enzymic reaction, and then placing reactants under a UV / visible light analyzer to test the descending speed of absorbance at the position where the dominant wave length is 340nm to determine the activity concentration of the monoamine oxidase.

The invention relates to a reagent (kit) for diagnosing / determining amino acid by using an enzyme colorimetric method and an enzyme link method, and also discloses a method for determining the concentration of the amino acid, a composition and components of the reagent, belonging to the technical field of medicine / food / environmental test determination. The reagent (kit) comprises the main components: a buffer solution, a reduced coenzyme, sodium bicarbonate (carbon dioxide), acetylphosphate, an amino acid oxidase, a pyruvic oxidase, a malic dehydrogenase and a stabilizing agent. A sample and the reagent are mixed according to a certain volume ratio to generate a series of enzymatic reactions, and reactants are placed under an ultraviolet / visible light analyzer for detecting the degree of absorbance decrease at the dominant wavelength of 340 nm, thereby measuring and calculating the concentration of the amino acid.

The invention relates to a reagent (kit) for diagnosing / determining amino acid by utilizing the techniques of an enzyme colorimetric method and an enzyme coupling method, and also relates to a method for determining the concentration of amino acid and a composition and components of the reagent, belonging to the technical field of detection and determination of medicine / foodstuff / environment. The reagent (kit) mainly comprises the following components: a buffer solution, reduced coenzyme, sodium hydrogen carbonate (carbon dioxide), acetyl coenzyme A, amino acid oxidase, pyruvic oxidase, malic dehydrogenase and a stabilizer. A sample and the reagent are mixed according to a certain volume ratio to generate a series of enzymic reactions, then a reactant is placed under an ultraviolet / visible light analyzer, and the declining degree of absorbance in the position of 340 nm of main wave length is detected so that the concentration of the amino acid is measured and calculated.

The invention relates to a reagent (kit) for diagnosing / determining amino acid by using an enzyme colorimetric method and an enzyme link method, and also discloses a method for determining the concentration of the amino acid, a composition and components of the reagent, belonging to the technical field of medicine / food / environmental test determination. The reagent (kit) comprises the main components: a buffer solution, a reduced coenzyme, sodium bicarbonate (carbon dioxide), acetylphosphate, an amino acid oxidase, a pyruvic oxidase, a lactic dehydrogenase and a stabilizing agent. A sample and the reagent are mixed according to a certain volume ratio to generate a series of enzymatic reactions, and reactants are placed under an ultraviolet / visible light analyzer for detecting the degree of absorbance decrease at the dominant wavelength of 340 nm, thereby measuring and calculating the concentration of the amino acid.

The invention relates to a reagent (kit) for diagnosing / determining amino acid by using an enzyme colorimetric method and an enzyme link method, and also discloses a method for determining the concentration of the amino acid, a composition and components of the reagent, belonging to the technical field of medicine / food / environmental test determination. The reagent (kit) comprises the main components: a buffer solution, a coenzyme, sodium bicarbonate (carbon dioxide), an acetylcoenzyme A, long-chain-acyl-coenzyme A, an amino acid oxidase, a pyruvic oxidase, an acyl-coenzyme A synthetase and a stabilizing agent. A sample and the reagent are mixed according to a certain volume ratio to generate a series of enzymatic reactions, and reactants are placed under an ultraviolet / visible light analyzer for detecting the degree of absorbance rise at the dominant wavelength of 340 nm, thereby measuring and calculating the concentration of the amino acid.