39 results about "Oxalate oxidase" patented technology

The invention discloses an oxalate oxidase with vitality under physiological PH condition with the gene sequence shown in SEQ ID NO.1-4 and the amino acid sequence shown in SEQ ID NO.5-8, and an application thereof, and particularly discloses an application in preparation of medicine for reducing the oxalic acid concentration of the oxalosis patients by using the oxalate oxidase, and belongs to the technical field of gene engineering. Because the oxalate oxidase has vitality when the physiological PH is 7.4, the medicine containing the oxalate oxidase can be prepared into injection or embedded by an enteric-coating material. The vitality of the oxalate oxidase with the PH of 7.4 is not lower than 20% of the maximum specific activity, and the oxalate oxidase with vitality under the PH of 7.4(PH7.0-8.0) is so far uniquely disclosed and verified. The found of the oxalate oxidase provides possibility for researching the recombinant oxalate oxidase medicine to treat the oxalosis. The medicine for preventing the oxalosis can be prepared by using the oxalate oxidase.

The invention relates to methods to prevent, treat, or slow the progression of a disorder associated with elevated oxalate concentration, the method comprising administering oxalate oxidase crystals, cross-linked crystals, or compositions containing those crystals to an individual. Oxalate oxidase crystals and compositions for administration to an individual are also provided, including stabilized crystals, such as cross-linked crystals of oxalate oxidase.

The invention discloses a reagent, a kit and a method for detecting the content of oxalic acid in urine and blood and relates to the field of scientific research and detection for medical oxalic acid. The reagent comprises a buffer solution of which the pH value is 4.0-7.5, Amplex red fluorescent dye of which the concentration is 1-50[mu]mol / L, horse radish peroxidase (HRP) of which the concentration is 0.1-2U / ml and oxalate oxidase of which the concentration is 0.01-1.0U / ML. The method comprises the following steps of collecting and purifying a sample to be detected to obtain a purified liquid to be detected; dividing the liquid to be detected into two parts; preparing the liquid to be detected into a calibrated liquid to be detected; dividing the liquid to be detected of each part into two groups, namely a determining group and a comparing group; adding an oxalic acid determining reagent in the determining group to obtain a fluorescent group to be detected; adding an oxalic acid background reagent in the comparing group to obtain a determined fluorescent value after comparing the groups to be detected, and obtaining the detection limit of the oxalic acid, namely 1.5-3mol / L. The reagent, the kit and the method disclosed by the invention, is used for scientific research and clinical medicine, and high throughout detection can be realized; the method is high in sensitivity and better in reliability.

The invention is a method for extracting oxalate oxidase from wheat bran and an enzyme preparing technique, washing the wheat bran by water, then homogenizing by buffer solution, and centrifuging and collecting deposit; using the same buffer solution or distilled water to drop-wash the deposit until the eluent is clear and then collecting the deposit; successively suspending the deposit with buffer solution containing 1mol / L NaCl, preserving the heat during water bath for 1-1.5 h and then centrifuging and collecting deposit; using buffer solution or distilled water to drop-wash the deposit until the eluent is clear and then collecting deposit; adding butter solution in the deposit, making boiling water bath for 15-20 minutes and centrifuging and collecting deposit, using buffer solution or distilled water to drop-wash the deposit until the eluent is clear and then collecting deposit; then suspending the deposit by water solution containing pepsin, preserving the heat during water bath for 1-1.5 h and then centrifuging and collecting deposit, using buffer solution or distilled water to drop-wash the deposit until the eluent is clear; and finally using vacuum condense-drying system to eliminate the water in the deposit, thus obtaining the oxalate oxidase.

The invention relates to a simple method for determining oxalic acid concentration. The method comprises the following steps: (1) reacting synthetic 3NO ligand with manganese perchlorate to prepare a manganese complex; (2) reacting the prepared metal manganese complex with standard hydrogen peroxide sample with concentration being 1-400mum, determining absorbance of the reaction product at the wavelength being 450 nm, and making a standard curve of absorbance of the standard hydrogen peroxide sample at the wavelength being 450 nm; (3) reacting oxalic acid sample having unknown concentration with excessive oxalate oxidase, and decomposing the oxalic acid into equal amount of hydrogen peroxide; and (4) reacting the product of the step (3) with the metal complex prepared in the step (1), determining the absorbance of the product at the wavelength being 450 nm, and comparing with the standard curve of the absorbance of the standard hydrogen peroxide sample at the wavelength being 450 nm to obtain the concentration of the oxalic acid in the sample. The method has the advantages of simple step, high flexibility, low cost and short time.

Novel oxalate oxidases are provided, which have suitable oxalate degrading activity near physiological pH (7.4). The properties of these OxOx make them potential drug candidates for use in reducing oxalate concentration in patients suffering from excess of oxalate. Especially due to the high activity at physiological pH, the OxOx's are suitable drug candidates for parenteral administration, i.e. to reduce the oxalate concentration in the plasma.

Methods for identifying one or more amino acid substitutions in an oxalate oxidase (OXOX) variant polypeptide that confer maintained or increased OXOX activity are described herein. Methods and compositions for increasing a plant's resistance to a pathogen using the modified OXOX variant polypeptides are provided. Transformed plants, plant cell, tissues, seed, and expression vectors are also provided.

The invention relates to an electrochemical biosensor for detecting oxalic acid concentration, and preparation and application thereof. Oxalic acid oxidase is fixed on the surface of part of reduction-oxidation grapheme, a dispensing method is adopted to enable immobilized oxalic acid oxidase to be decorated on the surface of a glassy carbon electrode, and the electrochemical biosensor based on decoration of oxalic acid oxidase and part of reduction-oxidation grapheme is formed. The glassy carbon electrode serves as a working electrode, and a difference volt-ampere pulse method is adopted to detect the oxalic acid concentration. The biosensor has good electro-catalysis activity on oxalic acid. Along with increase of the immobilization amount, the electro-catalysis activity is reinforced. The detection range of the electrode is wide, and the electrode has low detection lower limit and responds rapidly.

The invention relates to a manganese ion diagnostic / assay kit by utilizing indirect enzymatic recycling method, enzymatic-colorimetric method and enzyme coupling method technology, also relates to a method and principle for assaying concentration of manganese ions, and compositions of a reagent, and belongs to the technical field of medical / food / environmental test assay. The kit mainly comprises the following compositions: buffer solution, coenzyme, oxalic acid, acetic acid, ferrocytochrome b1, coenzyme A, oxalate oxidase, two different pyruvate dehydrogenases and a stabilizing agent; and a sample is mixed with the reagent in certain volume ratio to generate a series of enzymatic reactions, and a reactant is put under an ultraviolet / visual analyzer for detecting the rising speed of the absorbency on a 340nm position of the main wavelength, so that the concentration of the manganese ions is measured and calculated.

The invention relates to a manganese ion diagnosis / determination kit using the technology of an enzyme multiplication method, an enzyme colorimetric method and an ELISA method, a method for determining manganese ion concentration and composition and components of a reagent, and belongs to the technical field of test and determination of medical science, food and environment. The kit comprises the following main components: buffer solution, reduced coenzyme, oxalic acid, acetyl coenzyme A, ammonium chloride, oxalate oxidase, pyruvate dehydrogenase, 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 concentration of manganese ion.

The invention discloses an Escherichia coli expression system for producing oxalate oxidase, a production method of oxalate oxidase and application thereof. The recombinant expression plasmid in the E. coli expression system comprises: an oxalate oxidase gene, a chaperone gene, and a gene that promotes to pump manganese ions into E. coli cell-associated protein. By screening and testing the genesof different manganese ion-related proteins and their copy number, the type of a promoter for controlling expression of the molecular chaperone and the type of a culture medium used in the productionprocess of oxalate oxidase, an optimized expression combination of oxalate oxidase is obtained, and the expression activity of oxalate oxidase in E. coli exceeds 80 U / mL. The specific activity of theoxalate oxidase preliminarily purified by a supernatant of a cell disrupted liquid is greater than 20 U / mg, and the molecular weight of the monomer is about 25 kDa. The technical scheme of the invention has the advantages of simple production and purification process, high expression level and specific activity, easy industrial amplification and low cost, and is advantageous for industrial production and application of such enzymes.

The invention relates to a manganese ion diagnosis / determination kit using the technology of an enzyme multiplication method, an enzyme colorimetric method and an ELISA method, a method for determining manganese ion concentration and composition and components of a reagent, and belongs to the technical field of test and determination of medical science, food and environment. The kit comprises the following main components: buffer solution, reduced coenzyme, oxalic acid acetyl coenzyme A, oxalate oxidase, pyruvate dehydrogenase, lactate 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 concentration of manganese ion.

The invention relates to a kit for diagnosing / mensurating manganese ions by utilizing the technologies of the enzymic colorimetric method and the enzyme linked immunosorbent assay. The invention further relates to a method, a principle and the composition and the components of a reagent for mensurating the concentration of the manganese ions, and belongs to the technology field of medical / food / environmental inspection and measurement. The main components of the kit include a buffer solution, reduced coenzyme, oxalic acid, pyruvic acid, oxalate oxidase, malate dehydrogenase and a stabilizer. Through mixing a sample and the reagent by a certain volume ratio, a series of enzymatic reactions occur, then the reactant is placed under an ultraviolet / visible light analyzer, and the velocity of the decrease in absorbance at 340nm of the dominant wavelength is detected, thereby mensurating the concentration of the manganese ions.

Novel oxalate oxidases are provided, which have suitable oxalate degrading activity near physiological pH (7.4). The properties of these OxOx make them potential drug candidates for use in reducing oxalate concentration in patients suffering from excess of oxalate. Especially due to the high activity at physiological pH, the OxOx's are suitable drug candidates for parenteral administration, i.e. to reduce the oxalate concentration in the plasma.

The invention relates to a manganese ion diagnosis / determination kit using the technology of an enzyme colorimetric method and an ELISA method, a method for determining manganese ion concentration and composition and components of a reagent, and belongs to the technical field of test and determination of medical science, food and environment. The kit comprises the following main components: buffer solution, reduced coenzyme, oxalic acid, acetic acid, ferrocytochrome b1, oxalate oxidase, pyruvate dehydrogenase, lactate dehydrogenase, and a stabilizer. The method comprises the following steps: mixing samples 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 concentration of manganese ion.

The invention relates to a manganese ion diagnosis / determination kit using the technology of an enzyme colorimetric method and an ELISA method, a method for determining manganese ion concentration and composition and components of a reagent, and belongs to the technical field of test and determination of medical science, food and environment. The kit comprises the following main components: buffer solution, reduced coenzyme, oxalic acid, acetic acid, ferrocytochrome b1, ammonium chloride, oxalate oxidase, pyruvate dehydrogenase, 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 concentration of manganese ion.

The invention discloses a urine oxalic acid content detection kit and a detection method. The kit comprises a reagent R1 and a reagent R2. The reagent R1 is prepared from the following components: citric acid, DMAB and MBTH, the concentration of the citric acid in the reagent R1 is 0.01 to 1M, and the concentration of the DMAB in the reagent R1 is 0.1 to 0.5 mM; the concentration of the MBTH in the reagent R1 is 0.01 to 0.5 mM; the reagent R2 comprises the following components: a buffer solution with the pH value of 5.6, oxalate oxidase and peroxidase; the concentration of the buffer solution in the R2 reagent is 0.1 M, the concentration of the oxalate oxidase in the R2 reagent is greater than 50 U / L, and the concentration of the peroxidase in the R2 reagent is greater than 5000 U / L; and the R2 reagent is in a dry powder state and is redissolved by water when being used. The kit disclosed by the invention has a relatively high detection limit, can be used for detecting the content of oxalic acid in urine, has relatively high detection sensitivity, and has the advantages of simplicity and rapidness in operation.

The invention discloses a reagent, kit and method for detecting oxalic acid content in urine and blood, and relates to the field of scientific research and medical oxalic acid detection. The reagent includes a buffer solution with a pH value of 4.0-7.5 and a concentration of 1-50umol Amplex red fluorescent dye per L, horseradish peroxidase HRP with a concentration of 0.1~2U / ml and oxalate oxidase with a concentration of 0.01~1.0U / ml, the method includes the following steps: collecting and purifying the sample to be tested, After obtaining the purified test solution, divide it into several parts, prepare the standard test solution, and divide each part of the standard test solution into two groups: the measurement group and the comparison group, and add the oxalic acid determination reagent to the test group to obtain the fluorescence test group; Add oxalic acid background reagent to the comparison group to obtain the comparison test group, then measure the fluorescence value, and the detection limit of oxalic acid is 1.5-3nmol / L. The invention can realize high-throughput detection and can be used in scientific research and clinical medical detection. The method has high sensitivity and good reliability.

The invention relates to a manganese ion diagnosis / determination kit using the technology of an enzyme colorimetric method and an ELISA method, a method for determining manganese ion concentration and composition and components of a reagent, and belongs to the technical field of test and determination of medical science, food and environment. The kit comprises the following main components: buffer solution, reduced coenzyme, oxalic acid, acetic acid, ferrocytochrome b1, oxalate oxidase, pyruvate dehydrogenase, malic dehydrogenase, and a stabilizer. The method comprises the following steps: mixing samples 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 concentration of manganese ion.

The invention discloses a genetically engineered bacterium for recombinantly expressing oxalate oxidase, its construction method and application, and relates to the field of genetic engineering. The method for constructing the genetically engineered bacteria comprises the following steps: optimizing the oxalate oxidase gene derived from Cerexinus worms according to the codon preference of Trichoderma reesei to obtain the optimized gene of oxalate oxidase; knocking out Trichoderma reesei The pyr4 gene and mus53 gene in the genome were used to construct a mutant strain of Trichoderma reesei with simultaneous deletion of the pyr4 gene and mus53 gene; the optimized gene of oxalate oxidase was used to construct a recombinant expression vector; the vector for recombinant expression of oxalate oxidase was used to transform T. reesei A mutant strain of the mold pyr4 gene and mus53 gene deletion at the same time is obtained to obtain a genetically engineered bacterium expressing oxalate oxidase recombinantly. The genetically engineered bacterium of the invention can efficiently express the oxalate oxidase with natural activity, and is suitable for the research and development of recombinant oxalate oxidase medicines and the production of low-oxalate foods and beverages.

The invention relates to an electrochemical biosensor for detecting oxalic acid concentration, and preparation and application thereof. Oxalic acid oxidase is fixed on the surface of part of reduction-oxidation grapheme, a dispensing method is adopted to enable immobilized oxalic acid oxidase to be decorated on the surface of a glassy carbon electrode, and the electrochemical biosensor based on decoration of oxalic acid oxidase and part of reduction-oxidation grapheme is formed. The glassy carbon electrode serves as a working electrode, and a difference volt-ampere pulse method is adopted to detect the oxalic acid concentration. The biosensor has good electro-catalysis activity on oxalic acid. Along with increase of the immobilization amount, the electro-catalysis activity is reinforced. The detection range of the electrode is wide, and the electrode has low detection lower limit and responds rapidly.

The invention relates to a manganese ion diagnosis / determination kit using the technology of an enzyme colorimetric method and an ELISA method, a method for determining manganese ion concentration and composition and components of a reagent, and belongs to the technical field of test and determination of medical science, food and environment. The kit comprises the following main components: buffer solution, coenzyme, oxalic acid, oxalate oxidase, NAD(P)H oxidase 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 ascending speed of absorbance at the position where the dominant wave length is 340nm to determine the concentration of manganese ion.

The invention relates to a simple method for determining oxalic acid concentration. The method comprises the following steps: (1) reacting synthetic 3NO ligand with manganese perchlorate to prepare a manganese complex; (2) reacting the prepared metal manganese complex with standard hydrogen peroxide sample with concentration being 1-400 mum, determining absorbance of the reaction product at the wavelength being 450 nm, and making a standard curve of absorbance of the standard hydrogen peroxide sample at the wavelength being 450 nm; (3) reacting oxalic acid sample having unknown concentration with excessive oxalate oxidase, and decomposing the oxalic acid into equal amount of hydrogen peroxide; and (4) reacting the product of the step (3) with the metal complex prepared in the step (1), determining the absorbance of the product at the wavelength being 450 nm, and comparing with the standard curve of the absorbance of the standard hydrogen peroxide sample at the wavelength being 450 nm to obtain the concentration of the oxalic acid in the sample. The method has the advantages of simple step, high flexibility, low cost and short time.

The invention relates to a manganese ion diagnosis / determination kit using the technology of an enzyme multiplication method, an enzyme colorimetric method and an ELISA method, a method for determining manganese ion concentration and composition and components of a reagent, and belongs to the technical field of test and determination of medical science, food and environment. The kit comprises the following main components: buffer solution, reduced coenzyme, oxalic acid, acetyl coenzyme A, oxalate oxidase, pyruvate dehydrogenase, malic 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 concentration of manganese ion.

The invention discloses a urine oxalic acid content detection system and detection method. The detection system comprises a pretreatment kit and a detection kit; the pretreatment kit comprises activated carbon and a sample treatment solution, wherein the detection kit comprises a reagent R1 and a reagent R2; the R1 reagent is prepared from the following components: citric acid, DMAB and MBTH; the reagent R2 comprises the following components: a buffer solution with the pH value of 5.6, oxalate oxidase and peroxidase; the concentration of the buffer solution in the R2 reagent is 0.1 M, the concentration of the oxalate oxidase in the R2 reagent is greater than 50 U / L, and the concentration of the peroxidase in the R2 reagent is greater than 5000 U / L; and the R2 reagent is in a dry powder state and is redissolved by water when being used. The method has a high detection limit, can be used for detecting the content of oxalic acid in urine, has high detection sensitivity, and can eliminate interference of substances such as vitamin C in urine, so that the detection result is more accurate.

The invention relates to a manganese diagnosis / determination reagent kit which adopts the techniques of enzymatic colorimetric method and enzyme couple reaction, belonging to the technical field of medicine / food / environment test and determination. The invention also relates to the method principle of determining the manganese concentration, and the reagent composition and components. The component of the reagent kit mainly comprises buffer solution, oxalic acid, oxalate oxidase, peroxidase, reduced chromogen combination, and stabilizer. The method for determining the manganese concentration is as follow: oxidizing the colorless reduced chromogen combination into colored dye through a series of enzymatic reactions; determining the dye content at the 400 to 700 nm wavelength with a visible light analyzer; finally determining the manganese concentration directly.