309 results about "Ketoglutaric Acid" patented technology

The invention discloses an enzymic method for producing [alpha]-ketoglutaric acid. The method includes a step of carrying out a catalytic oxidation reaction, catalyzed by L-glutamic oxidase, to L-glutamic or a salt thereof in the presence of a hydrogen peroxide scavenger to obtain [alpha]-ketoglutaric acid. The method is short in production period, is high in product concentration and yield, is low in environment-protective pressure and is suitable for large-scale industrial production.

The invention is about a method of measuring the content of blood ammonia, and it also concerns the reagent box of blood ammonia diagnosis. This invention belongs to the field of medical testing and measuring technology. The reagent box is consisted of buffer solution, 2ú¡ketoglutarate, reduced coenzyme, adenosine triphosphate, phosphoenolpyruvate, glutamate dehydrogenase, glutamate kinase, pyruvate kinase, lactate dehydrogenase and stabilizer. Firstly, we cause an enzyme-coupled reaction through mixing the sample and the reagent according to a certain proportion of volume; secondly, put the final reactant under the biochemical analyzer and test the absorbance variational situation (speed) of dominant wavelength; then we can get the content of blood ammonia. By using this invention, we can get the necessary measuring result with high sensitiveness and fine precision through biochemical analyzer, and the result would not be contaminated by material of internal and exogenous sources. Thus, this method can be conveniently promoted and applied.

The invention discloses L-d-glutamic oxidase with substrate specificity and alpha-oxoglutarate produced by catalysis of the same, and belongs to the field of enzyme-method catalytic production of fine chemicals. The invention has the advantages that the L-d-glutamic oxidase which only has the substrate specificity to L-glutamic acid, L-sodium glutamate and glutamine is utilized, 20g/L L-sodium glutamate solution is added in fermented liquid which is fermented for 60 hours and contains the L-d-glutamic oxidase, carrying out conversion for 12 hours under the ventilating condition and the conditions that the temperature is 30 DEG C, the pH is 8.5 and the 5% (v/v) isopropyl alcohol, and measured by a high-performance liquid chromatography, the content of alpha-oxoglutarate reaches 14.5g/L.

The invention discloses an alpha-ketoglutarate producing yeast engineering strain and a construction method thereof, which belong to the field of genetic engineering. In the method, a molecular approach is adopted, and the yeast engineering strain Y.lipolytica ACL with improved ATP-citrate lyase activity is obtained by over-expressing an ATP-ATP-citrate lyase ACL coding gene from a mouse into a strain Yarrowia lipolytica WSH-Z06 for producing alpha-ketoglutarate by a fermentation method. Compared with a parent strain, the genetic engineering strain provided by the invention can intracellularly accumulate acetyl coenzyme A with the dry cell weight (DCW) of 0.89 mM/g by adopting glycerol as the unique carbon source so as to achieve the ACL activity of 3.56 U/mg protein improved by 7.5 times, achieve the alpha-ketoglutarat yield of 45.3 g/L which is 1.29 times that of parent protein and reduce the pyruvic acid yield to 17.2 g/L which is 68.8 percent of that of the parent strain, and has vast application prospect.

The invention relates to a method for producing alpha-ketoglutaric acid by virtue of an enzymic method. The method comprises the following steps: respectively synthesizing a glr gene and a daao gene and constructing expression vectors pET24a-glr and pET24a-daao; after double digestion of the expression vectors pET24a-glr and pET24a-daao respectively, connecting the expression vectors pET24a-glr and pET24a-daao to construct a double expression vector pET24a-glr-daao and transforming the double expression vector to escherichia coli to prepare a genetically engineered bacterium which simultaneously expresses glutamate racemase and D-amino acid oxidase; adding a wet thallus of the genetically engineered bacterium into water in a fermentation tank, then adding L-glutamic acid and catalase, and finally adjusting the pH by virtue of sodium hydroxide to prepare a reaction system for reaction; separating and purifying alpha-ketoglutaric acid converted in the reaction system after reaction. The method provided by the invention is low in cost, high in conversion rate (over 84%) and product concentration, and suitable for industrial application.

An alpha-ketoglutaric acid high-yield strain and a method for sieving and preparing alpha-ketoglutaric acid through strain by fermenting belong to the chemical engineering technique field. The strain of the invention is Yarrowia lipolytica WSH-Z06 and the preservation number is CCTCC NO: M207143. The strain is aneurine nutriment defect strain which is contrasted and sieved from the soil near the oil refinery according to the colony growth condition on the complete culture medium, basic culture medium and complementary culture medium flats, and then the aneurine nutriment defect strain is adopted by one-grade ferment to do shake flask culture one by one and sieve primarily. The fermentation liquor is analyzed by silica gel chromatography. The strain which keeps alpha-ketoglutaric acid colour development spot is transferred in the shake flask which is loaded with ferment culture medium to do ferment sieve again. The fermentation liquor is tested by high-effective liquid-phase method and produces the high-yield alpha-ketoglutaric acid strain compared with the production of alpha-ketoglutaric acid. The production of alpha-ketoglutaric acid can reach 39.3 g/L through optimizing the important component in the ferment culture medium and fermenting for 6 d.

The invention discloses a measuring method of Alanine Aminotransferase (ALT) and agent box in the external diagnostic technical domain, which comprises the following steps: reacting agent 1 and sample; generating hydrogen peroxide through reacting acetonic acid oxidase and endogenous acetonic acid; producing water and oxygen from hydrogen peroxide acted by catalase; reacting with agent 2; utilizing L-Ala and alpha-ketoglutaric acid starting serum ALT to react; detecting the activity of serum ALT of absorbance change of linear reaction.

The invention is a method of synthesizing alpha-ketoglutarate by fermenting microbes, promoting to produce a large amount of alpha-ketoglutarate in fermenting course by adding CaCO3 to the culture medium and increasing biotin concentration. in the course of fermenting candida glabrata CCTCC M202019 to producing pyruvic acid, the delay of time of adding CaCO3 will inhibit the generation of alpha-ketoglutarate and increase the carbon mole ratio of pyruvic acid to alpha-ketoglutarate (CPYR/CKG), and increase of CaCO3 concentration in the culture medium will promote accumulation of a large amount of alpha-ketoglutarate, when the CaCO3 concentration is 40g/L, it is most beneficial to alpha-ketoglutarate generation. Keeping the CaCO3 concentration in the culture medium but increasing biotin concentration in the culture medium so as to promote the continuous increase of the alpha-ketoglutarate concentration but continuous decrease of CPYR/ CKG value, and when biotin concentration is 60mum g/L, accumulated quantity of alpha- ketoglutarate is 23.5g/L. when Ca2+ exists, in-cell phosphoenolpyruvate carboxylase activity can be increased by 40%, and the activity of pyruvic acid dehydrogenase system does not change obviously. The increase of the Ca2+ and biotin concentration can remarkably the activity of enhance pyruvic acid dehydrogenase, thus making T.glabrata transfer from production of pyruvic acid by fermenting to synthesis of high-concentration alpha-ketoglutarate.

This invention relates to a test kit for diagnosing hepatic and biliary diseases. The test kit has such advantages as high stability, high accuracy and high sensitivity. The test kit comprises two reagents. Reagent 1 comprises oxidized thio coenzyme, buffer solution, preservative, and anti-interference agent. Reagent 2 comprises 3alpha-HSD, reduced coenzyme, buffer solution, preservative, complexing agent, polymer accelerator, stabilizer, and one of formate dehydrogenase-formic acid-NAD, glutamate dehydrogenase-glutamic acid-oxidized nicotinamide coenzyme, glucose-6-phosphate dehydrogenase-glucose-oxidized nicotinamide coenzyme, glucose dehydrogenase-xylose-oxidized nicotinamide coenzyme, lactate dehydrogenase-alpha-ketoglutarate-oxidized nicotinamide coenzyme, and glycerol dehydrogenase-ethylene glycol-oxidized nicotinamide coenzyme.

The invention discloses a construction method of dual-enzyme co-expression strains for producing [alpha]-ketoglutarate, and belongs to the technical fields of fermentation engineering and enzyme engineering. According to the construction method, on the basis of analyzing the production of the [alpha]-ketoglutarate from L-glutamic acid, a KatG dosage required by a transformation system is analyzed; and then, the co-enzyme co-expression strains, which are different in expression level, are constructed at a transcription level and a translation level, and the performances of the co-expression strains are evaluated by the production of the [alpha]-ketoglutarate through whole-cell transformation of L-glutamic acid at a shaker level, wherein transformation conditions are as follows: 110g/L of the L-glutamic acid, 2-2.5g/L of bacteria, a phosphate buffer system with pH value at 6.5, 30 DEG C, and 18-24h at 200rpm. The yield of the [alpha]-ketoglutarate from the optimum strain F006 can reach 107.2g/L or above, with a transformation rate of 98.4%, completely replacing exogenous catalase; bacterium concentration is increased, the yield of the [alpha]-ketoglutarate under the condition of 132g/L of a substrate can reach 127.1g/L and a transformation rate is 96.9%.

A method for detecting 2-oxoglutarate oxygenase activity, which method comprises: (i) contacting a 2-oxoglutarate oxygenase and a substrate of the 2-oxoglutarate oxygenase in the presence of 2-oxoglutarate; (ii) adding a derivatisation reagent capable of forming a fluorescent product with 2-oxoglutarate; (iii) detecting the fluorescent product produced by the reaction between the derivatisation reagent and 2-oxoglutarate, if any, thereby detecting 2-oxoglutarate oxgenase activity.

The invention discloses a method for producing a phenyllactic acid through whole-cell transformation of phenylalanine, and belongs to the technical field of enzyme engineering. The method successfullyconstructs an L-amino acid deaminase, L-lactic dehydrogenase and formate dehydrogenase co-expressed strain. Phenyllactic acid of 30 g/L is produced through the whole-cell transformation of phenylalanine, and the transformation rate is 100%. The establishment of a whole-cell transformation system solves the problems of complicated steps, low yield and environment pollution in the chemical synthesis of the phenyllactic acid as well as the problem of low transformation rate in the production of the alpha-oxoglutarate through the enzymatic conversion, the phenyllactic acid is produced through a one-step method at high yield without pollution, and a certain theoretical foundation is laid for the subsequent industrial production.

Purpose of the invention is to provide a reagent bar capable of half quantitative/ quantitative detecting whether content of glutamic-pyruvic transaminase (GPT) inside human body is normal or not rapidly, and relevant fabricating method. The reagent bar includes PVC hard strip for supporting reaction. The hard strip possesses carrier film of adsorbing L-alanine, pyruvate oxidase, and alpha-ketoglutarate. Belonging to fast test in micro scale, the disclosed product is one-off consumption material for testing. Range for detecting GPT is 0-1000U/L.

The invention relates to a kit for diagnosing/measuring glycine by utilizing the technologies of the enzymatic doubling amplification method, the enzymic colorimetry 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 measuring the concentration of the glycine, and belongs to the technical field of medical/food inspection and measurement. The main components of the kit include a buffer solution, coenzyme, a 2-ketoglutaric acid, guanosine monophosphate, glycine aminotransferase, glutamate dehydrogenase, guanosine monophosphate reduced enzyme 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 degree/velocity of the increase in absorbance at 340 nm of the dominant wavelength is detected, thereby measuring the active concentration of the glycine.

The invention discloses a high-efficiency liquid-phase chromatography detection method of L-arginine-alpha-ketoglutarate, and relates to the technical field of column chromatography. According to chromatographic condition, a chromatographic column adopts a C18 chromatographic column; a mobile phase adopts a 0.02mol/L to 0.08mol/L phosphate buffer solution with pH value of 3.0 to 4.0; the flow rate of the mobile phase is 0.5 to 1.3 ml/min; the detection wavelength is 200nm to 210nm; the temperature of the chromatographic column is 15 to 30 DEG C, and the content of L-arginine and the content of alpha-ketoglutarate in L-arginine-alpha-ketoglutarate or preparations of the L-arginine-alpha-ketoglutarate are determined by carrying out the high-efficiency liquid-phase chromatographic analysis. The content of L-arginine and the content of alpha-ketoglutarate in L-arginine-alpha-ketoglutarate or preparations of the L-arginine-alpha-ketoglutarate can be accurately determined by virtue of one-step high-efficiency liquid-phase chromatographic detection, and the two components can be well separated; moreover, the balance time of the C18 chromatographic column is short and is about 30 to 40 minutes, and the working efficiency can be improved.

The invention relates to an aspartate amino transferase (AST) detection kit and belongs to the technical field of clinic in vitro diagnostic reagents. The invention aims to provide the aspartate amino transferase detection kit with capability of effectively eliminating endogenous alpha-oxoglutarate interference and high stability in order to achieve more accurate and reliable determination results of aspartate amino transferase. The kit provided by the invention comprises a reagent 1 and a reagent 2, wherein the reagent 1 comprises the following components: Tris-HCl buffer solution, L-asparaginic acid, reduction coenzyme (NADH), malic dehydrogenase (MDH), enzyme protective agent, stabilizer and preservative, and the reagent 2 comprises the following components: Tris-HCl buffer solution, alpha-oxoglutarate, a stabilizer and a preservative. The enzyme protective agent and the stabilizer are added into the AST detection kit provided by the invention so that the stability of the aspartate amino transferase detection kit is promoted; the aspartate amino transferase detection kit is suitable for various full-automatic biochemical analyzers; the operation is simple and safe; the aspartate amino transferase detection kit has convenience in clinical application and popularization.

The invention discloses a composite dechlorination agent for removing high-concentration chlorine-containing wastewater and an application method thereof, which belong to the field of sewage treatment in environment protection. The composite dechlorination agent is prepared by compounding organic acids and inorganic salts, wherein the organic acids include two or more than two of nicotinic acid, citric acid, succinic acid, beta-ketoglutaric acid, and ketosuccinic acid; the inorganic salts include one or two of mercury sulfate, cadmium sulfate and copper sulfate; and the weight percentages of the organic acids and inorganic salts are 15-80% and 20-85% respectively. The application method comprises the steps of: adding the prepared composite dechlorination agent in percentage by weight of 0.05-0.5% into the high-concentration chlorine-containing wastewater and continuously stirring for 10-30min; adding a methylene dichloride solvent; standing for layering, separating, and subjecting obtained wastewater to air flotation treatment; and finally scraping off a layer of residue floating on the surface of the wastewater by a residue scraping machine to obtain the dechlorinated wastewater as the yielding water, wherein the dechlorinated wastewater can be lower than the national emission standard I. The composite dechlorination agent and the application method disclosed by the invention have the characteristics of simple technological process, smaller dosage of chemical agent, lower running expense, no sludge, high removal rate of chlorine ions in the wastewater, and the like; and the removal rate of chlorine ions in the wastewater can reach above 99.9%.

The invention discloses a method for extracting alpha-ketoglutaric acid from fermentation liquor. The method comprises the following steps: heating fermentation liquor containing the alpha-ketoglutaric acid for sterilization and decolorization, and drying mycoprotein; adding Ca(OH)2 into a filtration liquid, and performing centrifugal separation to obtain calcium 2-oxoglutarate and Ca(OH)2; adding H2SO4 into the calcium 2-oxoglutarate and Ca(OH)2, and performing centrifugal separation to obtain CaSO4 sediments; enabling the filtration liquid to flow into an ion exchange column for adsorption, and adding activated carbon into eluant for decolorization for 30 min; performing pressure reduction evaporation on the high-concentration alpha-ketoglutaric acid solution to obtain alpha-ketoglutaric acid crystals, and performing centrifugal separation to obtain an alpha-ketoglutaric acid crude product, washing the alpha-ketoglutaric acid crude product with methanol, removing impurities through washing, and performing centrifugation to remove methanol, so as to obtain the alpha-ketoglutaric acid, wherein the volume ratio of the alpha-ketoglutaric acid crude product to the methanol is 5:1. Through the adoption of the method, the problems in the prior art that pyruvic acid as a by-product during the extraction process of the alpha-ketoglutaric acid cannot be effectively separated and the discharge amount of waste liquid is high are solved.