59 results about "Glycerol kinase" patented technology

Recombinant organisms are provided comprising genes encoding a glycerol-3-phosphate dehydrogenase and / or a glycerol-3-phosphatase activity useful for the production of glycerol from a variety of carbon substrates. The organisms further contain disruptions in the endogenous genes encoding proteins having glycerol kinase and glycerol dehydrogenase activities.

The present invention provides a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family, particularly a bacterium belonging to genus Escherichia or Pantoea, which has been modified to have glycerol kinase in which feedback inhibition by fructose-1,6-bisphosphate is desensitized, thereby having enhanced ability to utilize glycerol.

[PROBLEMS] To provide lipase activity determination reagents which function by an enzymatic method. The reagents are easy to use in an ordinary clinical examination, have excellent handleability, and are excellent in accuracy and reproducibility. [MEANS FOR SOLVING PROBLEMS] Lipase activity is determined with any of reagents which comprise a low-concentration buffer and a diglyceride dissolved therein. The diglyceride is used as a substrate for lipase, whereby the liquid reagents can have long-term storage stability. One of the reagents converts a monoglyceride yielded by a lipase reaction into glycerol with the aid of monoglyceride lipase, and further contains glycerol kinase, pyruvate kinase, lactate dehydrogenase, reduced NAD, ATP, and phosphoenol pyruvate. Another reagent converts a monoglyceride yielded by a lipase reaction into glycerol with the aid of monoglyceride lipase, and further contains glycerol kinase, glucose, ADP-dependent hexokinase, glucose-6-phosphate dehydrogenase, oxidized NAD or oxidized NADP, and ATP. A further reagent converts a monoglyceride yielded by a lipase reaction into glycerol with the aid of monoglyceride lipase, and further contains glycerol kinase, glycerol-3-phosphate oxidase, peroxidase, and a dye which colors in the presence of hydrogen peroxide.

The invention relates to a kit and a method for determining triglyceride. The kit comprises a reagent group 1 and a reagent group 2, wherein the reagent group 1 comprises magnesium salts, triphosadenine, glycerokinase, glycerol lipase oxidase, peroxidase and chromogen; the reagent group 2 comprises lipoprotein lipases and 4-amino-antipyrine, wherein the chromogen is N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS) or comprises DAOS. The kit and the method provided by the invention have the advantage that the interference caused by calcium dobesilate and / or etamsylate existing in the sample during the triglyceride detection can be reduced.

The invention relates to the technical field of medical examination, and specifically relates to a triglyceride detecting kit. The kit comprises an agent R1 and an agent R2, wherein the agent R1 comprises glycerol kinase, lipoprotein lipase, peroxidase, composite chromogen and magnesium salt; the agent R2 comprises glycerophosphate oxidase, 4-ampyrone and triphosadenine; the composite chromogen comprises some of 3-methyl-N, N-aniline dipropyl sulfonate, N-ethyl-N-(3-sulfopropyl)-3-sodium methylaniline, N, N-bi(4-sulfobutyl)-3-methylaniline disodium salt, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-sodium methylaniline, and 2, 4, 6-tribromo-3-hydroxybenzoic acid. The kit in detecting of triglyceride is high in sensitivity, short in detecting time, and high in interference resistance.

To provide lipase activity determination reagents which function by the enzymatic method. The reagents are easy to use in an ordinary clinical examination, have excellent handleability, and are excellent in accuracy and reproducibility. Lipase activity is determined with any of reagents which comprise a low-concentration buffer and a diglyceride dissolved therein. The diglyceride is used as a substrate for lipase, whereby the liquid reagents can have long-term storage stability. One of the reagents converts a monoglyceride yielded by a lipase reaction into glycerol with the aid of monoglyceride lipase, and further contains glycerol kinase, pyruvate kinase, lactate dehydrogenase, reduced NAD, ATP, and phosphoenol pyruvate. Another reagent converts a monoglyceride yielded by a lipase reaction into glycerol with the aid of monoglyceride lipase, and further contains glycerol kinase, glucose, ADP-dependent hexokinase, glucose-6-phosphate dehydrogenase, oxidized NAD or oxidized NADP, and ATP. A further reagent converts a monoglyceride yielded by a lipase reaction into glycerol with the aid of monoglyceride lipase, and further contains glycerol kinase, glycero-3-phosphate oxidase, peroxidase, and a dye which colors in the presence of hydrogen peroxide.

The invention relates to a method for simultaneously detecting 17 genes. Sequences of mRNA of interleukin-1beta (IL-1beta, NM-000576, etc.) and sapiens glycerol kinase (NM-203391) are referred to on PUBMED; a reverse transcription primer and a PCR primer are designed; reverse transcription, PCR amplification and capillary electrophoresis are performed; and quantitative analysis is performed by a Gexp analysis system. The detection method not only comprises a quality control gene of a kanamycin resistance gene (Kan, R) for monitoring the reaction system, but also designs two amplified products for L-selectin which are used to detect the stability of the reaction system. The invention has the advantages of small sample size required, high sensitivity and accuracy. The method analyzes the properties and components of an atherosclerotic plaque by detecting the expression situation of peripheral blood related genes, and establishes a technical platform for batch screening of differential genes between cardiovascular diseases and controls.

The invention especially relates to the glycerol kinase mutant gene glpK of Bacillus subtilis and application thereof, belonging to the field of engineering bacteria. The base sequence of the mutant gene glpK is as shown in SEQ ID No. 1. An engineered bacterium constructed in the invention and containing the glycerol kinase mutant gene glpK (G810T) of Bacillus subtilis has biosafety and clear genetic background; and the included mutant gene glpK (G810T) can greatly improve the glycerol utilization of Bacillus subtilis, and under the condition of glycerol basic salt shake-flask culture, the included mutant gene glpK (G810T) increases the specific growth rate of the bacterium by 11% and allows maximum biomass to be 16% or more.

The invention provides a detection reagent for triglyceride and detection test paper for triglyceride. The detection reagent comprises 80-400KU / L lipoprotein lipase, 9-65KU / L glycerol kinase, 6-45KU / Lglycerol triphosphate oxidase, 2-25KU / L horse radish peroxidase, 0.8-1.2mmol / L 4-aminoantipyrine, 0.6-1mmol / L chromogen and 1.0-5mmol / L Mg<2+>. The detection reagent for triglyceride can remove interference and can measure the level of triglyceride quickly and accurately.

The present invention provides a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family, particularly a bacterium belonging to genus Escherichia or Pantoea, which has been modified to have glycerol kinase in which feedback inhibition by fructose-1,6-bisphosphate is desensitized, thereby having enhanced ability to utilize glycerol.

The invention belongs to the field of electrochemical enzyme biofuel cells, and discloses a biofuel cell anode of enzymatic glycerin and a preparation method and an application of the biofuel cell anode. The preparation method comprises the following steps: carrying out surface pretreatment on a base electrode, dripping a Nafion solution on the surface of the electrode to form a Nafion film; inserting the electrode into a Meldola's blue water solution for soaking, and fixing the Meldola's blue to the Nafion film through ion exchange; taking out the electrode, and cleaning and drying the electrode to obtain the electrode containing a mediator layer; and mixing a chitosan solution containing graphene, a glycerol kinase water solution and a glycerol-3-phosphate oxidase water solution evenly at the ratio, dripping the mixed solution on the surface of the electrode and drying the surface of the electrode to form the biofuel cell anode of the enzymatic glycerin. The product disclosed by the invention has the advantages of low cost, high catalyst and mediator charge capacity, good catalytic performance and the like, and has a good application prospect.

The invention provides a triglyceride determination kit. The kit comprises a liquid single reagent R1, the reagent R1 includes the following components with concentration: lipoprotein esterase, glycerol kinase, glycerol phosphate oxidase, peroxidase, piperazine-1,4-diethanesulfonic acid, NaOH, 4-aminoantipyrine, adenosine triphosphate, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium orN-ethyl-N-(2-hydroxy-3-propyl)-3'5-dimethoxyaniline sodium salt, sodium glutamate, magnesium acetate, flavin adenine dinucleotide, polyethylene glycol, and a betaine solution. The kit belongs to the technical field of biological detection, and the triglyceride determination kit provided by the invention significantly enhances the anti-interference ability while improving the stability of the reagent, and has good measurement accuracy for triglyceride.

Lipogenic yeasts bioengineered to overexpress genes for lipid production, and methods of use thereof. The yeasts are modified to express, constitutively express, or overexpress an acetyl-CoA carboxylase, an alpha-amylase, an ATP citrate lyase, a diacylglycerol acyltransferase, a fatty acid synthase, a glycerol kinase, a 6-phosphogluconate dehydrogenase, a glycerol-3-phosphate dehydrogenase, a malic enzyme, a fatty acyl-CoA reductase, a delta-9 acyl-CoA desaturase, a glycerol-3-phosphate acyltransferase, a lysophosphatidate acyltransferase, a glucose-6-phosphate dehydrogenase, a beta-glucosidase, a hexose transporter, a glycerol transporter, a glycoside hydrolase enzyme, an auxiliary activity family 9 enzyme, or combinations thereof. The yeasts in some cases are also modified to reduce or ablate activity of certain proteins. The methods include cultivating the yeast to convert low value soluble organic stillage byproducts into lipids suitable for biodiesel production and other higher value uses.

Methods of using crude glycerol to make fatty acids are provided, as well as integrated methods of converting glycerol waste from biodiesel production into more biodiesel. Bacteria and other microbes engineered to produce free fatty acids from glycerol are also provided.

The invention belongs to the technical field of electrochemical biosensors, and particularly discloses an enzyme biosensor for detecting glycerol based on carboxylated nano-zinc oxide and a preparation method and application thereof. The enzyme biosensor adopts a classical three-electrode system, a specific substance recognition film is solidified on a working electrode, and the substance recognition film is mainly prepared by mixing a carboxylated zinc oxide material layer, a glycerol kinase solution, a glycerol triphosphate oxidase solution and a chitosan solution. A three-electrode system is formed by using the modified working electrode, a reference electrode and a counter electrode so as to obtain the enzyme biosensor for detecting glycerol. The enzyme biosensor for detecting glycerolhas good electron transmissibility, can well transfer electrons generated by reaction, can realize selective detection of biomolecules, improves the reaction speed of the biosensor, and has good selectivity, reproducibility and stability.

The invention discloses a cyclophorase determination method for triglyceride in serum, and belongs to a method for measuring materials by measuring color change generated by reactions. A technical solution lies in that a reagent I contains effective components of glycerol kinase, 3-phosphoglycerol dehydrogenase, phosphoglycerol oxidase, peroxidase, adenosine triphosphate, NADH, 4-aminoantipyrine and 2,4-dichlorophen; and a reagent II only contains an effective component of lipoprotein lipase. Free glycerol in the serum is subjected to circular reactions with adenosine triphosphate, NADH, 4-aminoantipyrine and 2,4-dichlorophen in the reagent I under catalysis of glycerol kinase, 3-phosphoglycerol dehydrogenase, phosphoglycerol oxidase and peroxidase to generate quinoneimine; and after the reagent II is added, triglyceride is hydrolyzed to generate glycerol, and then the circular reaction is carried out. The content of triglyceride is calculated by an instrument based on quinoneimine generated by reactions in the reagent II, by using quinoneimine generated by reactions in the reagent I as a blank.

Lipogenic yeasts bioengineered to overexpress genes for lipid production, and methods of use thereof. The yeasts are modified to express, constitutively express, or overexpress an acetyl-CoA carboxylase, an alpha-amylase, an ATP citrate lyase, a diacylglycerol acyltransferase, a fatty acid synthase, a glycerol kinase, a 6-phosphogluconate dehydrogenase, a glycerol-3-phosphate dehydrogenase, a malic enzyme, a fatty acyl-CoA reductase, a delta-9 acyl-CoA desaturase, a glycerol-3-phosphate acyltransferase, a lysophosphatidate acyltransferase, a glucose-6-phosphate dehydrogenase, a beta-glucosidase, a hexose transporter, a glycerol transporter, a glycoside hydrolase enzyme, an auxiliary activity family 9 enzyme, or combinations thereof. The yeasts in some cases are also modified to reduce or ablate activity of certain proteins. The methods include cultivating the yeast to convert low value soluble organic stillage byproducts into lipids suitable for biodiesel production and other higher value uses.

Recombinant organisms are provided comprising genes encoding a glycerol-3-phosphate dehydrogenase and / or a glycerol-3-phosphatase activity useful for the production of glycerol from a variety of carbon substrates. The organisms further contain disruptions in the endogenous genes encoding proteins having glycerol kinase and glycerol dehydrogenase activities.

The invention discloses a recombinant bacterium for synthesizing butantriol oleate as well as a construction method and application of the recombinant bacterium, belonging to the technical field of gene engineering. For the recombinant bacterium, escherichia coli is used as an original strain, and a gene for encoding acyltransferase, a gene for encoding acyl CoA synthetase, a gene for encoding enoyl ACP reductase, a gene for encoding glycerol kinase, a gene for encoding 3-phosphoglycerol acyltransferase and a gene for encoding DAGP phosphatase are introduced into the escherichia coli to obtainrecombinant bacterium. Through fermentation, first synthesis of 1,2,4-butantriol oleate is realized. The recombinant bacterium constructed by the invention is fermented in an LB culture medium for 24hours, and the esterification of butantriol and sodium oleate can be realized to obtain the butantriol oleate, so that a new thought is provided for separation and purification of the butantriol.

The invention discloses a preparation method for D-type rare ketohexose. The preparation method comprises the steps that glycerin and pyrophosphoric acid are taken as substrates, and a multi-enzyme catalyst containing L-rhamnulose-1-phosphate aldolase, L-alpha-glycerophosphate oxidase, catalase, acid phosphatase and alditol oxidase is added to establish a multi-enzyme reaction system to perform anenzyme catalysis reaction, and products of the enzyme catalysis reaction are separated and purified. According to the preparation method, the glycerin and the pyrophosphoric acid are taken as raw materials instead of expensive D-glyceraldehyde or D-fructose, and therefore, the D-type rare ketohexose is low in production cost, and is suitable for large-scale production. According to the preparation method disclosed by the invention, glycerol kinase and adenosine triphosphate (ATP) are not needed in performing substrate phosphorylation to produce glycerol-3-phosphate without, thus greatly reducing the cost of producing the D-type rare ketohexose.

Disclosed is a method for selectively eliminating triglycerides in lipoproteins other than low density lipoprotein, which method allows one to provide a method for directly and differentially quantifying LDL-TG in a sample with excellent simplicity, specificity and accuracy using an automated analyzer or the like without performing a laborious operation of pretreatment such as centrifugation or electrophoresis. The method for eliminating triglycerides in lipoproteins other than low density lipoproteins includes allowing lipoprotein lipase, cholesterol esterase, glycerol kinase and glycerol-3-phosphate oxidase to act on a sample in the presence of a surfactant that acts on lipoproteins other than low density lipoprotein and / or a surfactant having LDL-protecting action, and eliminating hydrogen peroxide produced thereby.

The invention discloses a preparation method for L-type rare ketohexose. The preparation method comprises the following steps: with glycerol and pyrophosphoric acid as substrates, adding aldolase containing L-rhamnose-1-phosphate, glycerophosphate oxidase, catalase, acid phosphatase, equine dehydrogenase and NADH oxidase into the glycerol and pyrophosphoric acid so as to establish a multi-enzyme reaction system, and carrying out enzyme catalytic reaction; and separating an enzyme catalytic reaction product, and carrying out purifying. According to the invention, raw materials are low in price;glycerol triphosphate is produced without glycerol kinase and ATP for substrate phosphorylation; the cost of producing the L-type rare ketohexose is greatly reduced; equine dehydrogenase and NADH oxidase are combined to produce L-glyceraldehyde; and use of NAD+ is reduced.

Relating to a gene encoding a new glycerol kinase and a method for preparing the enzyme by gene recombination technique. A Glycerol kinase which has high resistance against preservative, a recombinant vector comprising a gene encoding the glycerol kinase, a transformant prepared by transforming a host cell with the recombinant vector, and a method for producing the glycerol kinase, including culturing the transformant to produce glycerol kinase, and collecting the resulting glycerol kinase.

The invention relates to a use of glycerol kinase in preparation of a drug for regulating lipid metabolism. The use reveals that glycerol kinase can catalyze glycerol into 3-phosphoglycerol for triglyceride synthesis, control the expression of SREBP-1c to adjust and control hepatic fatty acid and triglyceride synthesis and play an important role in maintenance of the blood lipid homeostasis. The lipid metabolism disorder mouse model has high glycerol kinase expression and thus through inhibiting the expression of glycerol kinase, the drug can significantly improve high fat food-induced fatty liver and hypertriglyceridemia. Therefore, glycerol kinase is a new target for preventing, alleviating or treating fatty liver and hypertriglyceridemia.

The invention discloses a preparation method of glycerol kinase, in particular comprising the following steps: S1, culturing in a fermentor: firstly, inoculating glycerol-preserving bacteria in a testtube LB culture medium, culturing in a shaking table at 37 DEG C and 200r / min for 10h; and secondly, culturing the glycerol-preserving bacteria in a shaking table at 37 DEG C and 200r / min for 10h. 12h; S2, inoculated in shake flask fermentation medium according to 2% inoculum, cultured in the same condition for 10h-12 hour as seed strain; S3, fermentor cultured until glucose is consumed completely, adding 6.0 g / L lactose to induce fermentation. The invention relates to the technical field of medical reagents. The preparation method of glycerol kinase can make the enzyme-producing characteristics of natural bacteria more stable and greatly enhance the activity of glycerol kinase, and realizes the expression of the fermentation and culture conditions of recombinant glycerol kinase r-GK engineering strain, the establishment of purification process and the quality control detection of the enzyme, these research results will be a solid foundation for the development and utilization of theenzyme.

The invention discloses a rapid blood fat detector and a detection method, the rapid blood fat detector comprises a test strip, an electrode plate and an electrochemical workstation, the test strip is provided with a siphon port for absorbing a solution to be detected, the test strip is connected with the electrode plate through a three-electrode system, and the electrode plate is connected with the electrochemical workstation for determination; the test strip comprises three types, namely a test strip for determining total cholesterol fixed with cholesterol oxidase, a test strip for determining high-density lipoprotein cholesterol fixed with cholesterol esterase and a test strip for determining triglyceride fixed with lipoprotein fatty acid and glycerol kinase, and by adopting a screen printing test strip, utilizing an electrochemical method and combining the catalytic action of enzyme on a substrate, high-sensitivity rapid detection of TG, TC and HDL-C values can be realized, then the LDL-C value is obtained through calculation, and rapid detection of four items of blood fat is realized.

The invention provides applications of a diacylglycerol kinase gamma (DGK gamma) and an activator thereof to preparation of medicament for treating liver cancer, also provides applications of diacylglycerol kinase gamma and an agent for detecting diacylglycerol kinase gamma to preparation of an agent for diagnosis of liver cancer with bad prognosis, and also provides applications of diacylglycerol kinase gamma or the agent for detecting diacylglycerol kinase gamma to preparation of the agent for predicating prognosis conditions of liver cancer.

he invention relates to yarrowia lipolytica YW100-1 and application thereof. The yarrowia lipolytica YW100-1 is obtained by overexpression of glycerol kinase GUT1, glycerol kinase GUT2, glycerol dehydrogenase GCY1, dihydroxyacetone kinase DAK1, dihydroxyacetone kinase DAK2 and NADH kinase POS5 in glycerol metabolism in the yarrowia lipolytica, and knockout of diacylglycerol acyhransferases genes DGA2 and 3-glycerophosphate dehydrogenase genes GPD2 in the yarrowia lipolytica. According to the yarrowia lipolytica YW100-1, when a bacterial strain takes glycerol as a carbon source, the yield of pyruvic acid is increased by 66.2% and reaches 121.2 g / L, and a good bacterial strain is provided for industrial production of the pyruvic acid.

The present invention relates to uses of glycerokinase as a glucose metabolism disorder treatment target. According to the present invention, the results show that glycerokinase can catalyze glycerol to generate 3-phosphoglycerol so as to participate in gluconeogenesis, can regulate the expressions of gluconeogenesis enzyme glucose-6-phosphatase (G6pase) and phosphoenolpyruvate carboxykinase (PEPCK) through an AKT-FOXO1 signaling pathway so as to regulate gluconeogenesis, and can provide important effects in the maintaining of the blood glucose homeostasis, such that the glycerokinase and the regulator thereof are the new target for blood glucose regulation and diabetes treatment.