215 results about "Phosphoenolpyruvic acid" patented technology

The invention discloses recombinant bacillus subtilis capable of increasing the yield of N-acetylneuraminic acid, and belongs to the field of genetic engineering. Supply of phosphoenolpyruvic acid in the synthesis pathway of N-acetylneuraminic acid is increased by taking bacillus subtilis (bacillus subtilis 168 delta nagP delta nagP delta gamP delta gamA delta nagA delta nagB delta 1dh delta pta::lox72; delta ctc::p43-Gna1, pP43NMK-AGE-NeuB) as an expression host and knocking out ptsG of a glucose specific enzyme EIICBA component of a phosphotransferase system, and therefore the synthesis passway is enhanced; compared with an original strain, the recombinant bacillus subtilis has the advantages that the yield of N-acetylneuraminic acid is increased to 660 mg/L from 190 mg/L, and a foundation is laid for improving N-acetylneuraminic acid production from bacillus subtilis in metabolic engineering.

The invention relates to a method for determining the content of blood ammonia, and also the reagent kit for blood ammonia diagnosis. The reagent kit comprises cushioning solution, adenosine triphosphate, phosphoenolpyruvate phosphatase, deacidized type coenzyme, ammonia kinase, pyruvate kinase, lactate 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 blood ammonia 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 relates to a liquid double reagent diagnostic reagent kit for determining the content of potassium ions in serum and blood plasma. By using the reaction principle that phosphoenolpyruvic acid (PEP) can be catalyzed by K+dependency pyruvate kinase (PK) to be converted into pyruvic acid and taking lactate dehydrogenase (LDH) as indicator enzyme by means of NADH, the reagent kit detects the reduction amount of the NADH at 340 nm and obtains the result that the change of the absorbance every minute (delta/min) is directly proportional to the content of K+, thereby establishing an enzyme coupling method for continuously determining the potassium ions. In addition, enzyme and a primer system required by slowly generating reduction type nicotinoyl coenzyme are added in a system andan enzyme-primer-coenzyme slow reaction system is formed in a reagent, so that the coenzyme can be slowly and circularly supplemented and the reagent can be stabilized when the concentration reaches a certain balance. The liquid double reagent diagnostic reagent kit is convenient and concise to use, carries out rapid detection on a common ultraviolet/visible light analyzer or a semiautomatic/fully-automatic chemical analyzer, needs no special or extra instrument and has low cost.

A method for constructing an ALA production bacterial strain, the method enhances the activity of related enzymes promoting the synthesis of oxaloacetate and in the 5-aminolevulinic acid (ALA) production bacterial strain, or introducing exogenous related enzymes promoting the synthesis of oxaloacetate, such as phosphoenolpyruvate carboxylase or pyruvate carboxylase, and/or reducing the activity of related enzymes in the downstream metabolic pathway of succinyl coenzyme A in the bacterial strain, such as succinyl coenzyme A synthetase or succinate dehydrogenase, and/or reducing the activity of phosphoenolpyruvate carboxylated kinase and/or malic enzyme. An ALA high-yield bacterial strain constructed by utilizing the method, and method for utilizing the bacterial strain to prepare ALA.

The present invention discloses an in-vitro cell-free protein synthesis system. The system comprises cell extract, carbohydrate and a phosphate compound, wherein the carbohydrate is maltodextrin, lactose, or a combination of the maltodextrin and glucose, or a combination of the lactose and the glucose, or a combination of the maltodextrin, the lactose and the glucose. ATP is provided for an in-vitro reaction by low-cost substances such as the glucose, the maltodextrin and the lactose instead of energy sources such as phosphoenolpyruvic acid, phosphocreatine and acetyl phosphate, so that whilethe cost is reduced, a mode of providing energy by slow release prolongs the reaction time and increases the yield of target protein.

The invention relates to a method for cell-free expression of signal protein and an expression system. The method includes joining a target gene of the signal protein on a pIX3.0 vector to obtain expression plasmids; extracting escherichia coli Rosetta (DE3) by S30 buffer solution to obtain escherichia coli extract; preparing an energy supply system comprising PEP (phosphoenolpyruvic acid)/PK (pyruvate kinase), CP (phosphocreatine)/CK (creatine kinase) and glucose; preparing an amino acid mixture and a saline solution; preparing lecithin/cholesterol liposome; adding the expression plasmids into a cell-free protein expression system comprising the escherichia coli extract, the energy supply system, the amino acid mixture, the saline solution and the lecithin/cholesterol liposome for expression. The method is capable of solving the problem of small signal protein expression quantity.

The invention discloses a corynebacterium glutamicum strain for production of 5-aminolevulinic acid and construction and application of corynebacterium glutamicum strain. A construction method includes: (1) deleting a lactic dehydrogenase coding gene 1dhA and acetic acid generation genes pta-ackA, pqo and cat in corynebacterium glutamicum to obtain a strain named CB4; inserting a strong sod promoter in front of a phosphoenolpyruvate carboxylase coding gene ppc in the strain CB4 to obtain a strain CB5; deleting a gene pck in the strain CB5 to obtain a strain CB6; (2) transferring plasmid pXA and plasmid pEP2<tuf>-rhtA into the strain CB6. The strain constructed according to the method is capable of generating 2.78g/L 5-aminolevulinic acid in a culture medium with 10g/L glucose serving as a carbon source, which lays a foundation for subsequent continuous feeding of a fermentation tank to increase yield of the 5-aminolevulinic acid.

The invention discloses recombinant bacteria for increasing the yield of succinic acid and a construction method thereof. The recombinant bacteria disclosed by the invention are obtained so as to increase the enzyme activities of phosphoenolpyruvate carboxylase (PPC) and phosphoenolpyruvate carboxykinase (PCK) in escherichia coli or mutant strains of the escherichia coli. Experiments prove that the expressions of ppc and pck genes of the E.coli are respectively regulated and controlled by using constitutive controlling elements with different expression intensity, and the law between the enzyme activities of the PPC and the PCK and the succinic acid production is explored; and on the basis, the PPC and PCK catalyzing enzymes are simultaneously used for giving play to respective catalyzing advantages through coordinated regulation of the ppc and pck genes of the E.coli, so that the yield and the conversion rate of the E.coli succinic acid are obviously increased.

The invention discloses recombinant corynebacterium glutamicum capable of being used for highly yielding L-phenylalanine and a method for producing the L-phenylalanine (Phe) by fermentation and belongs to the field of metabolic engineering. According to the invention, a corynebacterium glutamicum engineering strain C.glutamicum19AF/99TP capable of being used for highly yielding the L-Phe is obtained in a corynebacterium glutamicum type strain ATCC13032 by carrying out induction expression on the following four genes: a 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase gene, a chorismate mutase/prephenate dehydratase gene which has resistance to feedback inhibition, a transketolase gene and a phosphoenolpyruvic acid synthetase gene. A shikimic acid metabolic pathway of an L-Phe synthetic metabolic pathway is over-expressed in the C.glutamicum ATCC13032; a key enzyme gene of a chorismic acid metabolic pathway enables the yield of the L-Phe to reach 3.47g/L; two enzyme genes which combines an expression center metabolic pathway to improve a precursor enables the highest yield of the L-Phe to reach 4.86g/L; and the original strain ATCC13032 which is used as a contrast strain cannot detect accumulation of the L-Phe in the integral fermenting process.

The invention discloses a corynebacterium glutamicum for producing high-yield succinic acid by utilizing straw hydrolysate, and a construction and applications. The method comprises the following steps: (1) knocking out side products acetic acid and lactic acid formation pathway related genes from corynebacterium glutamicum ATCC 13032, and introducing anaplerotic pathway phosphoenolpyruvate carboxylase and pyruvate carboxylase and phosphopentose pathway transketoaldehydase and transketolase on a strong promoter overexpression chromosome; and introducing xylose transport gene on the chromosome; and (2) expressing pyruvate carboxylase, succinic acid export protein and citrate synthase as well as xylose isomerase and xylulokinase on the corynebacterium glutamicum obtained in the step (1). 98.6gL<-1> of succinic acid can be anaerobically produced after 22.5h by utilizing mixed glucose and xylose in straw hydrolysate, with the yield of 0.98g succinic acid/g total sugar. The final concentration, yield and productivity of succinic acid achieve higher level, and the corynebacterium glutamicum has good industrial potential.

The invention provides a carrier for enhancing aluminum-tolerance of a plant, and a method for establishing the same. The carrier is a plant expression vector having photoinduction promoters and phosphoenolpyruvate carboxylase (PEPC) genes. The method for establishing the carrier comprises the following steps: searching for the sequence of the full length gene of Synechococcus vulcanus PEPC in GenBank and designing a pair of primers with sequences as described in the specification; recovering and purifying PEPC full length gene segments and connecting the segments to a pUCm-T vector; establishing an entry vector pENTER*-PrbcS-PEPC; establishing a plant expression vector pH2-35S-PrbcS-PEPC. In the invention, the activity of citrate synthase of tabacoo with transgenic PEPC and CS genes is 2.4 to 2.6 times that of wild tobacco, and the activity of phosphoenolpyruvate carboxylase of such tabacco is 2.2 to 2.4 times that of wild tobacco. The special-purpose carrier provided in the invention can exert great influence on the improvement of aluminum-tolerance of a plant, and particularly, can significantly promote aluminum-tolerance of plants grown in acid red soil in southern China, thereby providing a novel approach for variety improvement of plants.

The invention discloses a method for knocking out pckA to promote synthesis of acetylglucosamine through bacillus subtilis and belongs to the field of genetic engineering. The method comprises the steps that BSGNK-PxylA-glmS-P43-GNA1 serves as original strains, phosphoenolpyruvate carboxylase (pckA) genes are knocked out through homologous recombination, the reaction that enolphosphopyruvate is converted into oxaloacetic acid in host bacterium cells is blocked, the carbon flux flowing to a tricarboxylic acid cycle is reduced, and accumulation of acetylglucosamine is promoted. In the process that a composite culture medium is used for fermentation, the acetylglucosamine yield of recombination bacillus subtilis with pckA knocked out reaches 29.27 g/L and is increased by 28.1% compared with that of control strains. The method lays a foundation for transforming bacillus subtilis to produce acetylglucosamine in metabolic engineering.

The invention belongs to the field of traditional Chinese medicine manufacturing, and relates to application of leonurine to preparation of a medicament for treating 2-type diabetes. Animal experiments prove that the fasting blood glucose of a 2-type diabetes mouse, i.e., a db/db mouse, can be lowered by the leonurine, and the tolerance of oral glucose is improved; and meanwhile, fasting plasma insulin is increased, the plasma triglyceride is reduced and the content of the plasma high-density lipoprotein is increased. Experiment results also show that the expression of liver glucose metabolic enzymes such as glucokinase, glucose-6-phosphatase and phosphoenolpyruvate carboxyl enzyme is adjusted by the leonurine in an Akt dependent mode; and the biological response of an inflammatory mediator, such as generation of TNF (Tumor Necrosis Factor)-alpha, degradation of IkB-alpha and subsequent phosphorylation of NF-kBp65, is suppressed. Through the leonurine, the inflammatory state of the 2-type diabetes can be corrected, and the symptoms of the 2-type diabetes can be improved; and the leonurine can be used as a treatment medicament to be applied to the treatment of the 2-type diabetes.

The invention discloses a genetically engineered bacterium for directly producing L-aspartic acid through fermentation. The classification naming of the genetically engineered bacterium is Escherichia coli CM-AS-115, and the preservation number of the genetically engineered bacterium is CCTCC NO: M 2016457. The bacterial strain relates to inactivation of multiple genes, evolution, metabolism and domestication are simultaneously carried out on the bacterial strain which knockouts the multiple genes, and a mutant strain, namely, CM-AS-105, which has a lower respiratory quotient under the aerobic condition and of which the highest dry cell weight is 60-70% of dry weight of an original strain W1485 is obtained; meanwhile, the bacterial strain further relates to over expressions of two genes, wherein the two genes comprise an enol phosphate type pyruvate carboxylase encoding gene (ppc) and an aspartase encoding gene (aspA), and the obtained bacterial strain is CM-AS-115. The genetically engineered bacterium for directly producing L-aspartic acid through fermentation achieves a way of completely adopting renewable biomass resources such as starch and cellulose as raw materials to ferment and prepare the L-aspartic acid, and the way is green and environmentally friendly.

The invention discloses a corynebacterium glutamicum engineering bacterium for anaerobic conversion to produce succinic acid, and a building method and application thereof, and belongs to the field of genetic engineering. A pyruvate carboxylase gene of the corynebacterium glutamicum and phosphoenolpyruvate carboxylase from escherichia coli are cloned to corynebacterium glutamicum (ATCC13032); a lactate dehydrogenase gene of the corynebacterium glutamicum is knocked out in a homologous recombination manner. By adopting the lactic dehydrogenase-defective corynebacterium glutamicum for coexpression of a carboxylase gene, anaerobic production of succinic acid is carried out in a cell reutilization manner, so that the yield of succinic acid can be greatly improved; the yield can be up to 75g/L; the conversion rate of saccharic acid is 75%; the corynebacterium glutamicum engineering bacterium has a good application prospect; a fermentation model, especially a fermentation model for cell reutilization is built according to the optimum condition for biological transformation of succinic acid; the acid-production performance in repeated batch transformation process of cells can be basically kept stable.

The invention discloses a corynebacterium glutamicum recombinant strain for producing 5-aminolevulinic acid. An establishing method comprises steps as follows: (1) a lactic dehydrogenase encoding gene ldhA and acetic acid producing genes pta-ackA, pqo and cat are knocked out from corynebacterium glutamicum ATCC 1303, and a strain CG4 is obtained; an sod promoter is inserted in front of a phosphoenolpyruvate carboxylase encoding gene in the CG4, and a strain CG5 is obtained; a phosphoenolpyruvate carboxykinase encoding gene pck is knocked out from the CG5, and a strain CG6 is obtained; (2) plasmids of an overexpressed 5-aminolevulinic acid synthase gene are transferred into the CG6, and a recombinant strain L is obtained; (3) 5-aminolevulinic acid transport protein plasmids are transferred into the L. The recombinant strain can promote 5-aminolevulinic acid to be transported outside corynebacterium glutamicum cells, and the yield of 5-aminolevulinic acid is 112.3% higher than that of a contrast strain.

The invention discloses recombinant bacteria for high-yield production of N-acetylneuraminic acid by utilizing manual double carbon sources and belongs to the field of genetic engineering. A gluconicacid kinase gene gntK and gluconic acid permease gntP are overexpressed through a strong promoter P43, and the manual double carbon sources including glucose and sodium gluconate are used for fermenting to realize efficient supply of phosphoenolpyruvic acid in an N-acetylneuraminic acid synthesis way and the synthesis way is strengthened. Compared with a starting strain, recombinant bacillus subtilis has the advantage that the yield of N-acetylneuraminic acid is improved to 1.52g/L from 1.02g/L; a foundation is laid for producing N-acetylneuraminic acid by carrying out further metabolic engineering modification on the recombinant bacillus subtilis.

The invention belongs to the technical field of biological engineering, and relates to a gene engineering bacterium strain for producing succinic acid, and a method for producing succinic acid by fermentation by using the same. The classification designation of the gene engineering bacterium strain for producing succinic acid is Escherichia coli BA305, and the preservation number is CCTCC NO:M2012102. The construction process mainly comprises the following steps: inactivating or knocking out phosphoenolpyruvic acid carboxylase gene and ptsG gene in a phosphate translocation system; and overexpressing the phosphoenolpyruvic acid carboxylase so that the recombinant colibacillus can efficiently utilize glucose, xylose, arabinose, levulose and other monosaccharides and efficiently utilize mixed saccharides in various proportions and cellulose hydrolysate, thereby greatly enhancing the synthesis efficiency of the succinic acid. The fermentation method adopts a two-stage fermentation mode: the aerobic stage can enhance the biomass, and the anaerobic stage is used for fermentation to produce the acid.

The invention relates to a corn C4 type phosphoenolpyruvate carboxykinase gene and an application thereof in wheat production. The gene is as indicated in SEQ ID No:1 of a sequence table, a gene clone vector is pMD19-pepc, an eucaryon expression vector is p3301-pepc, host bacteria is E.coli competent cell DH5 alpha, carboxylase is has an amino acid sequence as indicated by SEQ ID No:2. In the invention, the corn C4 type phosphoenolpyruvate carboxykinase is led into the wheat, functional analysis on transgenic plants indicates that net photosynthetic rate of the transgenic plants is greatly improved compared with the net photosynthetic rate of the plants which are not transgenic, enjoying an increase of 1.39 times. The research is so far the research in which the net photosynthetic rate of C3 crop is raised to the largest degree. The result of the functional analysis of the transgenic plants indicates that the related corn C4 type high light efficiency pepc gene can greatly improve the net photosynthetic rate of C3 crops such as wheat and the like and provides important technological and material support for high light efficiency transgenic breeding of crops such as wheat and the like.

The invention discloses a high-concentration glucose removal method for determining serum 1,5 anhydro-D-glucitol based on a pyranose oxidase method, which comprises the following steps: preparing a 2-morpholinoethanesulfonic acid (MES) buffer solution having a pH value of 6.0-9.0, wherein the MES buffer solution contains a certain amount of glucokinase (GK), glucose-6-phosphate dehydrogenase (G6PD), nicotinamide adenine dinucleotide phosphate (NADP), pyruvate kinase (PK), phosphoenolpyruvate (PEP) and adenosine triphosphate (ATP); and adding into a serum sample containing glucose (Glu), and reacting at 20-50 DEG C for 3-5 minutes, wherein the GK converts the Glu into glucose-6-phosphoric acid (G6P) in the presence of the ATP, and adenosine diphosphate (ADP) is generated at the same time; the G6P is further converted into glucose-6-phosphate lactone (G6PL) by the G6PD in the presence of the NADP+; and the ADP generated in the reaction is converted into ATP again under the action of thePK in the presence of the PEP. The glucose removal agent prepared by the method can be stably stored at 2-8 DEG C for 14 months, and can be used for a clinical biochemical autoanalyzer extensively used at present.

The invention belongs to the field of biology engineering technology, and relates to a genetic engineering strain for producing succinic acid by utilizing glucose and an acidogenic fermentation method of the genetic engineering strain. The genetic engineering strain for producing succinic acid by utilizing glucose is named as Escherichia coli BA205 and the preservation number is registered as CCTCC No.M2011447. In the construction process, Escherichia coli which is short of lactic dehydrogenase (LDH) gene and Pyruvate formate-lyase (PFL) gene activity is mainly used as an original strain; phosphoenolpyruvate carboxylase (PPC) gene is removed by utilizing a homologous recombination technology; and phosphoenolpyruvate carboxylase and nicotinic acid phosphoribosyl transferase are excessively co-expressed; therefore the synthesis efficiency of succinic acid is greatly increased. In the fermentation method, a two-stage fermentation manner is adopted, the biomass is improved in an aerobic stage and the acidogenic fermentation is carried out in an anaerobic stage.