76 results about "Glucose phosphate" patented technology

The invention provides a genetically engineered bacterium for synthesizing fucosyllactose as well as a preparation method and application of the genetically engineered bacterium. The genetically engineered bacterium is prepared by knocking out a glucose-1-phosphate undecanoate transferase gene wcaJ and a beta-galactosidase gene lacZ on the basis of an original strain, the invention relates to an overexpression gene of a phosphomannose gene manB, a phosphoguanine transferase gene manC, a GDP-mannose-4, 6-dehydratase gene gmd, a GDP-fucose synthetase gene WcaG and a beta-galactoside permease gene lacY, and further, the overexpression gene of an alpha-1, 2-fucosyltransferase or an alpha-1, 3-fucosyltransferase is also used for the overexpression of a phosphomannose gene manB, a phosphoguanine transferase gene manC, a GDP-mannose-4, 6-dehydratase gene gmd, a GDP-fucosyltransferase gene. According to the invention, the modified Escherichia coli probiotic Nissle1917 is preferably used as a host for biosynthesis of fucosyllactose, no endotoxin hidden danger exists, and a foundation is laid for industrial production of fucosyllactose.

The invention belongs to the technical field of chondroitin sulfate biosynthesis, and discloses a recombinant strain for high-yield chondroitin sulfate, application of the recombinant strain and a preparation method of the chondroitin sulfate. The recombinant strain provided by the invention can be used for simultaneously expressing UDP-glucosamine isomerase, UDP-glucose dehydrogenase, chondroitin polymerase, glucosamine synthetase, phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, glucose-6-phosphate isomerase and glucose phosphate mutase; according to the present invention, glucose-1-phosphate uridyl transferase, ATP sulfonase, sulfotransferase, pyruvate kinase and APS kinase are adopted, and the enzyme molecules are matched with the original carbon metabolism pathway in the recombinant strain cell to jointly form the enzyme-linked pathway having the efficient CSA synthesis ability, such that the high-yield and high-purity preparation of the CSA can be achieved; the method has the advantages of simple operation, high biological safety of the obtained product, easy realization of large-scale production and the like, and has a good application prospect.

The invention discloses a genetically engineered bacterium for producing uridine diphosphate glucose as well as a construction method and application of the genetically engineered bacterium, and belongs to the technical field of genetic engineering. The following genes are overexpressed in the genetically engineered bacterium: a pyrimidine nucleoside operon gene, a carbamyl phosphate synthetase gene, an aspartate carbamyl transaminase gene, an orotic acid phosphoribose transferase gene, a phosphoribose pyrophosphate synthase gene, a uridylate kinase gene and a glucophosphate mutase gene. A uridine triphosphate glucose-1-phosphate uridine group transferase gene; the genetically engineered bacterium does not express the following genes: a purine nucleotide repressor protein gene, a deoxyribonucleoside triphosphate reductase gene, a nucleoside triphosphate pyrophosphohydrolase gene, a cytidine triphosphate synthase gene, a pyrimidine nucleotidase gene, a nucleotidase gene, an ornithine carbamoyltransferase gene, a UDPG dehydrogenase gene and a lacI repressor protein gene. The genetically engineered bacterium can be used for efficiently synthesizing UDPG by taking glucose as a substrate.

The invention provides a method for increasing the yield of bacterial cellulose by utilizing low-dose ultraviolet radiation, and belongs to the field of microbial fermentation. The method comprises the following steps: inoculating a fermentation culture medium with a Taonella.mepensis seed solution, carrying out fermentation treatment, and carrying out ultraviolet radiation under set conditions in the fermentation process, so as to obtain a fermentation solution containing a bacterial cellulose membrane; and taking out the bacterial cellulose membrane in the fermentation liquor, and purifying the bacterial cellulose membrane to obtain the bacterial cellulose. On the basis of an HS culture medium formula, the ultraviolet irradiation time is regulated and controlled at the same time, so that the activity of cellulose synthesis related enzymes, such as the activity of UDP-glucose pyrophosphorylase and glucose phosphate mutase, is improved, the activity of glucose decomposition pathway related enzymes, such as pyruvate kinase and phosphofructokinase, is inhibited, and the activity of cellulose synthesis related enzymes is improved. The yield of the bacterial cellulose is further improved.

Engineering bacteria for expressing an alpha-glucose phosphate mutase gene, a glucose phosphate mutase gene, a difunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene and a mannose 6-phosphate phosphatase gene are used as a whole-cell catalyst, starch or a starch derivative is used as a substrate, phosphate and Mg < 2 + > are added, and the whole-cell engineering bacteria are obtained. A whole-cell catalysis system is constructed, and starch or starch derivatives can be efficiently catalyzed to synthesize D-mannose. According to the method, the D-mannose is prepared through whole-cell catalysis, and the method has the advantages of being low in cost, low in pollution, high in yield and the like and is suitable for industrial production of the D-mannose.

The invention provides application of acidophilous glycosyltransferase in salidroside production, and belongs to the technical field of biological engineering. The problem of producing salidroside under the acidic condition is solved. Comprising an application of acidophilous glycosyl transferase with an amino acid sequence as shown in SEQ ID NO.1 in salidroside production under an acidic condition and an acidophilous escherichia coli engineering strain for producing salidroside. The escherichia coli engineering strain overexpresses a mutant 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthetase gene aroGfbr, a cyclohexadiene dehydrogenase gene tyrC, a glucose phosphate mutant enzyme gene pgm and a UDP-glucose pyrophosphorylase galU, overexpresses a phenylpyruvate decarboxylase gene ARO10 derived from saccharomyces cerevisiae, and can be used for producing a mutant 3-deoxy-D-arabinoheptulose-7-phosphate mutant enzyme. The kit comprises an ethanol dehydrogenase gene ADH6 and a glycosyl transferase gene LrUGT85AF8. The method is mainly used for producing salidroside under an acidic condition.

The invention discloses a method for preparing inositol, particularly relates to a method for converting glucose into inositol through in-vitro multi-enzyme catalysis, and belongs to the field of enzyme catalysis preparation of inositol. According to the invention, the phosphoglucose synthase capable of realizing phosphorylation of glucose by using inorganic phosphate is screened, and the inositol can be generated through conversion by taking glucose as a substrate, so that a novel method for synthesizing the inositol through enzyme catalytic conversion is provided. In addition, the invention further provides hexokinase capable of improving the yield of inositol, and the conversion efficiency of glucose is greatly improved. Furthermore, the invention provides a multi-enzyme molecular machine containing the glucose phosphate synthase and / or hexokinase, the multi-enzyme molecular machine is applied to preparation of inositol, and the reaction steps are simple, green and pollution-free.

The invention provides a method for synthesizing D-psicose through fermentation of escherichia coli by using D-glucose and D-xylose. The method comprises the following steps: by taking escherichia coli as a host cell, over-expressing a galactose permease gene galP, a glucokinase gene glk, a 6-phosphate glucose isomerase gene pgi, a D-psicose-6-phosphate epimerase gene alsE with an SUMO tag and a D-psicose-6-phosphate phosphatase gene a6PP, and constructing a path for synthesizing D-psicose by taking D-glucose and D-xylose as substrates; the synthetic route of D-psicose is optimized by knocking out a D-glucose phosphorylation transport pathway gene ptsG, knocking out D-fructose-6-phosphokinase genes pfkA and pfkB, silencing a glucose-6-phosphate dehydrogenase gene zwf, overexpressing glutamate transhydrogenase genes gdh1 and gdh2, knocking out a UDP-glucose-4-epimerase gene galE and knocking out a transport protein key gene fryA in D-psicose; and finally, the optimal proportion of mixed sugar fermentation is explored, so that efficient synthesis of D-psicose from escherichia coli through mixed sugar fermentation of D-glucose and D-xylose is realized.

The invention belongs to the field of gene engineering, and particularly relates to application of a willow uridine diphosphate glucose-4-epimerase gene SpUGE1 in plant cell wall component remodeling, organelle cadmium content reduction and cadmium resistance improvement. According to the invention, the SpUGE1 gene in willow is extracted, and is transferred into arabidopsis thaliana by constructing a recombinant plasmid, so that the biological activity, Cd < 2 + > concentration and the like of wild arabidopsis thaliana and transgenic arabidopsis thaliana are compared. The result of the embodiment shows that the transgenic arabidopsis thaliana has certain tolerance to cadmium stress, the cell wall of the transgenic arabidopsis thaliana is obviously thickened, and the content of cadmium in organelles is obviously reduced.

The invention discloses an extraction method of mogroside V, and relates to the technical field of biology. Escherichia coli containing glycosyl transferases UGT-MS1 and UGT-MS2 is cultured, fermented, centrifuged and homogenized to obtain enzyme clear liquid, mogroside IIE, uridine diphosphate glucose, the enzyme clear liquid and salt are added into a phosphate buffer solution for enzyme catalysis to obtain conversion liquid, and the conversion liquid is subjected to enzyme purification to obtain the mogroside V. And finally, carrying out ultrafiltration, column loading and elution, then feeding into alkaline anion exchange resin, and concentrating and drying effluent to obtain mogroside V. The product is high in yield and environment-friendly.

The invention provides a method for eliminating the Crab effect of saccharomyces cerevisiae through sucrose phospholysis, which comprises the following steps: gradually performing targeted knockout on SUC2, MAL12, MAL32, MPH2, MPH3, IMA1, IMA2, IMA3, IMA4 and IMA5 genes in the saccharomyces cerevisiae by adopting a CRISPR / Cas9 system, and introducing exogenous genes PvSUF and LmSP to enable the saccharomyces cerevisiae to transfer sucrose into cells and perform phospholysis to obtain sugar and 1-phosphoric acid-glucose. On the basis of phospholysis of sucrose, the energy loss in the sucrose utilization process is reduced, the Crab effect is eliminated through flux shunt of central carbon metabolism, the constructed Crab negative strain does not pass evolution and has a good genetic background, and meanwhile, a synthesis path of acetyl CoA in cytoplasm is not removed, so that the cell growth pressure is reduced.

The invention provides a hydrogen production reaction system taking formaldehyde and water as co-substrates, which comprises a buffer solution, maltodextrin, formaldehyde, magnesium chloride, manganese chloride, NAD (Nicotinamide Adenine Dinucleotide), benzyl viologen, sodium phosphate, alpha-glucan phosphorylase, phosphoglucomutase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, 6-phosphoglucolactonase, diaphorase and hydrogenase. The enzyme is prepared from 1, 6-hexanediol phosphate synthase, hexanediol phosphate isomerase, glucose phosphate isomerase and 2, 6-hexanediol phosphate synthase. The invention also provides a corresponding hydrogen production method. Formaldehyde and water are utilized to generate hydrogen, so that excessive dependence on agricultural grain resources is avoided; meanwhile, high-energy formaldehyde is used for replacing traditional carbohydrates as a substrate, so that the energy barrier of co-substrate water is reduced, and the process of small molecular utilization of carbohydrates and hydrogen production is simplified; in addition, the method has the advantages of being not harsh in water quality condition and low in cost.

The application provides a myo-inositol-3-phosphate synthase mutant, which is obtained by mutating a myo-inositol-3-phosphate synthase with an amino acid sequence as shown in SEQ ID NO: 4 at positions 55, 57 and 273 respectively. The application also provides a preparation method of myo-inositol. The inventors have screened a wild-type myo-inositol-3-phosphate synthase and a myo-inositol monophosphatase with good performance, which can be used in cooperation with a glucan phosphorylase and a glucose phosphate mutase to catalyze starch to generate myo-inositol more efficiently. The myo-inositol-3-phosphate synthase mutant is applied to in-vitro non-fermentation biosynthesis of myo-inositol, combined with myo-inositol monophosphatases E1 and E5, and in a way of feeding whole cells, so that starch can be converted into myo-inositol in a one-pot method, without using NAD + coenzymes, and has a good industrial application prospect.

The invention relates to the technical field of biomedicine, in particular to a serum protein marker for ossification of ligamentum flavum and application of the serum protein marker. The serum protein marker comprises erythrocyte glucose phosphate isomerase and / or dystrophin associated glycoprotein 1. According to the method, a data-independent quantitative proteomics strategy is utilized to compare a normal healthy control (a patient who does not suffer from ligamentum flavum ossification and needs spinal surgery to take ligamentum flavum tissues) with a ligamentum flavum ossification patient; it is found that ligamentum flavum ossification-related proteins, namely erythrocyte glucose phosphate isomerase (GPI) and dystrophy protein-related glycoprotein 1 (DAG1), with the obviously-changed expression quantity in tissue are beneficial to diagnosis of patients with ligamentum flavum ossification and provide effective targets for development of treatment methods of the ligamentum flavum ossification.

The invention discloses a genetically engineered bacterium for producing heparinogen and a construction method and application thereof, the genetically engineered bacterium takes Escherichia coli Nissle 1917 as a chassis bacterium, and the construction method comprises the following steps: (1) knocking out endonuclease I coding gene endA in a chassis bacterium genome; (2) knocking out a 6-phosphofructokinase I coding gene pfkA in a chassis bacterium genome; (3) carrying out overexpression on a 6-phosphoglucosamine synthetase encoding gene glmS and / or a UDP-glucose-6-dehydrogenase encoding gene kfiD from a chassis bacterium genome; (4) removing the feedback inhibition of the 6-phosphate glucosamine on the GlmS so as to promote the synthesis of UDP-N-acetylglucosamine; and (5) knocking out the 6-phosphate glucosamine deaminase coding gene nagB in the chassis bacterium genome. The engineering strain obtained by the invention can more efficiently synthesize the proheparin, the shake flask titer reaches 397.9 mg / L, which is increased by about 3 times compared with a wild strain, and the engineering strain has certain industrial application value.

The method for catalytically preparing D-mannose provided in the application can effectively reduce the amount of by-products glucose and fructose, and greatly improve the conversion rate of D-mannose. Specifically, the engineering bacteria expressing isoamylase gene and the engineering bacteria expressing alpha-glucan phosphorylase gene, glucose phosphomutase gene, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene and mannose 6-phosphate phosphatase gene are used, starch or starch derivatives are used as substrates, phosphate buffer solution and Mg 2+ A preliminary catalytic system is constructed, and after the reaction is completed, there are unreacted substrates and maltodextrin in the reaction system, the saccharifying enzyme can hydrolyze the unreacted substrates and maltodextrin into glucose, the polyphosphate glucose kinase can convert all the glucose into phosphorylated glucose with the assistance of sodium hexametaphosphate, and then D-mannose is generated, thereby improving the yield and removing the by-products in the system.

This application relates to a phenytoin detection kit. Specifically, the glucose-6-phosphate dehydrogenase mutant of this application contains one mutation selected from the following compared to the wild-type glucose-6-phosphate dehydrogenase: D306C, D375C, or G426C. The detection kit prepared using the glucose-6-phosphate dehydrogenase mutant of this application exhibits high specificity, high sensitivity, ease of operation, short detection time, and accurate quantification, making it suitable for high-throughput detection.

The invention provides genetically engineered saccharomycetes capable of simultaneously producing baicalein and baicalin as well as a construction method and application of the genetically engineered saccharomycetes. According to the invention, by weakening or deleting a glucose 5-phosphate dehydrogenase gene (ZWF1) and phenylpyruvate decarboxylase genes ARO10 and PDC5 in a saccharomyces cerevisiae genome and enhancing the expression of a pre-benzoic acid dehydratase gene Pha2, the metabolic flow of a shikimic acid pathway is increased, the aldrin pathway is blocked, and the yield of the shikimic acid pathway is increased. Therefore, the yield of L-phenylalanine serving as a starting material for synthesizing baicalein and baicalin is increased; meanwhile, exogenous genes PAL, 4CL, CHS, CHI, FNSII, F6H, CPR and F7GAT are integrated onto the genome by virtue of a gene recombination technology, so that baicalein and baicalin are produced from the beginning by taking glucose and glycerol as a mixed carbon source without adding precursor substances such as exogenous L-phenylalanine or malonyl CoA and the like.

The application discloses a bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant and application thereof, and belongs to the technical field of enzyme engineering and biological catalysis. The application provides a bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant, wherein at least one of the glutamic acid at the 28th position, the arginine at the 83rd position, the valine at the 126th position, the isoleucine at the 161st position, the leucine at the 187th position, the phenylalanine at the 249th position, the leucine at the 319th position and the tyrosine at the 343rd position in the amino acid sequence shown in SEQ ID No. 1 is mutated. The application further improves the conversion efficiency of D-mannose, and overcomes the problem of a large amount of by-products.

This application relates to the use of conjugates in the preparation of diagnostic reagents. Specifically, the glucose-6-phosphate dehydrogenase mutant of this application contains one or a combination of mutations selected from the following, compared to the wild-type glucose-6-phosphate dehydrogenase: D306C, D375C, and G426C. Diagnostic kits prepared using the glucose-6-phosphate dehydrogenase mutant of this application exhibit high specificity, high sensitivity, ease of operation, short detection time, and accurate quantification, making them suitable for high-throughput detection.

The invention relates to a uridine diphosphate-glucose dehydrogenase mutant and an application of the uridine diphosphate-glucose dehydrogenase mutant in synthesis of uridine diphosphate-glucuronic acid. The amino acid sequence of the uridine diphosphate-glucose dehydrogenase mutant D7P166M / I169A is as shown in SEQ ID NO. 2, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO. 1. The invention also provides an application of the mutant in preparation of uridine diphosphate-glucuronic acid, and a method for preparing uridine diphosphate-glucuronic acid by using the mutant. Experimental determination shows that the melting temperature of the mutant is increased by 30 DEG C compared with that of a wild type, the activity is improved by 20%, and a powerful tool is provided for enzymatic synthesis of uridine diphosphate-glucuronic acid compounds. Compared with a conventional synthesis pathway, the method for synthesizing uridine diphosphate-glucuronic acid through immobilized enzyme flow provided by the invention can be used for stably and efficiently synthesizing for a long time, and is beneficial to reducing the cost and realizing industrial synthesis.

This application relates to the use of conjugates in the preparation of diagnostic reagents. Specifically, the glucose-6-phosphate dehydrogenase mutant of this application contains one or a combination of mutations selected from the following, compared to wild-type glucose-6-phosphate dehydrogenase: D306C, D375C, and G426C. Diagnostic kits prepared using the glucose-6-phosphate dehydrogenase mutant of this application exhibit high specificity, high sensitivity, ease of operation, short detection time, and accurate quantification, making them suitable for high-throughput detection.

The invention discloses a difunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant and application thereof, and belongs to the technical field of enzyme engineering and biological catalysis. The invention provides a difunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant and a preparation method of the difunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant. The amino acid sequence of the mutant is characterized in that at least one site of glutamic acid at the 28th site, arginine at the 83rd site, valine at the 126 site, isoleucine at the 161st site, leucine at the 187th site, phenylalanine at the 249th site, leucine at the 319th site and tyrosine at the 343th site of the amino acid sequence shown in SEQ ID No.1 is mutated. According to the method, the conversion efficiency of D-mannose is further improved, and meanwhile, the problem that a large number of by-products exist is solved.

This application relates to a digoxin detection kit. Specifically, the 6-glucose phosphate dehydrogenase mutant of this application comprises one or a combination of the following mutations compared to wild-type 6-glucose phosphate dehydrogenase: D306C, D375C, and G426C. The detection kit prepared using the 6-glucose phosphate dehydrogenase mutant of this application has strong specificity, high sensitivity, convenient operation, short detection time, and accurate quantitative determination, making it suitable for high-throughput detection.

This invention discloses a glucose phosphate mutase gene Rk PGM Its nucleotide sequence is shown in SEQ ID NO:1, and the amino acid sequence encoded by this gene is shown in SEQ ID NO:2; this gene was isolated from *Rhodotorula rubrum* (…). Rhodosporidium kratochvilovae YM25235 was used to ligate the gene to a vector and transfected into Rhodotorula rubrum cells. Experimental results showed that overexpression of Rk... PGM The gene will increase the level of extracellular polysaccharide synthesis in this strain; this invention modifies microorganisms through genetic engineering to improve their ability to secrete extracellular polysaccharides, providing a theoretical basis for increasing the yield of extracellular polysaccharides in Rhodotorula rubrum.

Provided in the present application are a glucose 6-phosphate isomerase OsPGIp1 protein and an encoding gene thereof, and use of the protein and a substance for regulating the expression of the encoding gene of the protein in regulating the height, grain weight, effective tiller number, seed-setting rate, or yield of a plant. The rice gene OsPGIp1 can be used for rice genetic breeding, germplasm resource screening, and mechanism research on breeding.

The invention relates to the technical field of genetic engineering, and discloses an engineering bacterium for high yield of D-pantothenic acid based on balanced sugar utilization, and a construction method and application thereof. Firstly, pfkB, eno and pgi genes of a glycolysis EMP pathway are weakened, zwf and pgl genes are overexpressed at the same time, and part of carbon flow flows into a PPP pathway in a push-pull mode; secondly, 6-phosphofructokinase II PfkB is knocked out, an initiation codon of an enolase gene eno is modified, and the enzyme activity of glucose phosphate isomerase Pgi is weakened; then, in order to enable the carbon flow to further flow to the PPP and ED paths, overexpressing common genes zwf and pgl of the PPP and ED, introducing edd and eda genes derived from zymomonas mobilis, and getting through the ED path in escherichia coli; thirdly, distributing redundant carbon flow of a PPP path to TCA circulation, and integrating an fxpk gene derived from bifidobacterium adolescentis and a pta gene derived from clostridium kruivmensis in a genome; and finally, through the combined action, the titer of the D-pantothenic acid reaches 4.80 g / L.

The invention discloses a genetically engineered bacterium for biosynthesis of inositol and application of the genetically engineered bacterium for biosynthesis of inositol, and the genetically engineered bacterium for biosynthesis of inositol is obtained by co-expressing a sweet orange inositol-3-phosphate synthase gene Csino3 and an escherichia coli inositol-1-monophosphate gene suhB in recombinant escherichia coli and then knocking out a glucose phosphate isomerase gene pgi. According to the technical scheme, the yield of inositol produced by the strain reaches 2.04 g / L and is increased by about 6.6 times compared with that before optimization, the inositol yield is greatly increased, the metabolic flux of glycolysis to glucose-6-phosphoric acid is effectively blocked, carbon metabolic flux is forced to be redirected to a pentose phosphate pathway, glucose-6-phosphoric acid is enriched, and a raw material is provided for inositol production. By blocking downstream branches of a glycometabolism pathway, enrichment of glucose-6-phosphate in cells is realized, so that sufficient precursor substances are provided for synthesis of inositol, and finally the yield of inositol is increased. The technical scheme is expected to be applied to construction of microbial cell factories, so that large-scale and low-cost production of inositol is realized, and theoretical support is provided for development of related industries.

This invention relates to the field of biotechnology, providing a novel NAD-dependent glucose-6-phosphate dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.2 or SEQ ID NO.3. The invention also provides the encoding gene of the above-mentioned gluconate-6-phosphate dehydrogenase, a recombinant expression vector including this gene, and a recombinant engineered bacterium. Furthermore, the invention provides a method for preparing the above-mentioned gluconate-6-phosphate dehydrogenase and its application in starch hydrogen production. This invention obtains a novel NAD-dependent NAD-dependent gluconate-6-phosphate dehydrogenase by mutating wild-type gluconate-6-phosphate dehydrogenase. + A mutant with better enzyme activity; this mutant is an NAD-dependent glucose-6-phosphate dehydrogenase, which can be used for hydrogen production from starch, effectively reducing the cost of hydrogen production and facilitating industrial production.

This application relates to the use of conjugates in the preparation of diagnostic reagents. Specifically, the glucose-6-phosphate dehydrogenase mutant of this application contains one or a combination of mutations selected from the following, compared to wild-type glucose-6-phosphate dehydrogenase: D306C, D375C, and G426C. Diagnostic kits prepared using the glucose-6-phosphate dehydrogenase mutant of this application exhibit high specificity, high sensitivity, ease of operation, short detection time, and accurate quantification, making them suitable for high-throughput detection.