45 results about "Polyphosphate kinase" patented technology

The invention belongs to the technical field of soil organic matter improvers, and discloses a soil organic matter improver based on a compound microbial agent and a preparation method thereof, and the soil organic matter improver comprises the following components: a compound enzyme, a modified chitosan microsphere complex, a modified polyelectrolyte, an inhibitor L-methionine sulfoximine, a substrate, an auxiliary factor and the compound microbial agent. A compound enzyme system is used as a core, soil ammonium ions are circularly and efficiently assimilated through glutamine synthetase / glutamate synthetase, and the soil ammonium ions are converted into organic nitrogen; and energy guarantee is provided by virtue of polyphosphate kinase. The modified chitosan microsphere complex is used for protecting and slowly releasing enzyme and a substrate, and controlled release of the inhibitor L-methionine sulfoximine is realized through the modified polyelectrolyte, so that carbon-nitrogen balance of soil is regulated and controlled. The system has a synergistic effect with a compound microbial agent, the problem of soil organic matter reduction caused by excessive nitrogen application is effectively solved, meanwhile, the crop root biomass is remarkably increased, and the soil organic matter content is increased.

The invention discloses a method for biological high-density synthesis of retinaldehyde and a synthetic strain thereof. The synthetic strain is obtained by expressing ATP citrate lyase ACL, acetyl-CoA synthase ACS, choline kinase CK, inositol polyphosphate kinase IPK and vitreoscilla hemoglobin VHb in host bacteria. Wherein the host bacterium is a pichia pastoris gene modified bacterial strain PP-B2C. The retinaldehyde production performance of the recombinant strain is verified on the basis of a precursor supply pathway, an IUP pathway and a dissolved oxygen level, and the retinaldehyde production capacity of the pichia pastoris is further improved. The construction method of the recombinant pichia pastoris is simple, the synthesis of retinaldehyde can be better promoted, and the retinaldehyde can be efficiently synthesized by the engineering strain by utilizing glucose and methanol through amplification fermentation of a 5L fermentation tank, so that the recombinant pichia pastoris is beneficial to industrial production.

The invention discloses an application of engineered citrobacter freundii in fermentation synthesis of a stable body. The engineered citrobacter freundii is citrobacter freundii overexpressing a polyphosphate kinase gene Ppk1. The stable body prepared by fermenting the engineered citrobacter freundii is rich in polyphosphates and polyamines, particularly, the content of the polyamines can be up to 21wt%, the polyphosphates in the stable body can store the polyamines in an ionic bond combination mode, the combined polyamines are insoluble in water, a natural anion coating is generated, and the stability of the stable body is improved. And the sustained-release tablet is not easily taken by intestinal epithelial cells, so that the sustained-release tablet has a certain sustained-release effect. The medicine prepared by taking the compound as a main active ingredient shows a remarkable curative effect in ulcerative colitis treatment, has the comprehensive effects of reducing oxidative stress, enhancing the anti-inflammatory ability of a body and regulating immunity, is suitable for patients of all ages, and has outstanding application value and market prospect.

The invention provides a yeast rich in polyphosphate and application of the yeast in synthesis of adenosine triphosphate, and the content of polyphosphate is 15-35 wt% based on the dry matter mass of the yeast rich in polyphosphate; according to the method disclosed by the invention, the yeast is subjected to phosphorus starvation culture and then is subjected to phosphorus supplementary culture, so that the yeast is enriched with polyphosphates, and the yeast rich in polyphosphates is obtained; then, the yeast rich in polyphosphate is used as a biocatalyst to synthesize ATP, that is, self-related enzyme systems, mainly including adenosine kinase, endo-polyphosphate kinase and excision-polyphosphate kinase, in the yeast rich in polyphosphate are used for replacing exogenous polyphosphate kinase and adenosine kinase, and in addition, ATP is synthesized by using the yeast rich in polyphosphate as a biocatalyst. According to the method for synthesizing ATP by using the yeast rich in polyphosphate as the biocatalyst, exogenous adenosine kinase and exogenous polyphosphate kinase do not need to be additionally added, the cost is low, the efficiency of converting adenosine into ATP is high, the batch-to-batch stability of the yeast is realized, and the enzyme activity of the self-related enzyme system is high.

The invention discloses a method for catalytically synthesizing adenosine triphosphate by using microcrystalline cellulose carrier purification immobilized enzyme, which comprises the following steps: preparing bacterial suspension by using escherichia coli wet thalli and Tris-HCL buffer solution, crushing, centrifuging, and taking supernate to obtain crude enzyme; adding a microcrystalline cellulose carrier and the wet thalli into the crude enzyme liquid, and washing the immobilized enzyme after immobilization to obtain the immobilized enzyme; the method comprises the following steps: adding immobilized enzyme adenosine kinase and immobilized polyphosphate kinase into a substrate system with the volume of 1L according to a weight ratio, and starting a catalytic reaction under the conditions that the pH is 5.5-7.5 and the temperature is 20-40 DEG C; after catalysis is finished, the immobilized enzyme adenosine kinase and the immobilized polyphosphate kinase are subjected to solid-liquid separation with reaction liquid through a Buchner funnel, the immobilized enzyme continues to be subjected to catalytic reaction, and the method for preparing the immobilized enzyme is simple in mode, low in cost, easy to industrially produce, high in stability and capable of being repeatedly used for many times, and products are easy to separate.

The invention relates to the field of polyphosphate kinase, and discloses polyphosphate kinase 2 as well as expression and application thereof. The invention provides a coding gene of polyphosphate kinase 2. The nucleotide sequence of the coding gene is shown as SEQ ID NO. 1. According to the polyphosphokinase 2, efficient soluble expression of the polyphosphokinase 2 is realized by modifying and optimizing a gene sequence, so that the polyphosphokinase 2 obtained by induced expression has relatively high catalytic activity on AMP / ADP and wide adaptability of taking cheap polyphosphoric acid as a phosphoric acid donor, and can stably realize efficient conversion from AMP / ADP to ATP; the method can be used for establishing a low-cost and high-efficiency ATP regeneration system.

The invention relates to a biosynthesis method of pyridoxal phosphate or pyridoxine phosphate, and belongs to the technical field of biosynthesis. In order to solve the existing problem of serious environmental pollution, the invention provides a biosynthesis method of pyridoxal phosphate or pyridoxine phosphate, which comprises the following steps: in the presence of polyphosphate, magnesium salt, ATP (adenosine triphosphate), pyridoxal kinase and polyphosphate kinase, performing phosphorylation reaction on pyridoxal or pyridoxine and acid salts thereof, thereby obtaining the pyridoxal phosphate or pyridoxine phosphate. The temperature of the phosphorylation reaction is controlled to be 30-38 DEG C, after the reaction is finished, protein separation is conducted on reaction liquid, mother liquor is separated, the compound pyridoxal phosphate or pyridoxine phosphate shown in the formula I is obtained, and the mother liquor is recycled and reused. According to the method, a large amount of ammonium acetophosphate does not need to be additionally added, operation is simpler, mother liquor can be recycled, a large amount of waste water is prevented from being discharged, environment friendliness is better achieved, and the yield is high.

The application discloses a polyphosphate kinase mutant, a preparation method and application thereof, and wild-type polyphosphate kinase from Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1, The active site is mutated by using a directed evolution method, and a polyphosphate kinase mutant with high activity in catalyzing synthesis of ATP is obtained, the relative activity of the mutant to a substrate ADP or AMP is much higher than that of a wild type, and the mutation is at least one of D307M, W408A and Y431P, or at least one of E304V, P331S and Y431P. The application realizes efficient synthesis from a cheap substrate to a high-value product, is helpful to industrial production of ATP, and has a good application prospect in the fields of medicine and the like.

The present invention relates to a method for producing α-sialyl-β-D-galactoside saccharides, particularly α-sialyl-(2→3)-β-D-galactoside saccharides and α-sialyl-(2→6)-β-D-galactoside saccharides, from a β-D-galactoside saccharide, a sialic acid donor, and an enzyme with β-galactoside α-sialyltransferase activity. The enzymes with β-galactoside α-sialyltransferase activity used herein do not exhibit catalytic activity towards the hydrolysis of cytidine 5'-monophospho-N-acetyl-neuraminic acid to cytidine and N-acetyl-neuraminic acid. The method can be performed in vitro and in vivo using a genetically engineered cell comprising a nucleic acid encoding said enzyme. Further, said process may be adapted to produce the sialic acid donor CMP-Neu5Ac from low-cost substrates N-acetyl-D-glucosamine (GlcNAc), pyruvate, a cytidine phosphate (CMP, CDP or CTP) and polyphosphate in a single reaction mixture with a set of optionally immobilized or optionally co-immobilized enzymes comprising N-acylglucoamine 2-epimerase (AGE), an N-acetylneuraminate lyase (NAL), an N-acylneuraminate cytidylyltransferase (CSS), optional a uridine kinase (UDK), a uridine monophosphate kinase and a polyphosphate kinase 3 (PPK3).

This invention discloses the application of engineered *Citrobacter freundii* in the fermentation synthesis of spermine. The engineered *Citrobacter freundii* is a strain overexpressing the polyphosphate kinase gene Ppk1. The engineered *Citrobacter freundii* is inoculated into a fermentation medium for fermentation culture to synthesize spermine. The synthesis and extraction method of this invention for producing spermine is simple to operate, highly reproducible, low in cost, and yields a high spermine content in the fermented bacterial cells, accounting for 12.42% of the bacterial dry weight. The extracted spermine has a purity as high as 90.26%.

The application belongs to the technical field of biology and specifically relates to a method for synthesizing D-tagatose through multi-enzyme cascade catalysis, which uses lactose as a substrate and utilizes beta-galactoside enzyme, L-arabinose isomerase, glucose isomerase, fructokinase, tagatose-1,6-diphosphate aldolase, polyphosphate kinase and phosphatase for synchronous cascade catalysis to synthesize D-tagatose. The method improves the conversion rate of D-tagatose by continuously converting the intermediate product D-glucose into D-tagatose, provides a new method for synthesizing D-tagatose from lactose, and provides certain theoretical basis and technical support for realizing the industrialized production of high-value-added D-tagatose.

The present disclosure discloses a method for efficient catalytic synthesis of PAPS based on constructing an ATP regeneration system, and belongs to the technical field of bioengineering. Efficient production of PAPS is realized through microbial recombination expression and artificial construction of PAPS bifunctional synthetase. On the basis, an ATP regeneration system coupling with polyphosphate kinase from Corynebacterium glutamicum and Mycobacterium tuberculosis can be used for recovering two byproducts: pyrophosphoric acid and ADP at the same time, the equivalent conversion of a substrate and a product is realized, the PAPS generated in a catalysis system has high purity, and the sulfonic acid group donation in most sulfonic acid transfer reactions can be realized.

This invention discloses an enzymatic method for preparing (S)-adenosylmethionine. The method uses adenosine diphosphate (ADP) and L-methionine as substrates, reacting them under the combined action of polyphosphate kinase (Ppk) and adenosylmethionine synthase (MAT) to obtain (S)-adenosylmethionine. This method produces a product with high chiral purity and high atom economy, making it more suitable for industrial production.

The present disclosure relates to biocatalytic ligation methods for generating oligonucleotides and fusion polypeptides for use in the methods. In particular, the disclosure relates to biocatalytic ligation methods that incorporate ATP regeneration and fusion polypeptides that include a polyphosphate kinase domain and an ATP-dependent nucleic acid ligase domain.

The invention discloses a vidarabine monophosphate biosynthesis method based on an ATP (adenosine triphosphate) regeneration system, and belongs to the technical field of biomedical engineering. According to the method, riboside arabinoside and ATP serve as reaction substrates, the target product riboside arabinoside monophosphate is synthesized through double-enzyme catalysis, double enzymes are deoxynucleoside kinase and polyphosphate kinase, the deoxynucleoside kinase is mainly used for catalyzing riboside arabinoside to synthesize riboside arabinoside monophosphate, and the polyphosphate kinase is used for catalyzing riboside arabinoside to synthesize riboside arabinoside monophosphate. Polyphosphokinase is mainly used for catalyzing ADP (adenosine diphosphate) and polyphosphoric acid or polyphosphate to regenerate to obtain ATP (adenosine triphosphate). The method can be used for a pure enzyme reaction system, a crude enzyme liquid reaction system or a resting cell catalysis system. The method disclosed by the invention is environment-friendly, realizes low-cost efficient conversion of a substrate through coupled ATP regeneration, plays a significant role in promoting biosynthesis of arabinoside monophosphate series nucleoside drugs, and has application potential and economic value.

The application discloses a polyphosphate kinase mutant and application thereof in catalyzing synthesis of glutathione, wherein the mutant is obtained by mutating the 22th amino acid in the amino acid sequence shown in SEQ ID NO:2.The application provides a polyphosphate kinase mutant from Cytophaga hutchinsonii, which has 2 times of catalytic activity compared to that before modification, and an ATP regeneration system formed by the mutant can be used in a glutathione synthesis reaction, and more than 19g of glutathione is generated in 2h, and the product generation rate is close to 90%.

The invention belongs to the field of enzyme engineering, and relates to efficient polyphosphate kinase. The amino acid sequence of the polyphosphate kinase is as shown in SEQ ID NO: 1. According to the method disclosed by the invention, PPK is excavated by combining sequence information, a protein language model and kinetic model prediction, and a high-activity candidate enzyme PPK2-15 derived from Phaeobacter italicus is obtained. The mutant PPK2-15-L107T is obtained through directed evolution, kcat is improved by about 5 times, Km is remarkably reduced, and the catalytic efficiency (kcat / Km) is improved by more than 10 times.

The invention belongs to the technical field of biological catalysis, and particularly relates to an anchor peptide mediated fusion protein and an immobilized enzyme biofilm reactor, the fusion protein comprises a functional enzyme protein and an anchor peptide mutant; the anchoring peptide mutant is obtained by mutating proline at the fifth position of an amino acid sequence as shown in SEQ ID No.2 into valine; the functional enzyme protein is nicotinamide ribokinase or polyphosphate kinase. The functional enzyme protein and the anchoring peptide mutant are connected by using the connecting peptide, and then the obtained fusion protein is anchored on the polypropylene hollow fiber membrane wire material, so that the obtained fusion protein has good anchoring effect and catalytic activity, the batch reaction yield stability can be obviously improved, and the preparation method is suitable for industrial production. An immobilized enzyme bio-membrane reactor is designed, and nicotinamide mononucleotide can be synthesized through biological catalysis.

The invention provides a method for catalytically producing uridine monophosphate and application, and belongs to the technical field of genetic engineering and microbial engineering. The uridine kinase CaUDK-F gene with the nucleotide sequence as shown in SEQ ID NO.1 is adopted, the uridine kinase CaUDK-F gene is overexpressed through escherichia coli and then combined with polyphosphate kinase PPK to conduct biological catalysis on a substrate uridine, uridylic acid can be efficiently produced, the concentration of uridylic acid in a catalysis system is high, separation and purification are convenient, and the technology is simple. Meanwhile, the uridine kinase CaUDK-F is subjected to site-specific mutagenesis by adopting a site-specific mutagenesis technology, and three key amino acid residue sites S85, L87 and E88 which influence enzyme activity are subjected to site-specific mutagenesis, so that the catalytic efficiency and the conversion rate of the uridine kinase are further improved; wherein the enzyme activity of the mutant S85A is increased to 2.6 times that of a wild enzyme, and when the mutant S85A catalyzes uridine to produce uridylic acid, the conversion rate is increased to 92% from 80% of the wild enzyme.

The application provides a polyphosphate kinase (PPK) mutant and a preparation method and application thereof, and belongs to the technical field of enzyme engineering. The polyphosphate kinase mutant is mutated on the amino acid sequence shown in SEQ ID NO. 1. The application also provides an engineering bacterium for expressing the polyphosphate kinase mutant and a construction method of the polyphosphate kinase mutant. The polyphosphate kinase mutant provided by the application has improved thermal stability compared with the wild type, and the enzyme activity can be maintained for more than 1 h under the condition of 37 DEG C, and under the same heat treatment condition, the enzyme activity is 1.5 times that of the wild type, and is more suitable for industrialization.

The present disclosure relates to biocatalytic ligation methods for producing an injectable formulation comprising an oligonucleotide; to biocatalytic ligation methods for producing sterile oligonucleotides; and to sterile fusion polypeptides for use in said methods. In particular, the present disclosure relates to biocatalytic ligation methods incorporating ATP regeneration and to sterile fusion polypeptides comprising a polyphosphate kinase domain and an ATP-dependent nucleic acid ligase domain.

The invention provides uridine kinase, a mutant thereof, a method for catalytically producing uridine monophosphate and application, and relates to the field of genetic engineering and microbial engineering. The uridine kinase CaUDK-T gene with the nucleotide sequence as shown in SEQ ID NO.1 is adopted, after escherichia coli is over-expressed, a substrate uridine is biologically catalyzed in combination with polyphosphate kinase PPK, and uridylic acid can be efficiently produced. Meanwhile, amino acid residues E148, S152 and V153 of uridine kinase CaUDK-T are replaced with glycine in a site-specific mutagenesis mode, mutants E148G, S152G and V153G are obtained through construction, the catalytic efficiency and conversion rate of uridine kinase are further improved, the enzyme activity of the uridine kinase mutant V153G is improved to 1.97 times that of wild enzyme, the conversion rate of 50 g / L uridine can reach 90%, efficient production of UMP is achieved, and the application prospect is wide. Therefore, the industrial production is promoted.

This invention discloses genetically engineered bacteria and their applications, belonging to the field of bioengineering technology. By constructing a system that enhances the expression of polyphosphate kinase 2-I, polyphosphate kinase 2-II, and peptide bond synthase, this invention establishes an ATP recycling system, effectively ensuring the continuous execution of enzyme-catalyzed reactions. Using ursodeoxycholic acid or glycine and taurine as substrates, the genetically engineered bacteria expressing these three enzymes generate tauroursodeoxycholic acid or glycoursodeoxycholic acid, increasing the yield of these enzymes and achieving efficient biosynthesis of tauroursodeoxycholic acid or glycoursodeoxycholic acid, demonstrating promising prospects for industrial application.

The invention relates to a recombinant yarrowia lipolytica strain with high yield of astaxanthin as well as a construction method and application thereof. The preparation method comprises the following steps: expressing geranyl geranyl diphosphate synthase CrtE, phytoene synthetase / lycopene cyclase CrtYB, phytoene desaturase CrtI, 3-hydroxy-3-methylglutaryl CoA reductase tHMGR, beta-carotene ketolase CrtW, beta-carotene hydroxylase CrtZ, ATP (adenosine triphosphate) citrate lyase ACL, acetyl-CoA synthase ACS, choline kinase CK and inositol polyphosphate kinase IPK in host bacteria, so as to obtain a recombinant bacterium; the expression cassettes of farnesyl diphosphate synthase ERG20 and isopentenyl pyrophosphate isomerase IDI are obtained. Meanwhile, a photocatalytic material O-g-C3N4 / PEI is added to receive visible light excitation and generate reducing electrons, synthesis of intracellular NADPH is promoted, and then accumulation of astaxanthin is promoted. And continuous fed-batch fermentation is carried out in a 5L fermentation tank, so that the production performance of yarrowia lipolytica is greatly improved, and a foundation is laid for industrial production of high-value terpenoids.

The present application relates to the field of biotechnology, and particularly to an enzyme composition and its application in preparing ceramide NP. The present application provides a new method for synergistically catalyzing the synthesis of ceramide NP by multiple enzymes; the method is based on enzyme directed evolution, and uses propenal and glycine as starting substrates, and under the action of mutant glycine aldolase, enaminoketol reductase, pentenoate ligase, olefin reductase, phosphite dehydrogenase and polyphosphate kinase, and in cooperation with a metal catalyst Grubbs Catalyst, to realize the synthesis of high-efficiency and high-purity ceramide NP; compared with the traditional microbial fermentation method, the synthesis path of the present application has the advantages of mild reaction condition, convenience for continuous production, short conversion period (<8h), low cost and high product purity (byproduct content <0.5%), and is suitable for the demand of industrial large-scale production.

The invention relates to a polyphosphate kinase mutant and application thereof. Specifically, a site-directed mutagenesis technology is utilized to carry out directed evolution on polyphosphate kinase from Cytophila huxinsonii, a plurality of polyphosphate kinase mutants with single-point or multi-point mutations are obtained after screening, and compared with a wild type, the enzyme activity and the thermal stability of the mutants are obviously improved, and the mutant can be used for preparing the polyphosphate kinase mutant with the single-point or multi-point mutations of the polyphosphate kinase mutant with the single-point or multi-point mutations of the polyphosphate kinase mutant with the single-point or multi-point mutations of the polyphosphate kinase mutant with the single-point or multi-point mutations of the polyphosphate kinase mutant. The polyphosphate kinase mutant disclosed by the invention can be widely applied to the fields of food industry, feed industry and medicines, and has a good application prospect in industrial production due to excellent thermal stability and improved reaction efficiency.

The invention relates to a method for producing hyaluronic acid through cell-free catalysis, and belongs to the technical field of biological catalysis. According to the invention, a system capable of being applied to cell-free catalytic production of hyaluronic acid is constructed, and genetically engineered bacteria are used for expressing N-acetyl hexosamine 1-kinase, glucosamine-1-phosphate acetyl transferase, glucuronide kinase, GlcA-1-P uridinetransferase, hyaluronic acid synthase, polyphosphate kinase, uridine monophosphate kinase and inorganic pyrophosphatase; according to the present invention, N-acetylglucosamine GlcNAc, glucuronic acid GlcA, ATP and UMP are adopted as substrates to perform catalytic production, UDP and ADP produced during the reaction process are converted into ATP and UTP, such that nucleotide circulation is achieved, raw material consumption is reduced, the final hyaluronic acid yield reaches 1.28 g / L, and an economic and efficient brand new path is provided for hyaluronic acid industrial production.

The present application provides an enzyme composition comprising sucrose phosphatase, UTP glucose-1-phosphate uridylyltransferase, polyphosphate kinase and the like, or a group of recombinant engineering bacteria expressing the above enzymes. By using the above enzyme composition or the recombinant engineering bacteria, UDP-glucose and its derivatives can be synthesized using sucrose as a raw material, which has application prospects in the field of biological manufacturing.

The present invention relates to a novel enzymatic method for producing nucleoside triphosphates (NTP), in particular modified NTPs, using polyphosphate kinase 2. The NTPs generated by the methods of the invention may be used in methods of producing polynucleotides, including oligonucleotides, for use in therapy.

The present application relates to the field of biological catalysis, and discloses a method for preparing creatine phosphate by immobilized enzyme catalysis, which comprises the following steps: mixing gelatin and a positive charge modifier to obtain a carrier liquid; adding creatine and magnesium salt to a crude creatine kinase solution for pre-incubation to realize conformational locking, then mixing with the carrier liquid and dropping into a low-temperature coagulation bath to solidify into gel microspheres, and then cross-linking and washing to obtain immobilized creatine kinase particles; finally, adding substrates, sodium hexametaphosphate, polyphosphate kinase and immobilized particles into a reaction system for reaction, and then separating and collecting the supernatant to obtain creatine phosphate. The present application protects the three-dimensional conformation of the active center of the enzyme by pre-incubation, uses the positive micro area of the carrier to electrostatically enrich the substrates to accelerate mass transfer, and introduces an auxiliary enzyme to construct an in-situ substrate regeneration system to re-phosphorylate the by-product adenosine diphosphate into adenosine triphosphate. The present application eliminates the feedback inhibition of the product, reduces the cost of raw materials, and improves the overall catalytic efficiency.