62 results about "Polyphosphate kinase" patented technology

The invention relates to a method for synthesis of glutathione in vitro, comprising the step of: generating ATP (adenosine triphosphate) from ADP (adenosine diphosphate) and polyphosphoric acid compound under the catalysis of polyphosphate kinase; under the catalysis of gamma-glutamylcysteine synthetase and glutathione synthetase, synthesizing glutathione in vitro by glutamic acid, cysteine and glycine with the energy provided by the ATP. Compared with the prior art, the method of the invention can bring enzymes into complete contact with a substrate so as to improve the mass transfer efficiency and the utilization rate of enzymes. And the reaction system of the method is simple and strong in controllability. In addition, the method can solve the problem of great investment of ATP during production and reduce the production cost.

The invention relates to a method for regenerating ATP(adenosine triphosphate). A heterogeneous source is used for expressing polyphosphate kinase, and under catalyzing of polyphosphate kinase, polyphosphates with the low polymerization degree are used as phosphoric acid donors for regenerating ATP. Compared with an existing ATP regeneration method, adopted polyphosphate kinase is a normal-temperature enzyme, can be combined with multiple enzymes for promoting reaction coupling, and adopts a tripolyphosphate, a tetrapolyphosphate and a hexametaphosphate as the phosphoric acid donors, these polyphosphates are low in polymerization degree, common, easy to obtain and low in price, on the basis of simplifying the ATP regeneration process, feasibility of ATP regeneration is improved, and the ATP regeneration cost is reduced; activity of a PPK enzyme is not inhibited by polyphosphate concentration, and possibility is provided for industrialized large-scale production.

The invention discloses a Citrobacter freundii with transformed phosphorus accumulating genes. Polyphosphate kinase Ppk1 genes derived from the Citrobacter freundii are led into the Citrobacter freundii. The DNA of the Citrobacter freundii only contains Ppk1 genes, and the control way of the Ppk1 genes and Ppx genes is dual-cis-trans cotranscription. The phosphorus accumulation ability of bacteria with the characteristics can be improved by means of the Ppk1 with host bacteria as a receptor to express the source. The invention further discloses a construction method of the Citrobacter freundii with the transformed phosphorus accumulating genes and the application of the Citrobacter freundii with the transformed phosphorus accumulating genes in waste water dephosphorization. The Citrobacter freundii with the transformed phosphorus accumulating genes has the advantage of being high in dephosphorization ability.

The invention discloses a construction approach for bacillus coli of transpolyphosphokinase gene, in which the said method includes gene-clone and carrier-construction; the clone includes extracting master DNA of bacillus coli; designing primers; augmenting ppk gene; restructuring the augmented object gene into clonic carrier pMD18-T and converting bacillus coli DH5 alpha; the construction includes adopting restriction enzymes of BamHI and HindIII bisenzyme to cut pMD18-T plasmid with ppk object gene and idle expression carrier pET-28a(+); directionally connecting the object gene by T4 to the expression carrier pET-28a(+), then converting the DH5 alpha; by the PCR and bisenzyme pressure methods screening and verifying the positive recon; extracting the recombination plasmid Pet28a-PPK and converting the acceptor strain BL21(DE3). The invention is of simple process and the obtained gene has efficient phosphorous removal ability.

The invention relates to a method for regenerating ATP using a rationally designed enzyme. Through rational design after sequence alignment, polyphosphate kinase from sinorhizobium meliloti is mutated and heterologously expressed; and under the catalysis of the polyphosphate kinase, the regeneration of ATP is realized by adopting polyphosphate with low polymerization degree as a phosphoric acid donor. Compared with existing ATP regeneration method, the polyphosphate kinase used in the invention is a normal-temperature enzyme which can react for coupling with multiple enzymes; the enzyme can take tetra-polyphosphate as a phosphoric acid donor; compared with phosphoric acid with high polymerization rate, the tetra-polyphosphoric acid is more common and easily available; and the feasibility of ATP regeneration is increased on the basis of simplifying the ATP regeneration process, and the cost of ATP regeneration is reduced. The activity of PPK enzyme is not inhibited by the polyphosphate concentration, and industrial large-scale production is possible.

The invention relates to the technical field of biology, and discloses an enzymatic synthesis method of beta-nicotinamide mononucleotide. According to the method, adenosine and phosphate serve as starting materials, D-ribose-1-phosphoric acid and nicotinamide ribose intermediates are generated under enzyme catalysis of an enzyme composition of PNP enzyme and NRK enzyme, NMN is obtained through an irreversible reaction finally, and the substrate conversion rate can be greatly increased; the whole reaction system only needs two enzymes to participate, and the by-product adenine can be recycled; meanwhile, only one molecule of ATP needs to be consumed while one molecule of NMN is generated, and ADP is regenerated into ATP by adding polyphosphate kinase, so that the cost of the whole process is greatly reduced.

The invention discloses a method for synthetizing L-carnosine by a one-step method and truncated L-carnosine synthetase. According to the method, beta-alanine, L-histidine, ATP and polyphosphate are used as raw materials, MgCl2 is used as an activator, L-carnosine synthetase and polyphosphate kinase are added, and through an enzymatic reaction under the condition of pH being 6.5-8.5 and the temperature being 30-45 DEG C, the carnosine is coupled, catalyzed and synthetized. According to the method disclosed by the invention, escherichia coli is used for respectively expressing the truncated L-carnosine synthetase (of which the amino acid sequence is as shown in SEQID No.3) and the polyphosphate kinase, through purification, mixing is performed, a dual-enzyme coupling system is formed, and the L-carnosine synthetase in truncated expression can catalyze the L-carnosine synthetized from beta-alanine and L-histidine; besides, accompanied with ATP dephosphorylation, ADP is formed, the polyphosphate kinase catalyzes polyphosphate transphosphorylation groups, and ATP is formed for the ADP, so that circulation and regeneration of the ATP can be realized; and through the dual-enzyme couplingsystem, a reaction is performed under the appropriate reaction condition, so that carnosine is obtained. The method disclosed by the invention has the advantages that the raw materials are low in cost, the enzymatic conversion time is short, the operation is simple and convenient, and the production cost is low.

The invention discloses a method for producing hydrogen by bacterial fermentation, which is implemented by culturing hydrogen-producing microorganism in a basic culture medium added with phosphate radical ions to produce hydrogen under the conditions of (1) complete anaerobic culture and (2) culture with the concentration being 0.1-7.6% according to percent by volume, and then culture under complete anaerobic condition. The phosphate radical ions are H2PO4, HPO4, PO4, pyrophosphate radical or polyphosphate radical ions. The concentration of the phosphate radical ions is 0.01-0.15mol/L in respect to the basic culture medium. The hydrogen producing microorganism is enteroaerogen. The enteroaerogen is enteroaerogen or enteroaerogen expressing polyphosphate kinase. The hydrogen production method of the invention can improve the conversion yield of hydrogen and reduce production cost.

The invention belongs to the technical field of bioengineering, and particularly relates to a recombinant genetically engineered bacterium for producing gastrodin, a construction method and application. According to the recombinant genetically engineered bacterium, glycosyl transferase is introduced into a host, glycosyl transferase is expressed through a host strain to catalyze a substrate to synthesize gastrodin, sucrose synthase and polyphosphate kinase are introduced into the host, synthesis and cyclic regeneration of UDPG are achieved, the amount of glycosyl donor of a glycosylation reaction is increased, a whole-cell catalytic conversion method is constructed, on the basis that no extra UPDG is added, the conversion rate of gastrodin reaches up to 95% or above, the gastrodin yield is increased, and the gastrodin production cost is reduced.

The invention discloses a method for producing spermidine by using cheap substrates and engineering bacteria, and belongs to the technical field of bioengineering. The method is characterized in thatrecombinant cells or a combination of recombinant cells expressing gamma-glutamyl kinase, glutamate-5-semialdehyde dehydrogenase, aspartokinase, aspartate-beta-semialdehyde dehydrogenase, amine dehydrogenase, L-2,4-diaminobutanoate decarboxylase, carboxyspermidine dehydrogenase, carboxyspermidine decarboxylase, glucose dehydrogenase, and polyphosphate kinase 2-I are constructed; and aspartic acidand arginine are catalyzed to synthesize the spermidine by utilizing the recombinant cells or the combination of the recombinant cells. According to the method, selected oxidoreductase can efficientlyuse NAD (NADH) as coenzyme, and the product feedback inhibition is avoided in a reaction process; and the method has a good industrial application prospect.

The invention discloses an enzymatic synthesis method of nicotinamide mononucleotide. The enzymatic synthesis method comprises the following steps: S1, taking ribose-5-phosphate, cytidine monophosphate, polyphosphate and nicotinamide as raw materials, and generating nicotinamide mononucleotide under the catalytic action of ribose phosphate pyrophosphorylase, polyphosphate kinase and nicotinamide ribose phosphate transferase. The applicant uses polyphosphate kinase to catalyze cytidine monophosphate to cyclically obtain cytidine triphosphate by using a phosphate group provided by the polyphosphate, and the cytidine triphosphate is used as a pyrophosphoric acid donor of the ribose phosphate pyrophosphorylase to promote the enzymatic reaction of the ribose-5 phosphate and the nicotinamide, thereby avoiding the use of expensive ATP (adenosine triphosphate). Besides, the cytidine monophosphate and the cytidine triphosphate in the enzymatic reaction process provided by the invention are almost insoluble in water under an acidic condition, so that impurities in the product can be quickly, simply and conveniently removed by directly adjusting the pH value after the reaction is finished, the purification process is simplified, and the separation and purification difficulty is reduced.

The invention relates to an in vitro cell-free expression system incorporating a novel inorganic polyphosphate-based energy regeneration system. In certain embodiments, the invention includes a cell-free expression system where the cellular energy source, ATP, is regenerated from inorganic polyphosphate using a dual enzyme system. In this embodiment, this dual enzyme system may include thermostable Adenosyl Kinase, and/or Polyphosphate Kinase enzymes.

The invention discloses a method for improving production capacity of an L-arginine strain through genetic engineering, which comprises the following steps: knocking out an argR gene in a corynebacterium glutamicum ATCC13032 genome to obtain a gene knockout strain ATCC13032 (delta argR); carrying out A26V and M31V mutation on the argB gene in the genome of the gene knockout strain to obtain a genemutation strain ATCC13032 (delta argR, argBmut (A26V M31V)); and mutating a base sequence of an upstream regulation region of an initiation codon of a polyphosphate kinase gene (NCgl2620) in a genomeof a gene mutation strain to obtain a gene engineering strain ATCC13032 (delta argR, argBmut (A26V M31V) and NCgl2620mut6). According to the method, the L-arginine production capacity of the strain can be improved by at least 15 times.

The ATP amplification method of the present invention is a method for amplifying and detecting a very trace amount of exogenous ATP by allowing a fusion protein (PPK-ADK) of a polyphosphate kinase and an adenylate kinase, the fusion protein not containing ADP, to act on a mixture of ATP, AMP, and a polyphosphate compound. The present invention also provides an ultrasensitive ATP amplification method by which ATP at a single cell level can be amplified and detected, and an ultrasensitive microbial assay based on this ATP amplification method.

The invention relates to an in vitro cell-free expression system incorporating a novel inorganic polyphosphate-based energy regeneration system. In certain embodiments, the invention includes a cell-free expression system where the cellular energy source, ATP, is regenerated from inorganic polyphosphate using a dual enzyme system. In this embodiment, this dual enzyme system may include thermostable Adenosyl Kinase, and/or Polyphosphate Kinase enzymes.