21 results about "ATP regeneration" patented technology

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.

This invention discloses a reaction tube for recombinase polymerase amplification (RPA), belonging to the field of nucleic acid detection. The reaction tube of this invention includes a tube body and a cap separate from the tube body. The tube body contains lyophilized bulbs or lyophilized powder containing premixed nucleic acid detection reagents, and the cap contains lyophilized powder containing an activator. The tube body and cap are sealed with a thin film. The lyophilized bulbs or lyophilized powder containing the premixed nucleic acid detection reagents include: recombinase, single-stranded DNA-binding protein, strand displacement DNA polymerase, buffer components, ATP, ATP regeneration components, and dNTPs. The lyophilized powder containing the activator includes: potassium ions, magnesium ions, and polyethylene glycol. This invention enables magnesium acetate to stably adhere to the cap, solving the problem of easy spillage of the lyophilized activator in the cap. In use, only the nucleic acid of the sample to be tested needs to be added; the operation is simple, easy to promote and apply, and suitable for batch detection.

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.

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

The invention discloses a high-yield cell-free protein synthesis system based on a cell crude extract and application of the high-yield cell-free protein synthesis system. The system comprises an escherichia coli extract, a DNA template, a buffer solution, an amino acid mixture, a nucleoside triphosphate mixture, an ATP regeneration system and antibiotics, the antibiotics are selected from compounds having inhibitory activity on metabolic processes except ribosome functions in prokaryotic microorganisms, including but not limited to one or more combinations of interfering DNA replication, DNA transcription, cell wall synthesis, energy metabolism, lipid synthesis or other non-translational related metabolic pathways. According to the method disclosed by the invention, the escherichia coli crude extract is used as a bacterial chassis, and non-essential metabolic pathways in a CFPS system are selectively inhibited by introducing different types of antibiotics, so that the yield of protein synthesis is increased.

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 present invention relates to the field of biosynthesis technology, and more particularly to a β-nicotinamide mononucleotide production system and a method for preparing β-nicotinamide mononucleotide in a dual-enzyme collaborative manner. The β-nicotinamide mononucleotide production system comprises an NRK-HS01 whole-cell catalyst, an ADK whole-cell catalyst, nicotinamide riboside, adenosine triphosphate, and MgCl2. The present invention couples the NRK-HS01 whole-cell catalyst with an ATP regeneration system (ADK whole-cell catalyst) to catalyze the conversion of nicotinamide riboside and adenosine triphosphate to produce NMN in a single step. This method achieves an NMN molar yield exceeding 99%, reduces ATP costs by 50%, and reduces feedback inhibition of the synthesis reaction caused by the accumulation of the byproduct ADP. This method also enables the recycling of ATP, thereby improving synthesis efficiency and saving production costs. This method is of great significance for the industrial development of biosynthesis of NMN.

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 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 discloses construction of a shewanella hydrogengens-CdS nanoparticle hybrid system based on a hydArpt gene and an application of the shewanella hydrogengens-CdS nanoparticle hybrid system. The preparation method comprises the following steps: connecting a hydArpt gene derived from Rhodopseudomonas paclitaxel coded hydrogenase to a vector pYYDT, carrying out ribosome optimization and transformation to obtain a plasmid pYYDT-hydArpt, introducing the recombinant plasmid pYYDT-hydArpt into wild shewanella oneidensis MR-1 to obtain a recombinant shewanella engineering bacterium SERP with high hydrogen production efficiency, and carrying out self-assembly to obtain a CdS / SERP high hydrogen production activity hybrid system. The CdS / SERP hybrid system constructed by the invention endows shewanella with light capturing ability, CdS nanoparticles provide photoelectrons for engineering bacteria to promote intracellular reducing power and ATP regeneration, and the hybrid system constructed by the invention can enhance the proton reduction rate of the engineering bacteria and improve the hydrogen production efficiency.

The present invention relates to the field of biotechnology, and in particular to an in vitro two-step enzymatic process for efficiently synthesizing GDP-L-fucose and its application. The present invention utilizes a bifunctional enzyme (Uniprot: Q58T34) having the amino acid sequence shown in SEQ ID NO: 1 as a template for site-directed mutagenesis to obtain variants M07 with high L-Fucose kinase activity and M10 with high GDP-L-Fucose pyrophosphorylase activity. The variants are then used to prepare GDP-L-Fucose using an ATP regeneration system in the presence of FKP and PPA enzymes using a two-step enzymatic process. The resulting GDP-L-Fucose has high yield and purity, low production cost and energy consumption, is suitable for large-scale production, and has broad application value.

The invention relates to a 3 '-phosphoadenosine-5'-phosphoryl sulfuric acid biosynthesis system based on ATP (adenosine triphosphate) regeneration and enzyme immobilization and application. The invention firstly provides an enzyme compound which comprises ATP (adenosine triphosphate) regeneration enzyme and PAPS (Phosphatidylcholine) synthetase; the ATP regeneration enzyme is immobilized PPK labeled by streptavidin-biotin; the PAPS synthetase is composed of immobilized ATPS labeled by streptavidin-biotin, APSK and PPA, and the immobilized ATPS labeled by streptavidin-biotin, the APSK and The mass ratio of the ATP regeneration enzyme to the PAPS synthetase is (1-1.5): 2. The invention provides a method for synthesizing 3 '-adenosine-5'-phosphoryl sulfuric acid by using an enzyme compound, so that high-efficiency and low-cost synthesis of PAPS is realized, and the problems of high cost and poor conversion efficiency of a traditional PAPS synthesis method are effectively solved. The invention further provides a 3 '-adenosine-5'-phosphoryl sulfuric acid biosynthesis system based on ATP regeneration and enzyme immobilization on the basis of the enzyme compound, and the system utilizes yeast cells as an ATP supply source to realize efficient conversion from adenosine to ATP.

The application provides a multi-dimensional information adaptive RNA cell-free synthesis system implementation method, comprising: obtaining a preset enzyme conformation adjustment parameter and a microenvironment pH regulation template according to a functional deviation degree, an activity offset amplitude and an ATP regeneration rate fluctuation of the ATP regenerating enzyme, generating an enzyme conformation dynamic regulation configuration, and determining a dynamic energy supply output mode matched with a current synthesis stage; based on real-time feedback after running of the dynamic energy supply output mode, collecting energy supply cycle frequency data, combining an intermediate product accumulation rate and an enzyme and stage adaptation index in the system, processing full-dimensional monitoring data of the energy feedback cycle, and judging an adaptation matching level of the ATP regenerating enzyme.

The invention discloses construction of a shewanella hydrogengens-CdS nano particle hybrid system based on a hydAson gene and an application of the shewanella hydrogengens-CdS nano particle hybrid system. The preparation method comprises the following steps: connecting a hydAson gene from Shewanella oneidensis coded hydrogenase to a vector pYYDT, carrying out ribosome optimization transformation on the vector pYYDT to obtain a plasmid pYYDT-hydAson, introducing the recombinant plasmid pYYDT-hydAson into wild shewanella oneidensis MR-1 to obtain a recombinant shewanella engineering bacterium SES with high hydrogen production efficiency, and carrying out self-assembly to obtain a CdS / SES high hydrogen production activity hybrid system. The CdS / SES hybrid system constructed by the invention endows shewanella with light capturing ability, CdS nanoparticles provide photoelectrons for engineering bacteria to promote intracellular reducing power and ATP regeneration, and the hybrid system constructed by the invention can enhance the proton reduction rate of the engineering bacteria and improve the hydrogen production efficiency.

The application belongs to the technical field of bioengineering, and particularly relates to a polyphosphate kinase mutant and application thereof in synthesis of beta-nicotinamide mononucleotide, wherein the polyphosphate kinase mutant is obtained by single-point or multi-point mutation of amino acids at positions 99, 186, 190 and 204 of a polyphosphate kinase with an amino acid sequence shown in SEQ ID NO. 1. The polyphosphate kinase derived from Lampropedia Hyalina DSM 16112 is subjected to molecular modification, and a plurality of mutants are obtained. The enzyme activity of the mutants is 1.5-14 times higher than that of the wild-type polyphosphate kinase, and the preference for AMP is 1.08-4.88 times higher than that of the wild-type polyphosphate kinase. The ATP regeneration system constructed by using the mutants can significantly reduce the use amount of the enzyme, reduce the fermentation capacity and cost, greatly shorten the reaction time, reduce the influence of AMP on the enzyme activity, and meet the demand of large-scale industrial production of NMN prepared by using the biological enzyme method, and has a wide application prospect.

The invention relates to the technical field of microorganisms, in particular to a microbial system for enhancing acetic acid metabolism and a construction method and application thereof. The microbial system comprises microorganisms capable of growing with acetic acid as a carbon source, and ACS genes are overexpressed in the microorganisms and used for converting acetic acid into acetyl coenzyme A; visual purple protein is expressed on a microbial cell membrane and is used for forming a transmembrane proton gradient under visible light irradiation by utilizing a light-driven proton pump function so as to drive ATP synthase to synthesize ATP; cdS nano-particles are loaded on the surfaces of the microorganisms or in the cells and are used for supplementing NADPH / NADH and assisting ATP regeneration. Through the synergistic effect of ACS gene overexpression, a light-driven proton pump and a CdS hybridization module in microorganisms, a light energy-ATP-NADPH dual-energy supply system is formed, and the problem of insufficient energy and reducing power in acetic acid metabolism is remarkably relieved.

This invention belongs to the field of biotechnology and discloses a method for the continuous preparation of β-nicotinamide mononucleotide (NMN). The invention covalently immobilizes four functional enzymes—ribokinase, phosphoribosylpyrokinase, nicotinamide phosphoribosyltransferase, and polyphosphate kinase—on an alkali-activated epoxy resin microsphere carrier to prepare a multi-enzyme composite catalyst. Using D-ribose, nicotinamide, and low-polymerization-degree polyphosphate as substrates, a small amount of ATP-initiating reaction solution is used to carry out continuous catalysis in a packed bed reactor. Polyphosphate kinase enables in-situ, zero-distance ATP regeneration, highly coupling ATP regeneration with NMN synthesis, significantly improving energy utilization and cascade catalytic efficiency. This invention solves the problems of high ATP consumption, non-recoverable enzymes, difficulty in continuous production, and limited diffusion of intermediate products in traditional enzymatic methods. The process is stable, green, safe, and suitable for industrial scale-up.

The invention belongs to the field of gene engineering, and particularly relates to a polyphosphate kinase mutant and application thereof. According to the invention, modification and screening are carried out on the basis of amino acids of wild type polyphosphate kinase PPK (polyP-polyphosphate kinase [EC: 2.7.4. 1]) from Mycobacterium tuberculosis, and the polyphosphate kinase with high enzyme activity, high catalytic efficiency and high ATP regeneration efficiency is obtained. The amino acid sequence of the polyphosphate kinase mutant is as shown in SEQ ID NO.11, and the polyphosphate kinase mutant is obtained by mutating valine at the 267th site of wild type polyphosphate kinase into glutamic acid and simultaneously mutating threonine at the 369th site into proline. Uridine monophosphate and cytidine monophosphate are produced through catalysis of the polyphosphokinase, the conversion yield is larger than 95%, meanwhile, cyclic regeneration of ATP is efficiently achieved, the ATP adding amount is reduced by 97%-98%, the product concentration is increased, the production cost is reduced, the catalysis application potential is greatly improved, and the important industrial application prospect is achieved.

The purpose of the present invention is to provide a method for producing a substance, whereby a substance can be produced simply and efficiently by an ATP-induced enzyme reaction coupled to an ATP regeneration reaction. A method for producing a substance according to one embodiment of the present invention is a method for producing a substance by an enzyme reaction using ATP, and is a method for carrying out the enzyme reaction and an ATP regeneration reaction in which ADP or AMP is reacted with a phosphoric acid donor to produce ATP in a reaction solution in the presence of a basic compound containing a divalent metal.

Bio-production processes that rely on biological activity can be very slow and inefficient, yet, the output of these processes may have significant value. Using biological enzymes outside their natural cellular environments offers a significant and largely untapped opportunity to enhance bio-production processes. By decoupling enzymes from their native contexts, one can modify both their sequences and structures in ways that are favorable for industrial applications. This system and method mixes substrate molecules, PPi, and enzymes that utilize PPi (PPi-dependent enzymes) in a reaction chamber and ends with stable-form products, including phosphorylated molecules.