11 results about "Uridine monophosphate" patented technology

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 uridine monophosphate specific glycoside hydrolase and application thereof in biosynthetic synthesis of pseudouridine. Nmygdh The function of the gene is verified by in-vitro enzyme activity experiment Nmygdh The recombinant protein has broad-spectrum and high-efficiency catalytic activity, and can efficiently catalyze the hydrolysis of glycosidic bonds in various nucleotides, such as uridine monophosphate (UMP), guanosine monophosphate (GMP), adenosine monophosphate (AMP) and cytidine monophosphate (CMP), and the activity of uridine monophosphate is the strongest. The application uses the protein to efficiently hydrolyze uridine monophosphate to obtain two substrates of pseudouridine, i.e. uracil and 5'-phosphoribose, and realizes the atom-economical and efficient biosynthetic synthesis of pseudouridine by combining the cascade reaction of pseudouridine glycosidase EcPsuG and dephosphorylase EcYjjG.

The present application belongs to the technical field of genetic engineering, and particularly relates to an engineered bacillus subtilis, a preparation method and application thereof. In the present application, in order to improve the supply of precursor cytidine triphosphate (CTP) in the process of fermentation of bacillus subtilis for producing cytidine, first, the feedback-inhibited CTP synthetase PyrG E156K is overexpressed to promote the conversion of uridine triphosphate to CTP; then, the repressor gene of the pyrimidine operon pyrR is knocked out to promote the synthesis of uridine monophosphate, and further promote the synthesis of CTP; finally, the pyrimidine nucleotide degradation genes pdp and cdd are knocked out to block the conversion of uridine / cytidine to uracil / cytosine and the conversion of cytidine to uridine, respectively, reduce the consumption of pyrimidine nucleotides, and further facilitate the intracellular accumulation of CTP. The yield of cytidine of the finally constructed engineering bacteria is about 12.6 times that of the original starting bacteria, and the results of the present application further prove the importance of CTP for cytidine synthesis.

This invention provides a kit for the simultaneous detection of nine flavor nucleotides in livestock and poultry products. The flavor nucleotides are cytidine monophosphate, uridine monophosphate, guanylic acid, inosine monophosphate, adenosine monophosphate, hypoxanthine, inosine, adenosine diphosphate, and adenosine triphosphate. The kit includes: a high-concentration extraction reagent, a standard curve solution, a high-concentration phosphate buffer solution, pH adjustment solution A, and pH adjustment solution B. The high-concentration extraction reagent is perchloric acid; the standard curve solution is a mixed solution of the nine flavor nucleotides; the high-concentration phosphate buffer solution is a solution of potassium dihydrogen phosphate and dipotassium hydrogen phosphate; pH adjustment solution A is sodium hydroxide; and pH adjustment solution B is phosphate. This invention also provides a method for the simultaneous detection of the nine flavor nucleotides using the above kit. This invention enables the simultaneous determination of the content of nine flavor nucleotides in livestock and poultry products. The method is simple, rapid, accurate, and sensitive, and can be used for batch determination, showing good prospects for practical application.

The invention relates to the technical field of analysis and detection, in particular to a method and a kit for simultaneously detecting uridine and uridine 5 '-monophosphate in serum by LC / MS-MS (Liquid Chromatography / Mass Spectrometry-Mass Spectrometry). The method comprises the following steps: S1, mixing a detection sample with an internal standard, and then adding a protein precipitant with the pH value of 2.7-3.5 to obtain a first mixed solution; the protein precipitant is an acetonitrile solution with the pH value of 2.7 to 3.5; s2, sequentially carrying out vortex oscillation and centrifugation on the first mixed solution, and then sequentially carrying out nitrogen blowing and redissolving treatment on supernate obtained by centrifugation to obtain a second mixed solution; and S3, sequentially carrying out vortex oscillation and centrifugation on the second mixed solution, and carrying out on-machine detection on a supernatant obtained by centrifugation. According to the method, the uridine and the uridine 5 '-monophosphate in the human serum can be accurately, efficiently and simultaneously detected, and the requirements of related laws and regulations on linearity, repeatability, accuracy, stability, specificity and the like of uridine and uridine 5'-monophosphate detection in the human serum are met.

The invention belongs to the technical field of crystallization, and relates to a method for crystallizing disodium uridinemonophosphate. Adjusting the pH value of the aqueous solution of the disodium uridinemonophosphate crude product, cooling, controlling the stirring speed, and adding an anti-solvent into the aqueous solution of the disodium uridinemonophosphate crude product; when the aqueous solution of the disodium uridineate crude product is turbid, stopping stirring, stopping adding the anti-solvent, and carrying out vacuum crystal growing; after crystal growing is finished, vacuum is removed, cooling continues, the stirring speed is controlled, stirring crystallization is conducted, and meanwhile an anti-solvent continues to be added; and after the anti-solvent is added, continuously controlling the stirring speed for stirring, and finally carrying out solid-liquid separation to obtain the uridine monophosphate disodium crystal. In the crystallization method of the disodium uridinemonophosphate crystal provided by the invention, the vacuum crystal growing and gradient temperature crystallization modes are introduced in the crystallization process of the disodium uridinemonophosphate, the obtained disodium uridinemonophosphate crystal is high in bulk density, good in fluidity and high in purity, and the problems that a later product is easy to agglomerate, uneven in loading capacity, overlarge in volume and the like are solved.

The invention discloses a UTP-glucose-1-phosphate uridine monophosphate transferase mutant and application thereof, and belongs to the technical field of biological engineering. The problem of low salidroside yield in the prior art is solved. The invention provides a UTP-glucose-1-phosphate uridylic acid transferase mutant, which is obtained by taking an amino acid sequence as shown in SEQ ID NO.2 as a starting sequence and mutating any one of the 6th site, the 126 site, the 134 site, the 140th site, the 177th site, the 180th site, the 214th site, the 249th site, the 284th site, the 289th site, the 296th site, the 298th site and the 300th site. The invention is mainly used for high-efficiency UTP-glucose-1-uridine phosphate transferase and application of the UTP-glucose-1-uridine phosphate transferase in de novo synthesis of salidroside.

The invention belongs to the technical field of gene engineering, and particularly relates to engineering modified bacillus subtilis, a preparation method and application thereof. In the invention, in order to improve the supply of precursor cytidine triphosphate (CTP) in the process of producing citicoline by fermentation of bacillus subtilis, firstly, CTP synthetase PyrGE156K with feedback inhibition removed is overexpressed, and the conversion from uridine triphosphate to CTP is promoted; then, a repressor protein gene pyrR of the pyrimidine operon is knocked out, synthesis of uridine monophosphate is promoted, and then synthesis of CTP is promoted; finally, the pyrimidine nucleotide degradation genes pdp and cdd are knocked out, conversion from uridine / cytidine to uracil / cytosine and conversion from cytidine to uridine are blocked respectively, consumption of pyrimidine nucleotide is reduced, and intracellular accumulation of CTP is facilitated. The citicoline yield of the finally constructed engineering bacterium is about 12.6 times of the yield of the original starting bacterium, and the result of the invention further proves the importance of CTP on citicoline synthesis.

The present disclosure provides methods, kits, and systems to efficiently identify the length of poly(A) tails, including exposing an RNA sample to a cleaving enzyme to generate a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP). Performing reverse-phase high-performance liquid chromatography (HPLC) on the mixture of nucleotide monophosphates. Determining a length of the poly(A) tail of the RNA sample by comparing a peak area of adenosine monophosphate (AMP) from the RNA sample to a peak area of at least one of a cytidine monophosphate (CMP), a uridine monophosphate (UMP), and a guanosine monophosphate (GMP) from the RNA sample.

The invention provides uridine monophosphate specific glucoside hydrolase and application of the uridine monophosphate specific glucoside hydrolase in biocatalytic synthesis of pseudouridine. The function of the Nmygdh gene is cloned and verified, in-vitro enzyme activity experiments prove that the Nmygdh recombinant protein has broad-spectrum and efficient catalytic activity and can efficiently catalyze hydrolysis of glycosidic bonds in various nucleotides, such as uridine monophosphate (UMP), guanosine monophosphate (GMP), adenosine monophosphate (AMP) and cytidine monophosphate (CMP), and the activity of the uridine monophosphate is the strongest. According to the invention, uridine monophosphate is efficiently hydrolyzed by using the protein to obtain two substrates of pseudouridine, namely uracil and 5 '-ribose phosphate, and pseudouridine glycosidase EcPsuG and dephosphorylase EcYjjG are combined for cascade reaction to realize atom-economical efficient biological catalytic synthesis of pseudouridine.

The invention belongs to the technical field of biosynthesis, and relates to a multi-enzyme catalysis system and method for synthesizing functional glycans. The system comprises a substrate module comprising nucleoside monophosphate, monosaccharide, polyphosphate and acceptor molecules; the nucleoside monophosphoric acid is one or more of uridine monophosphoric acid, adenosine monophosphoric acid, guanosine monophosphoric acid or cytidine monophosphoric acid; an enzyme module comprising a polyphosphate kinase, at least one remedial pathway synthetase, and at least one glycosyltransferase; the cofactor module comprises magnesium ions and optional inorganic pyrophosphatase; the system can realize in-situ regeneration of sugar nucleotide with nucleoside monophosphate as an initial cofactor and one-pot synthesis of target glycan. The invention systematically proposes that cheap NMP is used as a unique or main starting cofactor to replace expensive NTP or NDP for in-situ regeneration of various sugar nucleotides for the first time. Cofactor cost can be reduced by dozens of times, and a foundation is laid for large-scale production of glycans.