25 results about "Nucleoside phosphorylase" patented technology

Provided is a method for preparing purine nucleoside. The preparation method comprises catalyzing a uridine substrate and a substrate base by using a pyrimidine nucleoside phosphorylase and a purine nucleoside phosphorylase, and preparing and obtaining purine nucleoside. The uridine substrate comprises uridine, 2'-deoxyuridine or 2'-amino-2'-deoxyuridine, and the substrate base comprises a purine compound, wherein on the purine compound, R1 comprises -NH 2,-OCH 3 or -Cl, and R2 comprises -NH 2, -F or -Cl. The preparation method can solve the problem of low yield in the preparation of nucleoside compounds in the prior art, and is suitable for the field of enzymatic catalysis.

The invention discloses a synthesis method of 2 '-fluoro-2'-deoxyguanosine. According to the method, thymine nucleoside phosphorylase and purine nucleoside phosphorylase are used as biocatalysts, and 2 '-fluoro-2'-deoxyuridine and guanine are used as substrates to synthesize the 2 '-fluoro-2'-deoxyguanosine. Compared with a chemical method, the production cost is greatly reduced; meanwhile, the synthesis method can effectively solve the problem that the product is difficult to separate.

This invention relates to the finding that FAMIN (fatty acid metabolism-immunity nexus; LACC1, C13orf31)) is a trifunctional purine salvage enzyme that displays a unique combination of adenosine deaminase, purine nucleoside phosphorylase and methylthioadenosine phosphorylase activities. Methods of measuring FAMIN activity and screening for FAMIN modulators are provided that comprise determining the adenosine deaminase activity, purine nucleoside phosphorylase activity and / or methylthioadenosine phosphorylase activity of an isolated FAMIN protein.

The present invention provides a method and composition for the enzyme-catalyzed synthesis of nucleosides containing protecting groups. The method includes using pyrimidine nucleoside phosphorylase or uridine phosphorylase and purine nucleoside phosphorylase to catalyze the synthesis of nucleosides containing protecting groups from a substrate, the substrate including a substrate nucleoside, a substrate base, and a substrate phosphate, the substrate base containing a protecting group, the pyrimidine nucleoside phosphorylase including proteins having 80% or more identity to proteins P and NP shown in SEQ ID NO: 1, the uridine phosphorylase including proteins having 80% or more identity to protein UP shown in SEQ ID NO: 2, and the purine nucleoside phosphorylase including proteins having 80% or more identity to protein PNP shown in SEQ ID NO: 3, 7, or 8. This method solves the problem in the prior art that there is no biosynthetic method for producing nucleosides containing protecting groups, and is suitable for the field of enzyme catalysis.

Compositions and methods relating to a pharmacological therapy for a human genetic disease, specifically mitochondrial DNA depletion-deletions syndromes, and more specifically, those related to mutations in the GUK1 gene. The pharmacological therapy involves the administration of deoxyguanosine (dG), a purine nucleoside phosphorylase (PNP) inhibitor, including but not limited to forodesine, or both.

The invention discloses an application of cold adaptability purine nucleoside phosphorylase in biosynthesis of nucleoside compounds. According to the invention, the cold adaptability purine nucleoside phosphorylase ColPNP from Colwellia is screened and obtained, and the cold adaptability purine nucleoside phosphorylase ColPNP, ribose kinase EcRK from escherichia coli and phosphopentose mutase EcPPM from escherichia coli can jointly form a multi-enzyme cascade catalytic system to efficiently catalyze synthesis of nucleoside compounds. The cold adaptability purine nucleoside phosphorylase ColPNP screened by the invention has a relatively low optimum temperature which is similar to the optimum temperature of EcRK and EcPPM, can improve the synthesis efficiency of nucleoside compounds, and has a wide application prospect in the aspect of preparing virus infection and tumor nucleoside compounds.

Provided herein is a one-pot method of synthesising a C-nucleoside-5′-monophosphate such as pseudouridine-5′-monophosphate, from an N-nucleoside using a multi-step enzymatic pathway. The method comprises reacting the N-nucleoside with a phosphate source and nucleoside phosphorylase to form a pentose-1-phosphate intermediate, reacting the pentose-1-phosphate intermediate with a phosphomutase to form pentose-5-phosphate intermediate, and reacting the pentose-5-phosphate intermediate with a nucleoside-5′-phosphate C-glycosidase to form the C-nucleoside-5′-monophosphate.

To provide a mutant purine nucleoside phosphorylase capable of efficiently producing nicotinamide riboside.SOLUTION: Provided is a mutant purine nucleoside phosphorylase having one or more amino acid mutations in the amino acid sequence of a wild-type purine nucleoside phosphorylase having: (a) a specific amino acid sequence; (b) an amino acid sequence containing one or more amino acid substitutions, deletions, insertions or additions in the amino acid sequence of (a); or (c) an amino acid sequence having 90% or more sequence identity to the amino acid sequence of (a), and having a higher activity of producing nicotinamide riboside than the wild-type purine nucleoside phosphorylase.SELECTED DRAWING: None

The application discloses a monomerized pyrimidine nucleoside phosphorylase mutant and a construction method and application thereof, and belongs to the field of enzyme engineering and biological catalysis technology.The mutant is obtained by amino acid substitution mutation of wild-type pyrimidine nucleoside phosphorylase shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.The mutant is transformed into a stable monomer form by dimer interface remodeling and hinge region stabilization of the closed active conformation, the natural dimer PyNP is transformed into a stable monomer form, and the catalytic activity of the mutant to uridine, 2'-deoxyuridine and similar substrates is maintained in the monomer state.Compared with the wild-type enzyme, the mutant has lower oligomer dependence, better temperature stability, pH stability and storage stability, and can be used for biological catalytic synthesis of nucleosides and nucleoside analogs.

Provided in the present invention are a pyrimidine nucleoside phosphorylase mutant and a preparation method for 2'-fluoro nucleoside. The pyrimidine nucleoside phosphorylase mutant comprises: (a) a mutant protein generated on the basis of a pyrimidine nucleoside phosphorylase wild-type enzyme TtPyNP as shown in SEQ ID NO: 1, the mutation being selected from any one or more of of the following mutations: K80 etc.; or (b) a protein having 70% or more homology with the amino acid sequence defined in (a) and having purine nucleoside phosphorylase activity. The method can solve the problem in the prior art of low yield of 2'-fluoro nucleoside synthesized by enzyme catalysis, and is suitable for the field of enzyme catalysis.

This disclosure relates to the use of Purine Nucleoside Phosphorylase (PNP) inhibitors such as ulodesine and its salts, in the treatment and / or prevention of diseases associated with nicotinamide adenine dinucleotide (NAD+) depletion, including diseases of mitochondrial dysfunction (including neurodegeneration and peripheral neuropathies), the preservation of cognitive function and in muscular disorders such as sarcopenia; and in metabolic syndrome and associated conditions. In particular the disclosure provides the use of PNP inhibitors such as ulodesine in the treatment of neurodegenerative conditions such as Parkinson's disease and amyotrophic lateral sclerosis.

The present invention provides a pyrimidine nucleoside phosphorylase mutant and a method for producing 2'-fluoronucleosides. Here, the pyrimidine nucleoside phosphorylase mutant includes (a) a protein resulting from a mutation based on the pyrimidine nucleoside phosphorylase wild-type enzyme TtPyNP represented by SEQ ID NO: 1, wherein the mutation is selected from one or more mutations such as K80, or (b) a protein having 70% or more homology to the amino acid sequence limited in (a) and possessing purine nucleoside phosphorylase activity. This can solve the problem in conventional techniques of low production yield of 2'-fluoronucleosides synthesized under catalytic conditions and is applicable to the field of enzyme catalysis.

The invention provides a preparation method and application of deoxyadenosine, and relates to the technical field of biology. The preparation method of the deoxyadenosine provided by the invention comprises the following steps: mixing beta-thymidine, adenine, Tt pyrimidine nucleoside phosphorylase and Apmtap purine nucleoside phosphorylase in a buffer solution, and carrying out an enzyme catalytic reaction to prepare the deoxyadenosine, the amino acid sequence of the Tt pyrimidine nucleoside phosphorylase is as shown in SEQ ID NO. 1; the amino acid sequence of the Apmtap purine nucleoside phosphorylase is as shown in SEQ ID NO. 2; the temperature of the catalytic reaction is 60-90 DEG C, and the pH value is 6.5-7.5. The preparation method is simple in process, mild in reaction condition, green, environment-friendly and high in product conversion rate, and can be used for industrial production.

Provide is a purine nucleoside phosphorylase mutant and a method for preparing a 2'-fluoronucleoside. The purine nucleoside phosphorylase mutant includes: (a) a protein with a mutation based on the purine nucleoside phosphorylase wild-type enzyme GsPNP as shown in SEQ ID NO: 3, where the mutation is selected from any one or more of the following mutations: L148, P19, Q41, etc.; or (b) a protein that has greater than 70% identity to the amino acid sequence as defined in (a) and has a purine nucleoside phosphorylase activity. The present application solves the problem of low yield in enzyme-catalyzed synthesis of 2'-fluoronucleoside in the prior art and is suitable for the field of enzyme catalysis.

The present application provides a method for enzymatic synthesis of a nucleoside bearing a protecting group, and a composition. The method includes: using pyrimidine nucleoside phosphorylase or uracil nucleoside phosphorylase and purine nucleoside phosphorylase to catalyze a substrate to synthesize a nucleoside bearing a protecting group, where the substrate includes a substrate nucleoside, a substrate base and a substrate phosphate, and the substrate base bears a protecting group; the pyrimidine nucleoside phosphorylase includes a protein that is of more than 80% identity to a protein PyNP shown in SEQ ID NO: 1; the uracil nucleoside phosphorylase includes a protein that is of more than 80% identity to a protein UP shown in SEQ ID NO: 2; and the purine nucleoside phosphorylase includes a protein that is of more than 80% identity to a protein PNP shown in SEQ ID NO: 3, 7 or 8. The present application can solve the problem that there is no biosynthesis method to produce a nucleoside bearing a protecting group in the prior art, and thus is suitable for the field of the enzymatic synthesis.

The invention relates to the technical field of biology, in particular to a preparation and purification method of 2 '-fluoro-2'-deoxyguanosine. The invention discloses a preparation and purification method of 2 '-fluoro-2'-deoxyguanosine.The preparation method comprises the following steps that 2, 6-diaminopurine and 2 '-fluoro-2'-deoxyuridine generate 2 '-fluoro-2, 6-diaminopine-2'-deoxynucleoside under the catalysis of thymine nucleoside phosphorylase and purine nucleoside phosphorylase, filtering is conducted, and the 2 '-fluoro-2, 6-diaminopine-2'-deoxyguanosine is obtained. Then adenosine deaminase is added for catalysis, and 2 '-fluoro-2'-deoxyguanosine is generated; biosynthesis of the 2 '-fluoro-2'-deoxyguanosine is divided into two stages, the intermediate 2 '-fluoro-2, 6-diaminopine-2'-deoxynucleoside is synthesized firstly, and then deamination is performed to form the 2 '-fluoro-2'-deoxyguanosine, so that the problem that the conversion rate is extremely low when guanine with low solubility is taken as a substrate is solved.

The invention belongs to the fields of chemical drugs, neurosurgery and oncotherapy, and particularly relates to application of a purine nucleoside phosphorylase inhibitor (PNP inhibitor) Forodesine or a pharmaceutically acceptable salt thereof in treatment of brain tumors, such as application of Forodesine or a pharmaceutically acceptable salt thereof in inhibition of glioma growth, application of Forodesine in promotion of anti-tumor immunity and the like, and particularly relates to application of a purine nucleoside phosphorylase inhibitor (PNP inhibitor) Forodesine or a pharmaceutically acceptable salt thereof in treatment of brain tumors. The invention also relates to a novel glioma treatment method. Through a large number of exploratory experiments, the applicant of the invention finds for the first time that Forodesine or pharmaceutically acceptable salt thereof can inhibit the growth of glioma by enhancing anti-tumor immunity and has an inhibition effect on glioma cells. Therefore, Forodesine or the pharmaceutically acceptable salt thereof can be used as a potential drug for treating brain tumors.

The invention discloses a method for improving enzyme activity of nucleoside phosphorylase, which comprises the following steps of: inoculating activated escherichia coli engineering bacteria into a seed culture medium for primary strain culture, and then inoculating the primary strain obtained by culture into a fermentation culture medium according to a proportion for fermentation culture to prepare nucleoside phosphorylase enzyme liquid; in the fermentation culture process, the culture temperature before fed-batch sugar is 37 DEG C, the fed-batch sugar is carried out after bottom sugar is used up, the total concentration of glucose and lactose in the fed-batch sugar is 90 g / L, the sugar adding rate of the fed-batch sugar is controlled to be 225-325 ml / h, the induction temperature after the fed-batch sugar is 25-32 DEG C, and the dissolved oxygen in the fermentation process is 20-40%. Specific escherichia coli engineering bacteria serve as production strains, the enzyme activity of the nucleoside phosphorylase is improved by controlling the concentration of base sugar, controlling dissolved oxygen, controlling the fed-batch sugar adding ratio, the sugar adding rate and the induction temperature, the enzyme activity can reach 500 U / g or above, and the quality requirement for the enzyme activity in the I + G conversion process can be met.

Provided in the present invention are a purine nucleoside phosphorylase mutant and a preparation method for 2'-fluoro nucleoside. The purine nucleoside phosphorylase mutant comprises: (a) a mutant protein generated on the basis of a purine nucleoside phosphorylase wild-type enzyme GsPNP as shown in SEQ ID NO: 3, the mutation being selected from any one or more of the following mutations: L148, P19, Q41, etc.; or (b) a protein having 70% or more homology with the amino acid sequence defined in (a) and having purine nucleoside phosphorylase activity. The method can solve the problem in the prior art of low yield of 2'-fluoro nucleoside synthesized by enzyme catalysis, and is suitable for the field of enzyme catalysis.

Application of nucleoside phosphorylase genes PtNP10 / 11 / 12 in altering the high ammonium tolerance of Populus plants, which relates to the field of genetic engineering, and particularly to an application of nucleoside phosphorylase genes PtNP10 / 11 / 12 in altering the high ammonium tolerance of Populus plants. The present invention provides a new use of nucleoside phosphorylase genes, laying a foundation for cultivating new germplasm materials of forest trees with efficient nitrogen utilization. Application of nucleoside phosphorylase genes PtNP10 / 11 / 12 in altering the high ammonium tolerance of Populus plants. Under high ammonium stress, the growth vigor, aboveground biomass and photosynthetic capacity of the nucleoside phosphorylase gene function-deficient mutants are stronger than those of wild-type plants. The present invention is used for wood improvement and cultivating new germplasm materials of forest trees.

This invention belongs to the fields of chemical pharmaceuticals, neurosurgery, and oncology therapeutics, specifically relating to the application of purine nucleoside phosphorylase inhibitors (PNP inhibitors) such as Forodesine or its pharmaceutically acceptable salts in the treatment of brain tumors, including the use of Forodesine or its pharmaceutically acceptable salts in inhibiting glioma growth and promoting anti-tumor immunity. It also relates to a novel treatment method for gliomas. Through extensive exploratory experiments, the applicant of this invention has for the first time discovered that Forodesine or its pharmaceutically acceptable salts can inhibit glioma growth by enhancing anti-tumor immunity, exhibiting inhibitory effects on glioma cells. Therefore, Forodesine or its pharmaceutically acceptable salts can serve as potential drugs for the treatment of brain tumors.

The present invention discloses a preparation method and application of a liquid compound microecological preparation and its protective agent. The components of the protective agent include glucose, soybean meal, peptone, sodium sulfite, disodium hydrogen phosphate, citric acid, calcium carbonate, glycerol, sodium butyrate, sodium lactate, D-sodium propionate, and nucleoside phosphorylase. By adding a certain proportion of the protective agent to the liquid compound microecological preparation, the present invention can enable the bacteria in the liquid compound microecological preparation to have a longer preservation time, with a slow decline in the bacterial count during long-term preservation, ensuring a higher viable bacteria count and stronger bacterial vitality, and greatly promoting production activities.

This invention discloses a purine nucleoside phosphorylase mutant and its application in the preparation of nicotinamide ribomatate. The PNP mutant is a substitution of at least one amino acid residue from the wild-type PNPLf derived from *Leptodactylus fuscus*, specifically serine at position 244, threonine at position 247, and alanine at position 290, preferably the S244Y / T247W / A290F triple mutant. This invention also provides a one-pot enzymatic method for preparing NR malate, using inosine and nicotinamide as substrates. The PNP mutant catalyzes a phosphorylation-transglycosylation tandem reaction to generate NR, which is then reacted with malic acid in situ to form NR malate. The NR yield can reach over 92%, and the total yield of NR malate can reach over 82%. This method is green, efficient, highly selective, and simplified, avoiding the large-scale use of organic solvents, making it suitable for industrial production.

This invention relates to the field of microbial technology, specifically disclosing a fermentation method for producing threonine and the application of nucleoside phosphorylase activity in inactivated or weakened microorganisms in threonine production. The invention discovered that the activity of nucleoside phosphorylase in inactivated or weakened microorganisms can increase the yield of threonine produced by fermentation. Therefore, a fermentation method for producing L-threonine is proposed, which uses recombinant microorganisms for fermentation. Compared to the starting strain, the recombinant microorganisms exhibit reduced or inactivated nucleoside phosphorylase activity. The amino acid sequence of the nucleoside phosphorylase is shown in any one of SEQ ID No. 28-30. This invention provides a new and effective method for threonine production.

The invention relates to the technical field of biology, in particular to a thymine phosphorylase mutant and application thereof. The invention provides a thymine nucleoside phosphorylase mutant derived from Escherichia coli and an application of the thymine nucleoside phosphorylase mutant in catalytic synthesis reaction of nucleoside compounds. Compared with wild type thymine phosphorylase, the 14 thymine phosphorylase mutants with excellent performance provided by the invention show more excellent thermal stability and synthesis activity of catalyzing non-natural nucleoside, and can be applied to industrial production and manufacturing of non-natural nucleoside biosynthesis.