41 results about "Proline Hydroxylase" patented technology

The invention discloses a gene segment provided with coded and highly-active trans-4-hydroxyl-L-prolyl hydroxylase and an application thereof. Gene codons of trans-4-hydroxyl-L-prolyl hydroxylase (GenBank ID: BAA 20094.1) in Dactylosporangiumsp. RH1 are optimized to obtain a p4hyd gene; the optimized p4hyd gene is truncated to obtain a P4Hyd1-257 gene; recombinant plasmids including pET-M-3C-P4Hyd and pET-M-3C-P4Hyd1-257 are built respectively, prokaryotic expression is performed through escherichia coli, and trans-4-hydroxyl-L-proline is produced in a conversion manner by utilizing thalli as an enzyme source. An experimental result shows that L-proline is taken as a substrate, compared with pET-M-3C-P4Hyd, the highest conversion rate of trans-4-hydroxyl-L-proline generated by pET-M-3C-P4Hyd1-257 in the conversion manner is increased from 91.4% to 97.4%, the conversion time is shortened from 80 hours to 60 hours, so that the conversion rate is improved remarkably, and the conversion time is shortened remarkably. The gene segment provided with coded and highly-active trans-4-hydroxyl-L-prolyl hydroxylase and the application thereof can be used for producing L-proline by a bioconversion method, and have better industrial application prospect.

The invention discloses a trans-4-hydroxy-L-proline hydroxylase modifying gene with high transformation rate and application thereof. An expression carrier of an optimization gene is constructed by optimizing nucleotide sequence of trans-4-hydroxy-L-proline hydroxylase (GenBankID:D78338.1) in Dactylosporangiumsp.RH1; the thallus is collected as the enzyme source of the transformation reaction through the prokaryotic expression. The experiment result expresses that the transformation rate of generating the trans-4-hydroxy-L-proline by using L-proline as a substrate is as high as 91%. The gene can be applied to biologically generating trans-4-hydroxy-L-hydroxyproline and has good industrial application value.

The invention relates to a hydroxylation method of a recombinant human-derived collagen; a collagen is hydroxylated by co-expression of proline hydroxylase from cystis bacteria RH1 and a human-derivedcollagen gene; the proline hydroxylase of the cystis bacteria RH1 has a gene sequence shown in SEQ ID No.1 and has a corresponding amino acid sequence shown in SEQ ID No.2. The invention provides a hydroxylation method of the recombinant collagen, wherein the method is easy to realize and simple in preparation process. The recombinant collagen prepared by the method is more similar to natural collagen in amino acid composition and spatial structure, and has the activity more similar to that of the natural collagen. Compared with a recombinant collagen prepared by a conventional method, the recombinant human-derived collagen allows the application field to be expanded, allows more excellent materials to be chosen in the increasingly developed fields of medical devices, cosmetics and food,and is more beneficial.

The invention discloses a method for producing trans-4-hydroxyproline from glucose in a fermentation manner by virtue of recombinant Escherichia coli without adding exogenous L-proline. A recombinant plasmid carried by the recombinant Escherichia coli has a mutant gene proBA2 and hyp (4-hydroxyproline), wherein proBA2 is mutated on a glutamyl kinase encoding gene proB, and the inhibition effect of L-proline on glutamyl kinase encoded by the mutated gene is remarkably reduced. proBA2 and hyp are co-expressed, and trans-4-hydroxyproline can be produced directly from glucose in the fermentation manner without adding exogenous L-proline. The recombinant plasmid is obtained by recombining proBA2 and hyp onto the same expression plasmid or by co-transforming recombinant plasmids which contain the two genes respectively and have different resistances. The invention further discloses the application of the Escherichia coli to the production of hydroxyproline.

The invention discloses a high-efficiency conversion method for producing trans-4-hydroxy-L-proline by a bioconversion technology. The high-efficiency conversion method comprises a high-activity self-induced expression method of L-prolyl hydroxylase in recombinant engineered escherichia coli, types and concentrations of assistants used in an L-proline-to-trans-4-hydroxy-L-proline efficient conversion system, and conversion conditions of the conversion system. The high-efficiency conversion method provides an important scientific basis for establishing bioconversion-based industrial production of trans-4-hydroxy-L-proline.

The invention provides a recombinant humanized III-type collagen. The recombinant humanized III-type collagen has a triple helix structure, and has an amino acid sequence reducing a humanized III-typecollagen alpha-1 chain and including 1068 amino acids. The invention further provides a preparation method of the recombinant humanized III-type collagen, wherein the prolyl hydroxylase of PBCV-1 includes 209 amino acids, and the base sequence is optimized according to the codons of saccharomyces cerevisiae. Compared with the prior art, the preparation method has the advantages that the triple helix is formed in vitro from collagen chains by utilizing the prolyl hydroxylase; and the saccharomyces cerevisiae expression system adopted by the invention is applicable to large-scale amplificationwithout endotoxin or later removal, the preparation cost is low, and the protein expression quantity is further improved. The recombinant humanized III-type collagen is excellent in stability, the amino acid composition of the recombinant humanized III-type collagen is almost completely the same as that of the alpha-1 chain of a natural collagen, and no immunological rejection can be generated when the recombinant humanized III-type collagen is applied to a human body. The recombinant humanized III-type collagen is good in biocompatibility and can be widely applied to surgical treatment and cosmetic medical industry.

The invention relates to a 5-hydroxy-1,7-naphthyridine compound substituted by an aryloxy group or a hetero-aryloxy group, a preparation method and a pharmaceutical application thereof. In particular, the compound is represented as the formula (I), wherein R2, R3 independently are hydrogen; R1 is hydrogen or a C1-C3 alkyl group; Ar is an aromatic ring or a hetero-aromatic ring selected from naphthalene ring, pyridine ring, thiophene ring, furan ring and substituted benzene ring. The invention also relates to the preparation method of the compound, a medicine composition including the compound or a pharmaceutically acceptable salt thereof, and an application of the compound or the pharmaceutically acceptable salt thereof in preparation of a medicine for inhibiting HIF proline hydroxylase or a medicine for promoting generation of endogenous EPO.

The invention relates to the technical field of biological genetic engineering recombination, and particularly relates to a modified recombinant human III-type collagen mature peptide containing hydroxyproline and a preparation method and application thereof. The invention provides a gene for encoding the recombinant human III-type collagen mature peptide, and a proline hydroxylase expression vector pPZAStA-P4HA2-P4HB2 is connected with a nucleotide sequence of the gene for encoding the recombinant human III-type collagen mature peptide to obtain a recombinant vector, and the recombinant vector is transferred into a yeast expression system to obtain yeast capable of expressing the modified recombinant human III-type collagen mature peptide containing hydroxyproline. The yeast is used for expressing the modified recombinant human III-type collagen mature peptide containing hydroxyproline, and the recombinant human III-type collagen mature peptide is large in molecular weight and high in expression level, is closer to natural collagen, and shows good biological activity.

The invention discloses a method for producing trans-4-hydroxyl-L-proline by means of fermentation by the aid of recombinant corynebacterium acetoacidophilum. Recombinant plasmids carried by the recombinant corynebacterium acetoacidophilum have proline hydroxylase genes (P4H) capable of catalyzing proline to generate trans-4-hydroxyproline. The method has the advantage that the free L-proline can be converted into the trans-4-hydroxyl-L-proline by the aid of proline-4-hydroxylase in cells of the recombinant corynebacterium acetoacidophilum under the condition of existence of alpha-ketoglutaric acid and ferrous ions. The invention further discloses application of corynebacterium acetoacidophilum to producing the trans-4-hydroxyl-L-proline.

The invention relates to a dihydropyrazolone compound as well as a preparation method and medical application thereof, in particularly relates to a general formula (I) compound shown in the specification, as well as a preparation method thereof, a pharmaceutical composition containing the compound, and application of the compound as a proline hydroxylase (PHD) inhibitor, the compound and the pharmaceutical composition containing the compound can be used in treating and / or preventing of PHD -activity- related diseases such as cardiovascular diseases, chronic kidney disease, anemia, wounds, cancer, autoimmune diseases and the like. In the general formula (I), the definition of each substituent in the formula is the same as that in the specification.

The invention discloses a novel proline 3-hydroxylase. The proline 3-hydroxylase has an amino acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:4. The invention also discloses an expression vector and a host cell containing the enzyme or a coding gene thereof as well as applications of the expression vector and the host cell in production of trans-3-hydroxy-L-proline. The proline 3-hydroxylase disclosed by the invention has high catalytic performance, so that the trans-3-hydroxy-L-proline can be produced by taking L-proline or glucose as a substrate, the production cost is greatly reduced, and the industrial production of the trans-3-hydroxy-L-proline is further realized.

The invention discloses vegetable protein peptide powder and a preparation method thereof. The method comprises the steps: (1), uniformly mixing vegetable protein powder and water according to the mass ratio, and adjusting the pH value to 7.0-7.5; (2) adding a compound enzyme I, and carrying out heat preservation at the temperature of 40-50 DEG C, wherein the compound enzyme I comprises proline hydroxylase, neutral peptide chain endonuclease and trypsin; (3) after the enzymolysis is finished, heating to 92-98 DEG C, and carrying out heat preservation; (4) after cooling, adjusting the pH valueto 8.0-8.5, then adding a compound enzyme II into the vegetable protein liquid, and carrying out heat preservation at the temperature of 40-50 DEG C, wherein the compound enzyme II comprises plant proteolytic enzyme, papain and flavor enzyme; (5) after the enzymolysis is finished, heating to 92-98 DEG C, and carrying out heat preservation; and (6) after cooling, sequentially carrying out centrifugal separation, membrane separation desalination and freeze drying to obtain the vegetable protein peptide powder. According to the method, the protein peptide powder can be prepared from vegetable protein, and the tryptophan content is high.

The present invention provides engineered proline hydroxylase polypeptides for the production of hydroxylated compounds, polynucleotides encoding the engineered proline hydroxylases, host cells capable of expressing the engineered proline hydroxylases, and methods of using the engineered proline hydroxylases to prepare compounds useful in the production of active pharmaceutical agents.

The present invention provides engineered proline hydroxylase polypeptides for the production of hydroxylated compounds, polynucleotides encoding the engineered proline hydroxylases, host cells capable of expressing the engineered proline hydroxylases, and methods of using the engineered proline hydroxylases to prepare compounds useful in the production of active pharmaceutical agents.

The invention discloses a biosynthesis method for increasing the yield of trans-4-hydroxyproline by knocking out other metabolic pathways. A system for producing hydroxyl-L-proline by using the biosynthesis method refers to a production method that free L-proline is converted into trans-4-hydroxyl-L-proline from a living body by virtue of proline hydroxylase in biological cells in the presence of alpha-oxoglutarate and ferrous ions. The method for increasing the yield of hydroxyproline comprises a step of cutting off other metabolic pathways of a precursor substrate glutamic acid which competes with L-proline in biological cells, wherein other metabolic pathways refer to an arginine metabolic pathway. By using the method, the yield of the trans-4-hydroxyl-L-proline can be up to 921 mg / L.

The present invention provides engineered proline hydroxylase polypeptides for the production of hydroxylated compounds, polynucleotides encoding the engineered proline hydroxylases, host cells capable of expressing the engineered proline hydroxylases, and methods of using the engineered proline hydroxylases to prepare compounds useful in the production of active pharmaceutical agents.

The invention relates to the technical field of biological genetic engineering recombination, in particular to a recombinant human type III collagen mature peptide modified with hydroxyproline and its preparation method and application. The invention provides a gene encoding a recombinant human type III collagen mature peptide, and combines the proline hydroxylase expression vector pPZAStA-P4HA2-P4HB2 with the nucleotides of the gene encoding a recombinant human type III collagen mature peptide The recombinant vector obtained by sequence ligation is transferred into the yeast expression system to obtain a yeast that can express the mature peptide of recombinant human type III collagen modified with hydroxyproline, and use the yeast to express the recombinant human type III modified with hydroxyproline Collagen mature peptide, the recombinant human type III collagen mature peptide has a large molecular weight, a high expression level, is closer to natural collagen, and exhibits good biological activity.

The invention discloses a method for reforming proline 4-hydroxylase. According to the method, a section between specific sites of proline 4-hydroxylase to be reformed is subjected to whole replacement, and / or the specific sites are subjected to amino acid mutation, so that the activity and the heat stability of the obtained proline 4-hydroxylase can be significantly improved, and the proline 4-hydroxylase which is excellent in stability and activity can be obtained. The proline 4-hydroxylase disclosed by the invention can be applied to production of trans-4-hydroxyl-L-proline or downstream products by using the trans-4-hydroxyl-L-proline as a precursor.

The invention discloses a protein with an L-proline excretion function and application of the protein. The invention relates to a method for producing L-proline or hydroxyproline by using protein ThrE. The L-proline is produced by enhancing the activity of a polypeptide with an L-proline efflux function in a strain for producing L-proline. Alternatively, the activity of a polypeptide having an L-proline efflux function in an L-proline producing host cell is weakened and the activity of proline hydroxylase is enhanced to produce hydroxyproline.

The invention provides a trans-4-hydroxy-L-proline synthesis strain. The strain is bacillus cereus HBU-AI; the preservation unit is China General Microbiological Culture Collection Center (CGMCC) andthe preservation number is CGMCC No. 14164; the preservation date is May 17, 2017. The invention further provides an L-proline hydroxylase gene sequence of the strain and application of an L-proline hydroxylase gene to production of trans-4-hydroxy-L-proline. A result shows that the engineering strain containing the L-proline hydroxylase gene has a relatively high capability of synthesizing the trans-4-hydroxy-L-proline; when the strain is fermented for 52h, the concentration of hydroxyproline in a fermentation solution can reach 41.6g / L and the residual concentration of L-proline in a productis 0.8g / L.

The invention discloses a trans-4-hydroxy-L-proline hydroxylase modifying gene with high transformation rate and application thereof. An expression carrier of an optimization gene is constructed by optimizing nucleotide sequence of trans-4-hydroxy-L-proline hydroxylase (GenBankID:D78338.1) in Dactylosporangiumsp.RH1; the thallus is collected as the enzyme source of the transformation reaction through the prokaryotic expression. The experiment result expresses that the transformation rate of generating the trans-4-hydroxy-L-proline by using L-proline as a substrate is as high as 91%. The gene can be applied to biologically generating trans-4-hydroxy-L-hydroxyproline and has good industrial application value.

The invention relates to a DNA molecule, an expression vector, a recombinant bacterium and a construction method thereof, and a preparation method of hydroxyproline. The nucleotide sequence of the DNA molecule RBS1 is shown in SEQ ID NO.1. The RBS1 provided by the invention can improve the expression of proline hydroxylase coding gene.

The present invention provides a novel L-proline-4-hydroxylase, the amino acid sequence of which is shown in SEQ ID No.2, derived from Micromonospora sp. CNB394. The present invention also provides a genetically engineered bacterium E.coli HYP that efficiently expresses the above-mentioned L-proline-4-hydroxylase and synthesizes high-yield trans-4-hydroxyl-L-proline de novo. The genetically engineered bacterium has The advantages of good genetic stability and strong fermentative acid production ability, no plasmid, using the genome to express the above-mentioned novel L-proline-4-hydroxylase, using glucose and other cheap carbon sources as substrates to efficiently synthesize trans- 4-Hydroxy-L-Proline, the yield of trans-4-Hydroxy-L-Proline after 42 hours of fermentation is as high as 51g / L, which has a good industrial application prospect.

The invention discloses a gene segment provided with coded and highly-active trans-4-hydroxyl-L-prolyl hydroxylase and an application thereof. Gene codons of trans-4-hydroxyl-L-prolyl hydroxylase (GenBank ID: BAA 20094.1) in Dactylosporangiumsp. RH1 are optimized to obtain a p4hyd gene; the optimized p4hyd gene is truncated to obtain a P4Hyd1-257 gene; recombinant plasmids including pET-M-3C-P4Hyd and pET-M-3C-P4Hyd1-257 are built respectively, prokaryotic expression is performed through escherichia coli, and trans-4-hydroxyl-L-proline is produced in a conversion manner by utilizing thalli as an enzyme source. An experimental result shows that L-proline is taken as a substrate, compared with pET-M-3C-P4Hyd, the highest conversion rate of trans-4-hydroxyl-L-proline generated by pET-M-3C-P4Hyd1-257 in the conversion manner is increased from 91.4% to 97.4%, the conversion time is shortened from 80 hours to 60 hours, so that the conversion rate is improved remarkably, and the conversion time is shortened remarkably. The gene segment provided with coded and highly-active trans-4-hydroxyl-L-prolyl hydroxylase and the application thereof can be used for producing L-proline by a bioconversion method, and have better industrial application prospect.

The invention discloses a high-efficiency transformation method for producing trans-4-hydroxy-L-proline by a biotransformation method, which specifically includes a high-activity self-induced expression method of L-proline hydroxylase in recombinant E. The type, concentration and conversion conditions of the auxiliary agent in the high-efficiency conversion system of converting -proline into trans-4-hydroxy-L-proline, for the establishment of a biotransformation method for the industrial production of trans-4-hydroxy-L-proline Amino acid technology provides an important scientific basis.

The invention discloses the application of a prolyl hydroxylase inhibitor in the preparation of a drug for improving the symptoms of hepatolenticular degeneration disease. The prolyl hydroxylase inhibitor used in the invention can stabilize HIF‑1α and activate it. Transcriptional activity; the gray scale of the liver was weakened, and the expression of lipid metabolism factors was significantly reduced. H&E staining of the liver showed that prolyl hydroxylase inhibitors can effectively improve liver pathology and reduce liver copper accumulation; Expression of neuronal signaling and factors related to dopaminergic neuronal signaling was improved, and motor performance was improved.

The present invention pertains to the field of pharmaceutical chemicals, and relates to a substituted acethydrazide derivative, preparation method and use thereof; preferably, relates to a compound of Formula I or a pharmaceutically acceptable salt thereof. The compound of the present invention or a pharmaceutically acceptable salt thereof can effectively inhibit proline hydroxylase, stabilize HIF-α, particularly HIF-1α, and has potency in the manufacture of a medicament for treatment and / or prophylaxis and / or adjuvant therapy of anemia, acute ischemic reperfusion injury.

The invention discloses a method for producing trans-4-Hyp (trans-4-hydroxy-L-proline) through transformation of recombinant escherichia coli, and belongs to the technical field of biological engineering. A P4H enzyme derived from unulturated bacterium esnapd2 is cloned, related modification is performed on the basis of the P4H enzyme to obtain a mutant, and the recombinant Escherichia coli expressing the L-proline hydroxylase or the mutant thereof is constructed. The recombinant escherichia coli expressing the L-proline hydroxylase or the mutant of the L-proline hydroxylase is subjected to whole-cell transformation to produce Hyp, the yield of trans-4-Hyp after 24h of transformation is 5.38 g / L, and the transformation rate can reach 94.5%. A foundation is provided for industrial production of Hyp by microorganisms.

The present disclosure provides engineered proline hydroxylase polypeptides for producing hydroxylated compounds, polynucleotides encoding the engineered proline hydroxylases, hosts capable of expressing the engineered proline hydroxylases Cells, and methods of using the engineered proline hydroxylases to prepare compounds useful in producing active pharmaceutical agents. The present disclosure provides engineered proline hydroxylase biocatalysts, polynucleotides encoding the biocatalysts, methods of making them, and methods of using these engineered biocatalysts to make hydroxylated compounds.