22 results about "Proline Hydroxylase" patented technology

The invention relates to the technical field of collagen extraction, and provides a collagen extraction process for increasing hydroxyproline content, which sequentially comprises the steps of raw material pretreatment, deliming and degreasing, directional enzymolysis, gradient pressure-temperature synergistic extraction, purification, concentration and drying, a composite pretreatment agent containing a proline hydroxylase activator, EDTA-2Na and neutral protease is adopted in the raw material pretreatment; deashing and degreasing are combined with a chelating agent solution and ultrasonic treatment; a compound enzyme solution composed of trypsin, papain and proline hydroxylase is used for directional enzymolysis; a two-stage gradient pressure-temperature cooperation mode is adopted for extraction; purification is realized through combination of gel filtration chromatography and ion exchange chromatography; and finally carrying out freeze concentration and vacuum freeze drying to obtain the product. According to the process, through a multi-step synergistic effect, the content of hydroxyproline in collagen is remarkably increased, the product purity is high, natural functional characteristics are reserved, and the process is suitable for the fields of food, cosmetics, biological medicine and the like.

This invention belongs to the fields of genetic engineering and enzyme engineering, specifically relating to proline hydroxylase mutants and their application and methods in hydroxyproline production. This invention provides p4h... G141A p4h S85A p4h V126A p4h H125A p4h S205A p4h D122A p4h S211A Mutations at specific sites significantly increased the production of hydroxyproline and decreased the proportion of proline in the mutant enzyme. Further saturation mutations at these sites, combined with the dominant mutations, yielded strains with even better performance, such as p4h. S85Q‑V126A p4h S85A‑V126T p4h G141A‑H125S p4h G141A‑H125T Compared with traditional chemical methods, this process has lower production costs, is more environmentally friendly, and allows for large-scale continuous production.

The invention discloses recombinant collagen III + X as well as a preparation method and application thereof, and belongs to the technical field of biochemical engineering. The amino acid sequence of the recombinant collagen III + X provided by the invention is shown as SEQ ID NO: 1, and the recombinant collagen III + X is formed by repeating human III type collagen 387-467 amino acid fragments twice and connecting human X type collagen 521-680 amino acid fragments to the C end. Experiments prove that the recombinant III + X collagen and proline hydroxylase are co-expressed to form a stable triple helix structure, and the recombinant III + X collagen has great advantages in the aspects of cell migration, safety and the like, and can be used as a raw material for preparing wound dressings and skin care products.

The invention belongs to the technical field of bioengineering, and particularly relates to a kluyveromyces marxianus engineering strain for recombinant expression of hydroxylated human III-type collagen and application of the kluyveromyces marxianus engineering strain. The Kluyveromyces marxianus engineering strain of the human type III collagen is obtained by carrying out recombinant expression construction on proline hydroxylase and a human type III collagen gene in the Kluyveromyces marxianus; according to the invention, two truncation designs and codon optimization are carried out on the existing natural human III type collagen amino acid sequence to obtain the human III type collagen C3S1 and C3S2, and the recombinant secretory expression in the kluyveromyces marxianus engineering strain is realized. Like hydroxylation modification of the human III type collagen, the human III type collagen C3S1 and the human III type collagen C3S2 which are subjected to recombinant expression have proline hydroxylation characteristics. The recombinant human III-type collagen is prepared by using the kluyveromyces marxianus engineering strain, the method is simple, the production efficiency is high, and large-scale production is easy.

The application discloses an engineering bacterium for producing N-acetyl-trans-4-hydroxyproline and application thereof and belongs to the technical field of biotechnology.The application realizes fermentation synthesis of N-acetyl-trans-4-hydroxyproline by taking proline as a substrate through mutation of acyltransferase and introduction of exogenous proline-4-hydroxylase.The application has the advantages of low production cost, short fermentation period and high strain stability and has high economic benefits.The recombinant strain constructed by the application can convert 0.5M of proline into 19.64mg / L of N-acetyl-trans-4-hydroxyproline.

The invention relates to construction of a proline hydroxylase alpha subunit mutant from a human placenta and an expression vector of the proline hydroxylase alpha subunit mutant, and a mutant protein which is efficiently expressed in Ecoli BL21 (DE3) and has greatly improved hydroxylation efficiency and a construction body of the mutant protein are obtained by analyzing and screening a natural hydroxylase subunit.

The invention belongs to the technical field of gene recombination, and particularly relates to a recombinant human III-type triple helix collagen, a recombinant yeast strain for expressing the recombinant human III-type triple helix collagen and a construction method of the recombinant human III-type triple helix collagen. The amino acid sequence of the recombinant human III-type triple-helix collagen is shown as SEQ ID NO. 1, and the recombinant human III-type triple-helix collagen has triple-helix conformation close to a natural biological structure of collagen and has good application value and potential. According to the recombinant yeast strain disclosed by the invention, human-derived III-type collagen and proline hydroxylase are co-expressed in yeast cells without YPS1 protease, and meanwhile, a gene beneficial to vesicle transport and protein folding is transferred, so that hydroxylation of the recombinant collagen can be realized, a natural triple helix conformation is formed, and the recombinant yeast strain can be used for preparing a recombinant collagen product. The degradation problem of the collagen in the expression process can be solved, and the extracellular secretion expression of the triple helix collagen is realized.

Provided is a method for preparing a recombinant human collagen. The method comprises: constructing a genetically engineered bacterium which expresses a prolyl hydroxylase and a recombinant human collagen; culturing the genetically engineered bacterium and performing digestion treatment by means of pepsin to obtain a recombinant human collagen type I or type III. The prolyl hydroxylase comprises P4Ha2, P4Hb and vP4H, and by means of controlling the ratio of the copy numbers of the genes thereof, the recombinant human collagen and a natural human collagen are consistent in hydroxylation modification. Also provided is a large-scale production method for the recombinant human collagen. The prepared recombinant human collagen has completely the same sequence composition and length and substantially the same hydroxyproline content as the natural human collagen, and also has a stable full-length triple helix structure and biological activity without immunogenicity and potential risk of viruses.

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

The invention relates to the technical field of biological medicines, and provides a high VEGF protein / miR-210 extracellular vesicle induced by a serum-free combined culture medium and application of the high VEGF protein / miR-210 extracellular vesicle. According to the invention, high VEGF protein / miR-210 extracellular vesicles are obtained based on induction of a serum-free culture medium; the serum-free culture medium comprises a basic culture medium component combination: sugar-free RPMI1640 and MCDB131 in a volume ratio of 4: 1; the metabolic regulation additive is prepared by synergistically adding sodium pyruvate, cysteine and a proline hydroxylase inhibitor and taking alanine glutamine as an energy substitute; the growth factor is a combination of polyvinyl alcohol, 4-hydroxyethylpiperazine ethanesulfonic acid, ITS-X, epidermal growth factors and basic fibroblast growth factors, wherein the ITS-X, the epidermal growth factors and the basic fibroblast growth factors are added before use. CD63 and CD81 are specifically expressed on the surface of the prepared high VEGF protein / miR-210 extracellular vesicle, the high VEGF protein / miR-210 extracellular vesicle is rich in VEGF protein and miR-210, and the high VEGF protein / miR-210 extracellular vesicle is applied to targeted therapy of allergic rhinitis by inhibiting Th2 type immunoreaction.

This invention discloses a triple-helix recombinant human type III collagen, its preparation method, and its applications, belonging to the field of biotechnology. The triple-helix recombinant human type III collagen is obtained by linking a leader peptide, a linker sequence, recombinant human type III collagen, and a proline hydroxylase. The leader peptide and proline hydroxylase are linked to the N-terminus or C-terminus of the recombinant human type III collagen via the linker sequence, respectively. The triple-helix recombinant human type III collagen prepared by this invention is expected to have significantly higher biological activity than small molecule collagen peptides with random coil structures or incomplete small molecule fragments, better mimicking the function of endogenous collagen in the human body. As a raw material for pharmaceuticals or cosmetics, it shows application potential in promoting fibroblast proliferation and stimulating endogenous collagen synthesis.

The invention relates to the technical field of biological catalysis and enzyme engineering, and discloses a proline hydroxylase for specifically generating trans-3-hydroxy-L-proline, and the proline hydroxylase is a mutant obtained by a site-specific mutagenesis technology on the basis of a wild type proline hydroxylase amino acid sequence as shown in SEQ ID NO: 1. The regioselectivity of the mutant for catalyzing hydroxylation of L-proline to generate trans-3-hydroxy-L-proline is obviously higher than that of the wild type enzyme, and the generation amount of a by-product trans-4-hydroxy-L-proline is obviously inhibited or eliminated; the trans-3-Hyp is generated by enzyme catalysis through mutation of key sites, the selectivity of trans-3-Hyp generated by enzyme catalysis reaches 99% or above, complete inhibition of generation of trans-4-Hyp by-products is achieved, meanwhile, the product composition is extremely simplified, rapid and complete conversion of a substrate can be achieved through combined mutation (H63F / L250I) and enzyme under the optimized condition, and the production efficiency is far higher than that of wild type and most reported engineering enzymes.

The invention relates to the technical field of microbial engineering, and discloses an L-hydroxyproline-producing de novo synthesis type corynebacterium glutamicum and a construction method thereof, and the corynebacterium glutamicum strengthens the biosynthesis flux from glucose to L-proline by overexpressing a key enzyme gene of an endogenous proline synthesis pathway; by introducing an exogenous proline-4-hydroxylase gene, the accumulated L-proline is efficiently converted into the L-hydroxyproline; meanwhile, by knocking out key genes of a competitive pathway, generation of by-products such as lysine and threonine is reduced, and metabolic interference is relieved. The invention further provides a construction method of the engineering bacterium. The construction method comprises the steps of vector construction, gene knockout and overexpression, transformation, screening and the like. According to the method, an economical and feasible foundation is laid for industrial biological manufacturing of the L-hydroxyproline, the overall conversion efficiency from a carbon source to a final product is improved, and the utilization rate of raw materials and the yield of the L-hydroxyproline are improved.

The invention belongs to the technical field of gene engineering, and particularly relates to a proline hydroxylase controllable expression vector, a construction method of proline hydroxylase expression controllable engineering bacteria and application of the proline hydroxylase expression controllable engineering bacteria. The invention constructs a recombinant vector. The recombinant vector comprises a Trp promoter, a ribosome binding site, a multiple cloning site and a marker gene, wherein the nucleotide sequence of a part of gene segments of the Trp promoter is as shown in SEQ ID NO. 2. By adopting the recombinant vector, a controllable expression vector of the target protein can be further constructed, the expression level of the target protein can be accurately controlled by changing the tryptophan concentration in a culture medium, high tryptophan inhibits expression, and low tryptophan promotes expression. The regulation mechanism allows reduction of ineffective expression of the target protein in a thallus growth stage, reduces metabolic load, can realize real-time regulation in different stages, and provides a solution for efficient production of high-quality target protein.

The application discloses application of a PHD3 inhibitor in prevention and treatment of AIDS. Through immunological, biochemical and other multidisciplinary research means, the application analyzes the mechanism that a viral antisense protein ASP inhibits production of type I IFN to realize immune escape and establish latent infection in an HIV-1 infection process. Mechanically, after being expressed into a protein in a cell in the body, the ASP needs to undergo post-translational modification to exert its inhibiting effect, and after hydroxylation modification of a 47-position proline, the ASP can help to realize the ability of inhibiting the body immune response, and the post-translational modification depends on a host proline hydroxylase 3 (PHD3) to realize. Based on the mechanism-based research, it is found that a PHD3 inhibitor Molidustat offsets the hydroxylation of the ASP by PHD3, thereby blocking the immune evasion of HIV-1 and antagonizing infection, and showing a good development prospect of an AIDS treatment and prevention drug.

A combined pharmaceutical preparation of tafamidis or a pharmaceutically acceptable salt thereof and a proline hydroxylase inhibitor. The combined drug use of tafamidis and the proline hydroxylase inhibitor can activate the expression of an HIF-2α downstream target gene, thereby displaying a synergistic effect. In a cisplatin-induced renal anemia model, tafamidis can enhance the effect of the proline hydroxylase inhibitor in treating renal anemia.

The application discloses a kind of coding genes, the coding gene can encode human source III type collagen alpha 1 chain functional fragment and transdermal peptide TD-1, wherein the coding gene of human source III type collagen alpha 1 chain functional fragment and the coding gene of transdermal peptide TD-1 are expressed in series.The application also discloses a kind of recombinant vectors and a kind of recombinant bacteria, the coding gene and the coding gene of human source 4-proline hydroxylase are contained in the above-mentioned recombinant bacteria.The application also discloses the application of the above-mentioned coding gene, recombinant vector, recombinant bacteria in the production of high transdermal performance recombinant humanization III type collagen.The recombinant humanization III type collagen prepared by the application has similar structure with human source III type collagen, has good biological activity and stability, and has good transdermal ability, has good antioxidant, anti-carbonylation, inhibits tyrosinase activity, inhibits melanin production, promotes keratinocyte migration and promotes the ability of wound repair.

The invention discloses a triple-helix recombinant human type III collagen, a preparation method and an application, and belongs to the technical field of biology, the triple-helix recombinant human type III collagen is obtained by connecting a leading peptide, a Linker sequence, a recombinant human type III collagen and proline hydroxylase, the leader peptide and the proline hydroxylase are respectively connected to the N end or the C end of the recombinant human source III type collagen through a Linker sequence. The expected biological activity of the prepared triple-helix recombinant human-derived III-type collagen is far higher than that of small-molecule collagen peptide with a random coil structure or a small-molecule fragment with an incomplete structure, the functions of endogenous collagen of a human body are better simulated, and the triple-helix recombinant human-derived III-type collagen can be used as a medicine or cosmetic raw material. The application potential is shown in the aspects of promoting fibroblast proliferation, stimulating self collagen synthesis and the like.

Disclosed is crystal form M of the proline hydroxylase inhibitor daprodustat, wherein the X-ray powder diffraction thereof, expressed in 2θ angles and using Cu-Ka radiation, has characteristic peaks at 4.7±0.2°, 6.5±0.2°, and 6.8±0.2°. Disclosed are the preparation method for and the use of crystal form M. The crystal form M has good light stability, high temperature stability and high humidity stability, good solubility, and high purity.

This invention relates to the construction of a human placental-derived proline hydroxylase α subunit mutant and its expression vector. Through analysis and screening of the natural hydroxylase subunit, a mutant protein and its construct that are efficiently expressed in Ecoli BL21(DE3) with significantly improved hydroxylation efficiency were obtained.

The application discloses a recombinant collagen III+X, a preparation method and application thereof, and belongs to the technical field of bio-chemical industry. The recombinant collagen III+X provided by the application has an amino acid sequence shown as SEQ ID NO:1, is composed of a human collagen III 387-467 amino acid fragment repeated twice, and is connected with a human collagen X 521-680 amino acid fragment at a C terminal. Experiments prove that the recombinant collagen III+X can form a stable triple helix structure by co-expression with a proline hydroxylase, has great advantages in cell migration and safety, and can be used as a raw material for preparation of wound dressings and skin care products.

The invention belongs to the technical field of biological catalysis, and discloses a method for biosynthesizing hydroxypiperidinecarboxylic acid by using L-lysine. According to the method, a multi-enzyme cascade catalytic system is constructed, and the method comprises the following steps: firstly, carrying out concerted catalysis on L-lysine by using lysine 6-aminotransferase and imine reductase to generate L-piperidinecarboxylic acid; then, the L-piperidinecarboxylic acid is converted into 5-hydroxypiperidinecarboxylic acid through cascade connection of proline hydroxylase with position selectivity; meanwhile, glucose dehydrogenase (GDH) is introduced to construct an NADH cofactor cyclic regeneration system. The 5-hydroxypiperidinecarboxylic acid compound is synthesized from L-lysine by utilizing three-step enzyme catalysis and constructing a multi-enzyme cascade catalysis system, the method belongs to a biosynthesis method, the raw materials are easy to obtain, the operation is simple and convenient, the reaction condition is mild, the method is green and environment-friendly, an efficient method is provided for hydroxypiperidinecarboxylic acid synthesis, and the method is suitable for industrialization.