11 results about "Proline Hydroxylase" patented technology

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.

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 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.

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 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.

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.

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.