291 results about "Collagenase" patented technology

The invention discloses a collagenase mutant, a gene segment, a recombinant plasmid, a recombinant expression system, a collagen peptide, and a preparation method and application thereof, and belongs to the field of gene engineering and fermentation engineering. The collagenase mutant KT513 with high specific activity is obtained through recombinant expression in a trichoderma reesei host, and the enzyme activity of the collagenase mutant KT513 reaches 7423.18 U / mL. The optimum action pH of the enzyme is 3.0, the red snapper scale collagen can be subjected to enzymolysis in the pH range of 3.0-5.0, and the enzyme has excellent enzymolysis activity. After the collagenase mutant KT513 is subjected to enzymolysis at 40 DEG C for 4 hours, the extraction rate of fish scale collagen reaches 80.09%, and the prepared collagen peptide has good antioxidant activity. When the addition concentration is 10 mg / mL, the in-vitro scavenging activity of the prepared collagen peptide on hydroxyl radicals reaches 99.26% and is equivalent to that of ascorbic acid, and the collagen peptide has a wide application prospect in preparation of antioxidant preparations.

The invention discloses a temperature-controllable preparation method for tissue temperature dissociation into single cells and application, the temperature-controllable preparation method for tissue temperature dissociation into single cells comprises the following steps: S1, providing a tissue sample, and pre-treating the tissue sample to obtain a sample block; s2, mixing the sample blocks with a compound digestive enzyme, and filtering to obtain a single-cell suspension after digestion treatment is completed; wherein the compound digestive enzyme is prepared from collagenase, hyaluronidase and papain; and selectively, S3, carrying out red removing treatment. The invention not only provides an improved preparation method of the single-cell suspension, but also has higher efficiency on the premise of ensuring the yield and activity of single cells, and provides a new strategy and tool for cancer research and treatment by particularly enriching CSCs.

The invention provides a preparation method of non-denatured type II collagen capable of increasing bone mineral density and improving bone elasticity, and belongs to the technical field of biologication.The preparation method comprises the steps that an EDTA-citric acid buffer solution containing beta-mercaptoethanol is adopted for decalcifying cartilage, a supercritical CO2 extraction technology is adopted for degreasing, and the collagen is obtained; alkaline protease and collagenase II are adopted for substrate directional enzymolysis and gradient digestion; then the non-denatured type II collagen is separated by adopting three-phase centrifugal separation, and the non-denatured type II collagen with high extraction rate, high purity and high structural integrity can be obtained by adopting bionic H column chromatography purification.

The invention discloses bacillus subtilis for secretory expression of collagenase and application of the bacillus subtilis. The invention provides bacillus subtilis for secretory expression of collagenase and application of the bacillus subtilis in fat cell extraction, and after optimization of an expression element promoter and a signal peptide and large-scale fermentation of a fermentation tank, the highest enzyme activity can reach 669U. ML <-1 >. After purification, adipose-derived cells are successfully extracted and separated from mouse adipose cells, and after passage, the adipose-derived cells present the form of adipose cells. The method shows that the method has a good application prospect in the aspect of fat cell extraction.

It is an object of the present invention to provide a highly safe protease (collagenase) that is useful for food or medical applications, such as collagen tripeptide production or cartilage degradation, the intended use thereof, and the like. According to the present invention, provided is an enzyme agent for collagen tripeptide production, comprising, as an active ingredient, a collagenase consisting of the amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence equivalent thereto.

The invention discloses starch-collagen composite hydrogel as well as a preparation method and application thereof. Collagen is catalyzed by microbial transglutaminase to form a covalent cross-linked network, and meanwhile, hydroxypropyl starch is introduced to construct a physical interpenetrating network through molecular chain penetration and hydrogen-bond interaction; and chondroitin sulfate is further integrated to strengthen the network structure and biological activity through electrostatic interaction. The method is mild in condition and does not need a toxic chemical cross-linking agent, the obtained composite hydrogel has an interpenetrating double-network structure and has excellent mechanical properties, enzymatic degradation resistance and cell affinity, the compression modulus of the composite hydrogel is remarkably improved, the composite hydrogel can keep structural stability for a long time in a collagenase environment, cell adhesion and proliferation can be effectively promoted, and the composite hydrogel has a good application prospect. The hydrogel can be widely applied to tissue engineering scaffolds, wound dressings, drug sustained-release carriers and cartilage repair materials.

The present invention discloses a livestock and poultry manure biochar-based fertilizer, comprising the following raw materials in parts by weight: 10-42% of dilute protein solution and collagenase-modified livestock and poultry manure biochar, 20-40% of urea, 7-25% of diammonium phosphate, 7-24% of potassium chloride or potassium sulfate, 0.5-1% of modified starch, and 6-23% of bentonite. The livestock and poultry manure is selected from one of chicken manure, cow manure, and pig manure. After the biochar is modified with collagenase, the surface of the biochar has more phenolic hydroxyl groups, carboxyl groups, aldehyde groups, and hydroxyl functional groups, which are effective for NH4 + , PO4 3‑ , K + , has a stronger adsorption effect, collagenase modification makes the protein distribution more uniform, and the generated groups form a stable chemical bond with the biochar surface: it improves the dispersibility of the modified biochar in aqueous solution and reduces agglomeration; it has better chemical stability in acidic and alkaline environments.

The invention discloses a collagenase mutant, a gene segment, a recombinant plasmid, a recombinant expression system and application thereof, and a method for preparing the collagenase mutant and collagen tripeptide, and belongs to the field of gene engineering and fermentation engineering. The invention provides a collagenase mutant KT231 capable of realizing high-efficiency secretory expression in streptomyces S.lividans TK24 and a preparation method of the collagenase mutant KT231. The collagenase mutant KT231 can be used for efficiently carrying out enzymolysis on the skin of the narrow gadus at 60 DEG C, has excellent heat resistance and higher enzyme activity, and can be used for efficiently preparing the collagen tripeptide from the skin of the narrow gadus. The collagen peptide is compounded with commercially available papaya chymotrypsin, so that the proportion of collagen tripeptide in the fish skin enzymatic hydrolysate can be remarkably increased, and the fish skin enzymatic hydrolysate has no bitter taste. The method has wide application prospects in preparation of products, tissue engineering materials and wound healing accelerators for resisting wrinkles, preserving moisture, repairing damaged skin and improving bone and joint health.

The invention relates to brevibacillus parabrevis and application thereof in tissue isolation, and belongs to the technical field of biology. The brevibacillus parabrevis JN-6 disclosed by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC NO.29951, and the preservation date is March 7, 2024. The brevibacillus parabrevis JN-6 disclosed by the invention has the advantages that the preservation number is CGMCC NO.29951; the brevibacillus parabrevis provided by the invention has collagen degradation capability, and the produced collagenase can be used for adipose tissue separation and is further cultured into adipose-derived stem cells. When the produced collagenase is used for digestion and separation, the number of obtained cells can be 6.17 * 10 < 6 > / mL, the adipose-derived stem cells can be effectively differentiated, and the method has a good application prospect.

The invention relates to the technical field of cell separation and culture, in particular to a method for separating and culturing haematochrome cells of plectropomus leopardus. The separation method of the plectropomus leopardus haematochrome cells is created for the first time, the EDTA digestive juice is used for pre-digesting epidermis cells on the outer layers of the plectropomus leopardus fins, then the collagenase digestive juice is used for digesting connective tissues (pigmented cells) of internal dermis, digestive enzymes such as trypsin and other mechanical methods which have great damage to the cells are not used, and the method is suitable for large-scale production of the plectropomus leopardus haematochrome cells. The plectropomus leopardus haematochrome cell can reduce cell damage, can be effectively separated to obtain the plectropomus leopardus haematochrome cell which is high in purity, good in activity and capable of being cultured in vitro, and lays a foundation for subsequent experiments such as single cell sequencing, cell in-vitro culture and molecular function verification.

The invention discloses an isolated culture method of placenta decidua basalis stem cells. The isolated culture method comprises the following steps: acquiring decidua basalis tissues from a placenta; a trypsin solution is added into the bottom demolding tissue for pre-digestion, and a pre-digested bottom demolding tissue is obtained; adding a mixed enzymatic hydrolysate of trypsin and collagenase into the pre-digested decidua basalis tissue, carrying out digestion treatment under a magnetic stirring condition, sieving, centrifuging, and discarding supernate to obtain decidua basalis stem cells; adding a complete culture medium into decidua basalis stem cells, blowing, beating and uniformly mixing, and inoculating the decidua basalis stem cells into a culture dish according to the inoculation density of 2000-5000 cells / cm < 2 > for culturing to obtain P0-generation cells; carrying out subculture on the P0 generation cells; according to the separation culture method, external pollution is effectively reduced through refined cleaning and stripping steps, and then a one-step digestion method is adopted and combined with a magnetic stirring technology, so that the cell separation efficiency and purity are remarkably improved; meanwhile, the optimal inoculation density is explored through practice, and the cell culture effect and yield are directly improved.

This invention describes a wound dressing product for active continuous debridement of devitalized tissues in non-healing wounds including diabetic ulcers, pressure ulcers, burn injuries and other etiologies. The present invention pertains to the principle of continuous wound debridement which makes necrotic tissue more susceptible for removal and hence enhances progressive wound healing. The dressing contains an active ingredient, such as collagenase which serves to debride wounds in-situ. In the present invention purified Collagenase (90% pure) was deposited onto several wound dressing materials. A key feature of this invention is that the activity level of the Collagenase used was substantially preserved.

The invention relates to the technical field of medical cosmetology, in particular to a porous hollow glass bead capable of slowly releasing collagen and application of the porous hollow glass bead. The porous hollow glass bead capable of slowly releasing the collagen comprises a porous hollow glass bead body, the outer wall of the porous hollow glass bead body is provided with a pore channel structure penetrating through the exterior and an inner cavity of the hollow glass bead body, and the pore channel and the inner cavity of the porous hollow glass bead body are filled with collagen gel. The compressive strength of the hollow glass beads is utilized to improve the mechanical supporting effect, the collagen gel is subjected to catalytic decomposition under the action of collagenase in a body so as to achieve the slow-release effect, the outer walls of the hollow glass beads slow down the reaction time through physical isolation, and then the slow-release time is prolonged; meanwhile, ions such as Si and Ca released by the hollow glass beads can promote cells to secrete collagen, the biological activity is improved, and the three beneficial effects of physical support, collagen slow release and biological safety are achieved.

The invention relates to a method for extracting high-purity extracellular vesicles from liver tissues of pelteobagrus vachelli, which comprises the following steps of: firstly, mixing collagenase II and collagenase IV, performing enzymolysis on the tissues, adding a CaCl2 solution, incubating, and digesting the liver tissues into a tissue suspension; then removing cells and fragments through low-speed centrifugation, and filtering to remove impurities by using needle filters of 0.8 [mu] m and 0.22 [mu] m; and finally, further purifying by combining ultracentrifugation with a high-precision iodixanol density gradient centrifugation method, and merging density layers with high purity to obtain high-purity EVs. The method has the characteristics of high extraction purity, high efficiency and simplicity and convenience in operation, and has a wide application prospect.

The invention discloses an eggshell membrane peptide for enhancing bone mineral density as well as a preparation method and application thereof, belongs to the technical field of egg by-product processing, and particularly relates to a preparation method of the eggshell membrane peptide for enhancing bone mineral density, which comprises the following steps: carrying out vacuum freeze drying and crushing on an eggshell membrane to obtain eggshell membrane powder; mixing the eggshell membrane powder with deionized water to obtain an eggshell membrane solution; adding keratinase, performing enzymolysis, and taking supernate; adding collagenase, carrying out enzymolysis, and centrifuging to take supernate, so as to obtain primary enzymatic hydrolysate; adding alkaline phosphatase, performing enzymolysis and centrifugation, and taking supernate to obtain enzymatic hydrolysate; and performing ultrafiltration and drying to obtain the eggshell membrane peptide. According to the eggshell membrane peptide for enhancing the bone mineral density, keratin cross-linking is destroyed through enzymolysis of keratinase, active peptide capable of stimulating osteoblast proliferation is released through enzymolysis of collagenase, phosphopeptide calcium binding sites are further activated through enzymolysis of alkaline phosphatase, calcium salt deposition is promoted, and the eggshell membrane peptide for enhancing the bone mineral density is provided.

The present invention relates to a cartilage tissue single cell extraction kit and an extraction method thereof, the kit comprises: (1) a first enzyme for enzymatic hydrolysis, the first enzyme for enzymatic hydrolysis comprising one or more of trypsin, chondroitinase ABC and fucosidase; (2) a second enzyme for enzymolysis, wherein the second enzyme for enzymolysis comprises collagenase II, neutral protease II, hyaluronidase and Dnase I; and (3) a basic solution, wherein the basic solution comprises a basic culture medium and / or a buffer solution. The invention also provides a method for extracting single cells from cartilage tissues by using the kit. The cartilage tissue single-cell suspension prepared by the method has the advantages of high cell yield, high activity and the like, and has a wide application prospect.

The invention provides a preparation method of a hydrolyzed collagen product with surface amphipathicity as well as the product and application thereof, and the preparation method comprises the following steps: (1) taking hydrolyzed collagen enzymatic hydrolysate as a substrate, then preparing a fermentation culture medium with vegetable oil and sugar, and fermenting by adopting candida bumblebee; (2) after fermentation, adding lysozyme into the fermentation liquor for activating treatment, and then sterilizing, centrifuging and filtering; and (3) carrying out freeze-drying treatment on the filtered fermentation liquor to obtain the hydrolyzed collagen product with surface amphipathicity. The hydrolyzed collagen product prepared by the invention not only has low molecular weight and high permeability to skin, but also has water locking and moistening effects on the skin surface due to the amphiphilic sophorolipid on the surface, so that the moisture retention of the skin is better improved, and the product has excellent product characteristics and has wide market prospects.

The invention discloses a novel colorectal cancer organoid culture method, which obviously improves the activity of primary cells (more than or equal to 90%) and the forming efficiency of the organoid (the success rate is more than 95%) through alcohol gradient cleaning, collagenase-hyaluronidase combined digestion and stent-free culture technologies. The improved culture medium takes R-Spondin1 as a core, nicotinamide and a small-molecule inhibitor are combined, and long-term amplification of organoids (passage is more than or equal to 10 times) is supported. The method is suitable for individualized drug screening, tumor microenvironment simulation and drug resistance mechanism research, has the advantages of high efficiency, stability and low cost, and provides a reliable model for precise medical treatment of colorectal cancer.

The invention relates to a multifunctional hemostatic sponge as well as a preparation method and application thereof. The hemostatic sponge comprises a matrix formed by cross-linking methylacryloylated collagen and nano-hydroxyapatite modified by epigallocatechin gallate, the mass ratio of the methacrylated collagen to the nano-hydroxyapatite modified by the epigallocatechin gallate is (2 to 30): 1. By combining ColMA and HAP coated EGCG and optimizing the proportion, the obtained hemostatic sponge has a good collagenase degradation resisting effect and hemostatic, anti-inflammatory and antibacterial effects, tissue regeneration and repair can be promoted, the use comfort of a patient is higher, and the treatment effect is better.

The invention belongs to the technical field of biology, and particularly relates to bacillus velezensis HALZ676 as well as application and a method of the bacillus velezensis HALZ676 to cowhide fermentative degradation, the bacillus velezensis HALZ676 is separated from preserved pig trotters, and by analyzing biological characteristics, whole genome and metabonomics differences of the bacillus velezensis HALZ676, the cowhide can be efficiently degraded, and the cowhide can be efficiently degraded. It is found that peptide components smaller than 3000 Da in cow leather degradation liquid obtained through fermentation of the strain HALZ676 account for 68% or above, and the cowhide degradation liquid has remarkable antioxidant capacity and antibacterial capacity; the cowhide fermentation liquor is mainly used for promoting conversion and enrichment of antioxidant, anti-inflammatory and anti-cancer metabolites through a linoleic acid metabolic pathway, so that the antioxidant, anti-inflammatory and anti-cancer capacities of the cowhide fermentation liquor are enhanced; the cowhide fermented by the strain HALZ676 can enrich functional active components, and a theoretical basis and a promising strategy are provided for high-valued production of processing byproducts of livestock and poultry skin fermented by probiotic bacillus with high yield of collagenase.

The invention discloses a cod bone-derived high-activity calcium chelating peptide and a preparation method thereof, and belongs to the technical field of functional biological product preparation. The calcium chelating peptide provided by the invention is derived from a cod bone collagen enzymatic hydrolysate and is obtained through affinity chromatography separation of hydroxyapatite, the calcium chelating activity is 4.9-5.2 mu g / mg, and the calcium chelating peptide contains aspartic acid and glutamic acid enriched peptide fragments, through deep processing of cod bones, high-valued utilization of cod bone resources is realized, the high-activity calcium chelating peptide is prepared, and the calcium chelating peptide has a wide application prospect. And a high-quality raw material is provided for the development of a novel calcium supplement.

The invention provides a preparation method of a single-cell sequencing suspension fixedly preserved in situ. The invention provides digestive juice for preparing a single-cell suspension. The digestive juice is a solution composed of collagenase II and neutral protease. The invention also provides a kit containing the digestive juice. The invention provides application of the digestive juice to preparation of a kit and application of the kit to in-situ fixed preservation of cells and preparation of a single-cell suspension. The invention also provides a preparation method of the in-situ immobilized preserved cell and the single-cell suspension, and the single-cell suspension prepared by the preparation method.

The invention discloses a construction method of a novel ovarian endometriosis mouse model. Longitudinally cutting the uterus tissue of the donor mouse into small pieces; then putting the small uterus blocks into a collagenase solution for incubation; then removing collagenase from the small uterus blocks; attaching the small uterus block to the notch of the female mouse of which the envelope on the ovary surface is cut, so that the small uterus block covers the exposed ovary surface, pushing back into the abdominal cavity as soon as possible, nailing the skin and muscles by using a suturing nail, disinfecting and putting back into the mouse cage; after 24 hours of fasting, water can be freely drunk; and conventionally feeding after 24 hours. The method does not need preoperative / postoperative estradiol management, has the advantages of simplicity in operation, time saving, good effect, high molding success rate (about 90%) and the like, and is worthy of further popularization. The constructed ovarian endometriosis mouse model can evaluate the ovarian function condition and the influence of infertility, and overcomes the defect that the existing endometriosis mouse model lacks special ovarian types.

The invention aims to provide a preparation method of amnion-derived mesenchymal stem cells with high collagen expression. The preparation method comprises the following steps: step 1, reagents and materials: amnion tissues: obtaining a fresh amnion from a healthy puerpera after delivery; the PBS is used for cleaning amniotic tissues; collagenase: type I collagenase, which is used for digesting amniotic tissues to separate mesenchymal stem cells; a cell culture medium is a DMEM / F12 culture medium, and 10% of fetal calf serum and 1% of penicillin-streptomycin double antibodies are added; the transforming growth factor TGF-beta is used for promoting the expression of mesenchymal stem cell collagen; and the fibroblast growth factor FGF can assist in promoting cell proliferation and collagen expression. The invention aims to provide the preparation method of the amniotic membrane-derived mesenchymal stem cells for highly expressing collagen, and the amniotic membrane-derived mesenchymal stem cells can be widely applied to treatment of immune system diseases, reproductive system diseases, skin system diseases, skeletal system diseases, nervous system diseases, digestive system diseases, respiratory system diseases, circulatory system diseases and urinary system diseases. Particularly, the effects of immunosuppression and tissue repair are achieved.

The invention belongs to the field of biological material extraction, and particularly relates to a method for extracting collagen peptide from marine organisms, which comprises the following steps: S1, crushing marine organism raw materials, soaking the crushed marine organism raw materials in a citric acid buffer solution containing alginate oligosaccharide for swelling treatment, and then performing high-voltage pulse electric field treatment to obtain pretreated collagen slurry; and S2, carrying out first-stage enzymolysis treatment on the pretreated collagen slurry through alkaline protease, and then carrying out second-stage enzymolysis treatment through collagenase and flavourzyme. S3, performing separation treatment through a first ultrafiltration membrane to obtain first filtrate, performing deodorization treatment on the first filtrate through a first nanofiltration membrane of which the surface is coated with a beta-cyclodextrin adsorbent to obtain second filtrate, and performing separation treatment on the second filtrate through a second nanofiltration membrane to obtain third filtrate. And S4, concentrating, desalting and drying the third filtrate to obtain collagen peptide powder. Through the synergistic effect of multiple steps, the concentration of the molecular weight of the collagen peptide and the efficient deodorization are realized on the basis of retaining the active structure of the collagen.

The invention provides a bovine type I collagen terminal end peptide characteristic polypeptide, the bovine type I collagen terminal end peptide characteristic polypeptide comprises an N terminal end peptide characteristic polypeptide and a C terminal end peptide characteristic polypeptide, the amino acid sequence of the N terminal end peptide characteristic polypeptide is Q * LSYGYDE, and the amino acid sequence of the C terminal end peptide is LSFLPQPPQE. The invention also provides application of the bovine type I collagen terminal peptide marker polypeptide in detection of N-terminal peptide and C-terminal peptide of bovine type I collagen. The invention also provides a preparation method of a collagen enzymolysis product containing the bovine type I collagen terminal peptide characteristic polypeptide. The bovine type I collagen tail end terminal peptide marker polypeptide provided by the invention is a special terminal peptide at the tail end of bovine type I collagen. The terminal peptide marker polypeptide is used as a marker and can be used for quickly identifying the bovine type I collagen terminal peptide and accurately and quantitatively analyzing the bovine type I collagen terminal peptide. The qualitative and quantitative detection methods are simple to operate and high in sensitivity.

The present invention provides a composition comprising collagenase as an enzyme; calcium, histidine, and glycine as coenzymes, and a method for stabilizing enzymes, the method comprising the preparation of the composition by adding histidine and glycine to collagenase. According to the present invention, the enzyme composition and method for stabilizing enzymes can minimize or inhibit the agglutination of enzymes, maintain the concentration of freeze-dried enzymes even during long-term storage, and provide a formulation that has excellent rehydration time and hygroscopicity when restored to a liquid state, as well as superior enzyme activity.

The present invention relates to the technical field of cashmere preparation, and in particular to a preparation process of cashmere modified by natural plants, wherein the cashmere is treated by collagenase enzymolysis, soaked in water with loofah and ginkgo leaves, and then treated with a modifier, so that the cashmere has good softness, comfort and elasticity, and the strength and performance of the cashmere are significantly enhanced, and the anti-pilling is also significantly improved, and the cashmere has significant antibacterial properties, and the dyeing is more uniform. Moreover, when the weight ratio of the loofah extract and the ginkgo leaf extract in the modifier is 2.6:1, the softness, elasticity and anti-pilling effect of the cashmere are most significant, and once the weight ratio changes significantly, the performance of the cashmere is significantly reduced.

The invention relates to a collagenase-loaded LDH (layered double hydroxide) / EDTA (ethylene diamine tetraacetic acid) layered double hydroxide nano material as well as a preparation method and application thereof. The collagenase-loaded LDH / EDTA layered double hydroxide nano material is prepared from a zinc-aluminum layered double hydroxide LDH carrier, ethylenediamine tetraacetic acid EDTA inserted between layers of the LDH carrier through anion exchange, and collagenase loaded on the surface of the LDH carrier through an electrostatic adsorption effect. The invention further discloses a preparation method of the collagenase-loaded LDH / EDTA layered double hydroxide nano material for preparing the collagenase-loaded LDH / EDTA layered double hydroxide nano material for preparing the collagenase-loaded LDH / EDTA layered double hydroxide nano material.

The present application relates to a kind of anti-aging composition and its application of brightening and shrinking pores.The composition includes the following components: halocyprid extract, purple magnolia extract, desert rose leaf cell extract, rain lily labiatae flower extract and white lily extract, and the mass ratio of the halocyprid extract, purple magnolia extract, desert rose leaf cell extract, rain lily labiatae flower extract and white lily extract is (0.2-2):(0.001-0.1):(0.001-0.1):(0.001-0.1):(0.001-0.1).The composition of the present application can effectively improve the clearance rate of collagenase, maintain the elasticity and tightness of skin, have good anti-aging and pore-shrinking effect, and can also effectively remove hydroxyl radicals, have good whitening and brightening effect.