Glycosaminoglycan lysing enzyme and application thereof

Abstract

The present invention provides a glycosaminoglycan lyase and its application in agriculture and clinical medicine. The glycosaminoglycan lyase has the advantages of high catalytic efficiency, good stability, etc., and the glycosaminoglycan lyase can simultaneously cleave multiple glycosaminoglycans, such as hyaluronic acid and chondroitin sulfate (such as CS-A, CS-C or CS-D), and has broad application prospects in agriculture, clinical medicine, food health care and cosmetics.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a glycosaminoglycan lyase and its application in agriculture and clinical medicine. Background Art

[0002] Hyaluronic acid, also known as hyaluronic acid (HA), is a member of the glycosaminoglycan (also known as mucopolysaccharide) family and exists naturally in organisms. The disaccharide unit of natural hyaluronic acid is composed of β1-3-D-glucuronic acid and β1-4-N-acetyl-D-glucosamine. Hyaluronic acid is an important component of the cell matrix and various tissues. It has many important physiological functions through the synthesis of membrane proteins on the cell membrane, such as: regulating cell proliferation, migration and differentiation; natural moisturizing effect; lubricating joints and protecting cartilage; regulating protein synthesis; regulating inflammatory response; regulating immune function; promoting wound healing, etc. The unique viscoelasticity and biocompatibility of hyaluronic acid make it widely used in the biological and medical fields.

[0003] Recent studies have found that low molecular weight hyaluronic acid and hyaluronic acid oligosaccharides have more unique biological functions. Studies have shown that low molecular weight hyaluronic acid and hyaluronic acid oligosaccharides have the effects of inhibiting tumor proliferation, promoting wound healing, promoting bone and angiogenesis, and immunomodulation, and are easy to penetrate into the dermis, and are activators of immune cells and cytokines. In addition, low molecular weight hyaluronic acid and hyaluronic acid oligosaccharides have better antioxidant free radical effects, can effectively repair cell damage caused by ultraviolet rays, and can also penetrate into the epidermis of the skin to supplement the endogenous hyaluronic acid in the skin, thereby nourishing the skin and delaying aging. Therefore, small molecule hyaluronic acid has broad application prospects in clinical medicine, food health care and cosmetics.

[0004] At present, the degradation method of hyaluronic acid mainly contains physical method, chemical method and enzymatic degradation method.Physical method (heating, mechanical shearing, ultraviolet radiation, etc.) can be applied to the degradation of hyaluronic acid, but it is difficult to reduce the hyaluronic acid molecular weight to below 10kDa by physical method degradation, and the molecular weight distribution range of the prepared product is larger, and it is difficult to obtain uniform product.Chemical degradation method degradation of hyaluronic acid requires more violent reaction conditions, although it can reach the purpose of controlling product molecular weight to a certain extent, but due to the complex degradation conditions of different chemical reagents, it causes product properties to be easily affected and product purification is difficult, and there is the problem of waste liquid pollution.Enzymatic hydrolysis is a gentle method for degrading hyaluronic acid, and the product structure obtained by degradation is uniform, and the actual recovery rate of the product is high, and the purity is high, which is particularly suitable for preparing low molecular weight hyaluronic acid. Summary of the invention

[0005] The hyaluronidase currently on the market is mainly animal tissue-derived hyaluronidase, which has low activity, high cost, and animal virus safety risks. The main method for industrial production of small molecule hyaluronic acid is still chemical degradation, but there are problems such as structural damage and environmental pollution. Therefore, it is of great significance to screen hyaluronic acid lyase derived from microorganisms using abundant microbial resources in the soil for the industrial production of low molecular weight hyaluronic acid.

[0006] In view of the above-mentioned existing problems, the present invention screened and obtained a plant endophyte (Phytobacter sp.) HAD-10. The extracellular enzyme solution obtained by culturing the plant endophyte (Phytobacter sp.) HAD-10 can effectively cleave glycosaminoglycans. In addition, the sequence of the glycosaminoglycanase that plays a major role was clarified, and its cleavage activity was confirmed by experiments.

[0007] The first aspect of the present invention provides an extracellular enzyme solution or a preparation method thereof, wherein the extracellular enzyme solution is the extracellular supernatant obtained by culturing a plant endophytic bacterium (Phytobacter sp.) HAD-10, the extracellular supernatant is centrifuged to collect the precipitate, and then the precipitate is resuspended to obtain the extracellular enzyme solution, wherein the plant endophytic bacterium (Phytobacter sp.) HAD-10 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on February 22, 2024, with a deposit number of CGMCC No. 29875.

[0008] The storage address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The culturing includes using a culture medium.

[0010] The culture medium may be liquid, semi-solid or solid.

[0011] The culture medium is, for example, LB culture medium.

[0012] The culture temperature is 30-40°C.

[0013] The culture may be shake flask culture.

[0014] The genome of the plant endophyte (Phytobacter sp.) HAD-10 includes a sequence encoding SEQ ID NO: 3, preferably SEQ ID NO: 2.

[0015] The extracellular enzyme solution cleaves glycosaminoglycans.

[0016] Preferably, the glycosaminoglycan comprises one or more of hyaluronic acid, dermatan sulfate, keratan sulfate, heparin, heparan sulfate or chondroitin sulfate.

[0017] Further preferably, the chondroitin sulfate includes one or more of CS-A, CS-B, CS-C, CS-D, CS-E, CS-F, CS-H, CS-K, CS-L or CS-M.

[0018] A second aspect of the present invention provides a glycosaminoglycan lyase, wherein the glycosaminoglycan lyase cleaves glycosaminoglycans and the glycosaminoglycan lyase comprises SEQ ID NO:3.

[0019] Preferably, the nucleotide sequence encoding the glycosaminoglycan lyase comprises SEQ ID NO: 2 or a nucleotide sequence having more than 80% homology with SEQ ID NO: 2.

[0020] Preferably, the glycosaminoglycan comprises β1-3 glycosidic bonds and / or β1-4 glycosidic bonds.

[0021] Preferably, the glycosaminoglycan comprises one or more of hyaluronic acid, dermatan sulfate, keratan sulfate, heparin, heparan sulfate or chondroitin sulfate.

[0022] Further preferably, the chondroitin sulfate comprises one or more of CS-A, CS-B, CS-C, CS-D, CS-E, CS-F, CS-H, CS-K, CS-L or CS-M. In a specific embodiment of the present invention, the chondroitin sulfate comprises one or more of CS-A, CS-C or CS-D.

[0023] Preferably, the glycosaminoglycan lyase is derived from a microorganism, and more preferably, the microorganism comprises a plant endophyte (Phytobacter sp.).

[0024] In a specific embodiment of the present invention, the glycosaminoglycan lyase is derived from a plant endophyte (Phytobacter sp.) HAD-10, which has been deposited in the China General Microbiological Culture Collection Center (CGMCC) on February 22, 2024, with a deposit number of CGMCC No. 29875 and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0025] The third aspect of the present invention provides a nucleic acid encoding the above-mentioned glycosaminoglycan lyase.

[0026] Preferably, the nucleic acid comprises SEQ ID NO: 2 or a nucleotide sequence having more than 80% homology with SEQ ID NO: 2.

[0027] The fourth aspect of the present invention provides an expression vector, wherein the expression vector expresses the above-mentioned glycosaminoglycan lyase, or the expression vector contains the above-mentioned nucleic acid.

[0028] The fifth aspect of the present invention provides a recombinant cell, wherein the recombinant cell expresses the above-mentioned glycosaminoglycan lyase, or the recombinant cell contains the above-mentioned nucleic acid, or the recombinant cell contains the above-mentioned expression vector.

[0029] Preferably, the recombinant cell comprises one or more of Escherichia coli, yeast (such as Pichia pastoris), mammalian cells, insect cells, Streptococcus zooepidemicus or baculovirus;

[0030] Further preferably, the insect cells include any one or more of sf9 cells, sf21 cells, Hi-5 cells, S2 cells or Tn cells;

[0031] Further preferably, the mammalian cells include one or more of CHO cells, BHK cells, HEK293 cells, HT-1080 cells, CAP cells, PER.C6 cells, NS0 cells or Sp2 / 0 cells.

[0032] In a specific embodiment of the present invention, the recombinant cell is Escherichia coli, preferably Escherichia coli BL21 strain or its derivatives, such as any one of BL21 (DE3), BLR (DE3), Rosetta (DE3) or OrigamiB (DE3).

[0033] In a sixth aspect of the present invention, a method for preparing a glycosaminoglycan lyase is provided, the method comprising obtaining a sequence of the glycosaminoglycan lyase from the genome of a plant endophyte (Phytobacter sp.) HAD-10, then heterologously expressing the sequence, and then purifying the glycosaminoglycan lyase;

[0034] The plant endophyte (Phytobacter sp.) HAD-10 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on February 22, 2024, with the deposit number: CGMCC No. 29875;

[0035] The amino acid sequence of the glycosaminoglycan lyase comprises SEQ ID NO:3.

[0036] Preferably, the heterologous expression is prokaryotic expression or eukaryotic expression, such as expression in Escherichia coli.

[0037] Further preferably, the preparation method comprises introducing a nucleotide sequence encoding glycosaminoglycan lyase into a host cell, culturing and inducing its expression; preferably, a vector is used to introduce the nucleotide sequence encoding glycosaminoglycan lyase into Escherichia coli.

[0038] Preferably, the host cell is selected from one or more of Escherichia coli, yeast (such as Pichia pastoris), mammalian cells, insect cells, Streptococcus zooepidemicus or baculovirus;

[0039] Further preferably, the insect cells include any one or more of sf9 cells, sf21 cells, Hi-5 cells, S2 cells or Tn cells;

[0040] Further preferably, the mammalian cells include one or more of CHO cells, BHK cells, HEK293 cells, HT-1080 cells, CAP cells, PER.C6 cells, NS0 cells or Sp2 / 0 cells.

[0041] In a specific embodiment of the present invention, the host cell is Escherichia coli, preferably Escherichia coli BL21 strain or its derivatives, such as any one of BL21 (DE3), BLR (DE3), Rosetta (DE3) or OrigamiB (DE3).

[0042] Preferably, the nucleotide sequence encoding the glycosaminoglycan lyase comprises SEQ ID NO: 2 or a nucleotide sequence having more than 80% homology with SEQ ID NO: 2.

[0043] Preferably, the preparation method further comprises a purification step.

[0044] Preferably, the glycosaminoglycan lyase includes the glycosaminoglycan lyase described in the second aspect above.

[0045] Preferably, the vector comprises a vector backbone and a nucleotide sequence encoding glycosaminoglycan lyase.

[0046] The vector skeleton includes pET series vectors or pGEX series vectors.

[0047] Further preferably, the pET series vectors include but are not limited to pET-22b, pET-3a, pET-3d, pET-11a, pET-12a, pET-14b, pET-15b, pET-16b, pET-17b, pET-19b, pET-20b, pET-21a, pET-23a, pET-23b, pET-24a, pET-25b , pET-26b, pET-27b, pET-28a, pET-29a, pET-30a, pET-31b, pET-32a, pET-35b, pET-38b, pET-39b, pET-40b, pET-41a, pET-41b, pET-42a, pET-43a, pET-43b, pET-44a or pET-49b.

[0048] In a specific embodiment of the present invention, the preparation method comprises cloning a nucleotide sequence containing SEQ ID NO: 2 or having more than 80% homology with SEQ ID NO: 2 into a pET series vector, constructing a recombinant expression vector, and transforming the successfully constructed recombinant expression vector into Escherichia coli. Preferably, the preparation method further comprises a purification step.

[0049] The seventh aspect of the present invention provides a method for degrading glycosaminoglycans, which comprises mixing the above-mentioned glycosaminoglycan lyase, the above-mentioned extracellular enzyme solution, the glycosaminoglycan lyase obtained by the above-mentioned preparation method and the glycosaminoglycans to be degraded.

[0050] Preferably, the glycosaminoglycan comprises β1-3 glycosidic bonds and / or β1-4 glycosidic bonds.

[0051] Preferably, the glycosaminoglycan comprises one or more of hyaluronic acid, dermatan sulfate, keratan sulfate, heparin, heparan sulfate or chondroitin sulfate.

[0052] Preferably, the chondroitin sulfate includes one or more of CS-A, CS-B, CS-C, CS-D, CS-E, CS-F, CS-H, CS-K, CS-L or CS-M.

[0053] In a specific embodiment of the present invention, the chondroitin sulfate includes one, two or three of CS-A, CS-C or CS-D.

[0054] Preferably, the mixing reaction time is 1-5 h, preferably 1-3 h, such as 1, 2, 3, 4 or 5 h.

[0055] Preferably, the mixed reaction temperature is 30-40°C, preferably 35-40°C, for example 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40°C.

[0056] Preferably, the glycosaminoglycan is hyaluronic acid, and the degradation product includes small molecule hyaluronic acid.

[0057] Further preferably, the small molecule hyaluronic acid includes hyaluronic acid oligosaccharide or GalNAc monosaccharide.

[0058] The eighth aspect of the present invention provides a small molecule hyaluronic acid obtained by the above-mentioned method for degrading glycosaminoglycans.

[0059] Preferably, the small molecule hyaluronic acid includes hyaluronic acid oligosaccharide or GalNAc monosaccharide.

[0060] In a ninth aspect of the present invention, there is provided an application of the above-mentioned glycosaminoglycan lyase, the above-mentioned extracellular enzyme solution, the glycosaminoglycan lyase obtained by the above-mentioned preparation method, the above-mentioned nucleic acid, the above-mentioned expression vector or the above-mentioned recombinant cell, wherein the application comprises:

[0061] 1) Application in the cleavage of glycosaminoglycans or the preparation of small molecule hyaluronic acid;

[0062] 2) Application in the preparation of drugs for inhibiting tumor spread, promoting wound healing, promoting bone formation, promoting angiogenesis, regulating immunity, resisting oxidative free radicals or repairing cell damage;

[0063] 3) Application in food, health care or cosmetics fields.

[0064] Preferably, the glycosaminoglycan comprises β1-3 glycosidic bonds and / or β1-4 glycosidic bonds.

[0065] Preferably, the glycosaminoglycan comprises one or more of hyaluronic acid, dermatan sulfate, keratan sulfate, heparin, heparan sulfate or chondroitin sulfate;

[0066] Further preferably, the chondroitin sulfate comprises one or more of CS-A, CS-B, CS-C, CS-D, CS-E, CS-F, CS-H, CS-K, CS-L or CS-M. In a specific embodiment of the present invention, the chondroitin sulfate comprises one or more of CS-A, CS-C or CS-D.

[0067] Preferably, the small molecule hyaluronic acid comprises hyaluronic acid oligosaccharide or GalNAc monosaccharide;

[0068] Further preferably, the hyaluronic acid oligosaccharide comprises saturated hyaluronic acid oligosaccharide and / or unsaturated hyaluronic acid oligosaccharide;

[0069] Further preferably, the GalNAc monosaccharide includes saturated GalNAc monosaccharide and / or unsaturated GalNAc monosaccharide.

[0070] Preferably, the hyaluronic acid oligosaccharide comprises even-numbered oligosaccharides;

[0071] Further preferably, the even-numbered oligosaccharide includes one or more of hyaluronic acid disaccharide, hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, hyaluronic acid octasaccharide or hyaluronic acid decasaccharide.

[0072] Preferably, the hyaluronic acid oligosaccharides include odd-numbered oligosaccharides;

[0073] Further preferably, the odd-numbered oligosaccharide includes one or more of hyaluronic acid trisaccharide, hyaluronic acid pentasaccharide, hyaluronic acid heptasaccharide or hyaluronic acid nonasaccharide.

[0074] The tenth aspect of the present invention provides a use of SEQ ID NO: 3 in cleaving glycosaminoglycans.

[0075] The eleventh aspect of the present invention provides a drug, which comprises the above-mentioned glycosaminoglycan lyase, the above-mentioned extracellular enzyme solution, the glycosaminoglycan lyase obtained by the above-mentioned preparation method, the above-mentioned nucleic acid, the above-mentioned expression vector, the above-mentioned recombinant cell or the above-mentioned small molecule hyaluronic acid.

[0076] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0077] Preferably, the pharmaceutically acceptable excipients include one or a combination of two or more of a carrier (e.g., an adsorption carrier), an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a preservative, an antioxidant, a buffer, an antibacterial agent, a suspending agent, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, a suspending agent, a solubilizer, a thickener or a stabilizer.

[0078] Preferably, the drug may be in the form of any one of syrup, elixir, suspension, powder, granule, tablet, capsule, lozenge, aqueous solution, cream, ointment, lotion, gel or emulsion, or a combination of two or more thereof.

[0079] Preferably, the preparation type of the medicine is preferably a unit dose preparation. The amount of the active pharmaceutical ingredient in the unit dose preparation can be changed or adjusted from 0.001 mg to 1000 mg (preferably 0.1 mg to 500 mg, further preferably 30 mg to 100 mg, such as 0.001, 0.01, 0.1, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mg), depending on the specific application and effectiveness of the active ingredient. According to the needs of a specific embodiment, the composition may also include other suitable therapeutic agents.

[0080] The drug is suitable for enteral or parenteral administration.

[0081] Preferably, preparations suitable for enteral administration, such as oral administration, enteral administration or administration via catheter include one or more of an oral agent (e.g., capsules, sachets, pills, tablets, lozenges, powders, granules, aqueous or non-aqueous solutions or suspensions, water-in-oil or oil-in-water emulsions, elixirs or syrups), a rectal suppository or a rectal enema.

[0082] Preferably, preparations suitable for parenteral administration, such as by intravenous, intramuscular, intradermal and subcutaneous routes of administration, include aqueous and non-aqueous isotonic sterile injections, which may contain antioxidants, buffers, bacteriostats and solutes that make the preparation isotonic with the recipient's blood, and aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers and preservatives. The preparations may be present in unit doses or in multi-dose sealed containers such as ampoules and vials. Solutions and suspensions for injection may be prepared from sterile powders, granules and tablets of the types previously described.

[0083] The twelfth aspect of the present invention provides a cosmetic comprising the above-mentioned glycosaminoglycan lyase, the above-mentioned extracellular enzyme solution, the glycosaminoglycan lyase obtained by the above-mentioned preparation method, the above-mentioned nucleic acid, the above-mentioned expression vector, the above-mentioned recombinant cell or the above-mentioned small molecule hyaluronic acid.

[0084] Preferably, the cosmetics contain auxiliary materials acceptable in the cosmetics field.

[0085] Preferably, the excipients acceptable in the cosmetic field include solvents, solubilizers, preservatives, antioxidants, pH adjusters, penetration enhancers, oils, moisturizers, thickeners, chelating agents, skin feel regulators, surfactants, emulsifiers, amino acids, propellants / propellants, flavors, pigments, and other functional additives, or a combination of two or more thereof.

[0086] Preferably, the dosage form of the cosmetic comprises one or more of a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a facial cleanser, a liquid foundation (such as a powder liquid foundation, an emulsion liquid foundation or a wax liquid foundation), a facial mask, a massage cream or a spray.

[0087] The thirteenth aspect of the present invention provides a food, which comprises the above-mentioned glycosaminoglycan lyase, the above-mentioned extracellular enzyme solution, the glycosaminoglycan lyase obtained by the above-mentioned preparation method, the above-mentioned nucleic acid, the above-mentioned expression vector, the above-mentioned recombinant cell or the above-mentioned small molecule hyaluronic acid.

[0088] Preferably, the food further comprises auxiliary materials acceptable in the food field.

[0089] Preferably, the excipients acceptable in the food field include one or a combination of two or more of a carrier (such as an adsorption carrier), an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a suspension stabilizer or a preservative.

[0090] The types of food described in the present invention are not particularly limited and can be any known food, for example, dairy products, biscuits, cakes, beverages, health products, etc.

[0091] Preferably, the food is in at least one form selected from solid, dairy, solution, powder and suspension products.

[0092] The term "homology" as used in the present invention refers to that in terms of the use of amino acid sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs, while ensuring that the structures or functions are similar to those of known sequences, so that the sequences used have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36% compared to the sequences obtained by the prior art. , 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity.

[0093] The "and / or" described in the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A", "A and B" and "B". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0094] The term “include” or “comprising” described in the present invention is an open description, which includes the specified components or steps described and other specified components or steps that will not be substantially affected.

[0095] The "small molecule hyaluronic acid" described in the present invention refers to a hyaluronic acid with a molecular weight ranging from 5kDa to 50kDa, preferably less than 10kDa, such as hyaluronic acid oligosaccharide or GalNAc monosaccharide.

[0096] Beneficial effects of the present invention:

[0097] The present invention provides a glycosaminoglycan lyase, which has the advantages of high catalytic efficiency, good stability, etc., and the glycosaminoglycan lyase can simultaneously cleave multiple glycosaminoglycans, such as hyaluronic acid and chondroitin sulfate (such as CS-A, CS-C or CS-D), and has broad application prospects in agriculture, clinical medicine, food health care and cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0099] Figure 1 :Blast comparison results;

[0100] Figure 2 : Colony morphology of HAD-10 strain;

[0101] Figure 3 :Microscopic examination of HAD-10 strain;

[0102] Figure 4 : Absorbance of extracellular crude enzyme reaction solution of HAD-10 strain at A540;

[0103] Figure 5 :Sample evolution analysis of HAD-10 strain based on 16S rRNA;

[0104] Figure 6 :Sample evolution analysis of HAD-10 strain based on housekeeping genes;

[0105] Figure 7 : Agarose gel electrophoresis of the recombinant vector;

[0106] Figure 8 :Colony PCR images;

[0107] Fig. 9 :SDS-PAGE of HAlyase2;

[0108] Fig.10 : TLC characterization of the polysaccharide cleavage ability of HAlyase2. DETAILED DESCRIPTION

[0109] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0110] Example 1 Strain screening, identification and acquisition of hyaluronan lyase sequence

[0111] 1. Screen the microorganisms in the soil of the Qufu factory and obtain strains with the ability to degrade hyaluronic acid.

[0112] (1) The soil sample was resuspended in sterile water and inoculated into HA inorganic nitrogen source liquid culture medium. The culture was shaken at 37°C and 220 rpm for 2-3 days. The culture was repeated 2-3 times to observe the change in the viscosity of the culture medium.

[0113] (2) Dilute the bacterial solution in a gradient manner and take 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 200 μL of the gradient was respectively transferred to HA inorganic nitrogen source solid culture medium, and three parallel plates were set up for inverted culture at 37°C for 2-3 days.

[0114] (3) Use the BSA-acetic acid method to observe whether there are strains that produce clearing zones.

[0115] (4) Streak the positive colonies obtained in the initial screening on an LB plate for isolation and purification, and repeat this three times until the morphology of the single colonies on the plate is consistent.

[0116] (5) After purification, the single colonies were inoculated into HA as the sole carbon source inorganic salt culture medium for re-screening. After the purified strains were cultured in HA liquid culture medium for 3 hours, it was found that the HA liquid culture medium of the HAD-10 strain changed from a gel-like state to a fluid state within 1 hour, indicating that this strain has the ability to efficiently degrade HA.

[0117] 2. Sequence the 16S rDNA sequence of HAD-10 strain.

[0118] The whole genome of the HAD-10 strain was extracted; the 16S rDNA gene of the strain was amplified by PCR using universal primers 27F (SEQ ID NO: 4, 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO: 5, 5'-TACGACTTAACCCCAATCGC-3') of 16S rDNA, and the 16S rDNA sequence was obtained by sequencing as shown in SEQ ID NO: 1. At the same time, the 16S rDNA of the strain was BLASTed on the NCBI website to obtain homology information, and it was found that the 16S rDNA sequence of the strain had a sequence similarity of about 99% with Citrobacter bitternis, Phytobacter diazotrophicus and Phytobacter ursingi (see Figure 1 ).

[0119] The HAD-10 strain was identified as a plant endophyte (Phytobacter sp.) and was deposited in the General Microbiology Center of the China Microbiological Culture Collection (CGMCC) on February 22, 2024. The deposit number is: CGMCC No. 29875, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0120] 3. Observe the single colony morphology of HAD-10 strain and perform microscopic examination on it.

[0121] The HAD-10 strain was spread on the LB plate in gradient dilutions to observe the single colony morphology of the strain (the colony edge was wrinkled, the colony color was milky white, and the colony morphology was flat and round); Gram staining was performed on the strain, and microscopic examination revealed that the strain was a rod-shaped Gram-negative bacterium. The colony morphology diagram is shown in Figure 2 , see the microscopic examination results. Figure 3 .

[0122] 4. Extraction of extracellular crude enzyme solution of HAD-10 strain.

[0123] The HAD-10 strain was inoculated into 50 mL of LB liquid culture medium and cultured in a shake flask at 37°C; the extracellular supernatant of the strain was collected; 80% ammonium sulfate was added to the extracellular supernatant, centrifuged and the precipitate was collected; the precipitate was resuspended in PBS buffer to prepare HAD-10 extracellular crude enzyme solution; the extracellular crude enzyme concentration was detected using a BCA quantitative kit, and the enzyme concentration was 3.36 mg / mL.

[0124] 5. Determine the polysaccharide utilization capacity of HAD-10 extracellular crude enzyme solution.

[0125] 5 mg / mL hyaluronic acid (HA), chitosan (CH), chondroitin sulfate A (CS-A), chondroitin sulfate C (CS-C), chondroitin sulfate D (CS-D), heparin (Hep), and xanthan gum (XG) were used as substrates, and 1 mg / mL crude enzyme solution was added. The reaction was carried out at 37°C for 2 h, and the absorbance of the reaction solution at A540 was measured. It was found that the absorbance value of the reaction solution was larger than that of the control. It was found that the extracellular crude enzyme of strain HAD-10 had the ability to cleave / hydrolyze HA, CS-A, CS-C, and CS-D, see Figure 4 and Table 1.

[0126] Table 1

[0127] HA CS-A CS-C CS-D <![CDATA[A 540 Compare]]> 0.312 0.369 0.351 0.429 <![CDATA[A 540 Reactions]]> 0.964 0.544 0.455 0.562

[0128] 6. Conduct genetic evolution analysis on HAD-10 strain.

[0129] The MEGA software was used to construct a phylogenetic tree based on the 16S rRNA sequence and housekeeping gene of HAD-10 and the 16S rRNA and housekeeping gene sequences of other homologous strains to obtain the evolutionary relationship. The results of the evolutionary analysis based on 16S rRNA can be found in Figure 5 , based on the evolutionary analysis results of housekeeping genes, see Figure 6 .

[0130] 7. Perform whole genome sequencing on HAD-10 strain and discover the hyaluronan lyase gene.

[0131] The HAD-10 strain was sent to Shanghai Meiji Biotechnology Co., Ltd. for full gene sequencing to obtain the gene annotation of this strain. According to the gene annotation information, the gene sequence of hyaluronan lyase was obtained as shown in SEQ ID NO: 2. This lyase was named HAlyase2, and its protein sequence is shown in SEQ ID NO: 3.

[0132] Example 2 Heterologous expression, purification and enzyme activity detection of hyaluronan lyase HAlyase2 gene

[0133] 1. Heterologous expression of hyaluronan lyase HAlyase2 gene.

[0134] The HAlyase2 gene was cloned into the vector pET-15b, and the sequencing was correct. The HAlyase2-pET-15b recombinant plasmid was successfully constructed (see agarose gel electrophoresis for details). Figure 7 After the construction is successful, the plasmid is transformed into Escherichia coli BL21. The colony PCR results are shown in Figure 8 .

[0135] 2. Expression and purification of hyaluronan lyase protein.

[0136] In 50 mL LB medium (10 g / L trypsin, 5 g / L yeast extract and 10 g / L sodium chloride), add 100 μg / mL ampicillin (Amp), inoculate the E. coli culture containing the HAlyase2 recombinant plasmid, and culture it overnight at 37°C with vigorous shaking at 220 rpm. Inoculate the overnight culture into 1 L LB medium containing 100 μg / mL Amp and culture it at 37°C with 220 rpm until the OD reaches 0. 600 The value reached 0.6-0.8. Then IPTG (0.4mM) was added to induce the expression of HAlyase2, and cultured at 16℃, 180rpm for 16-20 hours. Bacteria were collected by centrifugation at 4℃, 5000×g for 10min. The bacteria were broken by ultrasound, and the cell debris was removed by centrifugation at 4℃, 24000×g for 30min. The supernatant was collected and purified by Ni column. The SDS-PAGE results of HAlyase2 can be found in Fig. 9 .

[0137] 3. Analysis of the polysaccharide cleavage ability of hyaluronan lyase HAlyase2.

[0138] 5 mg / mL hyaluronic acid (HA), chitosan (CH), chondroitin sulfate A (CS-A), chondroitin sulfate C (CS-C), chondroitin sulfate D (CS-D), heparin (Hep), and xanthan gum (XG) were used as substrates, and 1 mg / mL HAlyase2 was added. The reaction was carried out at 37°C. After 10 minutes, the reaction products were analyzed by TLC. It was found that HAlyase2 had high cleavage activity against HA and could also cleave CS-A, CS-C, and CS-D (see Fig.10 ), the cleavage products of the reaction are all disaccharides.

[0139] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0140] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0141] Some nucleotide sequences involved in this application:

[0142] SEQ ID NO: 1:

[0143]

[0144]

[0145] SEQ ID NO:3:

[0146] MSVTLERLDCRYLTPQPALDKAWLNDAIADVLSSIDAMLPRFSQTFPAASAQGGFYPPVEKVDWTEGFWTGLLWLAWEVTGEEKYRRVAEGFLDNFEERLDKQIKVDTHDLGFLYLLSCVNSWRLTGNTRARALALRAADLLYRRFNSTAGVIQAWGDLNDPARQGRMIIDCNLNVPLLFWAANETGNTDWRDAATRHLAQAARYLVREDASTFHTFYMDISNGEPLRGDTHQGFSDSSCWARGQAWGIYGFALGFQHTGNPALPELSRKLTHYFLNRLPEDGVCYWDLIFTAQDHAYRDTSAAAIAVCGMAELLKHLPLTDSLRPAYLNAISLMMRHLQAHYFAHNEDGLLREGVYNFGRDMGINVPNLWGDYFWFEALVRLSRFWTPYW

Claims

1. A method for degrading hyaluronic acid, characterized in that: The method comprises mixing an extracellular enzyme solution or a hyaluronan lyase with the hyaluronic acid to be degraded; The extracellular enzyme solution is used to culture plant endophytes ( Phytobacter sp. ) HAD-10 to obtain the extracellular supernatant, centrifuge the extracellular supernatant to collect the precipitate, and then resuspend the precipitate to obtain the extracellular enzyme solution, wherein the plant endophyte ( Phytobacter sp. )HAD-10 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 22, 2024, with the deposit number: CGMCC No.29875; The amino acid sequence of the hyaluronan lyase is SEQ ID NO:

3.

2. The method according to claim 1, characterized in that The mixing reaction time is 1-5h.

3. The method according to claim 1, characterized in that The mixed reaction temperature is 30-40°C.

4. The method according to claim 1, characterized in that: The culturing includes using a culture medium.

5. The method according to claim 4, characterized in that The culture medium includes LB culture medium.

6. The method according to claim 1, characterized in that The culture temperature is 30-40°C.

7. The method according to claim 1, characterized in that The plant endophyte ( Phytobacter sp. ) The genome of HAD-10 includes a gene encoding hyaluronan lyase.

8. The method according to claim 1, characterized in that The plant endophyte ( Phytobacter sp. ) The genome of HAD-10 includes a gene whose nucleotide sequence is shown in SEQ ID NO:

2.

9. The method according to claim 1, characterized in that: The preparation method of the hyaluronate lyase comprises the steps of: Phytobacter sp. ) The gene of hyaluronan lyase is obtained from the genome of HAD-10, and the gene is then heterologously expressed and then purified to obtain hyaluronan lyase.

10. The method according to claim 9, characterized in that The heterologous expression is prokaryotic expression or eukaryotic expression.

11. The method according to claim 9, characterized in that The heterologous expression is expression in Escherichia coli.

12. The method according to claim 9, characterized in that The preparation method comprises the steps of introducing the gene of hyaluronan lyase into host cells, culturing and inducing the expression of the gene.

13. The method according to claim 12, characterized in that The nucleotide sequence of the hyaluronan lyase gene is SEQ ID NO:

2.

14. The use of extracellular enzyme solution or hyaluronan lyase, characterized in that: The applications include: application in cracking hyaluronic acid or preparing small molecule hyaluronic acid; The extracellular enzyme solution is used to culture plant endophytes ( Phytobacter sp. ) HAD-10 to obtain the extracellular supernatant, centrifuge the extracellular supernatant to collect the precipitate, and then resuspend the precipitate to obtain the extracellular enzyme solution, wherein the plant endophyte ( Phytobacter sp. )HAD-10 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 22, 2024, with the deposit number: CGMCC No.29875; The amino acid sequence of the hyaluronan lyase is SEQ ID NO:

3.

15. The use according to claim 14, characterized in that: The culturing includes using a culture medium.

16. The use according to claim 15, characterized in that The culture medium includes LB culture medium.

17. The use according to claim 14, characterized in that: The culture temperature is 30-40°C.

18. The use according to claim 14, characterized in that: The plant endophyte ( Phytobacter sp. ) The genome of HAD-10 includes a gene encoding hyaluronan lyase.

19. The use according to claim 14, characterized in that: The plant endophyte ( Phytobacter sp. ) The genome of HAD-10 includes a gene whose nucleotide sequence is shown in SEQ ID NO:

2.

20. The use according to claim 14, characterized in that The preparation method of the hyaluronate lyase comprises the steps of: Phytobacter sp. ) The gene of hyaluronan lyase is obtained from the genome of HAD-10, and the gene is then heterologously expressed and then purified to obtain hyaluronan lyase.

21. The use according to claim 20, characterized in that The heterologous expression is prokaryotic expression or eukaryotic expression.

22. The use according to claim 20, characterized in that The heterologous expression is expression in Escherichia coli.

23. The use according to claim 20, characterized in that The preparation method comprises the steps of introducing the gene of hyaluronan lyase into host cells, culturing and inducing the expression of the gene.

24. The use according to claim 23, characterized in that The nucleotide sequence of the hyaluronan lyase gene is SEQ ID NO:

2.

25. The use according to claim 14, characterized in that The small molecule hyaluronic acid includes hyaluronic acid oligosaccharide or GalNAc monosaccharide.

26. The use according to claim 25, characterized in that The hyaluronic acid oligosaccharide includes saturated hyaluronic acid oligosaccharide and / or unsaturated hyaluronic acid oligosaccharide.

27. The use according to claim 25, characterized in that The GalNAc monosaccharide includes saturated GalNAc monosaccharide and / or unsaturated GalNAc monosaccharide.

28. The use according to claim 25, characterized in that The hyaluronic acid oligosaccharides include even-numbered oligosaccharides.

29. The use according to claim 28, characterized in that The even-numbered oligosaccharides include one or more of hyaluronic acid disaccharide, hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, hyaluronic acid octasaccharide or hyaluronic acid decasaccharide.

30. The use according to claim 25, characterized in that The hyaluronic acid oligosaccharides include odd-numbered oligosaccharides.

31. The use according to claim 30, characterized in that The odd-numbered oligosaccharides include one or more of hyaluronic acid trisaccharide, hyaluronic acid pentasaccharide, hyaluronic acid heptasaccharide or hyaluronic acid nonasaccharide.

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