CN110331178B

CN110331178B - Method for preparing micromolecular hyaluronic acid by enzyme cleavage method, obtained micromolecular hyaluronic acid and application thereof

Info

Publication number: CN110331178B
Application number: CN201910659515.5A
Authority: CN
Inventors: 江晓路; 张京良; 王鹏
Original assignee: Qingdao Marine Biomedical Research Institute Co Ltd
Current assignee: Qingdao Marine Biomedical Research Institute Co Ltd
Priority date: 2019-06-27
Filing date: 2019-07-22
Publication date: 2021-01-19
Anticipated expiration: 2039-07-22
Also published as: CN110331178A

Abstract

The invention provides a method for preparing micromolecular hyaluronic acid by an enzyme cleavage method, the obtained micromolecular hyaluronic acid and application thereof. The invention uses hyaluronidase prepared by fermentation of Arthrobacter globiformis HL6 with the preservation number of CCTCC NO: M2018452 or a hyaluronidase shown as SEQ ID NO: 1, degrading hyaluronic acid or a salt thereof with the molecular weight of more than 600kDa to prepare the hyaluronic acid or the salt thereof with the small molecular weight. The micromolecule hyaluronic acid prepared by the invention has high purity, is safe and nontoxic, has good moisturizing, percutaneous absorption, oxidation resistance and anti-inflammatory activity, is widely applied to the fields of food, cosmetics, medicines and the like, can be used as a standard substance for the structure and quality research of hyaluronic acid, and has wide research and application prospects.

Description

Method for preparing micromolecular hyaluronic acid by enzyme cleavage method, obtained micromolecular hyaluronic acid and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for preparing micromolecule hyaluronic acid by an enzyme cleavage method, the obtained micromolecule hyaluronic acid and application thereof.

Background

Hyaluronic Acid (HA), a high-molecular mucopolysaccharide formed by repeating and alternately connecting N-acetylglucosamine and D-glucuronic acid disaccharide units, is widely present in human bodies, is an important component of skin, vitreous body, joint synovial fluid and cartilage tissue, and HAs unique physicochemical properties and biological functions. It is widely used in the fields of food, cosmetics and medicine due to its high viscoelasticity and plasticity, super-strong water retention and permeability and good biocompatibility.

Recent studies have found that the biological activity of HA is directly related to its relative molecular weight, and HA of different molecular weights have different or even diametrically opposite biological activities. The high molecular weight HA HAs good viscoelasticity, moisture retention, lubrication and anti-inflammatory activity, and can be used in cosmetics, ophthalmic surgery, arthritis treatment, postoperative adhesion prevention, etc. The micromolecule HA HAs the functions of transdermal absorption, easy digestion, tumor resistance, promotion of endothelial cell proliferation, angiogenesis, wound healing, immunoregulation and the like, in recent years, the research of hyaluronic acid oligosaccharide (o-HA) gradually becomes a hotspot, and the research finds that the o-HA molecular fragment HAs the angiogenesis promoting activity in vivo and can also obviously promote the endothelial cell proliferation in vitro; the o-HA can protect granulation tissues from being damaged by oxygen free radicals, can effectively promote wound healing, can stimulate endothelial cells to identify injured parts, and can perform primary protection and repair on the wound parts; research shows that the o-HA also HAs the active functions of immunoregulation, tumor resistance and the like. The o-HA segment with remarkable activity can be used as a new resource for drug research and development and functional product development, so that the research for obtaining a large amount of high-purity o-HA segments HAs very important significance.

The preparation method of the small molecular HA mainly comprises a physical degradation method, a chemical degradation method, an enzymatic degradation method and a biological synthesis method. Many physical methods (heat, mechanical shear, ultraviolet radiation and ultrasonic disruption) are applicable to the degradation of hyaluronic acid, it is difficult to reduce the molecular weight of hyaluronic acid to below 10kDa by physical methods, and the molecular weight distribution range of the prepared product is large, and it is difficult to obtain HA having a specific molecular weight. TransformingThe chemical degradation method is used for degrading HA into oligosaccharide, a relatively violent reaction condition is needed, such as relatively high acid and alkali concentration, and the like, so that a high degradation degree can be achieved, the HA molecular structure can be damaged in the reaction process no matter acid hydrolysis, alkali hydrolysis or oxidative degradation, the structures of glucuronic acid and N-acetylglucosamine can be damaged while the glycosidic bond on the sugar chain is broken, such as acetyl groups are hydrolyzed off, monosaccharide six-membered rings are broken, and the like, so that the quality and the biological activity of oligosaccharide are remarkably influenced, the oligomeric hyaluronic acid prepared by the chemical degradation method is easy to brown, and the production process can pollute the environment. De Angelis et al synthesize 2-20 oligosaccharides using Pasteurella HA synthase (pmHAS), glucuronyl transferase and N-acetylglucosaminyltransferase, which is a biosynthetic reaction requiring strict reaction conditions and multiple reaction enzymes, and thus, the cost is high. The enzymolysis method is a mild method for degrading hyaluronic acid, has mild reaction conditions, is easy to control, does not cause environmental pollution, has uniform product structure, does not cause disaccharide structure fracture, has high actual recovery rate of the product, low product cost, high purity and good quality, and is particularly suitable for preparing low-molecular-weight hyaluronic acid. At present, commonly used hyaluronidase is mainly derived from animal extraction, TAWADA and the like degrade HA by hyaluronidase (hydrolase) extracted from bovine testes to prepare low-molecular oligomeric HA with monosaccharide residues 4-52; chinese patent CN 103484513B discloses a method for preparing HA oligosaccharides using a hyaluronidase (hydrolase) derived from leeches, the product being a saturated HA oligosaccharide. However, the hyaluronidase extracted from animals has low purity, potential risk of cross contamination of animal viruses and high price, and limits the application of the hyaluronidase in the preparation of small molecular hyaluronic acid. The hyaluronic acid lyase derived from microorganisms is completely different from the hyaluronic acid hydrolase, so that various defects of animal-derived hyaluronidase can be effectively avoided. The hyaluronidase produced by the microbial fermentation method has high activity and high purity, does not contain animal-derived viruses, and has unique advantages compared with animal-derived hyaluronidase. Chinese patent CN 102876748B discloses preparation of molecular weight 3000-10 by degrading air source bacillus hyaluronidase⁴Method for oligomeric hyaluronate salts of Da and oligomeric transparencies produced therebyA protonic acid salt and application. Chinese patent CN 103484513B discloses a method for preparing low-molecular hyaluronate with molecular weight of 10kDa-1000kDa by degrading air-sourced bacillus hyaluronidase, the low-molecular hyaluronate produced by the method and application of the low-molecular hyaluronate. The protective range is 3000-1000kDa low molecular hyaluronate.

Because the hyaluronidase on the market at present is mainly animal tissue source hyaluronidase, has the problems of low activity, high cost, animal virus safety risk and the like, and is not suitable for large-scale production of small molecular hyaluronic acid by an enzyme method, the main method for industrially producing the small molecular hyaluronic acid at present still has the problems of chemical degradation, structural damage, environmental pollution and the like, and the development of novel, safe and low-cost hyaluronidase is urgently needed to realize the production of the small molecular hyaluronic acid by the industrial enzyme method.

Disclosure of Invention

Through a great deal of research and creative work of the inventor, the method realizes that the Arthrobacter globiformis HL6 is fermented and produced to obtain the hyaluronidase with high enzyme activity, successfully obtains the enzyme production gene and the amino acid sequence of the hyaluronidase, constructs a recombinant vector and smoothly realizes the recombinant expression and the purification preparation in escherichia coli and bacillus subtilis (the national invention patent application number is 201811310938.8).

Aiming at the defects of the prior art, the invention provides a method for preparing small molecular hyaluronic acid by an enzyme cleavage method, the obtained small molecular hyaluronic acid and application thereof. The method for preparing the small-molecule hyaluronic acid by using the microbial hyaluronidase enzyme method has the advantages of simple process operation, mild conditions, no environmental pollution and animal-derived virus pollution, no damage to the product structure, good product uniformity and realization of environment-friendly industrial production of the small-molecule hyaluronic acid. The micromolecule hyaluronic acid prepared by the invention has high purity, is safe and nontoxic, has good moisturizing, percutaneous absorption, oxidation resistance and anti-inflammatory activity, can be widely applied to the fields of food, cosmetics, medicines and the like, can also be used as a hyaluronic acid oligosaccharide standard substance for the research of the structure and the quality of hyaluronic acid, and has wide research and application prospects.

The invention aims to solve the technical problem of providing a method for preparing micromolecular hyaluronic acid by an enzyme cleavage method.

The technical problem to be solved by the invention is to provide the application of the small-molecule hyaluronic acid.

In order to solve the technical problems, the invention adopts the following technical scheme:

a method for preparing small molecular hyaluronic acid by enzyme cutting comprises preparing hyaluronidase by fermenting Arthrobacter globiformis (Arthrobacter globiformis) HL6 with a preservation number of CCTCC NO: M2018452 or preparing small molecular hyaluronic acid by using an amino acid sequence shown as SEQ ID NO: 1, degrading hyaluronic acid with the molecular weight of more than 600kDa or a salt thereof, and preparing the small-molecular hyaluronic acid or the salt thereof.

The method for preparing the micromolecular hyaluronic acid by the enzyme cleavage method comprises the following steps:

1) preparing hyaluronic acid or salt solution thereof: adding hyaluronic acid or its salt with molecular weight of more than 600kDa into purified water to prepare solution with mass volume fraction of 0.05-20%;

2) enzymolysis: adjusting the temperature of the hyaluronic acid or its salt solution in step 1) to 20-50 deg.C, pH to 5-10, adding 100U/g-5 × 10⁵Degrading hyaluronic acid or its salt to 379Da-600kDa by U/g hyaluronidase to obtain enzymolysis solution;

3) inactivation: keeping the enzymolysis liquid in the step 2) at 55-100 ℃ for 5-30 minutes, and inactivating the added enzyme;

4) and (3) filtering: adding or not adding 0.01-2mol/L of soluble inorganic salt into the enzymolysis liquid in the step 3), stirring until the inorganic salt is completely dissolved, and filtering by using a filter membrane or a filter element to obtain a filtrate;

5) and (3) precipitation: slowly adding alcohol or ketone with the volume of 1.5-30 times of that of the filtrate into the filtrate obtained in the step 4), uniformly mixing, and precipitating to separate out the small molecular hyaluronic acid or the salt thereof;

6) and (3) dehydrating and drying: collecting the small molecular hyaluronic acid or the salt precipitate thereof in the step 5), adding alcohol or ketone which is the same as the alcohol or ketone in the step 5) for dehydration, and drying to obtain the small molecular hyaluronic acid or the salt thereof;

in the step 1), the hyaluronic acid salt is at least one of sodium salt, potassium salt, calcium salt, magnesium salt or zinc salt of hyaluronic acid;

in the step 2), the reaction temperature is preferably selected to be adjusted to 20 to 45 ℃ and the pH is preferably adjusted to 5.5 to 8. The acid or alkali used for adjusting the pH is at least one of hydrochloric acid, glacial acetic acid, nitric acid, sulfuric acid, phosphoric acid or sodium hydroxide and potassium hydroxide; adding 100U/g-5X 10 hyaluronic acid or its salt solution⁵U/g Arthrobacter globiformis (Arthrobacter globiformis) HL6 hyaluronidase or the amino acid sequence is shown as SEQ ID NO: 1, degrading hyaluronic acid or a salt thereof to a molecular weight of 379Da-600kDa, wherein the hyaluronidase has good degradability on the hyaluronic acid, and the hyaluronic acid with the required molecular weight can be obtained by only adding proper hyaluronidase and controlling the reaction time.

In the step 3), the temperature of the enzymolysis liquid is preferably adjusted to 60-80 ℃, and the heat preservation is carried out for 10-20 minutes to inactivate the hyaluronidase.

In the step 4), the easily soluble inorganic salt is at least one of sodium salt, potassium salt, calcium salt, magnesium salt or zinc salt; the filter membrane or the filter element can be a common filter membrane or filter element in the field, and the filter membrane or the filter element can be used in the invention as long as the material and the pore size meet the process requirements.

In the step 5), the alcohol or ketone is at least one of methanol, ethanol, propanol, butanol or acetone.

In the step 6), the alcohol or ketone is at least one of methanol, ethanol, propanol, butanol or acetone.

The invention also provides the small molecule hyaluronic acid or the salt thereof prepared by the method, wherein the small molecule hyaluronate is sodium salt, potassium salt, calcium salt, magnesium salt or zinc salt of the small molecule hyaluronic acid.

The micromolecular hyaluronic acid or the salt thereof prepared by the method has the advantages that the infrared spectrum is basically consistent with the European pharmacopoeia standard spectrum, the structure is complete, no group falls off, unsaturated C-C double bonds are generated between C4 and C5 of glucuronic acid, a characteristic absorption peak is generated near 232nm, a remarkable monitoring parameter is provided for the degradation process, the degradation process can be monitored according to the change of the absorbance at 232nm in the reaction process, the reaction process is easier to control, and the micromolecular hyaluronic acid or the salt thereof with different molecular weights can be accurately prepared by controlling the reaction time and the absorbance at 232nm of the solution under the condition of certain process parameters.

The small molecular hyaluronic acid or the salt thereof prepared by the method has the molecular weight range of 379Da-600kDa, has no toxicity to cells, has good biological activities of moisture retention, transdermal absorption, oxidation resistance, inflammation resistance and the like, and has good application prospects in cosmetics, medicines and foods.

The invention also provides a composition containing the small-molecule hyaluronic acid or the salt thereof prepared by the method.

The invention also provides a composition, which also comprises an acceptable auxiliary material or carrier in food, cosmetics or medicines.

The invention also provides a composition which can be a food, a cosmetic or a pharmaceutical.

In the present invention, if not specifically indicated, "small molecule hyaluronic acid or small molecule hyaluronate" is simply referred to as "small molecule hyaluronic acid or a salt thereof" and has a molecular weight in the range of 379Da to 600kDa, preferably in the range of 379Da to 200kDa, more preferably in the range of 379Da to 10kDa, and still more preferably in the range of 379Da to 3 kDa.

Compared with the prior art, the invention has the advantages and beneficial effects that: the invention relates to a hyaluronidase produced by fermenting Arthrobacter globiformis HL6 derived from sea or a hyaluronidase with an amino acid sequence shown as SEQ ID NO: 1, the hyaluronidase produced by microbial fermentation has high activity, high product purity, low impurity content and no potential animal virus hazard, is suitable for industrial production and applied to industrial production of the small molecular hyaluronic acid, is especially suitable for industrial preparation of the small molecular hyaluronic acid with the molecular weight less than 3kDa, can be as low as hyaluronic disaccharide, has mild reaction conditions, low production cost and no environmental pollution, and the enzymolysis product has 232nm characteristic absorption, thereby being convenient for the precise control of the production process and preparing the small molecular hyaluronic acid with different molecular weights.

The micromolecule hyaluronic acid or the salt thereof prepared by the invention has no phenomena of group falling, sugar ring breakage and the like, has complete product structure, high actual content of hyaluronic acid, high purity, safety and no toxicity, has good biological activities of moisture retention, transdermal absorption, oxidation resistance, anti-inflammation and the like, can be applied to the fields of food, cosmetics, medicines and the like, can also be used as a hyaluronic acid oligosaccharide standard product for the research of the structure and the quality of hyaluronic acid, and has wide research and application prospects.

Drawings

Embodiments of the invention are described in further detail below with reference to the attached drawing figures, wherein:

FIG. 1: and scanning the ESI-MS of the enzymolysis product. The abscissa is m/z and the ordinate is ion current intensity.

FIG. 2: hyaluronic acid ultraviolet scanning maps with different molecular weights. The abscissa is wavelength (nm) and the ordinate is absorbance.

FIG. 3: hyaluronic acid infrared scanning maps with different molecular weights. The abscissa is the wave number (cm)^-1) The ordinate is transmittance; wherein FIG. 3a is LMWHA; FIG. 3b is HMWHA; FIG. 3c is o-HA.

FIG. 4: effect of hyaluronic acid of different molecular weights on HUVEC cell proliferation. The abscissa represents the sample concentration (mg/mL) and the ordinate represents the relative proliferation rate (%).

FIG. 5: moisturizing activity of hyaluronic acid with different molecular weights. The abscissa is time (h) and the ordinate is moisture retention (%).

FIG. 6: hyaluronic acid with different molecular weights is absorbed in vitro through skin. The abscissa is time (h) and the ordinate is Qn (ug/cm)²)。

FIG. 7: antioxidant activity of hyaluronic acid of different molecular weights. The abscissa represents the sample concentration (mg/mL), the ordinate represents DPPH clearance (%) in FIG. 7(a), and the ordinate represents absorbance at 700nm in FIG. 7 (b).

FIG. 8: effect of hyaluronic acid of different molecular weights on TNF-a of inflammatory cells. The abscissa is the experimental group and the ordinate is the TNF-a concentration (pg/mL).

Detailed Description

The technical scheme of the invention is further illustrated by combining specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Unless otherwise specified, the concentrations in the present invention are mass-volume concentrations.

In the invention, the molecular weight of the small-molecular hyaluronic acid is determined by gel exclusion chromatography (GPC), and the product analysis of the oligomeric hyaluronic acid is carried out by Mass Spectrometry (MS) which selects an anion detection mode; the content measurement adopts a carbazole-sulfuric acid method. The method for measuring the activity of the hyaluronidase adopts a Chinese pharmacopoeia method (refer to the general rule 1207 of the Chinese pharmacopoeia 2015 edition: the hyaluronidase measuring method). The enzyme activity unit of the fermentation liquor is defined as: enzyme activity units (U/mL) per mL of fermentation broth.

The length of 16S rRNA of the bacterium HL6 which is separated from seawater and produces hyaluronidase is 1443bp, the bacterium has the highest similarity of 99.7 percent with Arthrobacter bacteria through the sequence comparison and identification of the 16S rRNA in NCBI database, and the bacterium is preliminarily identified as the Arthrobacter. The bacterial strain is subjected to morphological and physiological biochemical identification according to Bergey's handbook, and the result shows that the bacterial strain is cultured for 48 hours at 24 ℃, and the bacterial colony is round, colorless and transparent. Gram staining positive. Can utilize creatine, sarcosine, betaine and glycine as single carbon source, can liquefy gelatin and hydrolyze starch, but can not reduce NO₃To NO₂Biotin is required as a growth factor, ammonium sulfate and soil factor are not required as a growth factor, and leucine enzyme and glutaminase are positive, and xylose, mannose, ribose and raffinose cannot be utilized. Morphological and physiological and biochemical results show that the strain has significant difference with arthrobacter globiformis A152 reported in the invention with the application number of 201610115793.0. Combining 16rRNA sequence analysis, morphological and physiological biochemical identification results, the strain was named Arthrobacter globiformis HL 6. 16s rRNA

TTTTTAGAGTTTTTTTATTCGTGGCTCAGGATGAACGCTGGCGGCGTGCTTCACACATGCAAGTCGAACGATGATCCCAGCTTGMTGGGGGATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTTGACTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACCATCTGACGCATGTCATGGTGGTGGAAAGCTTTTGTGGTTTTGGATGGACTCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGTAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCTGTGAAAGACCGGGGCTCAACTCCGGTTCTGCAGTGGGTACGGGCAGACTAGAGTGCAGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCTGTAACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGGACATTCCACGTTTTCCGCGCCGTAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATGAACCGGAAAGACCTGGAAACAGGTGCCCCGCTTGCGGTCGGTTTACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGTTGCCAGCGCGTTATGGCGGGGACTCATAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGTTGCGATACTGTGAGGTGGAGCTAATCCCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCACGAAAGTTGGTAACACCCGAAGCCGGTGGCCTAACCCTTGTGGGGGGAGC

Preservation information: arthrobacter globiformis HL6, which was deposited in the China center for type culture Collection on year 07, month 05 of 2018, address: the preservation number of Arthrobacter globiformis HL6 in China, Wuhan university is as follows: CCTCC NO: M2018452.

The hyaluronidase of the invention comprises hyaluronidase which is produced by fermenting Arthrobacter globiformis (Arthrobacter globiformis) HL6 and amino acid sequences which are produced by fermenting bacteria, fungi and mammalian cells except Arthrobacter globiformis (Arthrobacter globiformis) HL6 and have amino acid sequences shown in SEQ ID NO: 1, or a hyaluronidase enzyme. Fermentation of Arthrobacter globiformis (Arthrobacter globiformis) HL6 produces hyaluronidase or fermentation of bacterial, fungal and mammalian cells other than Arthrobacter globiformis (Arthrobacter globiformis) HL6 produces amino acid sequences as shown in SEQ ID NO: 1 to the national patent application No. 201811310938.8, the entire contents of which are incorporated herein by reference.

Example 1 cloning of the Hyaluronan lyase Gene and construction of recombinant vector

By taking Arthrobacter globiformis (Arthrobacter globiformis) HL6 total DNA with the preservation number of CCTCC M2018452 as a template, amplifying hyaluronidase genes HyLs (without signal peptides) by using primers EC-F and EC-R containing restriction enzyme cutting sites through functional gene analysis of a database:

EC-F：GGACTAGTCATGTTCGCCAACCACGCCT(SEQ.ID.No.3)

EC-R：GGGGTACCCGGATACCGGGCGACGTTAGC(SEQ.ID.No.4)

wherein ACTAGT is the restriction site of endonuclease Spe I, and GGTACC is the restriction site of endonuclease Kpn I.

PCR was used for amplification, and the 50. mu.l reaction system was:

1 μ L of DNA template in 50 μ L of reaction system; EC-F (10. mu.M), 1. mu.L; EC-R (10. mu.M), 1. mu.L; dNTPs (2.5 mM each), 4. mu.L; taq (2U/. mu.L), 1. mu.L; 10 × Taq buffer, 5 μ L; ddH2O, to 50. mu.L.

The PCR amplification procedure was: pre-denaturation at 95 deg.C for 3min, denaturation at 96 deg.C for 30s, annealing at 60 deg.C for 45s, extension at 70 deg.C for 90s, and circulation at 70 deg.C for 10min for 30 times.

Carrying out 1% agarose electrophoresis on the PCR product, and carrying out sequencing verification to obtain an expression gene HyLs of Arthrobacter globiformis HL6 CCTCC M2018452 hyaluronidase, wherein the length is 2298bp, and the sequencing sequence is shown in a sequence table SEQ.ID.No. 2. The gene sequence shown in SEQ.ID.No.2 is compared with the whole genome sequence, and the two sequences are consistent. The PCR product was purified according to the protocol required for the Cycle-Pure Kit (OMEGA Bio-Tek Co.) purification Kit.

The vector pProEX-HTa and the PCR amplification gene are subjected to double enzyme digestion respectively, the digestion product is subjected to agarose electrophoresis, and the recovery is carried out according to the operation method required by a Gel extraction kit (OMEGA Bio-Tek Co.).

Connecting the fragment containing the gene sequence of the hyaluronic acid lyase gene with a vector pProEX-HTa by using T4 ligase to obtain a recombinant vector HTa-HyLs containing the hyaluronic acid lyase gene.

Example 2 fermentation of Arthrobacter globiformis (Arthrobacter globiformis) HL6 for preparation of hyaluronidase

Inoculating Arthrobacter globiformis (Arthrobacter globiformis) HL6 with the preservation number of CCTCC NO: M2018452 into a sterilized seed culture medium (the formula of the seed culture medium is 0.2g/L of sodium hyaluronate, 5g/L of glucose, 2g/L of peptone, 1.5g/L of dipotassium hydrogen phosphate and MgSO 2 g/L)₄0.5 g/L, pH 7.5), 32 ℃, 200r/min, and culturing for 24h to obtain seed liquid. Inoculating the seed culture solution into a sterilized fermentation medium (2 g/L sodium hyaluronate, 10g/L glucose, 5g/L peptone, 1.5g/L dipotassium hydrogen phosphate, MgSO 1%) according to 1% inoculation amount₄0.5 g/L, pH 7.5), 28 deg.C, 200r/min for 24h, centrifuging the fermentation liquid at 4 deg.C, 8000r/min for 10min, collecting the fermentation supernatant, and determining the activity of hyaluronidase to be 1 × 10⁵U/mL。

Example 3 recombinant expression of Hyaluronidase in E.coli

1. Escherichia coli strain DH 5. alpha. was selected, and after preparation of competent cells, heat shock transformation (42 ℃, 60s), incubation (37 ℃, 160rpm, 45min), transformants were selected on solid plates containing 75. mu.g/mL ampicillin sodium LB, and untransformed strains could not grow on the plates. PCR detection is carried out to obtain a positive clone strain, and Plasmid extraction is carried out by using a Plasmid Mini Kit (OMEGA Bio-Tek Co.) Kit to obtain recombinant plasmids HTa-HyLs.

Selecting an escherichia coli expression strain BL21(DE3), preparing competent cells, carrying out heat shock transformation (42 ℃, 60s) and incubation (37 ℃, 45min), transforming the extracted recombinant plasmid HTa-HyLs into an escherichia coli expression strain BL21(DE3), screening by a 75 mu g/mL ampicillin LB solid plate, culturing at 37 ℃ for 16h to obtain a transformant, selecting a single colony transformant, and carrying out PCR (polymerase chain reaction) detection to obtain a positive clone strain. Storing in glycerol at-80 deg.C.

The polypeptide containing the coding amino acid sequence shown as SEQ ID NO: 1 of the hyaluronidase Gene of Escherichia coli BL21(DE3) was inoculated in LB liquid medium (75. mu.g/mL ampicillin sodium), and cultured at 37 ℃ to OD₆₀₀When the concentration is 0.6-0.7, adding IPTG toInducing culture at 24 deg.C and 180r/min to final concentration of 0.5mM for 24h, centrifuging the fermentation liquid at 4 deg.C and 8000r/min for 10min, collecting the fermentation supernatant, and determining the enzyme activity of hyaluronidase to be 5 × 10⁶U/mL。

2. Recombinase purification

1) Performing affinity chromatography (GE) on the centrifuged fermentation supernatant by using a nickel column, performing primary separation and purification according to a nickel column purification instruction, and collecting a pure primary separation and purification product;

2) reusing the primary purified product obtained in the step 1)

Purifying the Fast Flow chromatographic column (GE company) again, firstly balancing the column by using 20mM Tris-HCl buffer solution with pH7.5, carrying out linear gradient elution by using 20mM Tris-HCl buffer solution (pH 7.5) with the concentration of 0-1mol/L sodium chloride after loading, detecting protein peaks at 280nm, collecting components according to pipes, measuring the hyaluronidase activity of the collected liquid of each pipe, and collecting active protein fractions;

3) further purifying the active protein fraction collected in the step 2) by Sephadex G100 Gel (GE), collecting the eluent by a tube, detecting the activity of the hyaluronidase, and collecting the active protein fraction to obtain the purified hyaluronidase. Carrying out SDS-PAGE electrophoresis on the purified hyaluronidase or obtaining a single protein band, wherein the molecular weight is about 80kDa and is basically consistent with the result of the sequence SEQ.ID.No.1 of the hyaluronidase presumed from the gene of the hyaluronidase in the sequence SEQ.ID.No. 2.

Example 4 preparation of Small molecule hyaluronic acid

Adding 1L of purified water into a 5L beaker, slowly adding 10g of hyaluronic acid with molecular weight of 2000kDa while stirring, stirring until the hyaluronic acid is completely dissolved, adjusting pH to 5.5 with hydrochloric acid, adjusting temperature to 42 ℃, and then adding 0.5mL (5X 10) of hyaluronidase prepared in example 2 into the hyaluronic acid solution⁴U), reacting for 6h while keeping the temperature, taking 50mL samples at intervals, keeping the temperature of the samples at 60 ℃ for 20min to inactivate enzyme, adding 0.5mol/L NaCl, stirring until the solution is completely dissolved, filtering the enzymolysis solution by using a 0.45 mu m filter membrane, adding 3 times of ethanol into the enzymolysis solution for precipitation, and collectingCollecting precipitate, washing with ethanol, dehydrating, and vacuum drying to obtain small molecular hyaluronic acid. The molecular weights of the samples at the various time points are shown in Table 1.

TABLE 1 enzymatic hydrolysis time and hyaluronic acid molecular weight

Sample (I)	3min	10min	30min	1h	2h	3h	4h	5h	6h
										Molecular weight (kDa)	550	205	95.2	46.9	28.3	18.4	12.7	7.7	3.8
PD	1.85	1.82	1.75	1.73	1.68	1.67	1.65	1.35	1.2

Example 5 preparation of Small molecule sodium hyaluronate

1L of purified water was added to a 5L beaker, 100g of sodium hyaluronate with a molecular weight of 1500kDa was slowly added with stirring until complete dissolution, the pH was adjusted to 7.0 with sodium hydroxide, the temperature was adjusted to 37 ℃, and then 1mL (5X 10) of the hyaluronidase prepared in example 3 (hyaluronic acid, Hyaluronidase, Hyaluronan, Hy⁶U), reacting for 6 hours under heat preservation, sampling 50mL at intervals, preserving the temperature of the sample at 70 ℃ for 15min to inactivate enzyme, adding 1mol/L NaCl, stirring until the solution is completely dissolved, filtering the enzymolysis solution by using a 0.45-micrometer filter membrane, adding 3 times of ethanol into the enzymolysis solution for precipitation, collecting the precipitate, washing by using ethanol, dehydrating, and drying in vacuum to obtain the micromolecular sodium hyaluronate. The molecular weights of the samples at the various time points are shown in Table 2.

TABLE 2 enzymatic hydrolysis time and molecular weight of sodium hyaluronate

Example 6 preparation of Small molecule Potassium hyaluronate

Adding 1L of purified water into a 5L beaker while stirringSlowly adding 1g sodium hyaluronate with molecular weight of 650kDa while stirring, stirring to dissolve completely, adjusting pH to 8.0 with potassium hydroxide, adjusting temperature to 30 deg.C, and adding 0.01mL (1 × 10) of hyaluronidase prepared in example 2 into potassium hyaluronate solution³U), reacting for 6 hours under heat preservation, sampling 50mL at intervals, preserving the temperature of the sample at 80 ℃ for 10min to inactivate enzyme, filtering the enzymolysis liquid by a filter element with the diameter of 0.45 mu m, adding 3 times of acetone into the enzymolysis liquid for precipitation, collecting the precipitate, washing by acetone, dehydrating, and drying in vacuum to obtain the micromolecular potassium hyaluronate. The molecular weights of the samples at the various time points are shown in Table 3.

TABLE 3 enzymatic hydrolysis time vs. molecular weight of Potassium hyaluronate

Sample (I)	3min	10min	30min	1h	2h	3h	4h	5h	6h
										Molecular weight (kDa)	590	500	380	325	250	225	201	185	150
PD	1.88	1.85	1.85	1.78	1.78	1.72	1.69	1.71	1.70

Example 7 preparation of hyaluronic acid oligosaccharides

Adding 1L of purified water into a 5L beaker, slowly adding 10g of sodium hyaluronate with molecular weight of 1500kDa while stirring, stirring until the sodium hyaluronate is completely dissolved, adjusting pH to 7.0 with sodium hydroxide, adjusting temperature to 37 ℃, and then adding 50mL (5X 10) of the hyaluronidase prepared in example 2 into the sodium hyaluronate solution⁶U), reacting for 8 hours in a heat preservation way, sampling 100mL in 4 hours and 8 hours respectively, preserving the temperature of the sample for 20min for enzyme deactivation at 60 ℃, stirring until the enzyme is completely dissolved, filtering the enzymolysis liquid by using a 0.45 mu m filter membrane, adding 20 times of ethanol into the enzymolysis liquid for precipitation, collecting the precipitate, then washing by using ethanol, dehydrating, and freeze-drying to obtain the hyaluronic acid oligosaccharide.

ESI-MS analysis is carried out on the enzymolysis product, a negative ion ionization mode is adopted, and the scanning range m/z is as follows: 100 + 2000, the mass spectrum scanning spectrum is shown in the attached figure 1, and the ESI-MS scanning result shows that: the sample after 4h enzymolysis mainly comprises four ion peaks with m/z of 175.03, 378.1, 757 and 779.2, which respectively represent unsaturated uronic acid (m/z:175.03), unsaturated hyaluronic disaccharide (m/z:378.1), unsaturated hyaluronic tetrasaccharide (m/z:757 and 779.2), and also comprises trace ion peaks with m/z of 599.0, 1180.4 and 1581.6, which respectively represent unsaturated hyaluronic trisaccharide (m/z:599.0), unsaturated hyaluronic hexasaccharide (m/z:1180.4) and unsaturated hyaluronic octasaccharide (m/z: 1581.6); and (3) performing enzymolysis for 8 hours, wherein a mass spectrogram of the sample mainly contains m/z:378.1, the unsaturated hyaluronidase of the invention is used for degrading hyaluronic acid in a cracking way, and the hyaluronic acid of the invention is used for degrading hyaluronic acid in a final product of unsaturated disaccharide, so that the action mode and the degradation product of the hyaluronic acid enzyme (hydrolase, saturated tetrasaccharide as a final product) and the leech hyaluronidase (hydrolase, saturated tetrasaccharide and hexasaccharide as a final product) extracted from animal tissues commonly at present are obviously different.

Performing ultraviolet scanning on a high molecular hyaluronic acid (HMWHA) sample before enzymolysis and a hyaluronic acid oligosaccharide (o-HA) sample after enzymolysis for 8 hours, wherein the ultraviolet scanning map is shown in the attached figure 2, and the ultraviolet scanning result shows that: the hyaluronic acid oligosaccharide prepared by the invention has characteristic absorption at 232nm, which indicates that unsaturated double bonds are generated, undegraded high molecular hyaluronic acid has no ultraviolet absorption at 232nm, and the online monitoring and control of the enzymolysis process can be realized by establishing the corresponding relation between the ultraviolet absorption value, the molecular weight and the viscosity of the enzymolysis liquid in the industrial production, so that low molecular hyaluronic acid or hyaluronic acid oligosaccharide with different molecular weights can be efficiently prepared.

Example 8 Infrared scanning analysis of enzymatic products

The molecular weight of hyaluronic acid (HMWHA) not degraded by hyaluronidase, the small molecular weight hyaluronic acid (LMWHA) prepared in example 4, and the hyaluronic acid oligosaccharide (o-HA) prepared in example 7 were measured at 4000cm^-1To 400cm^-1The infrared scanning spectrum is shown in the attached figure 3, and the infrared scanning shows that: the enzymolysis products all have characteristic absorption peaks of saccharides, and the degradation products with different molecular weights and hyaluronic acid before enzymolysis all have similar characteristic absorption peaks, and are compiled with 2010 by the State pharmacopoeia CommissionThe fuchsin external spectrum is concentrated, and the standard spectra of hyaluronic acid are consistent, which shows that the micromolecule hyaluronic acid and hyaluronic acid oligosaccharide obtained after enzymolysis keep complete structures, and no groups fall off in the enzymolysis process.

Example 9 cytotoxicity of Small molecule hyaluronic acid

HUVEC cells in logarithmic growth phase were seeded at 15000/well (180. mu.l/well) in a 96-well plate, and after 24 hours of culture, high molecular hyaluronic acid (HMWHA), small molecular hyaluronic acid (LMWHA) prepared in example 4, and hyaluronic acid oligosaccharide (o-HA) samples prepared in example 7 were added, 3 duplicate wells for each concentration. After 24 hours of drug action, the supernatant was aspirated, 20. mu.l of MTT medium was added again, after 4 hours, the supernatant was aspirated, 150. mu.l of DMSO was added for solubilization, the absorbance at 570nm was measured with a microplate reader, and the relative cell proliferation rate (RGR) was calculated

In the formula, A_iIs the absorbance of the sample, A₀Absorbance of the control group.

The influence of HMWHA, LMWHA and o-HA with different concentrations of 0.01-1 mg/mL on HUVEC cell proliferation is shown in the attached figure 4, and the experimental result shows that: HMWHA, LMWHA and o-HA with different concentrations of 0.01-1 mg/mL have no obvious influence on HUVEC cell proliferation, the relative cell proliferation rate is higher than 95%, the relative cell proliferation rate specified by pharmacopoeia is higher than 80%, and the toxicity level is 0-1, so that the small molecular hyaluronic acid and hyaluronic acid oligosaccharide prepared by the method are safe and nontoxic.

Example 10 moisturizing Activity of Small molecule hyaluronic acid

Sample solutions of polymeric hyaluronic acid (HMWHA), the small-molecular hyaluronic acid (LMWHA) prepared in example 4, and the hyaluronic acid oligosaccharide (o-HA) prepared in example 7, each having a mass volume concentration of 10%, were prepared, and the moisturizing rates of the samples were measured in a saturated sodium carbonate aqueous solution (relative humidity RH ═ 43%) and a dry silica gel (relative humidity RH ═ 0%) environment, respectively, and the results of the moisturizing activity of the samples are shown in fig. 5, and the results of the moisturizing activity experiments show that: the hyaluronic acid samples with different molecular weights have good moisturizing activities in a saturated sodium carbonate environment (relative humidity RH is 43%) or a dry silica gel environment (relative humidity RH is 0%), the moisturizing activities are reduced along with the reduction of the molecular weight of an enzymolysis sample, but the moisturizing activities are not obviously different from HMWHA, the moisturizing rate of the same sample in the dry silica gel environment is slightly lower than that in the saturated sodium carbonate environment, but the obvious difference is not generated, and the hyaluronic acid with different molecular weights prepared by enzymolysis has good moisturizing activities in different environments.

Example 11 transdermal absorption Properties of Small molecule hyaluronic acid

A modified Franz diffusion cell was used, with the skin surface facing upward, and 5mg/mL of a sample solution of hyaluronic acid (HMWHA) polymer, the hyaluronic acid (LMWHA) small molecule prepared in example 4, and the hyaluronic acid oligosaccharide (o-HA) prepared in example 7 was added to the supply cell at 1mL, and 0.85% physiological saline at 6.5mL was added to the receiving cell, and the experiment was performed at 360rpm and 37 ℃ for 2h, 4h, 6h, 8h, 10h, 12h, and 24h, respectively, by sampling 1mL and filling with a fresh receiving solution. The uronic acid content in the sample is measured, and the cumulative permeability per unit area is calculated according to the following formula:

wherein:

qn: cumulative permeation per unit area (μ g/cm) of the sample at time t²)；

A: a transdermal diffusion area;

cn: time t concentration measurements;

ci: a concentration measurement before time t;

v: receiving a pool volume;

vo: sample volume

J: and (4) making a curve of Qn to t to obtain a regression equation, wherein the slope is the transdermal rate constant.

The cumulative permeation amount per unit area of different samples is shown in figure 6, and the results of in vitro transdermal absorption experiments show that: due to the larger molecular weight, the unit area cumulative permeation amount of HMWHA is lower and is 0.057 mu g/cm in 12h²And the cumulative permeation quantity per unit area slowly increases with the time, and only reaches 0.072 mu g/cm in 24 hours²(ii) a The cumulative permeability per unit area of LMWHA and o-HA increases linearly with time, the cumulative permeability per unit area of o-HA is slightly higher than LMWHA, and the cumulative permeability per unit area of LMWHA and o-HA are obviously higher than HMWHA, and the cumulative permeability per unit area of LMWHA and o-HA is 0.65 mug/cm at 12h²(LMWHA) and 0.71. mu.g/cm²(o-HA), and the cumulative permeability per unit area continuously increases with time, reaching 1.07. mu.g/cm at 24 hours²(LMWHA) and 1.15. mu.g/cm²(o-HA) exhibits good long-lasting transdermal absorption. The transdermal absorption coefficient reflects the transdermal absorption rate, the 24-hour transdermal absorption coefficient of HMWHA is 0.0031, R²0.8924; the LMWHA has a 24-hour transdermal absorption coefficient of 0.0446, R²0.9935, good linearity; the 24-hour transdermal absorption coefficient of o-HA is 0.0479, R²0.9917, good linearity; the transdermal absorption coefficient further indicates that the transdermal absorption performance of o-HA and LMWHA is better than that of HMWHA.

Example 12 antioxidant Activity of Small molecule hyaluronic acid

Respectively measuring DPPH clearance and reducing power of different concentrations of 0.5-25mg/mL of High Molecular Weight Hyaluronic Acid (HMWHA), small molecular weight hyaluronic acid (LMWHA) prepared in example 4 and hyaluronic acid oligosaccharide (o-HA) samples prepared in example 7, representing the antioxidant activity of hyaluronic acid with different molecular weights, wherein the antioxidant activity result is shown in the attached figure 7, and the DPPH clearance experiment result shows that: the micromolecular hyaluronic acid sample prepared by the enzyme method HAs lower molecular weight and higher DPPH clearance, the sample o-HA with the same concentration is larger than LMWHA and larger than HMWHA, the DPPH clearance of the o-HA with the same concentration is improved by about 4 times compared with the HMWHA, and the experimental sample HAs good linear growth in the concentration range of 5-25 mg/mL; the reduction force experiment result shows that: the reducing power of HMWHA is low, the reducing power of LMWHA and o-HA prepared by enzymolysis is obviously increased, and particularly the increasing trend of o-HA is obvious; the antioxidant activity results of DPPH clearance rate and reducing power show that the micromolecule hyaluronic acid prepared by the enzyme method, especially hyaluronic acid oligosaccharide, has good antioxidant activity and good application prospect in cosmetics.

Example 13 anti-inflammatory Activity of Small molecule hyaluronic acid

Experiment was performed using Lipopolysaccharide (LPS) induced mouse RAW264.7 macrophage inflammation model.

Grouping samples:

blank control group: c; model group: m;

100ug/mL HMWHA:HH；50ug/mL HMWHA:HL；

100ug/mL o-HA:OH；50ug/mL HMWHA:OL；

RAW264.7 cells (8X 10) in logarithmic growth phase were taken⁵50 mu L of cells/well) are added into a 96-well plate, the plates are attached to the wall for 30min, LPS with the final concentration of 1 mu g/mL is firstly added into a model group (M) and a sample group to be treated for 1h to stimulate inflammation, HMWHA and o-HA with the concentrations of 100ug/mL and 50ug/mL are respectively added into the sample group, the model group is replaced by PBS, a blank control group (C) is not treated, the culture is carried out for 8h, cell supernatant is taken, cell debris is removed by centrifugation at 2000r/min for 15min, and the concentration of TNF-a in the cell supernatant is measured by an ELISA kit. The effect of hyaluronic acid with different molecular weights on inflammatory cell TNF-a is shown in figure 8, and the experimental result shows that: the concentration of the model group TNF-a is obviously higher than that of the blank control group, which indicates that the model is available; HMWHA and o-HA both have anti-inflammatory effects of inhibiting TNF-a production, however, the difference is that HMWHA HAs an inhibition capacity of more than 50ug/mL at a concentration of 100ug/mL, and o-HA HAs an inhibition capacity of more than 100ug/mL at a concentration of 50ug/mL, which may be related to the difference of anti-inflammatory mechanisms of the two; the inhibition effect of the sample o-HA with the same concentration on TNF-a is better than that of HMWHA, especially under the condition of low concentration, the o-HA HAs better anti-inflammatory effect, and can obviously reduce the sample dosage, reduce the cost and have better development and application prospect.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Sequence listing

<110> Qingdao Marine biological medicine institute, Inc

<120> method for preparing small molecular hyaluronic acid by enzyme cleavage method, small molecular hyaluronic acid obtained by the same and application thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 765

<212> PRT

<213> Arthrobacter globiformis (Arthrobacter globiformis)

<400> 1

Met Pro Ala Ala His Ala Thr Ala Ala Ala Gly Pro Gly Thr Ala Gly

1 5 10 15

Pro Ala Ala Leu Ala Ser Ala Thr Val Ala Gly Ile Thr Gly Ala Ala

20 25 30

Val Ile Gly Ala Gly Ala Pro Ala Pro Ala Ala Ala Val Thr Ala Leu

35 40 45

Ala Thr Leu Ala Ala Ala Ser Leu Ala Leu Leu Ala Ala Val Ser Gly

50 55 60

Ala Thr Ser Val Pro Thr Ala Leu Ser Pro Ala Leu Ala Ala Gly Met

65 70 75 80

Val Thr Thr Thr Thr Ala Leu Ser Gly Leu Ala Ala Ala Thr Ala Thr

85 90 95

Pro Thr Ala Ala Val Pro Gly Ala Ser Ala Val Leu Ala Ala Ile Leu

100 105 110

Ala Gly Leu Ala Ala Ala Ala Thr Leu Cys Thr His Ala Gly Ala Gly

115 120 125

Gly Val Gly Ala Thr Thr Ser Thr Gly Ile Gly Val Pro Ala Ala Leu

130 135 140

Ala Ala Ala Met Val Leu Leu His Ala Gly Leu Ser Ala Ala Gly Ile

145 150 155 160

Gly Ala Thr Ser Ala Ala Ile Ala His Pro Val Pro Ala Pro Thr Leu

165 170 175

Gly Pro Pro Pro Leu Ala Gly Leu Ile Thr Ser Val Gly Ala Ala Ala

180 185 190

Val Ala Leu Cys Gly Gly Val Ile Ile Ala Ser Leu Ala Gly Gly Ala

195 200 205

Pro Gly Leu Leu Ala His Ala Val Ala Gly Leu Ser Gly Val Thr Gly

210 215 220

Thr Val Thr Ser Gly Ala Gly Ile Pro Ala Ala Gly Ser Pro Ile Gly

225 230 235 240

His Ser Thr Thr Pro Thr Thr Gly Ser Thr Gly Val Val Leu Leu Thr

245 250 255

Gly Leu Ser Leu Leu Pro Ser Leu Leu Gly Gly Thr Ala Pro Gly Val

260 265 270

Ser Ala Pro Ser Ala Ser Ile Pro Pro Ala Ala Val Gly Gly Ser Pro

275 280 285

Ala Pro Val Met Ile Ala Gly Ala Met Ala Ala Ser Val Ala Gly Ala

290 295 300

Ser Ile Ser Ala Gly Ala Ala Thr Gly Thr Ala Leu Gly Ala Ser Ala

305 310 315 320

Ile Gly Ala Ile Leu Leu Leu Ala Ala Ala Met Ala Pro Ala Thr Ala

325 330 335

Thr Ala Thr Ala Gly Leu Cys Ala Gly Thr Ile Ala Ala Ala Thr Thr

340 345 350

Ala Pro Ile Leu Ala Gly Ala Ser Leu Pro Ala Thr Ala Leu Val Leu

355 360 365

Gly Leu Gly Ser Thr Gly Ile Ala Pro Val Ala Gly Ala Pro Gly His

370 375 380

Ala Leu Pro Pro Ala Met Ala Ala Thr Met His Ala Gly Pro Gly Thr

385 390 395 400

Ala Leu Ser Leu Ser Leu Ser Ser Ala Ala Ile Ala Thr Thr Gly Cys

405 410 415

Gly Ala Gly Gly Ala Ala Ala Gly Thr His Thr Gly Ser Gly Met Thr

420 425 430

Thr Pro Thr Thr Ser Ala Leu Gly Gly Thr Ala Ala Ala Pro Thr Ala

435 440 445

Thr Ala Ala Thr Ala Ala Leu Pro Gly Ile Thr Val Ala Thr Thr Pro

450 455 460

Leu Pro Ala Leu Val Gly Gly Gly Thr Gly Ala Ala Val Pro Ala Ala

465 470 475 480

Gly Thr Ser Gly Ala Thr Ala Leu Gly Gly Val Ala Ala Val Gly Gly

485 490 495

His Leu Val Gly Pro Gly Ala Thr Gly Leu Ser Ala Ala Leu Ser Thr

500 505 510

Pro Val Ser Gly Gly Ala Thr Val Cys Leu Gly Ala Ala Ile Thr Thr

515 520 525

Gly Ser Gly Ala Ala Val Gly Ser Ile Val Ala His Ala Ala Leu His

530 535 540

Gly Gly Ser Ala Thr Leu Thr Thr Ala Ala Gly Thr Ile Ala Gly Ser

545 550 555 560

Val Gly Ser Ala Gly Val Leu Ser Gly Gly Ala Thr Val His Leu Gly

565 570 575

Gly Pro Gly Gly Thr Ala Met Leu Ala Ala Ser Pro Leu His Val Leu

580 585 590

Ala Gly Thr Ala Ser Gly Ser Thr Ser Gly Val Ala Thr Ala Gly Ser

595 600 605

Thr Thr Val His Gly Ala Thr Pro Ala Thr Leu Thr Val Ala His Gly

610 615 620

Ala Gly Pro Ala Ala Gly Ser Thr Ala Thr Val Val Ala Pro Gly Ala

625 630 635 640

Ser Val Ala Leu Thr Ala Leu Leu Val Gly Gly Ala Leu Thr Ala Val

645 650 655

Ile Ala Ala Ala Thr Thr Ala Gly Ser Val Gly Pro Leu Ala Ser Leu

660 665 670

Thr Thr Ala Ala Thr Pro Thr Leu Pro Gly Met Ala Gly Ala Leu Gly

675 680 685

Ala Ser Gly Pro Ala Cys Val Val Pro Ser Ala His Gly Ala Gly Leu

690 695 700

Ser Leu Ala Pro Ser Gly Pro Thr Gly Leu Ala Ala Ser Leu Thr Leu

705 710 715 720

Thr Leu Pro Gly Gly Thr Thr Ser Ser Val Leu Gly Gly Thr Gly Thr

725 730 735

Leu Gly Thr Ala Ala Ala Gly Ala Ser Thr Val Thr Leu Ala Thr Ala

740 745 750

Gly Leu Ala Gly Gly Thr Leu Val Ile Thr Leu Ala Ala

755 760 765

<210> 2

<211> 2298

<212> DNA

<213> Arthrobacter globiformis (Arthrobacter globiformis)

<400> 2

atgttcgcca accacgcctg ggccgacgcg gaaccgggca ccgcggagtt cgcagcgctg 60

cgaagccgtt gggtggacca gatcacgggc cgcaacgtca tccaagccgg cgatccggac 120

tttgccaggg cggtgacagc gctgaacacc aaagccgctg actccttggc aaagctcaac 180

cgggtttcag gccgaacctc ggtctttacg gacttgtcct tcgccaagga tgcagagatg 240

gtcaccacgt acacgcgttt atcccagctc gctgctgcct gggcaacacc aacggccgcg 300

gtgtttggtg attccgcagt actggcagac atcaaggcgg gcctcgccga cgccaatacc 360

ctctgctacc acgctggcag ggaagaggtc ggcaactggt ggtcgtggga aatcggtgtg 420

ccccgtgcct tggccgacgc catggtgctt cttcacgccg agctgtccgc cgctgaaata 480

caggcctaca gtgcggcgat cgaccatttt gtgccggacc cttggctgca gttcccaccc 540

aagcgcggca agatcacctc cgtgggcgcc aaccgtgtgg acctgtgcca aggggtcatc 600

atccggtccc tcgctggaga agatccgggc aagctcaacc acgcagtcgc cggactcagc 660

caggtgtggc agtacgtcac cagtggtgac ggaatcttcc gggacggctc gtttatccaa 720

cacagcacca ccccgtacac gggctcctac ggggtggtcc tgctcaccgg attgtccaag 780

ttgttctccc tcctgggagg cacggcgttc gaagtttcgg acccctcgcg cagtattttc 840

ttcgacgcag tggagggttc gtttgcgccc gtcatgatca acggggccat ggccgattcc 900

gtgcgcggca ggagtatcag ccgcgaggcc aacaccggct acgacctggg ggcatcggcc 960

atcgaagcca ttctgctgct ggcccgggcc atggatccag ctactgccac acgatggaga 1020

gggctgtgcg cgggatggat tgcgcgcaat acgtaccggc ccatcctcgc aggggccagc 1080

ctgcccagga ctgcattggt gaaggagctt cagtcaacgg gtatcgcacc ggtggcagaa 1140

gcccccgggc acaggctctt ccctgcgatg gaccgcacca tgcaccgggg acccggctgg 1200

gcattgtcgc tctccctgtc cagcaaccgc atcgcctggt acgaatgcgg caatggcgag 1260

aacaaccgcg gctatcacac gggttccggc atgacgtact tctatacgtc cgatctcggc 1320

caatacgatg acgcgttctg ggccacagcc aactacaacc gccttccggg catcaccgtg 1380

gacaccactc cgttgccgga caaggtggag ggtgaatggg gtgccgccgt tcctgcgaat 1440

gaatggagtg gcgccacggc gcttggcggg gttgccgccg tcggacaaca cctggtggga 1500

ccgggccgca cgggcctgtc cgccaggaag tcctggtttg tcagcggcga ggccactgtc 1560

tgcctcggcg ccgacatcac cactggttcc ggggccaggg tggaaagcat cgttgaccac 1620

cgcaacctcc accagggcag caatacactc acgacggcgg caggcaccat cgccggatcg 1680

gtcggcagtg ctgaggtact gagcgaagaa cgctgggttc atttggaggg tttcggaggc 1740

tacgccatgc tggacgattc cccgcttcac gtgctccggg aaacccgatc aggcagctgg 1800

tccggggtca acaccaacgg cagcaccacc gtccaccagc gcacctttgc caccctctac 1860

gtagaccacg gcgccggacc tgctgcgggc agctatgcct atgtggttgc tccgggcgct 1920

tctgtgaacc tgacccggaa gctggtgcag ggggacaaat accgggtgat ccgcaacgat 1980

acaacggcac agtccgtgga gttcaaggca tcgaagacca cggcagcaac cttctggaag 2040

cccgggatgg cgggggatct gggtgcgtcc gggcctgctt gcgtggtgtt ctccaggcac 2100

ggaaatgagt tgagcctggc gttcagtgag ccaacgcaga aggctgccag cctcacgctg 2160

accctgcccc agggcacatg gtccagcgtg ctggaaggca cgggcacact ggggaccgac 2220

gcagacggcc ggagtacggt gacccttgat acggccggcc tgaatggcca gacgaaggtc 2280

atcacactgc ggcgctaa 2298

<210> 3

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggactagtca tgttcgccaa ccacgcct 28

<210> 4

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggggtacccg gataccgggc gacgttagc 29

<210> 5

<211> 1145

<212> RNA

<213> Arthrobacter globiformis (Arthrobacter globiformis)

<400> 5

agagacgggc caggagaacg cggcggcggc cacacagcaa gcgaacgaga cccagcgmgg 60

gggaagggcg aacggggaga acacggagaa ccgcccgacc gggaaagccg ggaaacgggc 120

aaaccggaag accacgacgc agcagggggg aaagcggggg aggaccgcgg ccacagcggg 180

ggggaaggcc accaaggcga cgacgggagc cggccgagag gggaccggcc acacgggacg 240

agacacggcc cagacccacg ggaggcagca gggggaaagc acaagggcgc aagccgagca 300

gcgacgccgc ggagggagac ggcccgggga aaccccagag ggaagaagcg aaaggacgga 360

ccgcagaaga agcgccggca acacggccag cagccgcgga aacgagggcg caagcgaccg 420

gaaagggcga aagagccgag gcgggcgcgc gcggaaagac cggggccaac ccggcgcagg 480

ggacgggcag acagaggcag aggggagacg gaaccgggag cgggaaagcg cagaacagga 540

ggaacaccga ggcgaaggca ggccgggcga acgacgcgag gagcgaaagc aggggagcga 600

acaggaagaa cccggagcca gccgaaacgg ggcacagggg ggggacacca cgccgcgccg 660

agcaacgcaa aggccccgcc ggggagacgg ccgcaaggca aaaccaaagg aagacggggg 720

cccgcacaag cggcggagca gcggaaacga gcaacgcgaa gaaccaccaa ggcgacagaa 780

ccggaaagac cggaaacagg gccccgcgcg gcggacaggg ggcagggcgc agccggcgga 840

gaggggaagc ccgcaacgag cgcaaccccg caggccagcg cgaggcgggg accaaggaga 900

cgccggggca accggaggaa ggggggacga cgcaaacaca gccccagcgg gccacgcagc 960

acaaggccgg acaaaggggc gaacggaggg gagcaaccca aaaagccggc cagcggaggg 1020

gcgcaaccga ccccagaagc ggagcgcaga acgcagacag caacgcgcgg gaaacgcccg 1080

ggccgacaca ccgcccgcaa gcacgaaagg gaacacccga agccggggcc aacccggggg 1140

ggagc 1145

Claims

1. The method for preparing the small molecular hyaluronic acid by the enzyme cleavage method is characterized in that the hyaluronidase prepared by fermenting Arthrobacter globiformis (Arthrobacter globiformis) HL6 with the preservation number of CCTCC NO: M2018452 or the amino acid sequence of the hyaluronidase is shown as SEQ ID NO: 1, degrading hyaluronic acid with molecular weight of more than 600kDa or a salt thereof by the hyaluronidase to prepare the hyaluronic acid with small molecular weight or the salt thereof, and comprises the following steps:

2) enzymolysis: adjusting the temperature of the hyaluronic acid or salt solution thereof in the step 1) to 20-50 ℃, adjusting the pH to 5-10 by using acid and alkali, and preparing the hyaluronic acid or salt solution thereof into a solutionAdding the solution into the reactor at a ratio of 100U/g to 5X 10⁵Degrading hyaluronic acid or its salt to 379Da-600kDa by U/g hyaluronidase to obtain enzymolysis solution;

4) and (3) filtering: adding or not adding 0.01-2mol/L of soluble inorganic salt into the enzymolysis liquid in the step 3), stirring until the inorganic salt is completely dissolved, and filtering by using a microporous filter membrane or a filter element to obtain a filtrate;

6) and (3) dehydrating and drying: collecting the small molecular hyaluronic acid or the salt thereof precipitate in the step 5), adding alcohol or ketone which is the same as the alcohol or ketone in the step 5), dehydrating, and drying to obtain the small molecular hyaluronic acid or the salt thereof.

2. The method of claim 1, wherein: the nucleotide sequence shown as SEQ ID NO: 1 is encoded by a hyaluronidase comprising a polypeptide comprising a sequence encoding SEQ ID NO: 1, or a recombinant bacterium, yeast or cell of the hyaluronidase gene shown in 1.

3. The method of claim 1, wherein: in the step 2), the reaction temperature is adjusted to be 20-45 ℃ and the pH value is adjusted to be 5.5-8.

4. The method of claim 1, wherein: and in the step 3), adjusting the temperature of the enzymolysis liquid to be 60-80 ℃, and keeping the temperature for 10-20 minutes to inactivate the hyaluronidase.

5. The method of claim 1, wherein: further comprising one or more of the following conditions: the hyaluronate used in the step 1) is at least one of sodium salt, potassium salt, calcium salt, magnesium salt or zinc salt of hyaluronic acid; in the step 2), the acid is at least one of hydrochloric acid, glacial acetic acid, nitric acid, sulfuric acid or phosphoric acid, and the alkali is at least one of sodium hydroxide or potassium hydroxide; in the step 4), the soluble inorganic salt is at least one of sodium salt, potassium salt, calcium salt, magnesium salt or zinc salt; in the step 5) and the step 6), the alcohol or ketone is at least one of methanol, ethanol, propanol, butanol or acetone.