CN110331178A - A kind of enzyme cutting method prepares the method for micromolecule hyaluronic acid and gained micromolecule hyaluronic acid is applied with it - Google Patents

Abstract

The method of micromolecule hyaluronic acid is prepared the present invention provides a kind of enzyme cutting method and gained micromolecule hyaluronic acid is applied with it.The present invention degrades to molecular weight greater than 600 kDa hyaluronic acids or its salt using the hyaluronidase or the hyaluronidase as shown in SEQ ID NO:1 of the Arthrobacter globiformis HL6 fermentation preparation of deposit number CCTCC NO:M 2018452, prepare small-molecular-weight hyaluronic acid or its salt, this method technological operation is simple, mild condition, non-environmental-pollution and the pollution of animal origin virus, to product structure without destruction, product uniformity is good, realizes the environmentally friendly industrialized production of micromolecule hyaluronic acid.Micromolecule hyaluronic acid prepared by the present invention is with high purity, safe and non-toxic, with good moisturizing, Transdermal absorption, anti-oxidant, anti-inflammatory activity, it is widely used in food, cosmetics and medicine and other fields, also structure and quality research of the standard items for hyaluronic acid be can be used as, there is wide research application prospect.

Description

A method for preparing small-molecule hyaluronic acid by enzymatic cleavage and the obtained small-molecule hyaluronic acid Acids and their applications

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for preparing small-molecule hyaluronic acid by enzymatic cleavage, the obtained small-molecule hyaluronic acid and its application.

Background technique

Hyaluronic acid (HA) is a macromolecular mucopolysaccharide composed of N-acetylglucosamine and D-glucuronic acid disaccharide units repeatedly linked alternately. An important component of synovial fluid and cartilage tissue, it has unique physical and chemical properties and biological functions. Due to its high viscoelasticity and plasticity, super strong water holding capacity and permeability, and good biocompatibility, it is widely used in the fields of food, cosmetics and medicine.

Recent studies have found that the biological activity of HA is directly related to its relative molecular weight, and HA with different molecular weights has different or even opposite biological activities. High molecular weight HA has good viscoelasticity, moisture retention, lubrication and anti-inflammatory activity, and can be used in cosmetics, eye surgery, arthritis treatment, postoperative anti-adhesion, etc. Small molecule HA has the functions of transdermal absorption, easy digestion, anti-tumor, promoting endothelial cell proliferation, promoting angiogenesis, promoting wound healing and immune regulation. In recent years, the research on hyaluronic acid oligosaccharide (o-HA) has gradually become a hot spot , the study found that o-HA molecular fragments have pro-angiogenic activity in vivo, and can also significantly promote the proliferation of endothelial cells in vitro; o-HA can protect granulation tissue from the damage of oxygen free radicals, and can effectively promote wound healing. Stimulate endothelial cells to recognize the injured site, and perform preliminary protection and repair on the wound site; studies have shown that o-HA also has active functions such as immune regulation and anti-tumor. O-HA fragments with significant activity can be used as new resources in drug development and functional product development, so the research on obtaining a large number of high-purity o-HA fragments is of great significance.

The preparation methods of small molecule HA mainly include physical degradation, chemical degradation, enzymatic degradation and biosynthesis. Many physical methods (heating, mechanical shearing, ultraviolet radiation and ultrasonic crushing) can be applied to the degradation of hyaluronic acid. It is difficult to reduce the molecular weight of hyaluronic acid to below 10kDa through physical degradation, and the molecular weight distribution of the prepared product is relatively wide. Large, it is difficult to obtain HA with a specific molecular weight. The chemical degradation method to degrade HA into oligosaccharides requires more severe reaction conditions, such as higher acid and alkali concentrations, to achieve a high degree of degradation. Whether it is acid hydrolysis, alkali hydrolysis or oxidative degradation, it will be destroyed during the reaction process. The molecular structure of HA breaks the glycosidic bond on the sugar chain, and at the same time, the structure of glucuronic acid and N-acetylglucosamine will also be destroyed, such as the acetyl group is hydrolyzed, the monosaccharide six-membered ring is broken, etc. The quality and biological activity of sugar have a significant impact, and the oligomeric hyaluronic acid prepared by chemical degradation is also prone to browning, and the production process will pollute the environment. De Angelis et al. used Pasteurella HA synthase (pmHAS), glucuronosyltransferase and N-acetylglucosamine transferase to synthesize 2-20 oligosaccharides. This reaction is a biosynthetic reaction, and the reaction requires strict reaction conditions. Moreover, multiple reaction enzymes are required, and the cost is relatively high. Enzymatic hydrolysis is a mild method for degrading hyaluronic acid. Its reaction conditions are mild, easy to control, and will not cause environmental pollution. The product structure is uniform and will not cause disaccharide structure breakage. The actual recovery rate of the product is high, the product cost is low, and the purity is high. , good quality, especially suitable for the preparation of low-molecular-weight hyaluronic acid. At present, the commonly used hyaluronidase is mainly derived from animal extraction. TAWADA and others use hyaluronidase (hydrolase) extracted from bovine testis to degrade HA to prepare low-molecular-weight oligomeric HA with monosaccharide residues 4-52; Chinese patent CN 103484513B A method for preparing HA oligosaccharides using leech-derived hyaluronidase (hydrolase) is disclosed, and the product is saturated HA oligosaccharides. However, due to the low purity of hyaluronidase extracted from animals, the potential risk of animal virus cross-contamination, and the high price limit its application in the preparation of small molecule hyaluronic acid. As a completely different enzyme from hyaluronan hydrolase, microbial-derived hyaluronan lyase can effectively avoid various defects of animal-derived hyaluronidase. The hyaluronic acid lyase produced by microbial fermentation has high enzyme activity, high purity, and does not contain animal-derived viruses, which has unique advantages over animal-derived hyaluronidase. Chinese patent CN 102876748 B discloses a method for preparing oligomeric hyaluronate with a molecular weight of 3000-10 ⁴ Da by degradation of air-sourced Bacillus hyaluronidase, and the resulting oligomeric hyaluronate and its use. Chinese patent CN 103484513 B discloses a method for preparing low-molecular-weight hyaluronate with a molecular weight of 10kDa-1000kDa by degrading air-sourced Bacillus hyaluronidase, and the produced low-molecular-weight hyaluronate and its use. The protection range is low molecular weight hyaluronate in the molecular weight range of 3000-1000kDa.

Since the currently marketed hyaluronidases are mainly hyaluronidase enzymes derived from animal tissues, they have problems such as low activity, high cost, and safety risks of animal viruses, and are not suitable for large-scale production of small-molecule hyaluronic acid by enzymatic methods. The main method of industrial production of small molecule hyaluronic acid is still chemical degradation, but there are problems such as structural damage and environmental pollution. It is urgent to develop a new, safe and low-cost hyaluronidase to realize industrial enzymatic production of small molecule hyaluronic acid.

Contents of the invention

After a lot of research and creative work by the inventors, the fermentative production of Arthrobacter globiformis HL6 to obtain hyaluronidase with high enzyme activity was achieved, the hyaluronidase enzyme-producing gene and amino acid sequence were successfully obtained, and the recombinant vector was constructed and successfully realized. Recombinant expression and purification preparation in Escherichia coli and Bacillus subtilis (national invention patent application number: 201811310938.8).

Aiming at the deficiencies of the prior art, the present invention provides a method for preparing small-molecule hyaluronic acid by enzymatic cleavage, and the obtained small-molecule hyaluronic acid and its application. The microbial hyaluronidase enzymatic method used in the present invention prepares small molecule hyaluronic acid, the process is simple, the conditions are mild, there is no environmental pollution and animal source virus pollution, no damage to the product structure, the product uniformity is good, and the small molecule hyaluronic acid is realized. Acid environment-friendly industrial production. The small molecule hyaluronic acid prepared by the present invention has high purity, safety and non-toxicity, and has good moisturizing, transdermal absorption, anti-oxidation and anti-inflammatory activities, and can be widely used in the fields of food, cosmetics and medicine, and can also be used as hyaluronic acid The oligosaccharide standard is used in the research on the structure and quality of hyaluronic acid, and has broad research and application prospects.

The technical problem to be solved by the present invention is to provide a method for preparing small molecule hyaluronic acid by enzymatic cleavage.

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

In order to solve the above technical problems, the present invention adopts the following technical solutions to achieve:

A method for preparing small-molecule hyaluronic acid by enzymatic cleavage, comprising using the hyaluronidase or amino acid sequence prepared by fermentation of Arthrobacter globiformis HL6 with the preservation number CCTCC NO: M 2018452 as shown in SEQ ID NO: 1 The hyaluronidase is a method for degrading hyaluronic acid or a salt thereof with a molecular weight greater than 600kDa, and preparing a small molecule hyaluronic acid or a salt thereof.

The method for preparing small molecule hyaluronic acid by enzymatic cleavage of the present invention comprises the following steps:

1) Prepare hyaluronic acid or its salt solution: add hyaluronic acid or its salt with a molecular weight greater than 600kDa to purified water, and prepare a solution with a mass volume fraction of 0.05-20%;

2) Enzyme hydrolysis: adjustment step 1) The temperature of the hyaluronic acid or its salt solution is 20-50°C, the pH is 5-10, and 100U/g-5×10 ⁵ is added to the solution made of hyaluronic acid or its salt U/g hyaluronidase, degrade hyaluronic acid or its salt to a molecular weight of 379Da-600kDa to obtain an enzymatic solution;

3) Inactivation: keep the enzymatic hydrolysis solution in step 2) at 55-100°C for 5-30 minutes, and inactivate the added enzyme;

4) Filtration: Add or not add 0.01-2mol/L soluble inorganic salt to the enzymolysis solution in step 3), stir until completely dissolved, and filter with a filter membrane or filter element to obtain a filtrate;

5) Precipitation: Slowly add alcohol or ketone 1.5-30 times the volume of the filtrate to the filtrate of step 4), mix well, and precipitate out small molecule hyaluronic acid or its salt;

6) Dehydration and drying: collect the precipitation of small and medium-sized hyaluronic acid or its salt in step 5), add the same alcohol or ketone as in step 5) to dehydrate, and dry to obtain small-molecular hyaluronic acid or its salt;

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

In the above step 2), it is preferred to adjust the reaction temperature to 20-45°C and pH to 5.5-8. The acid or alkali used to adjust the pH is at least one of hydrochloric acid, glacial acetic acid, nitric acid, sulfuric acid, phosphoric acid or sodium hydroxide, potassium hydroxide; add 100U/g-5×10 ⁵ U/g Arthrobacter globiformis HL6 hyaluronidase or the hyaluronidase whose amino acid sequence is shown in SEQ ID NO: 1, degrades hyaluronic acid or its salts to a molecular weight of 379Da-600kDa, hyaluronic acid The enzyme has good degradability to hyaluronic acid, only need to add the appropriate hyaluronidase, control the length of the reaction time to obtain the required molecular weight hyaluronic acid.

In the above step 3), it is preferable to adjust the temperature of the enzymatic hydrolysis solution to 60-80° C. and keep it warm for 10-20 minutes to inactivate the hyaluronidase.

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

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

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

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

The infrared spectrum of the small molecule hyaluronic acid or its salt prepared by the above method is basically consistent with the standard spectrum of the European Pharmacopoeia, the structure is complete, no group falls off, and an unsaturated C=C double bond is generated between C4-C5 of glucuronic acid. It has a characteristic absorption peak near 232nm, which not only provides a significant monitoring parameter for the degradation process, but also can monitor the degradation process according to the change of 232nm absorbance in the reaction process, and it is easier to control the reaction process. Under the condition of certain process parameters, by controlling the reaction time and the 232nm absorbance value of the solution to accurately prepare small molecule hyaluronic acid or its salts with different molecular weights.

The small molecule hyaluronic acid or its salt prepared by the above method has a molecular weight range of 379Da-600kDa, has no toxicity to cells, and has good biological activities such as moisturizing, transdermal absorption, anti-oxidation, and anti-inflammation. It has a very good application prospect in food.

The present invention also provides a composition comprising the small molecule hyaluronic acid or its salt prepared by the above method.

The present invention also provides a composition, which also includes an auxiliary material or carrier acceptable in foods, cosmetics or medicines.

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

In the present invention, unless otherwise specified, "small molecule hyaluronic acid or small molecule hyaluronate" is simply referred to as "small molecule hyaluronic acid or its salt", and its molecular weight ranges from 379Da to 600kDa, and the preferred molecular weight range is 379Da-200kDa, a further preferred molecular weight range is 379Da-10kDa, an even more preferred molecular weight range is 379Da-3kDa.

Compared with the prior art, the advantages and beneficial effects of the present invention are: the present invention utilizes the hyaluronidase produced by the fermentation of Arthrobacter globiformis HL6 from marine sources or the hyaluronic acid whose amino acid sequence is shown in SEQ ID NO: 1 Acidase digestion to prepare small molecule hyaluronic acid. The hyaluronidase produced by microbial fermentation has high activity, high product purity, low impurity content and no potential animal virus hazards. It is suitable for industrial production and applied to the production of small molecule hyaluronic acid. Industrialized production, especially suitable for industrialized preparation of small molecule hyaluronic acid with a molecular weight of less than 3kDa, the lowest can reach hyaluronic acid disaccharide, mild reaction conditions, low production cost, no environmental pollution, and the enzymatic hydrolysis product has a characteristic absorption of 232nm, which is convenient for the production process. Precise control to prepare small molecule hyaluronic acid with different molecular weights.

The small-molecule hyaluronic acid or its salt prepared by the present invention has no phenomena such as group shedding, sugar ring breakage, etc., the product structure is complete, the actual content of hyaluronic acid is high, the purity is high, safe and non-toxic, and it has good moisturizing, transdermal absorption, Antioxidant, anti-inflammatory and other biological activities can be used in food, cosmetics and medicine and other fields, and can also be used as hyaluronic acid oligosaccharide standard for hyaluronic acid structure and quality research, which has broad research and application prospects.

Description of drawings

Below in conjunction with accompanying drawing, specific embodiment of the present invention is described in further detail, wherein:

Figure 1: ESI-MS scanning pattern of enzymatic hydrolysis products. The abscissa is m/z, and the ordinate is the ion current intensity.

Figure 2: UV scanning spectra of hyaluronic acid with different molecular weights. The abscissa is the wavelength (nm), and the ordinate is the absorbance.

Figure 3: Infrared scanning spectra of hyaluronic acid with different molecular weights. The abscissa is the wave number (cm ^-1 ), and the ordinate is the transmittance; Figure 3a is LMWHA; Figure 3b is HMWHA; Figure 3c is o-HA.

Figure 4: Effects of hyaluronic acid with different molecular weights on the proliferation of HUVEC cells. The abscissa is the sample concentration (mg/mL), and the ordinate is the relative proliferation rate (%).

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

Figure 6: In vitro transdermal absorption of hyaluronic acid with different molecular weights. The abscissa is time (h), and the ordinate is Qn (ug/cm ² ).

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

Figure 8: Effects of hyaluronic acid with 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 ways

The technical solutions of the present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the concentrations described in the present invention are all mass volume concentrations.

In the present invention, the molecular weight of the small molecule hyaluronic acid is determined by gel exclusion chromatography (GPC), and the oligomeric hyaluronic acid is analyzed by mass spectrometry (MS), and the anion detection mode is selected for the mass spectrometer; the content determination adopts carbazole-sulfuric acid Law. The hyaluronidase enzyme activity assay method adopts the Chinese Pharmacopoeia method (refer to the general rule 1207 of the Chinese Pharmacopoeia 2015 edition: hyaluronidase assay method). The enzyme activity unit of fermentation broth is defined as: enzyme activity unit per milliliter of fermentation broth (U/mL).

In the present invention, a hyaluronidase-producing bacterium HL6 was isolated from seawater, and its 16S rRNA length was 1443bp. After comparing and identifying the 16S rRNA sequence in the NCBI database, it had the highest similarity of 99.7% with Arthrobacter bacteria, and was initially identified as Arthrobacter. According to the morphological, physiological and biochemical identification of the strain according to Bergey's Bacteria Handbook, the results showed that the strain was cultured at 24°C for 48 hours, and the colony was round, colorless and transparent. Gram stain positive. Can use creatine, sarcosine, betaine and glycine as a single carbon source, can liquefy gelatin and hydrolyzed starch, but cannot reduce NO ₃ to NO ₂ , needs biotin as growth factor, does not need ammonium sulfate, soil factor as growth factor Factors, leucinase, glutaminase positive, can not use xylose, mannose, ribose and raffinose. Morphological, physiological and biochemical results show that this strain is significantly different from the Arthrobacter globosa A152 reported in the invention with application number 201610115793.0. Combined with 16rRNA sequence analysis, morphological and physiological and biochemical identification results, the strain was named Arthrobacter globiformis HL6. 16s rRNA

TTTTTAGAGTTTTTTTATTCGTGGCTCAGGATGAACGCTGGCGGCGTGCTTCACACATGCAAGTCGAACGATGATCCCAGCTTGMTGGGGGATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTTGACTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACCATCTGACGCATGTCATGGTGGTGGAAAGCTTTTGTGGTTTTGGATGGACTCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGTAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCTGTGAAAGACCGGGGCTCAACTCCGGTTCTGCAGTGGGTACGGGCAGACTAGAGTGCAGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCTGTAACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGGACATTCCACGTTTTCCGCGCCGTAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATGAACCGGAAAGACCTGGAAACAGG TGCCCCGCTTGCGGTCGGTTTACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGTTGCCAGCGCGTTATGGCGGGGACTCATAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGTTGCGATACTGTGAGGTGGAGCTAATCCCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCACGAAAGTTGGTAACACCCGAAGCCGGTGGCCTAACCCTTGTGGGGGGAGC。

Preservation information: Arthrobacter globiformis HL6, the strain was deposited in the China Type Culture Collection Center on July 5, 2018, address: Wuhan, China, Wuhan University, the preservation number of Arthrobacter globiformis HL6 is: CCTCC NO: M 2018452.

The hyaluronidase of the present invention includes the hyaluronidase derived from the fermentation of the marine bacterium Arthrobacterglobiformis HL6, and also includes the fermentation of bacteria, fungi, and mammalian cells other than Arthrobacterglobiformis HL6. The hyaluronidase produced has the amino acid sequence shown in SEQ ID NO:1. Arthrobacter globiformis HL6 fermented to produce hyaluronidase or the amino acid sequence produced by fermentation of bacteria, fungi, and mammalian cells other than Arthrobacter globiformis HL6 is hyaluronidase as shown in SEQ ID NO: 1 For the specific method of massnidase, refer to the national invention patent application with application number 201811310938.8, the entire content of which is hereby incorporated by reference into the present invention.

Example 1. Cloning of hyaluronan lyase gene and construction of recombinant vector

Using the total DNA of Arthrobacter globiformis HL6 with the preservation number CCTCC M 2018452 as a template, through the functional gene analysis of the database, amplify with primers EC-F and EC-R containing restriction endonuclease sites Hyaluronan lyase gene HyLs (without signal peptide):

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

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

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

PCR is used for amplification, and the 50 μl reaction system is:

In a 50 μL reaction system containing DNA template, 1 μL; EC-F (10 μM), 1 μL; EC-R (10 μM), 1 μL; dNTP (2.5 mM each), 4 μL; Taq (2U/μL), 1 μL; 10× Taq buffer, 5 μL; ddH2O, added to 50 μL.

The PCR amplification program was: pre-denaturation at 95°C for 3 min, denaturation at 96°C for 30 s, annealing at 60°C for 45 s, extension at 70°C for 90 s, 30 cycles, and 10 min at 70°C.

The PCR product was subjected to 1% agarose electrophoresis and sequence verification, and the expression gene HyLs of Arthrobacterglobiformis HL6CCTCC M 2018452 hyaluronan lyase was obtained, with a length of 2298bp. The sequencing sequence is shown in the sequence table SEQ.ID.No.2. The gene sequence shown in SEQ.ID.No.2 was compared with the whole genome sequence, and both were consistent. The PCR product was purified according to the operation method required by the Cycle-Pure Kit (OMEGA Bio-Tek Co.) purification kit.

The vector pProEX-HTa and the PCR amplified gene were subjected to double-enzyme digestion, and the digested products were subjected to agarose electrophoresis, and recovered according to the operation method required by the Gel extraction kit (OMEGA Bio-Tek Co.).

T4 ligase is used to connect the sequence fragment containing the hyaluronan lyase gene with the vector pProEX-HTa to obtain the recombinant vector HTa-HyLs containing the hyaluronan lyase gene.

Example 2. Preparation of Hyaluronidase by Fermentation of Arthrobacter globiformis HL6

The preservation number is CCTCC NO: M 2018452 Arthrobacter globiformis HL6 is inoculated into the sterilized seed culture medium (seed culture medium formula is: sodium hyaluronate 0.2g/L, glucose 5g/L, peptone 2g/L L, dipotassium hydrogen phosphate 1.5g/L, MgSO ₄ 0.5g/L, pH 7.5), 32°C, 200r/min, cultivated for 24h to obtain seed liquid. Inoculate the seed culture liquid into the sterilized fermentation medium according to the inoculation amount of 1% (sodium hyaluronate 2g/L, glucose 10g/L, peptone 5g/L, dipotassium hydrogen phosphate 1.5g/L, MgSO ₄ 0.5g/L L, pH 7.5), 28°C, 200r/min for 24h, the fermentation broth was centrifuged at 4°C, 8000r/min for 10min, the fermentation supernatant was collected, and the hyaluronidase activity was determined to be 1×10 ⁵ U/mL.

Example 3. Recombinant expression of hyaluronidase in Escherichia coli

1. Select Escherichia coli strain DH5α, prepare competent cells, heat shock transformation (42°C, 60s), and incubate (37°C, 160rpm, 45min), and screen transformants on LB solid plates containing 75 μg/mL ampicillin sodium. Transformed strains cannot grow on plates. After PCR detection, positive cloned strains were obtained, and the plasmid was extracted with the Plasmid Mini Kit (OMEGA Bio-Tek Co.) kit to obtain the recombinant plasmid HTa-HyLs.

Select the Escherichia coli expression strain BL21(DE3), and transform the extracted recombinant plasmid HTa-HyLs into the Escherichia coli expression strain BL21 after preparing competent cells, heat shock transformation (42°C, 60s), and incubation (37°C, 45min). (DE3), screened on 75 μg/mL ampicillin LB solid plate, cultured at 37°C for 16 hours to obtain transformants, selected single colony transformants for PCR detection, and obtained positive clone strains. Store in glycerol at -80°C.

Inoculate the recombinant Escherichia coli BL21 (DE3) containing the hyaluronidase gene encoding the amino acid sequence shown in SEQ ID NO: 1 in LB liquid medium (75 μg/mL ampicillin sodium), and cultivate it at 37°C until the _OD600 is 0.6 At ~0.7, add IPTG to a final concentration of 0.5mM, induce culture at 24°C, 180r/min for 24h, centrifuge the fermentation broth at 8000r/min for 10min at 4°C, collect the fermentation supernatant, and measure the activity of hyaluronidase It is 5×10 ⁶ U/mL.

2. Recombinase purification

1) The fermentation supernatant after centrifugation is firstly subjected to affinity chromatography (GE company) with a nickel column, followed by preliminary separation and purification according to the nickel column purification instructions, and the pure preliminary separation and purification product is collected;

2) The product of step 1) was initially purified with QFF- Fast Flow chromatographic column (GE company) is purified again, first with 20mM, the Tris-HCl damping fluid equilibrium column of pH7.5 is used to contain concentration after loading with the 20mM Tris-HCl (pH of 0-1mol/L sodium chloride) 7.5) The buffer solution is subjected to linear gradient elution, the protein peak is detected at 280nm, the components are collected in tubes, the hyaluronidase activity of the collected solution in each tube is measured, and the active protein fraction is collected;

3) Further purify the active protein fraction collected in step 2) with Sephadex G100 gel (GE), the eluent is 20mM pH 7.0 phosphate buffer, collect by tube, detect hyaluronidase activity, and collect active protein The protein fraction is purified hyaluronan lyase. Perform SDS-PAGE electrophoresis on the purified hyaluronidase, or obtain a single protein band with a molecular weight of about 80kDa, which is inferred from the hyaluronan lyase sequence SEQ.ID.No.2 hyaluronan lyase gene in the sequence table .ID.No.1 results are basically the same.

Example 4. Preparation of Small Molecular Hyaluronic Acid

Add 1L of purified water to a 5L beaker, slowly add 10g of hyaluronic acid with a molecular weight of 2000kDa while stirring, stir until completely dissolved, adjust the pH to 5.5 with hydrochloric acid, adjust the temperature to 42°C, and then add the example to the hyaluronic acid solution 2 Prepare 0.5mL of hyaluronidase (5×10 ⁴ U), incubate for 6 hours, take 50mL of samples at intervals, incubate the sample at 60°C for 20 minutes to inactivate the enzyme, add 0.5mol/L NaCl, stir until completely dissolved, filter with 0.45μm filter membrane Filter the enzymolysis solution, add 3 times of ethanol to the enzymolysis solution to precipitate, collect the precipitate, then wash with ethanol, dehydrate, and vacuum dry to obtain small molecule hyaluronic acid. The molecular weights of samples at different time points are shown in Table 1.

Table 1. Enzyme hydrolysis time and molecular weight of hyaluronic acid

sample 3min 10min 30min 1h 2 hours 3 hours 4h 5h 6 hours 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 Molecular Sodium Hyaluronate

Add 1L of purified water to a 5L beaker, slowly add 100g of sodium hyaluronate with a molecular weight of 1500kDa while stirring, stir until it is completely dissolved, adjust the pH to 7.0 with sodium hydroxide, adjust the temperature to 37°C, and then pour into the sodium hyaluronate solution Add 1 mL of hyaluronidase (5×10 ⁶ U) prepared in Example 3 to the solution, incubate for 6 hours, take 50 mL of samples at intervals, incubate the sample at 70°C for 15 minutes to inactivate the enzyme, add 1 mol/L NaCl, stir until completely dissolved, and use 0.45 μm Filter the enzymolysis solution with a filter membrane, add 3 times of ethanol to the enzymolysis solution to precipitate, collect the precipitate, then wash with ethanol, dehydrate, and vacuum dry to obtain small molecule sodium hyaluronate. The molecular weights of samples at different time points are shown in Table 2.

Table 2. Enzyme hydrolysis time and molecular weight of sodium hyaluronate

Example 6. Preparation of Small Molecule Potassium Hyaluronate

Add 1L of purified water to a 5L beaker, slowly add 1g of sodium hyaluronate with a molecular weight of 650kDa while stirring, stir until it is completely dissolved, adjust the pH to 8.0 with potassium hydroxide, adjust the temperature to 30°C, and then pour into the potassium hyaluronate solution Add 0.01mL (1×10 ³ U) of hyaluronidase prepared in Example 2, incubate for 6 hours, take 50mL samples at intervals, incubate the sample at 80°C for 10 minutes to inactivate the enzyme, filter the enzymolyzed solution with a 0.45 μm filter element, pour into the enzymolyzed solution Add 3 times of acetone to precipitate, collect the precipitate, then wash with acetone, dehydrate, and vacuum dry to obtain small molecule potassium hyaluronate. The molecular weights of samples at different time points are shown in Table 3.

Table 3. Enzyme hydrolysis time and molecular weight of potassium hyaluronate

sample 3min 10min 30min 1h 2 hours 3 hours 4h 5h 6 hours 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

Embodiment 7. Preparation of hyaluronic acid oligosaccharides

Add 1L of purified water to a 5L beaker, slowly add 10g of sodium hyaluronate with a molecular weight of 1500kDa while stirring, stir until it is completely dissolved, adjust the pH to 7.0 with sodium hydroxide, adjust the temperature to 37°C, and then pour into the sodium hyaluronate solution Add 50 mL of the hyaluronidase (5×10 ⁶ U) prepared in Example 2 to the mixture, incubate for 8 hours, take 100 mL of samples at 4 hours and 8 hours respectively, incubate the samples at 60°C for 20 minutes to inactivate the enzyme, stir until completely dissolved, and filter with a 0.45 μm filter membrane Filter the enzymolysis solution, add 20 times of ethanol to the enzymolysis solution to precipitate, collect the precipitate, then wash with ethanol, dehydrate, and freeze-dry to obtain hyaluronic acid oligosaccharides.

ESI-MS analysis was carried out on the enzymatic hydrolysis product, using negative ion ionization mode, scanning range m/z: 100-2000, mass spectrometry scanning spectrum is shown in Figure 1, ESI-MS scanning results show that: in the sample of enzymatic hydrolysis for 4 hours, there are mainly m/z z is 175.03, 378.1, 757 and 779.2 four kinds of ion peaks, representing unsaturated uronic acid (m/z: 175.03), unsaturated hyaluronic acid disaccharide (m/z: 378.1), unsaturated hyaluronic acid four Sugar (m/z: 757 and 779.2), also contains a small amount of ion peaks with m/z 599.0, 1180.4 and 1581.6, representing unsaturated hyaluronic acid trisaccharide (m/z: 599.0), unsaturated hyaluronic acid Hexasaccharide (m/z: 1180.4) and unsaturated hyaluronic acid octasaccharide (m/z: 1581.6); the mass spectrum of the sample after enzymatic hydrolysis for 8 hours shows that the sample mainly contains unsaturated hyaluronic acid disaccharide with m/z: 378.1 (ΔDiHA), indicating that the main end product of bacterial hyaluronidase degrading hyaluronic acid of the present invention is unsaturated hyaluronic acid disaccharide, hyaluronidase of the present invention is hyaluronan lyase, degrades hyaluronic acid in a cleavage manner , degrades hyaluronic acid with unsaturated disaccharide as the end product, currently common animal tissue extracts hyaluronidase (hydrolase, saturated tetrasaccharide as end product) and leech hyaluronidase (hydrolase, saturated tetrasaccharide and six sugar as the end product) both in the mode of action and degradation products are significantly different.

The high molecular hyaluronic acid (HMWHA) sample before enzymolysis and the 8-hour enzymolysis hyaluronic acid oligosaccharide (o-HA) sample were subjected to ultraviolet scanning, and the ultraviolet scanning pattern is shown in Figure 2. The ultraviolet scanning results show that: Hyaluronic acid oligosaccharides have characteristic absorption at 232nm, indicating the generation of unsaturated double bonds. Undegraded polymer hyaluronic acid has no 232nm ultraviolet absorption. The corresponding relationship realizes the online monitoring and control of the enzymatic hydrolysis process, and efficiently prepares low-molecular hyaluronic acid or hyaluronic acid oligosaccharides with different molecular weights.

Example 8. Enzymatic hydrolysis product infrared scanning analysis

For the high molecular hyaluronic acid (HMWHA) that was not degraded by hyaluronidase, the small molecular hyaluronic acid (LMWHA) prepared in Example 4, and the hyaluronic acid oligosaccharide (o-HA) prepared in Example 6 at 4000 cm ^- Infrared scanning is carried out in the wavelength range of ¹ to 400cm ^-1 , the infrared scanning spectrum is shown in attached drawing 3, the infrared scanning shows that: the enzymatic hydrolysis products all have the characteristic absorption peaks of carbohydrates, and the degradation products of different molecular weights are different from the hyaluronic acid before enzymatic hydrolysis All have similar characteristic absorption peaks, and are consistent with the standard spectrum of hyaluronic acid in the infrared spectrum of drugs compiled by the National Pharmacopoeia Committee in 2010, indicating that the small molecule hyaluronic acid and hyaluronic acid oligosaccharides obtained after enzymatic hydrolysis maintain structural integrity, and the enzymatic hydrolysis No groups were shed during the process.

Example 9. Cytotoxicity of small molecule hyaluronic acid

HUVEC cells in the logarithmic growth phase were inoculated on a 96-well plate at 15,000 cells/well (180 μl/well). After culturing for 24 hours, high molecular weight hyaluronic acid (HMWHA), small molecular hyaluronic acid prepared in Example 4 ( LMWHA) and the hyaluronic acid oligosaccharide (o-HA) sample prepared in Example 6, each concentration was provided with 3 duplicate holes. After 24 hours of drug action, the supernatant was sucked away, and then MTT medium containing 20 μl was re-added. After 4 hours, the supernatant was sucked away, and 150 μl DMSO was added to dissolve it. The absorbance value at 570 nm was measured with a microplate reader, and the relative cell proliferation rate was calculated. (RGR)

In the formula, _Ai is the absorbance of the sample, and _A0 is the absorbance of the control group.

The effects of HMWHA, LMWHA and o-HA at different concentrations of 0.01 to 1 mg/mL on the proliferation of HUVEC cells are shown in Figure 4. The experimental results show that HMWHA, LMWHA and o-HA at different concentrations of 0.01 to 1 mg/mL have no effect on the proliferation of HUVEC cells. There is no obvious effect, and the relative value-added rate of cells is higher than 95%. The Pharmacopoeia stipulates that the relative value-added rate of cells is greater than 80%, and its toxicity level is 0-1. safe and non-toxic.

Example 10. Moisturizing activity of small molecule hyaluronic acid

Sample solutions of high molecular weight hyaluronic acid (HMWHA), small molecule hyaluronic acid (LMWHA) prepared in Example 4, and hyaluronic acid oligosaccharide (o-HA) prepared in Example 6 were prepared respectively, with a mass volume concentration of 10%. Measure the moisture retention rate of the sample under the environment of saturated sodium carbonate aqueous solution (relative humidity RH=43%) and dry silica gel (relative humidity RH=0%) respectively. Under the environment of saturated sodium carbonate (relative humidity RH=43%), or in the environment of dry silica gel (relative humidity RH=0%), hyaluronic acid samples with different molecular weights all have good moisturizing activity. , the moisturizing activity has decreased, but there is no significant difference compared with HMWHA. The moisture retention rate of the same sample in the dry silica gel environment is slightly lower than that in the saturated sodium carbonate environment, but there is no significant difference, indicating that the hyaluronic acid with different molecular weights prepared by enzymatic hydrolysis of the present invention has different molecular weights in different It has good moisturizing activity in all environments.

Example 11. Transdermal absorption performance of small molecule hyaluronic acid

Adopt the improved Franz diffusion cell, the skin surface is upward, add 5mg/mL high molecular weight hyaluronic acid (HMWHA), the small molecular weight hyaluronic acid (LMWHA) prepared in Example 4 and the hyaluronic acid prepared in Example 6 to the supply pool oligosaccharide (o-HA) sample solution 1mL, add 6.5mL of 0.85% normal saline to the receiving tank, control the rotation speed at 360rpm, conduct the experiment at 37°C, and sample 1mL at 2h, 4h, 6h, 8h, 10h, 12h and 24h , and replenished with fresh acceptor solution. Measure the content of uronic acid in the sample, and calculate the cumulative penetration per unit area based on this, the calculation formula is as follows:

in:

Qn: Cumulative penetration per unit area of the sample at time t (μg/cm ² );

A: Transdermal diffusion area;

Cn: measured value of concentration at time t;

Ci: measured value of concentration before time t;

V: receiving pool volume;

Vo: sampling volume

J: transdermal rate constant, the regression equation is obtained by plotting Qn against t, and the slope is the transdermal rate constant.

The cumulative penetration per unit area of different samples is shown in Figure 6. The results of in vitro transdermal absorption experiments show that: due to the large molecular weight, the cumulative permeation per unit area of HMWHA is low, 0.057μg/cm ² in 12 hours, and with time The cumulative permeation per unit area increased slowly, and only reached 0.072μg/cm ² in 24 hours; the cumulative permeation per unit area of LMWHA and o-HA increased linearly with the prolongation of time, and the cumulative permeation per unit area of o-HA was slightly higher Compared with LMWHA, and both of them are significantly higher than HMWHA, the cumulative permeation per unit area at 12h is 0.65μg/cm ² (LMWHA) and 0.71μg/cm ² (o-HA), and as time goes on, the unit area Cumulative permeation increased continuously, reaching 1.07μg/cm ² (LMWHA) and 1.15μg/cm ² (o-HA) respectively in 24 hours, showing good long-lasting transdermal absorption. The transdermal absorption coefficient reflects the transdermal absorption rate, and the 24-hour transdermal absorption coefficient of HMWHA is 0.0031, R ² : 0.8924; the linearity is poor, and the 24-hour transdermal absorption coefficient of LMWHA is 0.0446, R ² : 0.9935, and the linearity is good; o The 24-hour transdermal absorption coefficient of -HA is 0.0479, R ² : 0.9917, and the linearity is good; the transdermal absorption coefficient further indicates that the transdermal absorption performance of o-HA and LMWHA is better than that of HMWHA.

Example 12. Antioxidative activity of small molecule hyaluronic acid

The samples of high molecular hyaluronic acid (HMWHA), small molecular hyaluronic acid (LMWHA) prepared in Example 4 and hyaluronic acid oligosaccharide (o-HA) prepared in Example 6 were measured respectively at different concentrations of 0.5-25 mg/mL The DPPH scavenging rate and reducing power are used to characterize the antioxidant activity of hyaluronic acid with different molecular weights. The results of antioxidant activity are shown in Figure 7. The results of DPPH scavenging experiments show that the lower the molecular weight of the small molecule hyaluronic acid sample prepared by enzymatic method, the lower the DPPH scavenging effect. The higher the rate, the same concentration of samples o-HA>LMWHA>HMWHA, the DPPH clearance rate of the same concentration of o-HA is about 4 times higher than that of HMWHA, and the experimental samples have a good linear increase in the concentration range of 5-25mg/mL ; The results of reducing power experiments show that: the reducing power of HMWHA is low, and the reducing power of LMWHA and o-HA prepared by enzymatic hydrolysis increases significantly, especially the upward trend of o-HA is obvious; the results of antioxidant activity of DPPH clearance rate and reducing power show that the enzyme The small molecule hyaluronic acid prepared by the method, especially the hyaluronic acid oligosaccharide has good antioxidant activity and has a good application prospect in cosmetics.

Example 13. Anti-inflammatory activity of small molecule hyaluronic acid

The lipopolysaccharide (LPS)-induced inflammation model of mouse RAW264.7 macrophages was used for experiments.

Sample grouping:

Blank control group: C; model group: M;

100ug/mL HMWHA:HH; 50ug/mL HMWHA:HL;

100ug/mL o-HA:OH; 50ug/mL HMWHA:OL;

Take RAW264.7 cells in the logarithmic growth phase (8×10 ⁵ cells/well, 50 μL) and add them to a 96-well plate, adhere to the wall for 30 minutes, and add LPS with a final concentration of 1 μg/mL to the model group (M) and sample group first. 1h, to stimulate inflammation, add HMWHA and o-HA with a concentration of 100ug/mL and 50ug/mL to the sample group respectively, replace it with PBS in the model group, and leave the blank control group (C) without any treatment, culture for 8h, take the cell supernatant, Centrifuge at 2000r/min for 15min to remove cell debris, and measure the concentration of TNF-a in the cell supernatant with an ELISA kit. The effect of different molecular weight hyaluronic acid on inflammatory cell TNF-a is shown in Figure 8. The experimental results show that the concentration of TNF-a in the model group is significantly higher than that of the blank control group, indicating that the model is available; both HMWHA and o-HA have the ability to inhibit TNF-a However, the difference is that the inhibitory ability of HMWHA is greater than 50ug/mL when the concentration is 100ug/mL, while o-HA is greater than 100ug/mL when the concentration is 50ug/mL, which may be different from the anti-inflammatory mechanism of the two Relevant; the inhibitory effect of o-HA on TNF-a in the same concentration samples is better than that of HMWHA, especially at low concentrations, indicating that o-HA has better anti-inflammatory effects, and can significantly reduce the amount of samples and reduce costs. It has better development and application prospects.

The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still understand the foregoing embodiments. Modifications are made to the stated technical solutions, or equivalent replacements are made to some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed in the present invention.

sequence listing

<110> Qingdao Marine Biomedical Research Institute Co., Ltd.

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Claims (10)

1. a kind of method that enzyme cutting method prepares micromolecule hyaluronic acid, which is characterized in that with deposit number for CCTCC NO:M The hyaluronidase or amino acid of 2018452 Arthrobacter globiformis (Arthrobacter globiformis) HL6 fermentation preparation Sequence hyaluronidase as shown in SEQ ID NO:1 is greater than 600kDa hyaluronic acid to molecular weight or its salt is degraded, and makes Standby small-molecular-weight hyaluronic acid or its salt comprising the steps of:

1) hyaluronic acid or its salting liquid are prepared: hyaluronic acid or its salt that molecular weight is greater than 600kDa being added into purified water, It is configured to the solution that quality volume fraction is 0.05-20%；

2) digesting: regulating step 1) temperature of hyaluronic acid or its salting liquid is 20-50 DEG C, it the use of acid-base accommodation pH is 5-10, 100U/g-5 × 10 are added in the solution being made into hyaluronic acid or its salt⁵Hyaluronic acid or its salt drop in the hyaluronidase of U/g Molecular weight 379Da-600kDa is solved, enzymolysis liquid is obtained；

3) inactivate: by enzymolysis liquid in step 2) 55-100 DEG C holding 5-30 minutes, the enzyme of addition is inactivated；

4) it filters: being added or be added without 0.01-2mol/L ease of solubility inorganic salts to enzymolysis liquid in step 3), stir to completely molten Solution, with miillpore filter or filter element filtering, obtains filtrate；

5) it precipitates: being slowly added to 1.5-30 times of filtrate volume of alcohol or ketone into the filtrate of step 4), be uniformly mixed, Precipitation Micromolecule hyaluronic acid or its salt；

6) dehydrate: collection step 5) small molecular hyaluronic acid or its salt precipitating, thereto be added it is identical with step 5) Alcohol or ketone are dehydrated, dry micromolecule hyaluronic acid or its salt.

2. according to the method described in claim 1, it is characterized by: the hyaluronidase as shown in SEQ ID NO:1 by Recombinant bacteria, yeast or cell containing hyaluronic acid enzyme gene shown in coding SEQ ID NO:1 generate.

3. according to the method described in claim 1, it is characterized by: the step 2) in adjust 20-45 DEG C of reaction temperature, pH5.5-8。

4. according to the method described in claim 1, it is characterized by: the step 3) is middle to adjust 60-80 DEG C of enzymolysis liquid temperature, guarantor Warm 10-20 minutes inactivates hyaluronidase.

5. according to the method described in claim 1, it is characterized by also including the one or mores in following condition: step 1) hyaluronate used in is at least one of sodium salt, sylvite, calcium salt, magnesium salts or the zinc salt of hyaluronic acid；Step 2) Described in acid be at least one of hydrochloric acid, glacial acetic acid, nitric acid, sulfuric acid or phosphoric acid, the alkali is sodium hydroxide or potassium hydroxide At least one of；Ease of solubility inorganic salts described in step 4) is at least one of sodium salt, sylvite, calcium salt, magnesium salts or zinc salt； In step 5) and step 6), the alcohol or ketone are at least one of methanol, ethyl alcohol, propyl alcohol, butanol or acetone.

6. micromolecule hyaluronic acid made from method of any of claims 1-5 or its salt, it is characterised in that: described The molecular weight ranges of micromolecule hyaluronic acid or its salt are 379Da-600kDa.

7. micromolecule hyaluronic acid according to claim 6 or its salt, which is characterized in that the micromolecule hyaluronic acid salt For the sodium salt of micromolecule hyaluronic acid, sylvite, calcium salt, magnesium salts or zinc salt.

8. micromolecule hyaluronic acid according to claim 6 or 7 or its salt preparation improve moisture of skin, anti-aging, Application in anti-oxidant, anti-inflammatory food, cosmetics or drug.

9. a kind of composition, it is characterised in that: include the described in any item micromolecule hyaluronic acids of right 6-8 or its salt.

10. composition according to claim 9, it is characterised in that: the composition also includes food, cosmetics or drug In acceptable auxiliary material or carrier.