WO2020034953A1 - 一种双酶水解制备透明质酸奇数寡糖的方法 - Google Patents

一种双酶水解制备透明质酸奇数寡糖的方法 Download PDF

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WO2020034953A1
WO2020034953A1 PCT/CN2019/100409 CN2019100409W WO2020034953A1 WO 2020034953 A1 WO2020034953 A1 WO 2020034953A1 CN 2019100409 W CN2019100409 W CN 2019100409W WO 2020034953 A1 WO2020034953 A1 WO 2020034953A1
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hyaluronic acid
odd
polymerization
solution
sugar
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康振
何静
黄浩
陈坚
堵国成
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江南大学
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals

Definitions

  • the invention relates to a method for preparing an odd-numbered oligosaccharide of hyaluronic acid by double-enzyme hydrolysis, and belongs to the technical field of biological engineering.
  • Hyaluronic acid is formed by the connection of D-glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) through ⁇ -1,3 glycosidic bonds and ⁇ -1,4 glycosidic bonds.
  • HA degradation hyaluronic acid oligosaccharides
  • o-HAs hyaluronic acid oligosaccharides
  • a common method of biodegradation is enzymatic hydrolysis.
  • hydrolytic enzymes bovine testis-type hyaluronidase (BTH) and leech type hyaluronidase (LHase) are the most specific to hyaluronic acid (HA) and have the highest specific hydrolysis efficiency.
  • BTH bovine testis-type hyaluronidase
  • LHase leech type hyaluronidase
  • Bovine testicular hyaluronidase hydrolyzes ⁇ -1,4 glycosidic bonds in HA to form an even number of oligosaccharides with glucosamine as the reducing end; leech type hyaluronidase (LHase) hydrolyzes ⁇ -1,3 in HA
  • LHase leech type hyaluronidase
  • the glycosidic bond produces an even number of oligosaccharides with glucuronic acid as the reducing end.
  • the two enzymes act on two different glycosidic bonds of hyaluronic acid, respectively, to produce two series of hyaluronic acid oligosaccharides with different reducing ends.
  • the technical problem to be solved by the present invention is to provide a method for preparing a hyaluronic acid oligosaccharide with an odd degree of polymerization.
  • a method for preparing an odd-numbered degree of hyaluronic acid oligosaccharide by hydrolysis of bovine testis-type hyaluronidase and leech type hyaluronidase is used.
  • the present invention provides a method for preparing hyaluronic acid oligosaccharides with an odd degree of polymerization.
  • the specific technical solution includes the following steps:
  • Hyaluronic acid having a molecular weight of 10 5 to 10 6 Da is used as a substrate, and hyaluronic acid type hyaluronidase (LHase) and bovine testicular type hyaluronidase (BTH) are used. Hydrolytic enzymes act on the substrate to obtain a series of mixtures of different degrees of polymerization of hyaluronic acid oligosaccharides;
  • the hydrolysis order of the two enzymes is not limited.
  • the method includes the following steps:
  • Hydrolyzed hyaluronic acid Hyaluronic acid with a molecular weight of 10 5 to 10 6 Da is used as a substrate. Hydrolyzed hyaluronidase LHase is first added to hydrolyze. After the enzyme is destroyed, bovine testicular hyaluronidase is added. BTH hydrolyzes and inactivates the enzyme to obtain a series of mixtures of different degrees of polymerization of hyaluronic acid oligosaccharides;
  • the method includes the following steps:
  • Hydrolyzed hyaluronic acid Use hyaluronic acid with a molecular weight of 10 5 to 10 6 Da as a substrate, first add bovine testis-type hyaluronidase BTH to hydrolyze, and then inactivate the enzyme, then add leech type hyaluronidase LHase is hydrolyzed and inactivated to obtain a series of mixtures of hyaluronic acid oligosaccharides with different odd degrees of polymerization;
  • the method includes the following steps:
  • Hyaluronic acid with a molecular weight of 10 5 to 10 6 Da is used as a substrate, and bovine testis-type hyaluronidase BTH and leech-type hyaluronidase LHase are added for hydrolysis and inactivation to obtain A series of mixtures of different degrees of polymerization of hyaluronic acid oligosaccharides;
  • the specific steps of separating and purifying the hyaluronic acid oligosaccharide obtained in step (1) by using a gel-filled ion exchange column are: loading, equilibrating, loading, and eluting 2. Collect the product according to the product peak, concentrate, desalinize, and freeze dry to obtain an odd degree of polymerization of hyaluronic acid oligosaccharide.
  • the hyaluronic acid reacts with the enzyme in the form of a solution.
  • concentration of the hyaluronic acid solution is 10-20 mg / mL, and the solvent is water.
  • the final concentration of the added leech hyaluronidase in step (1) is 5000 to 7000 U / mL, and the final concentration of the bovine testicular hyaluronidase is 1000 to 4000U / mL.
  • the action time of the leech type hyaluronidase in step (1) is 1 to 15 hours, and the action time of the bovine testicular type hyaluronidase is 1 to 15 hours.
  • the gel-filled ion exchange column is an ion exchange column filled with Q agarose gel HP (QHP).
  • the equilibrium solution used for the equilibrium is a Tris-HCl solution having a pH of 7 to 9, and the eluent used for elution is a 50 to 300 mM NaCl solution configured by the equilibrium solution. .
  • the elution volume is 2 to 20 times the column volume, preferably 10 to 20 times.
  • the elution process uses linear elution.
  • the linear elution is to first use an eluent with a concentration of 0, and gradually increase the eluent to the corresponding concentration (configured by the equilibrium solution). NaCl solution at a concentration of 50 to 300 mM).
  • the desalination method is any one of molecular exclusion gel column or dialysis bag desalination.
  • the molecular exclusion gel column is a Superdex 30 Increase 10/300 GL gel column, and the size of the dialysis bag is 0.5-1.0KD.
  • the method mainly includes the following steps:
  • a hyaluronic acid aqueous solution having a concentration of 5 to 20 mg / mL and a molecular weight of 10 5 to 10 6 Da is used as a substrate; first, a hydrolyzed hyaluronidase (LHase) having a final concentration of 5000 to 7000 U / mL is added to hydrolyze 1 to 10 hours; after the above enzymes are inactivated, bovine testicular hyaluronidase (BTH) with a final concentration of 1000 to 4000 U / mL is added to hydrolyze for 1 to 10 hours, the enzymes are boiled and filtered through a 0.22 ⁇ m filter membrane;
  • LHase hydrolyzed hyaluronidase
  • BTH bovine testicular hyaluronidase
  • the equilibrium solution is a Tris-HCl buffer solution with a pH of 6 to 8.
  • the eluent is the concentration configured by the equilibrium solution. It is a 50-300 mM NaCl solution.
  • the specific operation is as follows: the column is packed with Q agarose gel HP packing, equilibrated with an equilibrium solution, and the flow rate of the equilibrium solution is 2 to 5 mL / min. Then, the sample is loaded and eluted.
  • the strategy is linear elution with a NaCl solution with a concentration from 0 to 50 to 300 mM, the elution volume is 10 to 20 times the column volume, and the flow rate of the eluent is 2 to 6 mL / min.
  • the product is collected and concentrated, and is subjected to molecular exclusion coagulation
  • Hyaluronic acid oligosaccharides with an odd degree of polymerization in the product can be obtained by desalting with a gel column or a dialysis bag and freeze-drying.
  • the method mainly includes the following steps:
  • aqueous hyaluronic acid solution having a concentration of 5 to 20 mg / mL and a molecular weight of 10 5 to 10 6 Da is used as a substrate; firstly, a bovine testicular hyaluronidase (BTH) with a final concentration of 1000 to 4000 U / mL is added to hydrolyze 1 ⁇ 10 hours; after the above enzymes are inactivated, hydrolyzed hyaluronidase (LHase) with a final concentration of 5000 to 7000 U / mL is added to hydrolyze for 1 to 10 hours, the enzyme is boiled, and filtered through a 0.22 ⁇ m filter membrane;
  • BTH bovine testicular hyaluronidase
  • LHase hydrolyzed hyaluronidase
  • the equilibrium solution is a Tris-HCl buffer solution with a pH of 6 to 8.
  • the eluent is the concentration configured by the equilibrium solution. It is a 50-300 mM NaCl solution.
  • the specific operation is as follows: the column is packed with Q agarose gel HP packing, equilibrated with an equilibrium solution, and the flow rate of the equilibrium solution is 2 to 5 mL / min. Then, the sample is loaded and eluted.
  • the strategy is linear elution with a NaCl solution with a concentration from 0 to 50 to 300 mM, the elution volume is 10 to 20 times the column volume, and the flow rate of the eluent is 2 to 6 mL / min.
  • the product is collected and concentrated, and is subjected to molecular exclusion coagulation
  • Hyaluronic acid oligosaccharides with an odd degree of polymerization in the product can be obtained by desalting with a gel column or a dialysis bag and freeze-drying.
  • the method mainly includes the following steps:
  • aqueous hyaluronic acid solution having a concentration of 5 to 20 mg / mL and a molecular weight of 10 5 to 10 6 Da is used as a substrate; bovine testicular hyaluronidase (BTH) and Hydrolyzed hyaluronidase (LHase) with a final concentration of 5000 to 7000 U / mL is hydrolyzed for 1 to 10 hours, boiled to destroy the enzyme, and filtered through a 0.22 ⁇ m filter membrane;
  • BTH bovine testicular hyaluronidase
  • LHase Hydrolyzed hyaluronidase
  • the equilibrium solution is a Tris-HCl buffer solution with a pH of 6 to 8.
  • the eluent is the concentration configured by the equilibrium solution. It is a 50-300 mM NaCl solution.
  • the specific operation is as follows: the column is packed with Q agarose gel HP packing, equilibrated with an equilibrium solution, and the flow rate of the equilibrium solution is 2 to 5 mL / min. Then, the sample is loaded and eluted.
  • the strategy is linear elution with a NaCl solution with a concentration from 0 to 50 to 300 mM, the elution volume is 10 to 20 times the column volume, and the flow rate of the eluent is 2 to 6 mL / min.
  • the product is collected and concentrated, and is subjected to molecular exclusion coagulation
  • Hyaluronic acid oligosaccharides with an odd degree of polymerization in the product can be obtained by desalting with a gel column or a dialysis bag and freeze-drying.
  • the invention also provides an odd-numbered degree of polymerization hyaluronic acid oligosaccharide.
  • the structure of the odd-numbered degree of hyaluronic acid oligosaccharide is:
  • n 1, 2, 3.
  • the odd-numbered degree of polymerization hyaluronic acid oligosaccharide is prepared by any one of the methods for preparing an odd-numbered degree of polymerization hyaluronic acid oligosaccharide.
  • a novel N-type odd-numbered hyaluronic acid oligosaccharide is prepared by the present invention, which not only enriches the diversity of the oligosaccharide structure, but also carries out the relationship between the development of different structural types of hyaluronic acid oligosaccharide and the occurrence and development of diseases, etc. It is of great significance.
  • Figure 1 a: LCMS ion chromatogram of the hydrolysis product obtained in Example 1; b: LCMS ion chromatogram of the hydrolysis product obtained in Example 2; c: LCMS ion chromatogram of the hydrolysis product obtained in Example 3; d: LCMS ion chromatogram of the hydrolysate obtained in Example 4.
  • Figure 2 Mass spectrum of the target product in the hydrolysis solution: (a) 3N sugar (HA 3 NN ); (b) 3A sugar (HA 3 AA ); (c) 5N (HA 5 NN ); (d) 5A sugar ( HA 5 AA ); (e) 7N (HA 7 NN ); (f) 7A sugar (HA 7 AA ).
  • Fig. 3 List of molecular structure diagrams of odd-numbered hyaluronic acid oligosaccharides and their general formulas, in which (a) 3N sugar (HA 3 NN ) structure diagram; (b) 3A sugar (HA 3 AA ) structure diagram; (c ) 5N sugar (HA 5 NN ) structure diagram; (d) 5A sugar (HA 5 AA ) structure diagram; (e) 7N sugar (HA 7 NN ) structure diagram; (f) 7A sugar (HA 7 AA ) structure diagram ( ); (G) is an N-type odd-numbered hyaluronic acid oligosaccharide general formula and an A-type odd-numbered hyaluronic acid oligosaccharide general formula.
  • Figure 4 a: separation of hyaluronic acid disaccharide, 3A sugar (HA 3 AA ), 3N sugar (HA 3 NN ), tetrasaccharide (HA4), 5A sugar (HA 5 AA ), 5N sugar (HA 5 NN ), hexasaccharide (HA6); b: separation of hyaluronic acid 5A sugar (HA 5 AA ), 5N sugar (HA 5 NN ), hexasaccharide, 7A sugar (HA 7 AA ), 7N sugar (HA 7 NN ), octose (HA8).
  • the leech type hyaluronidase is obtained by recombinant expression of the leech type hyaluronidase gene from Pichia pastoris genetically engineered bacteria in this laboratory.
  • the nucleotide sequence is shown in the sequence SEQ ID NO.1; bovine testicular type hyaluronidase Purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.
  • Molecular exclusion gel column Superdex 30 Increase 10/300 GL column (column size 10mm ⁇ 300-310mm), column volume 24mL, column efficiency> 43 000N / m; typical pressure drop of packed bed 3.0MPa, column hardware pressure limit 5.0MPa.
  • the purity of the product was measured using the carbazole sulfuric acid method.
  • Example 1 Preparation of Odd Hyaluronic Acid 3A Sugar (HA 3 AA ), 3N Sugar (HA 3 NN ), 5A Sugar (HA 5 AA ), 5N (HA 5 NN ) Sugar 1
  • the macromolecular hyaluronic acid powder with a molecular weight of 10 5 to 10 6 Da was dissolved in pure water at a concentration of 10 mg / mL and fully dissolved overnight; the hydrolysis process was first added with hyaluronic acid LHase to make the final concentration of 5000U / mL, hydrolyzed at 37 ° C for 10 hours, and boiled the hydrolysis solution at 100 ° C to inactivate the enzyme to terminate the reaction. This is the first hydrolysis reaction.
  • bovine testicular hyaluronidase BTH with a final concentration of 4000 U / mL was added to hydrolyze at 37 ° C for 10 hours, and the enzyme was boiled at 100 ° C to remove the impurities, and ultra-high performance liquid chromatography was used in series.
  • the polarized time of flight mass spectrometer (MALDI SYNAPT MS) analysis and identification of the hydrolysate (the results are shown in Figure 1a and Figure 2a, b, c, d), and the molecular structure diagram ( Figure 3) was analyzed and speculated.
  • Odd sugar 3A sugar (HA 3 AA ), 3N sugar (HA 3 NN ), 5A sugar (HA 5 AA ), 5N sugar (HA 5 NN ), and even sugar disaccharide (HA2), tetrasaccharide (HA4), six Sugar (HA6).
  • the sugars collected by the ion column were: 3N sugar, disaccharide (HA2), 5N sugar, four Sugar (HA4), 3A sugar, hexasaccharide (HA6), 5A sugar, see Figure 4a.
  • the sample was lyophilized to obtain the odd sugars 3A sugar (HA 3 AA ), 3N sugar (HA 3 NN ), 5A sugar (HA 5 AA ), and 5N sugar (HA 5 NN ).
  • the yield of sugar was 75% to 90%, as measured by the carbazole sulfate method, and all of them had a high purity.
  • the macromolecular hyaluronic acid powder with a molecular weight of 10 5 to 10 6 Da was dissolved in pure water at a concentration of 10 mg / mL and fully dissolved overnight; the BTH was added at the same time to make the final enzyme concentration of 4000 U / mL and the final concentration of 5000U / mL LHase was hydrolyzed at 37 ° C for 10 hours, and the hydrolyzate was boiled at 100 ° C to inactivate the enzyme to terminate the reaction, remove impurities, and analyze and identify by ultra performance liquid chromatography with a quadrupole time-of-flight mass spectrometer (MALDI SYNAPT MS).
  • MALDI SYNAPT MS quadrupole time-of-flight mass spectrometer
  • Hydrolysates see Figures 1b, 2a, b, c, and d. Analysis and identification of the hydrolysis products are mainly odd sugars 3A sugar (HA 3 AA ), 3N sugar (HA 3 NN ), 5A sugar (HA 5 AA ), and 5N sugar. (HA 5 NN ) and even sugar disaccharides (HA2), tetrasaccharides (HA4), and hexasaccharides (HA6). It can be seen from the peak size that the yield of the odd oligosaccharide in this example is different from that in Example 1.
  • the separation step is the same as in Example 1. Finally, the sample is lyophilized to obtain the odd sugars 3A sugar (HA 3 AA ), 3N sugar (HA 3 NN ), 5A sugar (HA 5 AA ), and 5N sugar (HA 5 NN ).
  • the purity of the above oligosaccharides measured by the carbazole sulfate method is 75% to 90%, all of which reach a higher purity.
  • Example 3 Preparation of Odd Hyaluronic Acid 3A Sugar (HA 3 AA ), 3N Sugar (HA 3 NN ), 5A Sugar (HA 5 AA ), 5N (HA 5 NN ) Sugar 3
  • the hydrolysis order of the two enzymes is changed, that is, firstly hydrolyzed with bovine testis-type hyaluronidase (BTH) and then hydrolyzed with hyaluronine-type hyaluronidase.
  • BTH bovine testis-type hyaluronidase
  • hyaluronine-type hyaluronidase The remaining operation steps and conditions are the same as in Example 1.
  • the products were analyzed by ultra performance liquid chromatography with a quadrupole time-of-flight mass spectrometer (MALDI SYNAPT MS).
  • MALDI SYNAPT MS quadrupole time-of-flight mass spectrometer
  • IC ion current chromatograms
  • Odd sugar 3A sugar (HA 3 AA ), 3N sugar (HA 3 NN ), 5A sugar (HA 5 AA ), 5N sugar (HA 5 NN ) and even sugar disaccharide (HA2), tetrasaccharide (HA4), hexasaccharide (HA6). It can be seen from the peak size that the yield of the odd oligosaccharides in this example is different from that in Examples 1 and 2.
  • the separation step is the same as in Example 1. Finally, the sample is lyophilized to obtain the odd sugars 3A sugar (HA 3 AA ), 3N sugar (HA 3 NN ), 5A sugar (HA 5 AA ), and 5N sugar (HA 5 NN ).
  • the purity of the above oligosaccharides measured by the carbazole sulfate method is 75% to 90%, all of which reach a higher purity.
  • the macromolecular hyaluronic acid powder with a molecular weight of 10 5 to 10 6 Da was dissolved in pure water at a concentration of 10 mg / mL and fully dissolved overnight; firstly, LHase was added to make the final enzyme concentration of 5000 U / mL, and the solution was hydrolyzed at 37 ° C. For 6 hours, the hydrolyzate was boiled at 100 ° C to inactivate the enzyme to terminate the reaction. This is the first hydrolysis reaction.
  • BTH was added at a final concentration of 4000 U / mL to hydrolyze at 37 ° C for 6 hours, and the enzyme was boiled at 100 ° C to eliminate the enzyme, and the impurities were removed by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.
  • MALDI SYNAPT MS analysis and identification of the hydrolysate, see Figure 1d, 2c, d, e, f), and analysis and speculation of its molecular structure ( Figure 3).
  • Solution A was used to balance one column volume with a flow rate of 3 mL / min; solution B was subjected to linear elution so that the NaCI concentration in the eluent was 200 mM, the elution volume was 3 column volumes and the flow rate was 3 mL / min. Peak.
  • the sample solution was concentrated and then desalted through a Superdex 30Increase 10 / 300GL gel column. The product was detected by MALDI SYNAPT MS. The collected peaks were identified as 5N (HA 5 NN ) sugar, 7N sugar (HA 7 NN ), and hexasaccharide.
  • the purity of the above oligosaccharides measured by the carbazole sulfate method is 70% to 85%, all of which have reached a higher purity.
  • the hydrolysis order of the two enzymes is changed, that is, firstly hydrolyzed with LHase, and then with BTH.
  • the rest of the operating steps and conditions are the same as in Example 4, and the odd sugar 5A sugar (HA 5 AA ), 5N (HA 5 NN ) sugar, 7A sugar (HA 7 AA ), 7N sugar (HA 7 NN ), the purity of the above oligosaccharides measured by the carbazole sulfate method is 70% to 85%, all of which have a higher purity.
  • the hydrolysis order of the two enzymes is changed, that is, LHase and BTH are used for hydrolysis at the same time.
  • the remaining operation steps and conditions are the same as those in Example 4, and the odd sugar 5A sugar (HA 5 AA ) and 5N (HA 5 NN ) can also be prepared.
  • the sugars, 7A sugars (HA 7 AA ), 7N sugars (HA 7 NN ), the purity of the above oligosaccharides measured by the carbazole sulfate method were 70% to 85%, and all reached high purity.
  • the macromolecular hyaluronic acid powder with a molecular weight of 10 5 to 10 6 Da was dissolved in pure water at a concentration of 20 mg / mL, and was fully dissolved overnight; the hydrolysis process was preceded by the addition of a leech type hyaluronidase LHase to a final enzyme concentration of 7000 U. / mL, hydrolyzed at 37 ° C for 3 hours, and boiled the hydrolyzate at 100 ° C to inactivate the enzyme to terminate the reaction. This is the first hydrolysis reaction.
  • bovine testicular hyaluronidase BTH with a final concentration of 1000 U / mL was added for hydrolysis for 15 hours, and the enzyme was boiled at 100 ° C to eliminate the impurities.
  • the analysis and identification of the hydrolysate indicated that the main sugar was 3A sugar. (HA 3 AA ), 3N sugar (HA 3 NN ), 5A sugar (HA 5 AA ), 5N sugar (HA 5 NN ), and even sugar disaccharides, tetrasaccharides, and hexasaccharides.
  • the separation step is the same as in Example 1. Finally, the sample is lyophilized to obtain the odd sugars 3A sugar (HA 3 AA ), 3N sugar (HA 3 NN ), 5A sugar (HA 5 AA ), and 5N sugar (HA 5 NN ). .
  • a macromolecular hyaluronic acid powder having a molecular weight of 10 5 to 10 6 Da was dissolved in pure water at a concentration of 2 mg / mL, and the rest of the operating steps and conditions were the same as those in Example 1.
  • An ultra high performance liquid chromatography was used in tandem with a quadrupole. Analysis and identification of the hydrolysate by time-of-flight mass spectrometry (MALDI SYNAPT MS), an odd number of hyaluronic acid oligosaccharides can be detected, but the content is too low, and the preparation efficiency is low.
  • MALDI SYNAPT MS time-of-flight mass spectrometry
  • a macromolecular hyaluronic acid powder having a molecular weight of 10 5 to 10 6 Da was dissolved in pure water at a concentration of 50 mg / mL, and the rest of the operating steps and conditions were the same as in Example 1.
  • a quadrupole was connected in series using ultra high performance liquid chromatography.
  • Time-of-flight mass spectrometry (MALDI SYNAPT MS) analysis identified the hydrolysate. The substrate could not be effectively hydrolyzed and no odd number of hyaluronic acid oligosaccharide was obtained.

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Abstract

一种双酶水解制备透明质酸奇数寡糖的方法，属于生物工程技术领域。以大分子透明质酸为底物，通过控制水蛭型透明质酸酶(LHase)和牛睾丸型透明质酸酶(BTH)两种透明质酸水解酶的添加量和反应时间，同时制备得到两种不同结构的奇数聚合度透明质酸寡糖(三、五、七糖)。提出来一种新的、简单易行的制备奇数聚合度透明质酸寡糖的方法，为奇数聚合度透明质酸寡糖的功能及特性研究奠定了基础。

Description

一种双酶水解制备透明质酸奇数寡糖的方法 技术领域
本发明涉及一种双酶水解制备透明质酸奇数寡糖的方法，属于生物工程技术领域。
背景技术
透明质酸(HA)是一种由D-葡糖醛酸(GlcUA)和N-乙酰氨基葡萄糖(GlcNAc)通过β-1,3糖苷键连接，并以β-1,4糖苷键连接形成的无支链双糖重复单元的粘性多糖(Mr＝10 5～10 6Da)，是动物组织细胞间质的主要成分，是持水能力最强的天然物质。由于其独特的性质广泛应用于医药、临床诊治、化妆品和食品保健行业。近些年来发现不同分子质量的HA具有不同的生物活性，透明质酸寡糖(o-HAs,Mr＝10kDa以内)具有免疫活性、促进血管内皮细胞增殖和逆转肿瘤细胞多药耐药性等作用。
随着透明质酸寡糖(o-HAs)应用越来越广泛，近年来，国内外开始重视HA的降解及其降解产物的制备研究。目前，HA降解的方法主要有物理降解、化学降解、生物降解3大方法。其中生物降解因条件温和、无毒无害应用最为广泛。生物降解常用的方法是酶法水解。水解酶中对透明质酸(HA)专一性最佳、水解效率最高的有牛睾丸型透明质酸酶(BTH)、水蛭型透明质酸酶(LHase)两种。牛睾丸型透明质酸酶(BTH)水解HA中β-1,4糖苷键生成以氨基葡萄糖为还原性末端的偶数寡糖；水蛭型透明质酸酶(LHase)水解HA中β-1,3糖苷键生成以葡糖醛酸为还原性末端的偶数寡糖。两种酶分别作用于透明质酸的两种不同的糖苷键，生成两种还原端不同的透明质酸偶数寡糖系列。
由于以往酶法制备透明质酸寡糖多为单酶水解，产物绝大多数为偶数聚合度寡糖，所以其在医药等方面的应用研究也主要针对偶数聚合度透明质酸寡糖。奇数聚合度透明质酸寡糖(即聚合度为奇数的透明酸寡糖)作为一大类透明质酸寡糖，其制备及功能研究几乎为空白。因此，研究奇数聚合度寡糖的制备，对于透明质酸寡糖家族的研究发展、透明质酸寡糖新功能的探索应用，具有重要意义。
发明内容
本发明要解决的技术问题是提供一种奇数聚合度透明质酸寡糖的制备方法。尤其是利用牛睾丸型透明质酸酶和水蛭型透明质酸酶两种酶水解制备奇数聚合度透明质酸寡糖的方法。
本发明提供一种奇数聚合度透明质酸寡糖的制备方法，具体技术方案包括以下步骤：
(1)水解透明质酸：以分子量为10 5～10 6Da的透明质酸作为底物，使用水蛭型透明质酸酶(LHase)和牛睾丸型透明质酸酶(BTH)两种透明质酸水解酶作用于底物，得到一系列不 同奇数聚合度透明质酸寡糖的混合物；
(2)分离不同聚合度透明质酸寡糖：利用填充了凝胶的离子交换柱将步骤(1)得到的透明质酸寡糖进行分离纯化，具体操作为：用平衡液处理离子交换柱、洗脱液洗脱并按照目的产物的洗脱峰收集产物，产物经过浓缩、脱盐即可得到奇数聚合度的透明质酸寡糖；
其中，所述两种酶的水解顺序不限。
在本发明的一种实施方式中，所述方法包括以下步骤：
(1)水解透明质酸：以分子量为10 5～10 6Da的透明质酸作为底物，先加入水蛭型透明质酸酶LHase进行水解，灭酶后，再加入牛睾丸型透明质酸酶BTH进行水解，灭酶，得到一系列不同奇数聚合度透明质酸寡糖的混合物；
(2)分离不同聚合度透明质酸寡糖：利用凝胶填充的离子交换柱将步骤(1)得到的透明质酸寡糖进行分离纯化，得到不同的奇数聚合度透明质酸寡糖。
在本发明的一种实施方式中，所述方法包括以下步骤：
(1)水解透明质酸：以分子量为10 5～10 6Da的透明质酸作为底物，先加入牛睾丸型透明质酸酶BTH进行水解，灭酶后，再加入水蛭型透明质酸酶LHase进行水解，灭酶，得到一系列不同奇数聚合度透明质酸寡糖的混合物；
(2)分离不同聚合度透明质酸寡糖：利用填充了凝胶的离子交换柱将步骤(1)得到的透明质酸寡糖进行分离纯化，得到不同的奇数聚合度透明质酸寡糖。
在本发明的一种实施方式中，所述方法包括以下步骤：
(1)水解透明质酸：以分子量为10 5～10 6Da的透明质酸作为底物，同时加入牛睾丸型透明质酸酶BTH和水蛭型透明质酸酶LHase进行水解，灭酶，得到一系列不同奇数聚合度透明质酸寡糖的混合物；
(2)分离不同聚合度透明质酸寡糖：利用凝胶填充的离子交换柱将步骤(1)得到的透明质酸寡糖进行分离纯化，得到不同的奇数聚合度透明质酸寡糖。
在本发明的一种实施方式中，所述利用凝胶填充的离子交换柱将步骤(1)得到的透明质酸寡糖进行分离纯化的具体步骤为：装柱、平衡、上样、洗脱、根据产物峰收集产物，浓缩、脱盐、冷冻干燥得到奇数聚合度透明质酸寡糖。
在本发明的一种实施方式中，所述透明质酸以溶液形式与酶进行反应，透明质酸溶液的浓度为10～20mg/mL，其溶剂为水。
在本发明的一种实施方式中，步骤(1)中所述水蛭型透明质酸酶的添加终浓度为5000～7000U/mL，所述牛睾丸型透明质酸酶的添加终浓度为1000～4000U/mL。
在本发明的一种实施方式中，步骤(1)中所述水蛭型透明质酸酶作用时间为1～15h，所述牛睾丸型透明质酸酶作用时间为1～15h。
在本发明的一种实施方式中，所述的填充了凝胶的离子交换柱为填充了Q琼脂糖凝胶HP(QHP)的离子交换柱。
在本发明的一种实施方式中，所述平衡所用的平衡液为pH为7～9的Tris-HCl溶液，洗脱所用的洗脱液为由平衡液配置的浓度为50～300mM的NaCl溶液。
在本发明的一种实施方式中，洗脱体积为2～20倍的柱体积，优选10～20倍。
在本发明的一种实施方式中，洗脱过程采用线性洗脱，所述线性洗脱为先用浓度为0的洗脱液，逐渐增大到相应浓度的洗脱液(由平衡液配置的浓度为50～300mM的NaCl溶液)进行洗脱。
在本发明的一种实施方式中，所述脱盐方法为分子排阻凝胶柱或透析袋脱盐中的任一种。
在本发明的一种实施方式中，所述分子排阻凝胶柱为Superdex 30 Increase 10/300 GL凝胶柱，所述透析袋规格为0.5-1.0KD。
在本发明的一种实施方式中，所述方法主要包括以下步骤：
(1)水解透明质酸
以浓度为5～20mg/mL、分子量10 5～10 6Da的透明质酸水溶液为底物；水解过程首先添加终浓度为5000～7000U/mL的水蛭型透明质酸酶(LHase)水解1～10小时；将上述酶灭活后，添加终浓度为1000～4000U/mL的牛睾丸型透明质酸酶(BTH)水解1～10小时，煮沸灭酶，0.22μm滤膜过滤；
(2)分离透明质酸寡糖
利用离子交换柱分离步骤(1)0.22μm滤膜过滤得到的滤液中的透明质酸寡糖，平衡液为pH＝6～8的Tris-HCl缓冲液，洗脱液为由平衡液配置的浓度为50～300mM的NaCl溶液；具体操作为：采用Q琼脂糖凝胶HP填料装柱，利用平衡液平衡，平衡液流速为2～5mL/min，然后，上样、洗脱，其中，洗脱策略为浓度从0到50～300mM的NaCl溶液进行线性洗脱，洗脱体积为10～20倍的柱体积，洗脱液流速为2～6mL/min，收集并浓缩产物，通过分子排阻凝胶柱或透析袋进行脱盐，冷冻干燥即可得到产物中的奇数聚合度的透明质酸寡糖。
在本发明的一种实施方式中，所述方法主要包括以下步骤：
(1)水解透明质酸
以浓度为5～20mg/mL、分子量10 5～10 6Da的透明质酸水溶液为底物；水解过程首先添加终浓度为1000～4000U/mL的牛睾丸型透明质酸酶(BTH)水解1～10小时；将上述酶灭活后， 添加终浓度为5000～7000U/mL的水蛭型透明质酸酶(LHase)水解1～10小时，煮沸灭酶，0.22μm滤膜过滤；
(2)分离透明质酸寡糖
利用离子交换柱分离步骤(1)0.22μm滤膜过滤得到的滤液中的透明质酸寡糖，平衡液为pH＝6～8的Tris-HCl缓冲液，洗脱液为由平衡液配置的浓度为50～300mM的NaCl溶液；具体操作为：采用Q琼脂糖凝胶HP填料装柱，利用平衡液平衡，平衡液流速为2～5mL/min，然后，上样、洗脱，其中，洗脱策略为浓度从0到50～300mM的NaCl溶液进行线性洗脱，洗脱体积为10～20倍的柱体积，洗脱液流速为2～6mL/min，收集并浓缩产物，通过分子排阻凝胶柱或透析袋进行脱盐，冷冻干燥即可得到产物中的奇数聚合度的透明质酸寡糖。
在本发明的一种实施方式中，所述方法主要包括以下步骤：
(1)水解透明质酸
以浓度为5～20mg/mL、分子量10 5～10 6Da的透明质酸水溶液为底物；水解过程同时加入添加终浓度为1000～4000U/mL的牛睾丸型透明质酸酶(BTH)和终浓度为5000～7000U/mL的水蛭型透明质酸酶(LHase)水解1～10小时，煮沸灭酶，0.22μm滤膜过滤；
(2)分离透明质酸寡糖
利用离子交换柱分离步骤(1)0.22μm滤膜过滤得到的滤液中的透明质酸寡糖，平衡液为pH＝6～8的Tris-HCl缓冲液，洗脱液为由平衡液配置的浓度为50～300mM的NaCl溶液；具体操作为：采用Q琼脂糖凝胶HP填料装柱，利用平衡液平衡，平衡液流速为2～5mL/min，然后，上样、洗脱，其中，洗脱策略为浓度从0到50～300mM的NaCl溶液进行线性洗脱，洗脱体积为10～20倍的柱体积，洗脱液流速为2～6mL/min，收集并浓缩产物，通过分子排阻凝胶柱或透析袋进行脱盐，冷冻干燥即可得到产物中的奇数聚合度的透明质酸寡糖。
本发明还提供了一种奇数聚合度透明质酸寡糖，所述奇数聚合度透明质酸寡糖的结构为：
Figure PCTCN2019100409-appb-000001
其中，n＝1，2，3。
在本发明的一种实施方式中，所述奇数聚合度透明质酸寡糖由上述任一种制备奇数聚合度透明质酸寡糖的方法制备得到。
本发明的有益效果：
(1)通过利用两种水解酶先后作用的新型制备方法，可以同时制备得到两种类型的奇数透明质酸寡糖(A型和N型，还原端分别为GlcUA和GlcNAc)；反应条件温和，反应过程安全；通过对反应时间、加酶量的控制，可以实现目标聚合度奇数透明质酸寡糖的制备；制 备得到的产物较纯。
(2)本发明制备得到了新的N型奇数透明质酸寡糖，不仅丰富了寡糖结构的多样性，而且对于开展不同结构类型的透明质酸寡糖与疾病的发生、发展的关系等具有十分重要的意义。
附图说明
图1：a:实施例1得到的水解产物的LCMS离子色谱图；b:实施例2得到的水解产物的LCMS离子色谱图；c:实施例3得到的水解产物的LCMS离子色谱图；d:实施例4得到的水解产物的LCMS离子色谱图。
图2：水解液中目标产物的质谱图：(a)3N糖(HA 3 NN)；(b)3A糖(HA 3 AA)；(c)5N(HA 5 NN)；(d)5A糖(HA 5 AA)；(e)7N(HA 7 NN)；(f)7A糖(HA 7 AA)。
图3：奇数透明质酸寡糖的分子结构图列举以及其结构通式，其中，(a)3N糖(HA 3 NN)结构图；(b)3A糖(HA 3 AA)结构图；(c)5N糖(HA 5 NN)结构图；(d)5A糖(HA 5 AA)结构图；(e)7N糖(HA 7 NN)结构图；(f)7A糖(HA 7 AA)结构图()；(g)为N型奇数透明质酸寡糖通式和A型奇数透明质酸寡糖通式。
图4：a：阴离子交换柱分离透明质酸二糖、3A糖(HA 3 AA)、3N糖(HA 3 NN)、四糖(HA4)、5A糖(HA 5 AA)、5N糖(HA 5 NN)、六糖(HA6)；b：阴离子交换柱分离透明质酸5A糖(HA 5 AA)、5N糖(HA 5 NN)、六糖、7A糖(HA 7 AA)、7N糖(HA 7 NN)、八糖(HA8)。
具体实施方式
水蛭型透明质酸酶为本实验室由毕赤酵母基因工程菌重组表达水蛭型透明质酸酶基因得到的，核苷酸序列如序列SEQ ID NO.1所示；牛睾丸型透明质酸酶从西格玛奥德里奇(上海)贸易有限公司购买。
水解产物混合寡糖的分离制备方法：层析柱：阴离子交换柱HiTrap QHP(20mL)；流动相：Tris-HCl(pH＝8.0)；洗脱液：0～300mmol/LNaCl；流速:3mL/min；检测波长：210nm。分子排阻凝胶柱：Superdex 30 Increase 10/300 GL column(柱尺寸10mm×300-310mm)，柱体积24mL，柱效率>43 000N/m；填料床的典型压降3.0MPa，柱硬件压力限制5.0MPa。
产物纯度的检测方法使用咔唑硫酸法检测。
实施例1：奇数透明质酸3A糖(HA 3 AA)、3N糖(HA 3 NN)、5A糖(HA 5 AA)、5N(HA 5 NN)糖的制备1
(1)大分子透明质酸的水解
将分子量为10 5～10 6Da的大分子透明质酸粉末，以10mg/mL的浓度溶解于纯水中，过夜 充分溶解；水解过程先加入水蛭型透明质酸酶LHase使酶终浓度为5000U/mL，37℃水解10小时，100℃煮沸水解液使酶灭活终止反应，此为第一步水解反应。上述水解液将上述酶灭活后，添加终浓度为4000U/mL的牛睾丸型透明质酸酶BTH在37℃水解10小时，100℃煮沸灭酶，除去杂质，利用超高效液相色谱串联四极杆飞行时间质谱联用仪(MALDI SYNAPT MS)分析鉴定水解产物(结果见图1a和图2a,b,c,d)，并分析推测其分子结构图(图3)，分析鉴定水解产物主要为奇数糖3A糖(HA 3 AA)、3N糖(HA 3 NN)、5A糖(HA 5 AA)、5N糖(HA 5 NN)和偶数糖二糖(HA2)、四糖(HA4)、六糖(HA6)。
(2)透明质酸寡糖的分离
配制A液(平衡溶液):pH＝8、50mM Tris-HCI缓冲液；B液(洗脱溶液)：由A液配制的80mM的NaCl缓冲溶液，通过以下条件进行透明质寡的分离：以3mL/min的速度用1个柱体积的A液平衡HiTrap QHP(5mL)离子交换柱(高径3:1)；用1mL/min流速上样。用A液洗去未结合的样品，洗脱3个柱体积，流速3mL/min；B液进行线性洗脱(先用浓度为0的B液，逐渐增大到由A液配制的80mM的NaCl的B液进行洗脱)，洗脱体积为1个柱体积，流速为3mL/min，依次收集所得到的峰。浓缩得到样液，再通过Superdex 30 Increase 10/300 GL column凝胶柱进行脱盐，产物通过MALDI SYNAPT MS检测，离子柱收集到的糖依次为：3N糖、二糖(HA2)、5N糖、四糖(HA4)、3A糖、六糖(HA6)、5A糖，见图4a。最后将样品冻干，即可得到奇数糖3A糖(HA 3 AA)、3N糖(HA 3 NN)、5A糖(HA 5 AA)、5N糖(HA 5 NN)，通过硫酸咔唑法测定寡糖的得率，通过硫酸咔唑法测得上述寡糖的纯度为75％～90％，均达到较高的纯度。
实施例2：奇数透明质酸3A糖(HA 3 AA)、3N糖(HA 3 NN)、5A糖(HA 5 AA)、5N(HA 5 NN)糖的制备2
(1)大分子透明质酸的水解
将分子量为10 5～10 6Da的大分子透明质酸粉末，以10mg/mL的浓度溶解于纯水中，过夜充分溶解；水解过程同时加入BTH使酶终浓度为4000U/mL和终浓度为5000U/mL的LHase，37℃水解10小时，100℃煮沸水解液使酶灭活终止反应，除去杂质，利用超高效液相色谱串联四极杆飞行时间质谱联用仪(MALDI SYNAPT MS)分析鉴定水解产物(见图1b、2a,b,c,d)，分析鉴定水解产物主要为奇数糖3A糖(HA 3 AA)、3N糖(HA 3 NN)、5A糖(HA 5 AA)、5N糖(HA 5 NN)和偶数糖二糖(HA2)、四糖(HA4)、六糖(HA6)。通过峰的大小可以看到，本例中的奇数寡糖的得率与实施例1中的不同。
(2)不同聚合度透明质酸寡糖的分离
分离步骤与实施例1相同，最后将样品冻干，即可得到奇数糖3A糖(HA 3 AA)、3N糖(HA 3 NN)、5A糖(HA 5 AA)、5N糖(HA 5 NN)，通过硫酸咔唑法测得上述寡糖的纯度为75％～90％，均达到较高的纯度。
实施例3：奇数透明质酸3A糖(HA 3 AA)、3N糖(HA 3 NN)、5A糖(HA 5 AA)、5N(HA 5 NN)糖的制备3
(1)大分子透明质酸的水解
此实施例将两种酶的水解顺序改变，即先用牛睾丸型透明质酸酶(BTH)、后用水蛭型透明质酸酶水解，其余操作步骤和条件和实施例1相同，制备得到的产物用超高效液相色谱串联四极杆飞行时间质谱联用仪(MALDI SYNAPT MS)分析，离子流色谱图(IC)见图1c，图2a,b,c,d，分析鉴定水解产物主要为奇数糖3A糖(HA 3 AA)、3N糖(HA 3 NN)、5A糖(HA 5 AA)、5N糖(HA 5 NN)和偶数糖二糖(HA2)、四糖(HA4)、六糖(HA6)。通过峰的大小可以看到，本例中的奇数寡糖的得率与实施例1、2中的不同。
(2)透明质酸寡糖的分离
分离步骤与实施例1相同，最后将样品冻干，即可得到奇数糖3A糖(HA 3 AA)、3N糖(HA 3 NN)、5A糖(HA 5 AA)、5N糖(HA 5 NN)，通过硫酸咔唑法测得上述寡糖的纯度为75％～90％，均达到较高的纯度。
实施例4：奇数透明质酸5A糖(HA 5 AA)、5N糖(HA 5 NN)、7A糖(HA 7 AA)、7N糖(HA 7 NN)、的制备
(1)大分子透明质酸的水解
将分子量为10 5～10 6Da的大分子透明质酸粉末，以10mg/mL的浓度溶解于纯水中，过夜充分溶解；水解过程先加入LHase使酶终浓度为5000U/mL，37℃水解6小时，100℃煮沸水解液使酶灭活终止反应，此为第一步水解反应。上述水解液将上述酶灭活后，添加终浓度为4000U/mL的BTH在37℃水解6小时，100℃煮沸灭酶，除去杂质，利用超高效液相色谱串联四极杆飞行时间质谱联用仪(MALDI SYNAPT MS)分析鉴定水解产物,见图1d、2c,d,e,f)，并分析推测其分子结构图(图3)。主要为奇数糖5A糖(HA 5 AA)、5N(HA 5 NN)糖、7A糖(HA 7 AA)、7N糖(HA 7 NN)和偶数糖六糖(HA6)、八糖(HA8)。
(2)透明质酸寡糖的分离
配制A液(平衡溶液):pH＝8，浓度50mM Tris-HCI缓冲液；B液(洗脱溶液):由A液配制的200mM的NaCl缓冲溶液，通过以下条件进行透明质酸寡糖的分离：以3mL/min的速度用1个柱体积的A液平衡HiTrap QHP(20mL)离子交换柱(高径3:1)；用1mL/min流速 上样。用A液平衡1个柱体积，流速3mL/min；B液进行线性洗脱，使洗脱液中NaCI浓度为200mM，洗脱体积为3个柱体积，流速为3mL/min，依次收集所得到的峰。浓缩得到样液，再通过Superdex 30Increase 10/300GL凝胶柱进行脱盐，产物通过MALDI SYNAPT MS检测，鉴定收集到的峰分别为5N(HA 5 NN)糖、7N糖(HA 7 NN)、六糖、5A糖(HA 5 AA)、八糖、7A糖(HA 7 AA)，见图4b，通过硫酸咔唑法测得上述寡糖的纯度为70％～85％，均达到较高的纯度。
实施例5：奇数透明质酸5A糖(HA 5 AA)、5N(HA 5 NN)糖、7A糖(HA 7 AA)、7N糖(HA 7 NN)、的制备
此实施例将两种酶的水解顺序改变，即先用LHase水解、后用BTH水解，其余操作步骤和条件和实施例4相同，同样可制备奇数糖5A糖(HA 5 AA)、5N(HA 5 NN)糖、7A糖(HA 7 AA)、7N糖(HA 7 NN)，通过硫酸咔唑法测得上述寡糖的纯度为70％～85％，均达到较高的纯度。
实施例6：奇数透明质酸5A糖(HA 5 AA)、5N(HA 5 NN)糖、7A糖(HA 7 AA)、7N糖(HA 7 NN)、的制备
此实施例将两种酶的水解顺序改变，即同时使用LHase和BTH水解，其余操作步骤和条件和实施例4相同，同样可制备奇数糖5A糖(HA 5 AA)、5N(HA 5 NN)糖、7A糖(HA 7 AA)、7N糖(HA 7 NN)，通过硫酸咔唑法测得上述寡糖的纯度为70％～85％，均达到较高的纯度。
实施例7
将分子量为10 5～10 6Da的大分子透明质酸粉末，以20mg/mL的浓度溶解于纯水中，过夜充分溶解；水解过程先加入水蛭型透明质酸酶LHase使酶终浓度为7000U/mL，37℃水解3小时，100℃煮沸水解液使酶灭活终止反应，此为第一步水解反应。上述水解液将上述酶灭活后，添加终浓度为1000U/mL的牛睾丸型透明质酸酶BTH水解15小时，100℃煮沸灭酶，除去杂质，分析鉴定水解产物可知主要为奇数糖3A糖(HA 3 AA)、3N糖(HA 3 NN)、5A糖(HA 5 AA)、5N糖(HA 5 NN)和偶数糖二糖、四糖、六糖。
(2)透明质酸寡糖的分离
分离步骤与实施例1相同，最后将样品冻干，即可得到奇数糖3A糖(HA 3 AA)、3N糖(HA 3 NN)、5A糖(HA 5 AA)、5N糖(HA 5 NN)。
对比例1：透明质酸浓度的影响
将分子量为10 5～10 6Da的大分子透明质酸粉末，以2mg/mL的浓度溶解于纯水中，其余操作步骤和条件与实施例1相同，利用超高效液相色谱串联四极杆飞行时间质谱联用仪(MALDI SYNAPT MS)分析鉴定水解产物，能检测到奇数透明质酸寡糖，但是含量过低， 制备效率低。
对比例2：透明质酸浓度的影响
将分子量为10 5～10 6Da的大分子透明质酸粉末，以50mg/mL的浓度溶解于纯水中，其余操作步骤和条件与实施例1相同，利用超高效液相色谱串联四极杆飞行时间质谱联用仪(MALDI SYNAPT MS)分析鉴定水解产物，底物未能被有效水解未得到奇数透明质酸寡糖。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2019100409-appb-000002
Figure PCTCN2019100409-appb-000003
Figure PCTCN2019100409-appb-000004
Figure PCTCN2019100409-appb-000005

Claims (10)

  1. 一种制备奇数聚合度透明质酸寡糖的方法，其特征在于，所述方法包括以下步骤：
    (1)水解透明质酸：使用水蛭型透明质酸酶LHase与牛睾丸型透明质酸酶BTH两种透明质酸水解酶分别作用于分子量为10 5～10 6Da的透明质酸得到不同奇数聚合度的透明质酸寡糖的混合物；
    (2)分离不同聚合度的透明质酸寡糖：利用填充了凝胶的离子交换柱将步骤(1)得到的不同奇数聚合度的透明质酸寡糖的混合物进行分离纯化，分离纯化过程具体为：用平衡液处理离子交换柱、洗脱液洗脱并按照目的产物的洗脱峰收集产物，产物经过浓缩、脱盐即可得到奇数聚合度的透明质酸寡糖；
    其中，所述两种酶的水解顺序不限。
  2. 根据权利要求1所述的一种制备奇数聚合度透明质酸寡糖的方法，其特征在于，所述透明质酸以溶液形式与酶进行反应，透明质酸溶液的浓度为10～20mg/mL，其溶剂为水。
  3. 根据权利要求2所述的一种制备奇数聚合度透明质酸寡糖的方法，其特征在于，所述步骤(1)中所述水蛭型透明质酸酶的添加终浓度为5000～7000U/mL，作用时间为1～15h；所述牛睾丸型透明质酸酶的添加终浓度为1000～4000U/mL，作用时间为1～15h。
  4. 根据权利要求1-3任一所述的一种制备奇数聚合度透明质酸寡糖的方法，其特征在于，所述步骤(2)中的脱盐方法为凝胶柱或透析袋脱盐中的任一种。
  5. 根据权利要求1-4任一所述的一种制备奇数聚合度透明质酸寡糖的方法，其特征在于，所述步骤(2)中填充了凝胶的离子交换柱为填充了Q琼脂糖凝胶HP的阴离子交换柱。
  6. 根据权利要求1-5任一所述的一种制备奇数聚合度透明质酸寡糖的方法，其特征在于，所述步骤(2)中平衡液为pH为7～9的Tris-HCl溶液，洗脱液为由平衡液配置的浓度为50～300mM的NaCl溶液。
  7. 根据权利要求1-6任一所述的一种制备奇数聚合度透明质酸寡糖的方法，其特征在于，所述步骤(2)中洗脱过程采用线性洗脱，洗脱体积为10～20倍的柱体积。
  8. 根据权利要求1-7任一所述的一种制备奇数聚合度透明质酸寡糖的方法，其特征在于，所述方法主要包括以下步骤：
    (1)水解透明质酸
    以浓度为5～20mg/mL、分子量为10 5～10 6Da的透明质酸水溶液为底物；水解过程首先添加终浓度为5000～7000U/mL的水蛭型透明质酸酶水解1～10h；将上述酶灭活后，添加终浓度为1000～4000U/mL的牛睾丸型透明质酸酶水解1～10h，煮沸灭酶，0.22μm滤膜过滤；
    (2)分离透明质酸寡糖
    利用离子交换柱分离步骤(1)0.22μm滤膜过滤得到的滤液中的透明质酸寡糖，平衡液 为pH＝6～8的Tris-HCl缓冲液，洗脱液为由平衡液配置的浓度为50～300mM的NaCl溶液；具体操作为：采用Q琼脂糖凝胶HP填料装柱，利用平衡液平衡，平衡液流速为2～5mL/min，然后，上样、洗脱，其中，洗脱策略为线性洗脱，洗脱体积为10～20倍的柱体积，洗脱液流速为2～6mL/min，收集并浓缩产物，通过凝胶柱进行脱盐，冷冻干燥即可得到产物中的奇数聚合度的透明质酸寡糖。
  9. 根据权利要求8所述的一种制备奇数聚合度透明质酸寡糖的方法，其特征在于，交换步骤(1)中两种酶的使用顺序。
  10. 权利要求1～9任一所述的一种制备奇数聚合度透明质酸寡糖的方法在医药领域或化妆品领域中的应用。
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101283979A (zh) * 2008-06-05 2008-10-15 上海交通大学 促进伤口愈合的透明质酸小片段药膏的制备方法
CN102876748A (zh) * 2012-04-13 2013-01-16 华熙福瑞达生物医药有限公司 酶切法制备寡聚透明质酸盐的方法及所得寡聚透明质酸盐和其应用
CN104610467A (zh) * 2015-01-27 2015-05-13 江南大学 一种透明质酸四糖和六糖的分离方法
CN105524188A (zh) * 2015-12-14 2016-04-27 中国海洋大学 一种透明质酸奇数寡糖单体及其制备方法
JP2016141697A (ja) * 2015-01-29 2016-08-08 学校法人東京電機大学 修飾ヒアルロン酸及び／又はその塩、並びにその製造方法
CN106518934A (zh) * 2016-11-21 2017-03-22 苏州大学 不饱和透明质酸奇数寡糖及其制备方法
CN109097421A (zh) * 2018-08-14 2018-12-28 江南大学 一种双酶水解制备透明质酸奇数寡糖的方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5902795A (en) * 1992-06-16 1999-05-11 Trustees Of Tufts College Oligosaccharides reactive with hyaluronan-binding protein and their methods of use
WO2013172923A1 (en) * 2012-05-15 2013-11-21 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Uses of antagonists of hyaluronan signaling

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101283979A (zh) * 2008-06-05 2008-10-15 上海交通大学 促进伤口愈合的透明质酸小片段药膏的制备方法
CN102876748A (zh) * 2012-04-13 2013-01-16 华熙福瑞达生物医药有限公司 酶切法制备寡聚透明质酸盐的方法及所得寡聚透明质酸盐和其应用
CN104610467A (zh) * 2015-01-27 2015-05-13 江南大学 一种透明质酸四糖和六糖的分离方法
JP2016141697A (ja) * 2015-01-29 2016-08-08 学校法人東京電機大学 修飾ヒアルロン酸及び／又はその塩、並びにその製造方法
CN105524188A (zh) * 2015-12-14 2016-04-27 中国海洋大学 一种透明质酸奇数寡糖单体及其制备方法
CN106518934A (zh) * 2016-11-21 2017-03-22 苏州大学 不饱和透明质酸奇数寡糖及其制备方法
CN109097421A (zh) * 2018-08-14 2018-12-28 江南大学 一种双酶水解制备透明质酸奇数寡糖的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NEBINGER, P. ET AL.,: "High-Performance Liquid Chromatographic Analysis of Even- and Odd-Numbered Hyaluronate Oligosaccharides,", JOURNAL OF CHROMATOGRAPHY, vol. 265, no. 1, 31 December 1983 (1983-12-31), pages 19 - 25, XP026483488, ISSN: 0021-9673, DOI: 20190903133928Y *

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