Preparation method and application of hirudo hyaluronidase expressed by pichia pastoris

Abstract

The invention discloses a preparation method and application of hirudo hyaluronidase expressed by pichia pastoris, and belongs to the technical field of bioengineering.The preparation method comprises the steps of fermentation, pretreatment, one-step chromatographic purification and filtration. The pretreatment comprises the following steps: mixing and stirring a hirudo hyaluronidase fermentation solution and calcium chloride, adjusting the pH value to 7.2-7.6, centrifuging, collecting a supernatant, carrying out suction filtration by a filter membrane, taking a filtrate, filtering by a primary ultrafiltration membrane, and taking a permeate as a loading solution; the one-step chromatographic purification comprises the step of carrying out one-step chromatographic purification on the loading solution to obtain a chromatographic purification solution; according to the method disclosed by the invention, the hyaluronidase with high enzyme activity can be obtained, and the purified hyaluronidase with high enzyme activity can be used for carrying out enzymolysis on macromolecular sodium hyaluronate to prepare low-molecular-weight and oligomeric sodium hyaluronate.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, and in particular to a preparation method and application of leech hyaluronidase expressed in Pichia pastoris. Background Art

[0002] Hyaluronic acid (HA), also known as hyaluronic acid, is a vital component of the human body. It is an unbranched, high-molecular-weight, acidic mucopolysaccharide composed of repeating disaccharide units of N-acetylglucosamine (GlcNAc) and D-glucuronic acid (GlcA) linked by β-1,4- and β-1,3-glycosidic bonds. It possesses strong hydrophilicity and excellent moisturizing properties. It is widely present in biological tissues such as skin, cartilage, joints, and vitreous humor, and has important physiological functions. Based on its molecular weight, HA can be divided into high-molecular-weight HA (HMWHA) and low-molecular-weight HA (LMWHA). HMWHA can inhibit endothelial cell migration, exhibit anti-angiogenic activity, and promote wound healing. LMWHA can promote endothelial cell differentiation, resist apoptosis, promote the migration of cartilage and endothelial cells, and also exhibit anti-inflammatory properties. Hyaluronic acid, with its unique physicochemical properties, has played a vital role in medical and pharmaceutical research.

[0003] Currently, low-molecular-weight hyaluronic acid is primarily produced by degrading macromolecular hyaluronic acid into low-molecular-weight hyaluronic acid through physical, chemical, and enzymatic degradation methods. Physical degradation methods generally fail to produce ultra-low-molecular-weight hyaluronic acid. Chemical degradation methods may contain residual chemical reagents and may modify the aldehyde or hydroxyl groups in the hyaluronic acid monomers, disrupting the hyaluronic acid structure.

[0004] Because the enzymatic hydrolysis method is highly specific, has mild reaction conditions, and the structure of the polysaccharide remains unchanged, different molecular weight hyaluronic acids can be obtained by controlling different degradation times, making it an ideal method for preparing low-molecular-weight hyaluronic acid. Hyaluronidase (HAase) is a glycosidase that can degrade high-molecular-weight hyaluronic acid into low-molecular-weight or oligomeric hyaluronic acid. Some hyaluronidases also have the ability to degrade chondroitin and heparin. In addition to being used to prepare low-molecular-weight or oligomeric hyaluronic acid, hyaluronidase can also be used as a drug dispersant to degrade the hyaluronic acid of tissues, increase the permeability of tissues, promote the diffusion of injections, and cause less pollution.

[0005] According to different hydrolysis mechanisms, hyaluronidase can be divided into three categories: testicular hyaluronidase, leech hyaluronidase and microbial hyaluronidase.

[0006] At present, there has been no breakthrough in the industrialization of hyaluronidase. There is no commercial enzyme on the market for the degradation of specific low-molecular-weight hyaluronic acid, and the source of hyaluronidase is very limited. The current commercialized bovine testicular hyaluronidase is produced by extracting from animal tissues. On the one hand, there are problems of limited sources and a large number of impurities, which can easily cause immune reactions; on the other hand, the high price greatly limits their application.

[0007] Microbial hyaluronidase is significantly superior to traditional animal-derived enzymes in terms of yield, purity, safety, and controllability, and has greater scalability advantages. Currently, there are two main ways to express microbial hyaluronidase: extracellular secretion, such as the production of hyaluronidase by Pichia pastoris and Bacillus fermentation; and intracellular expression, such as the production of hyaluronidase by Citrobacter rodentium and Escherichia coli fermentation. Hyaluronidase expressed by Pichia pastoris is an extracellular enzyme that does not require cell wall destruction, eliminating the tedious process of high-pressure crushing and a series of processes such as centrifugation after crushing. Its purification process is simpler than intracellular expression.

[0008] Chinese patent CN117448305A discloses a method for producing leech hyaluronidase by yeast fermentation and its application. The invention centrifuges the fermentation broth to obtain the supernatant, elutes the purified protein through a nickel column, and collects the elution peak to obtain a crude enzyme solution. After purification, the enzyme activity of the fermentation supernatant increases from 2.414×10 6 U / mL increased to 4.698×10 6 Although the enzyme activity in the fermentation supernatant is high in this invention, the improvement of the enzyme activity after purification is not high, indicating that the chromatography purification method of the present invention is not applicable to leech hyaluronidase.

[0009] Chinese patent CN112501088A discloses a Bacillus, a hyaluronidase produced thereby, and a production method. The invention centrifuges the cultured hyaluronidase fermentation broth at 10,000-15,000 rpm for 10-20 minutes to remove the precipitate to obtain a supernatant, precipitates the supernatant with ammonium sulfate, and then collects the crude protein by centrifugation or filtration; the collected crude protein is re-dissolved with phosphate buffer, and small molecule impurities are removed by ultrafiltration or dialysis to obtain purified hyaluronidase. The process is cumbersome, and ammonium sulfate precipitation is used in the purification, which requires a large amount of ammonium sulfate and is not suitable for large-scale preparation of hyaluronidase.

[0010] Chinese patent CN118086253A discloses a hyaluronic acid hydrolase, its gene and its engineered bacteria and production method. The invention removes the precipitate after centrifugation of the hyaluronidase fermentation broth through a tubular centrifuge to obtain a supernatant. The supernatant is filtered at low temperature using a ceramic membrane with a pore size of 50nm, and the pH is adjusted to 3.5-4.5 using phosphoric acid. The supernatant is precipitated with ammonium sulfate, the crude protein precipitate is collected and redissolved with purified water, and then purified using Ni-NTA affinity purification. Finally, it is desalted and concentrated with an ultrafiltration membrane, and freeze-dried to obtain a finished product. The enzyme activity is detected to be 42 million U / g. The process of the invention is cumbersome, and the enzyme activity after purification is not improved; the invention uses purified water for redissolution and desalination in multiple steps, which may be the main reason for the loss of enzyme. Summary of the Invention

[0011] In response to the deficiencies in the prior art, the present invention provides a method for preparing leech hyaluronidase expressed in Pichia pastoris and its application, which can obtain high-enzyme activity hyaluronidase. The purified high-enzyme activity hyaluronidase can also be used to enzymatically hydrolyze macromolecular sodium hyaluronate to prepare low molecular weight and oligomeric sodium hyaluronate.

[0012] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows: A method for preparing leech hyaluronidase expressed by Pichia pastoris, comprising: fermentation, pretreatment, one-step chromatography purification, and filtration; The fermentation comprises inoculating a Pichia pastoris seed liquid into a BMGY medium, culturing at 28-32° C. and 150-250 rpm for 22-26 hours, centrifuging, washing the cells, adding the cells to a composite medium, and culturing at 28-32° C. and 150-250 rpm for 95-100 hours, maintaining the pH at 5.4-5.6 during the culturing period, adding a primary mixed solution with a volume fraction of 1.2% after culturing for 23-25 hours, and adding a secondary mixed solution with a volume fraction of 1.1-1.3% after culturing for 46-50 hours, and obtaining a leech hyaluronidase fermentation liquid after the culturing is completed; In the fermentation, the Pichia pastoris is Pichia pastoris GS115; The preparation method of the Pichia pastoris seed liquid comprises streaking Pichia pastoris on a YPD solid culture medium, culturing at 28-32° C. for 44-46 hours, taking a single colony and inoculating it into 40-60 mL of YPD liquid culture medium, and culturing at 28-32° C. and 150-250 rpm for 23-15 hours to obtain the Pichia pastoris seed liquid; When the Pichia pastoris seed solution was inoculated into the BMGY medium, the volume fraction of the Pichia pastoris seed solution was 10%; The amount of BMGY medium used is 40-60 mL; The centrifugal speed is 7000-9000 rpm and the time is 25-35 min; Use sterile water to wash the cells; The amount of complex culture medium used is 30-50 mL; The YPD solid medium comprises: 9.5-10.5 g / L yeast powder, 19-21 g / L peptone, 19-21 g / L glucose, 19-21 g / L agar powder, pH = 6.8-7.2; The YPD liquid culture medium comprises: 9.5-10.5 g / L yeast powder, 19-21 g / L peptone, 19-21 g / L glucose, and a pH of 6.8-7.2. The components of the BMGY medium are: yeast extract 9.5-10.5 g / L, peptone 19-21 g / L, K2HPO4 2.8-3.2 g / L, KH2PO4 11.6-12 g / L, YNB 3.3-3.5 g / L, ammonium sulfate 9.5-10.5 g / L, biotin 3.8×10 4 -4.2×10 4 g / L, glycerol 9.5-10.5 g / L, pH = 5.8-6.2; The components of the composite culture medium are: yeast extract 9.5-10.5 g / L, peptone 19-21 g / L, K2HPO4 2.8-3.2 g / L, KH2PO4 11.6-12 g / L, YNB 3.3-3.5 g / L, ammonium sulfate 9.5-10.5 g / L, biotin 3.8×10 4 -4.2×10 4 g / L, methanol 9.5-10.5g / L, betaine 0.9-1.1g / L, polyethylene glycol 400 0.9-1.1g / L, pH=5.3-5.7; The primary mixed solution is composed of glycerol, PTM1, and polyethylene glycol 6000, wherein the mass ratio of glycerol, PTM1, and polyethylene glycol 6000 is 100:1.1-1.3:3.8-4.2; The secondary mixed liquid is composed of methanol, PTM1, and polyethylene glycol 6000, wherein the mass ratio of methanol, PTM1, and polyethylene glycol 6000 is 100:1.1-1.3:3.8-4.2; The pretreatment comprises mixing the leech hyaluronidase fermentation broth with calcium chloride, adjusting the pH to 7.2-7.6, collecting the supernatant by centrifugation, filtering through a filter membrane, taking the filtrate, and then filtering through a first-level ultrafiltration membrane, and taking the permeate as the sample solution; During the pretreatment, the centrifugation speed is 7000-9000 rpm and the time is 25-35 min; The filter membrane used in the membrane filtration is a cellulose membrane with a filtration accuracy of 0.22 μm; The filter membrane used during the first-level ultrafiltration membrane filtration is a polyethersulfone ultrafiltration membrane with a filtration accuracy of 200kDa; When the leech hyaluronidase fermentation liquid and calcium chloride are mixed and stirred, the volume mass ratio of the leech hyaluronidase fermentation liquid to calcium chloride is 1L:0.9-1.1g, and the mixing and stirring time is 1-1.5h; The one-step chromatography purification is to perform one-step chromatography purification on the sample solution to obtain a chromatography purified solution; In the one-step chromatography purification, the chromatography column used is an XK series chromatography column, and the filler used is IMAC Sepharose 6 FF filler; The chromatographic purification procedure was as follows: equilibrium, NiA buffer 5CV; sample loading; rinsing, NiA buffer 10CV; elution, 30% NiB buffer 5CV, and collection of the elution peak for later use; The NiA buffer is composed of 19-21 mM phosphate buffer, 48-52 mM imidazole, 290-310 mM NaCl, and a pH of 7.3-7.5. The NiB buffer is composed of 19-21 mM phosphate buffer, 490-510 mM imidazole, 290-310 mM NaCl, and a pH of 7.3-7.5. The sample volume of the loading solution is 2000-4000mL; The filtration step is to filter the chromatographic purified liquid through a secondary ultrafiltration membrane, and the concentrated liquid is taken as the enzyme solution of leech hyaluronidase; In the filtration, the filter membrane used in the secondary ultrafiltration membrane filtration is a polyethersulfone ultrafiltration membrane with a filtration accuracy of 20 kDa.

[0013] An application of the aforementioned leech hyaluronidase is to heat water to 36-38°C, add leech hyaluronidase enzyme solution and macromolecular sodium hyaluronate, and keep the temperature at 36-38°C under sealed conditions to obtain an enzymatic hydrolyzate, and then post-treat the enzymatic hydrolyzate to obtain hyaluronic acid oligosaccharides; The enzyme activity of the leech hyaluronidase solution is 4.3×10 7 -4.7×10 7 U / mL; The volume mass ratio of water to macromolecular sodium hyaluronate is 500-1000mL:100g; The volume-to-mass ratio of leech hyaluronidase solution to macromolecular sodium hyaluronate is 1-3 mL:100 g; The post-treatment comprises filtering, ultrafiltration and spray drying the enzymatic hydrolysate; The filtration comprises adding diatomaceous earth to the enzymatic hydrolyzate, stirring evenly, and filtering with a filter paper until the feed liquid is clear; The mass ratio of the macromolecular sodium hyaluronate to diatomaceous earth is 1400:900-1100; the ultrafiltration is performed using an organic ultrafiltration membrane for separation, and the permeate is collected. During separation, the material temperature is controlled at 30-35° C. and the pressure is controlled at 0.4-0.5 MPa. When the solid content is above 5%, the permeate is collected; The filtration accuracy of the organic filtration membrane is 1wDa; The inlet air temperature of the spray drying is 170-180°C, and the outlet air temperature is 70-80°C.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) The preparation method of leech hyaluronidase expressed by Pichia pastoris of the present invention comprises the following steps: using a composite culture medium in fermentation, wherein betaine and polyethylene glycol are added to the composite culture medium; betaine and polyethylene glycol 400 can improve the permeability of Pichia pastoris GS115 and promote the production of leech hyaluronidase; polyethylene glycol 6000 is added to the mixed solution added during culture; polyethylene glycol 6000 has a large molecular weight and can reduce the permeability of cells and control the fermentation process; in pretreatment, the leech hyaluronidase fermentation liquid is mixed with calcium chloride; on the one hand, the calcium ions in the calcium chloride can improve the enzymatic activity of the hyaluronidase in the leech hyaluronidase fermentation liquid; on the other hand, the calcium ions can produce a chelation effect with polyethylene glycol, promote the preliminary separation of polyethylene glycol, and improve the purification effect, thereby obtaining an enzyme activity of 4.5×10 7 U / mL leech hyaluronidase enzyme solution; (2) The method for preparing the leech hyaluronidase expressed by Pichia pastoris of the present invention has a simple process and is suitable for industrial mass production of hyaluronidase. The purified hyaluronidase enzyme solution has high enzyme activity and is suitable for industrial production of low molecular weight and oligomeric sodium hyaluronate. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The SDS-PAGE electrophoresis test diagram of the samples taken in each step of Comparative Example 1 is shown; In the figure, lane 1 is the loading solution, lane 2 is the flow-through solution, lane 3 is the rinse solution, lane 4 is the 60% B eluate, and lane M is the standard protein; Figure 2 The SDS-PAGE electrophoresis test diagram of the samples taken in each step of Comparative Example 2 is shown; In the figure, lane 1 is the loading solution, lane 2 is the flow-through solution, lane 3 is the rinse solution, lane 4 is the 60% B eluate, and lane M is the standard protein; Figure 3 The SDS-PAGE electrophoresis test diagram of the samples taken at each step in Comparative Example 3 is shown; In the figure, lane 1 is the loading solution, lane 2 is the flow-through solution, lane 3 is the rinse solution, lane 4 is the 60% B eluate, and lane M is the standard protein; Figure 4 These are the liquid chromatograms of the reaction solution in Experiment A at reaction times of 0 h, 3 h, 5 h, 7 h, and 8 h. DETAILED DESCRIPTION

[0016] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific embodiments of the present invention are now described.

[0017] The recombinant Pichia pastoris strain in the Examples and Comparative Examples is Pichia pastoris GS115 expressing leech hyaluronidase, which was obtained from Jiangnan University.

[0018] Example 1 A method for preparing leech hyaluronidase expressed in Pichia pastoris 1. Fermentation: Pichia pastoris GS115 was streaked onto YPD solid medium and cultured at 30°C for 45 hours. A single colony was inoculated into 50 mL of YPD liquid medium and cultured at 30°C and 200 rpm for 24 hours to obtain a seed solution. The seed solution was inoculated into 50 mL of BMGY medium at a volume fraction of 10%, and cultured at 30°C and 200 rpm for 24 hours. The precipitate was collected after centrifugation at a speed of 8000 rpm for 30 minutes. The cells were washed with sterile water and added to 40 mL of composite medium. The culture was incubated at 30°C and 200 rpm for 96 hours. The pH was continuously monitored and adjusted during the culture period to maintain the pH at 5.5. After 24 hours of culture, a primary mixed solution with a volume fraction of 1.2% was added. After 48 hours of culture, a secondary mixed solution with a volume fraction of 1.2% was added. The leech hyaluronidase fermentation liquid was obtained upon completion of the culture. The YPD solid medium contains 10 g / L yeast powder, 20 g / L peptone, 20 g / L glucose, and 20 g / L agar powder, with a pH of 7. The YPD liquid culture medium contains 10 g / L yeast powder, 20 g / L peptone, and 20 g / L glucose, with a pH of 7. The components of the BMGY medium are: yeast extract 10 g / L, peptone 20 g / L, K2HPO4 3 g / L, KH2PO4 11.8 g / L, YNB 3.4 g / L, ammonium sulfate 10 g / L, biotin 4×10 4 g / L, glycerol 10g / L, pH=6; The composition of the composite culture medium is: yeast extract 10g / L, peptone 20g / L, K2HPO4 3g / L, KH2PO4 11.8g / L, YNB 3.4g / L, ammonium sulfate 10g / L, biotin 4×10 4g / L, methanol 10g / L, betaine 1g / L, polyethylene glycol 4001g / L, pH=5.5; The primary mixed solution is composed of glycerol, PTM1, and polyethylene glycol 6000, wherein the mass ratio of glycerol, PTM1, and polyethylene glycol 6000 is 100:1.2:4; The secondary mixed solution is composed of methanol, PTM1, and polyethylene glycol 6000, wherein the mass ratio of methanol, PTM1, and polyethylene glycol 6000 is 100:1.2:4; 2. Pretreatment: First, the leech hyaluronidase fermentation broth was mixed with calcium chloride at a volume-to-mass ratio of 1 L:1 g, stirred at room temperature for 1 hour, then the pH was adjusted to 7.4, and the supernatant was collected by centrifugation at a speed of 8000 rpm for 30 minutes; the supernatant was filtered through a cellulose membrane with a filtration accuracy of 0.22 μm, and the filtrate was taken. The supernatant was then filtered through a first-stage ultrafiltration membrane with a polyethersulfone ultrafiltration membrane with a filtration accuracy of 200 kDa, and the permeate was taken as the sample solution; 3. One-step chromatography purification: The sample solution is subjected to one-step chromatography purification to obtain a chromatography purified solution; The chromatography column used was an XK series chromatography column, and the filler used was IMAC Sepharose6 FF filler; The chromatographic purification procedure was as follows: equilibration, NiA buffer 5CV; sample loading; rinse, NiA buffer 10CV; elution, 30% NiB buffer 5CV, collecting the elution peak for later use; and sampling at each step for enzyme activity testing. The enzyme activity test results are shown below: The sample volume of the sample solution during loading was 2000 mL;

[0019] The NiA buffer is composed of 20 mM phosphate buffer, 50 mM imidazole, 300 mM NaCl, and a pH of 7.4. The NiB buffer is composed of 20 mM phosphate buffer, 500 mM imidazole, 300 mM NaCl, and a pH of 7.4. 4. Filtration: The chromatographic purified liquid is filtered through a secondary ultrafiltration membrane, and the concentrated liquid is taken as the leech hyaluronidase enzyme liquid. The filter membrane used in the secondary ultrafiltration membrane filtration is a polyethersulfone ultrafiltration membrane with a filtration accuracy of 20 kDa.

[0020] The enzyme activity of the leech hyaluronidase solution obtained in this example was 4.5×10 7 U / mL, and a purity of 98.2%. The method of this embodiment is suitable for industrial scale-up and industrial production of hyaluronidase.

[0021] Comparative Example 1 Based on Example 1, in the second pretreatment step, the pH adjustment of the leech hyaluronidase fermentation broth to 7.4 was omitted. That is, the leech hyaluronidase fermentation broth was directly centrifuged to collect the supernatant, and then filtered with a filter membrane; Change the chromatographic purification step in step 3 to: The sample solution was subjected to one-step chromatography purification using XK series chromatography columns and IMAC Sepharose 6 FF filler; The chromatography procedure was as follows: equilibration, NiA buffer, 5CV; sample loading; rinse, NiA buffer, 10CV; elution, 60% NiB buffer, 5CV, collecting the elution peak for later use; and sampling at each step for enzyme activity testing. The enzyme activity test results are shown below: The sample volume of the sample solution during loading was 2000 mL; The NiA buffer is composed of 20 mM phosphate buffer, 50 mM imidazole, 500 mM NaCl, and a pH of 7.4. The NiB buffer is composed of 20 mM phosphate buffer, 500 mM imidazole, 500 mM NaCl, and a pH of 7.4. SDS-PAGE electrophoresis was performed on the samples taken at each step in this embodiment. Figure 1 As shown. Figure 1 It can be seen that there are two problems in this comparative example: first, a large amount of target protein is present in lane 2, i.e., the flow-through; second, target protein is detected in lane 3, i.e., the rinse, indicating that the protein binding is not strong and the buffer conditions need to be optimized.

[0022] Comparative Example 2 Based on Comparative Example 1, in the second pretreatment step, the pH of the leech hyaluronidase fermentation liquid was adjusted to 7.4 before the leech hyaluronidase fermentation liquid was filtered through a membrane; Sampling at each step was performed on SDS-PAGE electrophoresis, such as Figure 2 shown.

[0023] Depend on Figure 2 It can be seen that the target protein was not detected in the flow-through of lane 2 in this example, indicating that the problem of protein non-binding can be greatly improved by optimizing the pH of the leech hyaluronidase fermentation broth.

[0024] Comparative Example 3 Based on Comparative Example 2, the concentration of NaCl in the NiA buffer and NiB buffer used in the one-step chromatography was adjusted from 500 mM to 300 mM.

[0025] Sampling at each step was performed on SDS-PAGE electrophoresis, such as Figure 3 As shown; Depend on Figure 3 It can be seen that the target protein was not detected in the flow-through of lane 2, and only a trace amount of target protein was detected in the eluate of lane 3, indicating that the problem of non-binding or weak binding of the target protein can be greatly improved by optimizing the pH of the fermentation broth and adjusting the concentration of NaCl in NiA buffer and NiB buffer.

[0026] Comparative Example 4 On the basis of Comparative Example 1, the composition of the composite culture medium in the first fermentation step was changed to: yeast extract 10 g / L, peptone 20 g / L, K2HPO4 3 g / L, KH2PO4 11.8 g / L, YNB 3.4 g / L, ammonium sulfate 10 g / L, biotin 4 × 10 4 g / L, methanol 10g / L, betaine 1g / L, pH=5.5; The first-stage mixed solution was changed to consist of glycerol and PTM1, where the mass ratio of glycerol to PTM1 was 100:1.2; The secondary mixed liquid was changed to consist of methanol and PTM1, where the mass ratio of methanol to PTM1 was 100:1.2; In the second pretreatment step, the permeate was directly used as the loading solution, without mixing the permeate with calcium chloride in a volume-to-mass ratio of 1 L:0.2 g and stirring at room temperature for 1 h.

[0027] The enzyme activity of the leech hyaluronidase solution finally obtained in this comparative example was 2.0×10 7 U / mL. This indicates that the use of polyethylene glycol and calcium chloride can improve the enzymatic activity of the leech hyaluronidase solution.

[0028] Test Example 1 Degradation Product Analysis In order to analyze the enzymatic hydrolysis process and hydrolysis end products of leech hyaluronidase, the purified leech hyaluronidase was reacted with sodium hyaluronate substrate. Samples were taken at intervals during the reaction, and then HPLC was used to analyze the hydrolysis of HA oligosaccharides at each stage, as follows: Test A: Add 500 mL of pure water to a 1 L beaker, heat the water bath to 37° C., first add 3 mL of the leech hyaluronidase solution prepared in Example 1, and then add 100 g of sodium hyaluronate substrate (molecular weight 50 wDa) in one go; seal the beaker with plastic wrap, mix well, and react at 37° C., sampling every 1 h. After the reaction is completed, heat in a 100° C. metal bath for 5 min to terminate the reaction to obtain a reaction solution.

[0029] Test B: Test B is a control test, specifically, 500 mL of pure water was added to a 1 L beaker, and the water bath was heated to 37 ° C. The leech hyaluronidase solution prepared in Example 1 was first heat-inactivated to obtain a heat-inactivated enzyme solution, and then 3 mL of the heat-inactivated enzyme solution was added thereto, and 100 g of sodium hyaluronate substrate (molecular weight 50 wDa) was added at one time; the beaker was sealed with plastic wrap, mixed and reacted at 37 ° C., sampling was performed every 1 h, and the reaction was terminated by heating in a 100 ° C metal bath for 5 min to obtain a control reaction solution.

[0030] The degradation products at 0 h, 3 h, 5 h, 7 h, and 8 h in experiment A and at 0 h, 3 h, 5 h, 7 h, and 8 h in experiment B were analyzed by high performance liquid chromatography (HPLC): HPLC used an Agilent 1260 high performance liquid chromatograph with a UV detector, the chromatographic column was YMC-Pack PolyamineⅡ (250×4.6 mml.DS-5 μm, 12 nm), the mobile phase was 0.1 M sodium dihydrogen phosphate aqueous solution and acetonitrile, the column temperature was 30°C, the detection wavelength was 210 nm, the eluent was a mixture of 90% (v / v) 0.1 M sodium dihydrogen phosphate aqueous solution and 10% (v / v) acetonitrile, and the elution was at 0.5 mL / min for 50 min.

[0031] The liquid chromatograms of the degradation products at 0h, 3h, 5h, 7h, and 8h of reaction in test A are as follows: Figure 4 shown by Figure 4 It can be seen that leech hyaluronidase can degrade hyaluronic acid into hyaluronan disaccharide (HA2), hyaluronan tetrasaccharide (HA4), hyaluronan hexasaccharide (HA6), hyaluronan octasaccharide (HA8), hyaluronan decasaccharide (HA10), etc.; and as the reaction time increases, the degradation products are mainly hyaluronan tetrasaccharide (HA4) and hyaluronan hexasaccharide (HA6). These results show that the molecular weight of the enzymatic cleavage products can be controlled by adjusting the amount of leech hyaluronidase added and the enzymatic cleavage time, thereby obtaining low-molecular-weight hyaluronan oligosaccharides of different molecular weights.

[0032] In Experiment B, the liquid chromatograms of the degradation products at 3 h, 5 h, 7 h, and 8 h of reaction were the same as the liquid chromatogram of the degradation products at 0 h of reaction, indicating that the heat-inactivated enzyme solution did not have the effect of degrading hyaluronic acid.

[0033] Application Example 1 Preparation of small molecule sodium hyaluronate 1. Enzymatic hydrolysis: Add 14 L of pure water to a stainless steel barrel, heat in a water bath at 37°C, first add 14 mL of the purified leech hyaluronidase solution obtained in Example 1, then add 1400 g of macromolecular sodium hyaluronate (molecular weight 100 wDa) at once, seal with plastic wrap, and heat at 37°C for 8 h to obtain an enzymatic hydrolyzate. 2. Pretreatment of the enzymatic hydrolysate: Add 1 kg of diatomaceous earth to the enzymatic hydrolysate, stir evenly, and filter through filter paper until the liquid is clear to obtain the pretreated enzymatic hydrolysate; 3. Organic ultrafiltration membrane separation: Use 1wDa organic ultrafiltration membrane to separate the pretreated enzymatic hydrolysate and collect the permeate. During separation, the material temperature is controlled at 30-35℃ and the pressure is controlled at 0.4-0.5MPa. Use a handheld content meter to test the solid content. When the solid content is above 5%, collect the permeate. 4. Spray drying: spray drying the permeate, controlling the air inlet temperature at 175°C and the air outlet temperature at 75°C during spray drying to obtain low molecular weight hyaluronic acid oligosaccharides.

[0034] It can be seen from the results of Test Example 1 that in this application example, the molecular weight of the enzymatic cleavage product can be controlled by adjusting the amount of leech hyaluronidase solution added and the enzymatic cleavage time in the first step of the enzymatic hydrolysis reaction to obtain low-molecular-weight hyaluronic acid oligosaccharides of different molecular weights.

Claims

1. A method for preparing leech hyaluronidase expressed in Pichia pastoris, characterized in that: include: fermentation, pretreatment, one-step chromatography purification, and filtration; The pretreatment comprises mixing the leech hyaluronidase fermentation broth with calcium chloride, adjusting the pH to 7.2-7.6, collecting the supernatant by centrifugation, filtering through a filter membrane, taking the filtrate, and then filtering through a first-level ultrafiltration membrane, and taking the permeate as the sample solution; The one-step chromatography purification is to perform one-step chromatography purification on the sample solution to obtain a chromatography purified solution; In the one-step chromatography purification, the chromatography procedure is as follows: equilibration, NiA buffer 5CV; sample loading; rinsing, NiA buffer 10CV; elution, 30% NiB buffer 5CV, collecting the elution peak for later use; The NiA buffer is composed of 19-21 mM phosphate buffer, 48-52 mM imidazole, 290-310 mM NaCl, and a pH of 7.3-7.

5. The NiB buffer comprises: 19-21 mM phosphate buffer, 490-510 mM imidazole, 290-310 mM NaCl, and a pH of 7.3-7.

5.

2. The method for preparing the leech hyaluronidase expressed by Pichia pastoris according to claim 1, characterized in that: The fermentation comprises inoculating a Pichia pastoris seed liquid into a BMGY culture medium, culturing at 28-32° C. and 150-250 rpm for 22-26 hours, centrifuging and washing the bacteria, adding the bacteria into a composite culture medium, and culturing at 28-32° C. and 150-250 rpm for 95-100 hours, maintaining the pH at 5.4-5.6 during the culture period, adding a primary mixed solution with a volume fraction of 1.2% after culturing for 23-25 hours, and adding a secondary mixed solution with a volume fraction of 1.1-1.3% after culturing for 46-50 hours, and obtaining a leech hyaluronidase fermentation liquid after the culture is completed.

3. The method for preparing the leech hyaluronidase expressed by Pichia pastoris according to claim 2, characterized in that: In the fermentation, the Pichia pastoris is Pichia pastoris GS115; The preparation method of the Pichia pastoris seed liquid comprises streaking Pichia pastoris on a YPD solid culture medium, culturing at 28-32° C. for 44-46 hours, taking a single colony and inoculating it into 40-60 mL of YPD liquid culture medium, and culturing at 28-32° C. and 150-250 rpm for 23-15 hours to obtain the Pichia pastoris seed liquid; When the Pichia pastoris seed solution was inoculated into the BMGY medium, the volume fraction of the Pichia pastoris seed solution was 10%; The amount of BMGY medium used is 40-60 mL; The centrifugal speed is 7000-9000 rpm and the time is 25-35 min; Use sterile water to wash the cells; The amount of complex culture medium used is 30-50 mL.

4. The method for preparing the leech hyaluronidase expressed by Pichia pastoris according to claim 2, characterized in that: During the fermentation, the YPD solid medium comprises the following components: yeast powder 9.5-10.5 g / L, peptone 19-21 g / L, glucose 19-21 g / L, agar powder 19-21 g / L, pH = 6.8-7.2; The YPD liquid culture medium comprises: 9.5-10.5 g / L yeast powder, 19-21 g / L peptone, 19-21 g / L glucose, and a pH of 6.8-7.

2. The components of the BMGY medium are: yeast extract 9.5-10.5 g / L, peptone 19-21 g / L, K2HPO4 2.8-3.2 g / L, KH2PO4 11.6-12 g / L, YNB 3.3-3.5 g / L, ammonium sulfate 9.5-10.5 g / L, biotin 3.8×10 4 -4.2×10 4 g / L, glycerol 9.5-10.5 g / L, pH = 5.8-6.2; The components of the composite culture medium are: yeast extract 9.5-10.5 g / L, peptone 19-21 g / L, K2HPO4 2.8-3.2 g / L, KH2PO4 11.6-12 g / L, YNB 3.3-3.5 g / L, ammonium sulfate 9.5-10.5 g / L, biotin 3.8×10 4 -4.2×10 4 g / L, methanol 9.5-10.5g / L, betaine 0.9-1.1g / L, polyethylene glycol 400 0.9-1.1g / L, pH=5.3-5.7; The primary mixed solution is composed of glycerol, PTM1, and polyethylene glycol 6000, wherein the mass ratio of glycerol, PTM1, and polyethylene glycol 6000 is 100:1.1-1.3:3.8-4.2; The secondary mixed liquid consists of methanol, PTM1, and polyethylene glycol 6000, wherein the mass ratio of methanol, PTM1, and polyethylene glycol 6000 is 100:1.1-1.3:3.8-4.

2.

5. The method for preparing the leech hyaluronidase expressed by Pichia pastoris according to claim 1, characterized in that: During the pretreatment, the centrifugation speed is 7000-9000 rpm and the time is 25-35 min; The filter membrane used in the membrane filtration is a cellulose membrane with a filtration accuracy of 0.22 μm; The filter membrane used during the first-level ultrafiltration membrane filtration is a polyethersulfone ultrafiltration membrane with a filtration accuracy of 200kDa; When the leech hyaluronidase fermentation liquid and calcium chloride are mixed and stirred, the volume mass ratio of the leech hyaluronidase fermentation liquid to calcium chloride is 1L:0.9-1.1g, and the mixing and stirring time is 1-1.5h.

6. The method for preparing the leech hyaluronidase expressed by Pichia pastoris according to claim 1, characterized in that: In the one-step chromatography purification, the chromatography column used is an XK series chromatography column, and the filler used is IMAC Sepharose 6FF filler; The loading volume of the sample solution is 2000-4000mL.

7. The method for preparing leech hyaluronidase expressed in Pichia pastoris according to claim 6, characterized in that: The filtration step is to filter the chromatographic purified liquid through a secondary ultrafiltration membrane, and the concentrated liquid is taken as the enzyme solution of leech hyaluronidase; In the filtration, the filter membrane used in the secondary ultrafiltration membrane filtration is a polyethersulfone ultrafiltration membrane with a filtration accuracy of 20 kDa.

8. A use of the leech hyaluronidase prepared according to any one of claims 1 to 7, characterized in that: The water is heated to 36-38° C., and leech hyaluronidase enzyme solution and macromolecular sodium hyaluronate are added. The solution is kept warm at 36-38° C. under sealed conditions to obtain enzymatic hydrolyzate, and the enzymatic hydrolyzate is then post-processed to obtain hyaluronic acid oligosaccharide.

9. The use of leech hyaluronidase according to claim 8, characterized in that: The enzyme activity of the leech hyaluronidase solution is 4.3×10 7 -4.7×10 7 U / mL; The volume mass ratio of water to macromolecular sodium hyaluronate is 500-1000mL:100g; The volume-to-mass ratio of the leech hyaluronidase solution to the macromolecular sodium hyaluronate is 1-3 mL:100 g.

10. The use of leech hyaluronidase according to claim 8, characterized in that: The post-treatment comprises filtering, ultrafiltration and spray drying the enzymatic hydrolysate; The filtration comprises adding diatomaceous earth to the enzymatic hydrolyzate, stirring evenly, and filtering with a filter paper until the feed liquid is clear; The mass ratio of the macromolecular sodium hyaluronate to diatomaceous earth is 1400:900-1100; the ultrafiltration is performed using an organic ultrafiltration membrane for separation, and the permeate is collected. During separation, the material temperature is controlled at 30-35° C. and the pressure is controlled at 0.4-0.5 MPa. When the solid content is above 5%, the permeate is collected; The filtration accuracy of the organic filtration membrane is 1wDa; The inlet air temperature of the spray drying is 170-180°C, and the outlet air temperature is 70-80°C.

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