Extraction and purification method of high-activity hirudin based on combination of composite enzymolysis and chromatography

Abstract

The invention relates to the technical field of biological extraction, in particular to a method for extracting and purifying high-activity hirudin based on combination of composite enzymolysis and chromatography, which comprises the following steps: sample pretreatment: dissecting leeches to take salivary gland tissues at the heads of the leeches, and freeze-drying and crushing the salivary gland tissues to obtain salivary gland tissue powder; performing extraction: performing enzymolysis extraction on the salivary gland tissue powder by adopting a compound enzyme to obtain a crude extracting solution; the method comprises the following steps: carrying out primary enrichment by using macroporous adsorption resin to obtain a first purified solution, carrying out cation exchange chromatography refining to obtain a second purified solution, carrying out affinity chromatography high purification to obtain affinity chromatography high purification, and carrying out concentration and freeze-drying on the purified product to obtain the high-purity hirudin product. Compared with the prior art, the method has the advantages that the salivary gland tissue powder is subjected to enzymolysis with the compound enzyme at low temperature and purified, so that the purity of the hirudin is 95% or above, and the antithrombin activity of a product determined by a thrombin inhibition test is higher than 15000 ATU / mg.

Description

Technical Field

[0001] This invention relates to the field of bioextraction technology, specifically to a method for the extraction and purification of highly active hirudin based on a combined enzymatic hydrolysis-chromatography approach. Background Technology

[0002] Hirudin is a polypeptide secreted by the salivary glands of leeches. Hirudin forms an irreversible complex with thrombin in a 1:1 ratio, thereby achieving anticoagulant, antithrombotic, and blood circulation-promoting effects. It is one of the key raw materials for preparing drugs to treat or prevent thrombotic diseases, thus making hirudin crucial. Currently, recombinant hirudin produced using gene-engineering methods has high yields, but some recombinant hirudin suffers from problems such as the loss of sulfated tyrosine residues, resulting in its anticoagulant, antithrombotic, and blood circulation-promoting effects being far inferior to those of natural hirudin. Existing methods for extracting natural hirudin are as follows: Patent No. CN20181000563.8 discloses a method for extracting hirudin. Specifically, it discloses placing A parts by mass of leech powder and B parts by mass of physiological saline into an extraction container for extraction for a preset time to obtain an extraction mixture. After centrifuging the mixture, a preset volume of supernatant is collected. The problems are twofold: first, hirudin is tightly bound to other proteins and cellular components in saline gland tissue, making it difficult for physiological saline to completely release hirudin from leech powder; second, physiological saline is a highly polar solvent, which extracts a large amount of unwanted water-soluble impurities during hirudin extraction, resulting in low purity hirudin.

[0003] Patent No. CN201010526681.7 discloses a method for extracting highly anticoagulant hirudin from natural leeches. Specifically, it discloses a method of stimulating live leeches with a stimulating solution prepared using 5g sodium chloride, 0.3g acetylcysteine ​​hydrochloride monohydrate, 5g lysine, 100U of neutral protease, 150U of papain, and 1000mL of deionized water. This stimulates the leeches to secrete a fluid, which is then centrifuged at 1600-20000 r / min to obtain crude hirudin. However, the concentration of hirudin in the leech secretion is extremely low, potentially leading to a very low extraction yield. Furthermore, high-speed centrifugation is used to extract hirudin, but this separation method cannot effectively remove other proteins and enzymes from the product, resulting in low purity hirudin.

[0004] In summary, existing methods for extracting hirudin from leeches using whole sap or powder result in low yields and extremely low purity of hirudin due to the complexity of the raw materials and limitations in the extraction process. Furthermore, existing methods for extracting hirudin from leech secretions result in extremely low hirudin concentrations in the secretions and problems with the extraction process, leading to very low extraction rates and purity of hirudin.

[0005] Therefore, it is of great significance to develop an efficient, mild, green extraction and purification technology that can preserve the natural conformation and biological activity of hirudin to the greatest extent. Summary of the Invention

[0006] The purpose of this invention is to provide a method for the extraction and purification of highly active hirudin based on a combined enzymatic hydrolysis-chromatography approach, aiming to solve the problems of complex composition of natural hirudin extraction targets or low hirudin concentration, as well as low extraction yield and low extraction purity caused by defects in the extraction process.

[0007] To achieve the above objectives, this invention provides a method for the extraction and purification of highly active hirudin based on a combined enzymatic hydrolysis-chromatography approach, wherein the extraction and purification method includes: S1, Sample pretreatment: Dissect the leech and take the salivary gland tissue from its head. Freeze-dry and pulverize the salivary gland tissue to obtain salivary gland tissue powder. S2, Extraction: The salivary gland tissue powder was extracted by enzymatic hydrolysis with a compound enzyme to obtain a crude extract; S3, repeated purification: S31, Preliminary enrichment with macroporous adsorption resin: The crude extract is loaded into an HPD-100 macroporous adsorption resin column. After loading, it is washed with 2-3 column volumes of deionized water and eluted with 20%-30% ethanol aqueous solution. The elution peak is collected to obtain the first purified solution. S32, Cation exchange chromatography purification: The first purified solution is loaded onto a CM Sepharose Fast Flow cation exchange column. After loading, the column is washed with equilibration buffer until the baseline is stable. Then, the column is eluted with a linear NaCl gradient and the elution peaks in the 0.3-0.4M NaCl range are collected to obtain the second purified solution. S33, affinity chromatography for high purification: The second purification solution is loaded onto an affinity column coupled with thrombin, washed thoroughly with equilibration buffer, eluted with glycine-hydrochloric acid buffer, and the collection solution is neutralized with Tris base to obtain the purified product. S4, the purified product is concentrated and freeze-dried to obtain a high-purity hirudin product.

[0008] Preferably, in the above technical solution, in step S1, the salivary gland tissue from the head of the leech is dissected, and the salivary gland tissue is freeze-dried and pulverized to obtain salivary gland tissue powder, specifically as follows: (1) Live leeches were starved for a week, and after being anesthetized at low temperature, the leeches were quickly dissected to obtain salivary gland tissue from their heads.

[0009] (2) After rinsing the head salivary gland tissue with pre-cooled phosphate buffer and draining it, the head salivary gland tissue was placed in an ultra-low temperature freezer at -80℃ for quick freezing and then vacuum freeze-drying. The freeze-dried tissue was then ultra-finely pulverized to obtain salivary gland tissue powder.

[0010] Preferably, in the above technical solution, the phosphate buffer is PBS, the concentration of the PBS is 0.01M, and the pH value of the solution is 6.5.

[0011] Preferably, in the above technical solution, in step S2, the step of using a compound enzyme to extract the salivary gland tissue powder to obtain a crude extract, the compound enzyme is composed of 0.5%-1.5% neutral protease, 0.2%-0.8% bromelain, and 0.1%-0.5% hyaluronidase by weight percentage.

[0012] Preferably, in the above technical solution, the enzymatic hydrolysis conditions for extracting the salivary gland tissue powder by the compound enzyme are a temperature of 35-45℃, a pH value of 6.5-7.0, a reaction time of 2-4h, and slow stirring at 50-100rpm.

[0013] Preferably, in the above technical solution, the solution after enzymatic hydrolysis is rapidly heated to 80-85°C and maintained for 10-13 minutes to inactivate enzyme activity, and then immediately cooled in an ice bath. It is then centrifuged at 4-6°C and 8000-10000 rpm for 18-22 minutes, and the supernatant is collected as the crude extract.

[0014] Preferably, in the above technical solution, the equilibration buffer is an acetate-ammonium acetate buffer with a concentration of 0.02M and a pH value of 5.0-6.0.

[0015] Preferably, in the above technical solution, in step S31, the preparation method of the HPD-100 macroporous adsorption resin column is as follows: S311, HPD-100 macroporous adsorption resin is soaked in 95% ethanol for 24 hours, stirred 3-4 times during the period, and the resin effluent is rinsed with deionized water to remove ethanol. Then it is soaked in 5% hydrochloric acid solution for 2 hours and rinsed with deionized water until neutral. Finally, it is soaked in 5% sodium hydroxide solution for 2 hours and rinsed with deionized water until neutral to obtain HPD-100 macroporous adsorption resin with a water content of 45-55%. S312, the HPD-100 macroporous adsorption resin from step S311 is placed into an elution column to obtain an HPD-100 macroporous adsorption resin column.

[0016] Preferably, in the above technical solution, in step S4, the process of concentrating and freeze-drying the purified product to obtain a high-purity hirudin product specifically involves: concentrating and desalting the purified product using an ultrafiltration tube with a molecular weight cutoff of 3 kDa, followed by vacuum freeze-drying.

[0017] Compared with the prior art, the beneficial effects of this application are as follows: (1) The principle of high extraction efficiency and good activity retention of the present invention is: hyaluronidase decomposes tissue cell walls and intercellular matrix, reducing mass transfer resistance; neutral protease and bromelain gently hydrolyze binding proteins, releasing hirudin; the three enzymes work together and adjust the enzymatic hydrolysis conditions to achieve efficient and gentle release of hirudin, avoiding the destruction of hirudin activity by severe chemical conditions. (2) From dissection to purification, key steps are all carried out at low temperature to maximize the protection of the natural conformation and biological activity of hirudin; multiple purifications improve the purity of the product to over 95%, and the antithrombin activity of the product is higher than 15,000 ATU / mg as determined by the thrombin inhibition test. The principle is as follows: First, macroporous resin enrichment replaces the traditional organic solvent precipitation, which is safe and environmentally friendly, and can effectively remove pigments and sugars; Second, the high isoelectric point (pI ~3.9) of hirudin is used for cation exchange chromatography, which can effectively separate impurities with different charge properties and achieve good purification effect; Finally, affinity chromatography utilizes its specific binding with thrombin to achieve "one-step purification" to ultra-high purity, which is suitable for pharmaceutical-grade products with extremely high purity requirements. Detailed Implementation

[0018] The technical solutions in the embodiments of this invention are clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0019] (1) Screening test of enzyme preparation components of the present invention: This invention screens the optimal protease species from enzyme preparations such as papain, bromelain, acidic protease, neutral protease, trypsin, alkaline protease, hyaluronidase, and pepsin. The specific method is as follows: Live leeches are starved for one week, then anesthetized at low temperature, and their head salivary gland tissue is rapidly dissected. The head salivary gland tissue is rinsed with pre-cooled PBS phosphate buffer (0.01M, pH 6.5) and drained. The drained head salivary gland tissue is then rapidly frozen in an ultra-low temperature freezer at -80℃, vacuum freeze-dried, and ultra-finely pulverized to obtain 8g of salivary gland tissue powder. The salivary gland tissue powder is suspended in 10 times its volume of PBS extract (0.02M, pH 6.8) to obtain a suspension. 2% enzyme preparation is then added to the suspension for enzymatic hydrolysis. The results are shown in Table 1 below.

[0020] Table 1. Crude extracts of salivary gland tissue powder treated with different 2% enzyme preparations. Enzyme preparation name Enzymatic hydrolysis conditions Crude extract (mg) 2% acidic protease Temperature 45℃, pH 4.0, time 2 hours 108.64 2% neutral protease Temperature 45℃, pH 6.5, time 2 hours 183.78 2% alkaline protease Temperature 50℃, pH 9.0, time 2 hours 179.41 2% Papain Temperature 55℃, pH 5.0, time 2 hours 196.68 2% Bromelain Temperature 50℃, pH 6.5, time 2 hours 124.78 2% Hyaluronidase Temperature 37℃, pH 7.0, time 2 hours 104.16 2% pepsin Temperature 37℃, pH 2.5, time 2 hours 139.96 2% trypsin Temperature 37℃, pH 6.5, time 2 hours 154.28 The screening experiments above showed that the crude extraction effects were ranked as follows: papain, neutral protease, alkaline protease, trypsin, pepsin, bromelain, acidic protease, and hyaluronidase.

[0021] Furthermore, through multiple screening tests, the combination of neutral protease, bromelain, and hyaluronidase was determined to yield 253.82 mg of crude extract when enzymatically hydrolyzed for 2 hours at 40°C and pH 6.5. Among the many combinations, the crude extract yield was the best.

[0022] (2) Effect of enzyme formulation ratio on crude extract content The design method for setting neutral protease (0.1%, 0.5%, 1%, 1.5%, 2%), bromelain (0.1%, 0.5%, 1%, 1.5%, 2%) and hyaluronidase (0.1%, 0.5%, 1%, 1.5%, 2%) is shown in Table 2 below. The crude extracts obtained by enzymatic hydrolysis of 8g of salivary gland tissue powder under the conditions of 40℃ and pH 6.5 for 2h are shown in Table 2.

[0023] Table 2. Effect of enzyme formulation ratio on crude extract content Serial Number neutral protease Bromelain Hyaluronidase Crude extract (mg) 1 0.1% 0.1% 0.1% 95.76 2 0.1% 0.5% 0.5% 101.32 3 0.1% 1% 1% 117.48 4 0.1% 1.5% 1.5% 125.85 5 0.1% 2% 2% 147.53 6 0.5% 0.1% 0.5% 267.38 7 0.5% 0.5% 1% 235.15 8 0.5% 1% 1.5% 241.74 9 0.5% 1.5% 2% 258.97 10 0.5% 2% 0.1% 247.48 11 1% 0.1% 1% 252.28 12 1% 0.5% 1.5% 243.81 13 1% 1% 2% 220.17 14 1% 1.5% 0.1% 252.43 15 1% 2% 0.5% 241.58 16 1.5% 0.1% 1.5% 210.65 17 1.5% 0.5% 2% 225.73 18 1.5% 1% 0.1% 283.54 19 1.5% 1.5% 0.5% 256.41 20 1.5% 2% 1% 221.65 21 2% 0.1% 2% 187.77 22 2% 0.5% 0.1% 173.24 23 2% 1% 0.5% 158.76 24 2% 1.5% 1% 144.58 25 2% 2% 1.5% 141.77 Table 2 shows that the effects of 0.5-1.5% neutral protease, 0.1-1% bromelain, and 0.1-0.5% hyaluronidase are better than other ratios.

[0024] (3) Effect of enzymatic hydrolysis conditions on the content of crude extract The present invention sets the temperature (35℃, 40℃, 45℃, 50℃), pH value (6.0, 6.5, 6.5, 7.5), and reaction time (1h, 2h, 3h, 4h) to enzymatically hydrolyze 8g of salivary gland tissue powder under the above enzymatic hydrolysis conditions to obtain crude extracts as shown in Table 3.

[0025] Table 3. Effects of enzymatic hydrolysis conditions of the compound enzyme on the content of crude extract. Serial Number temperature pH value reaction time Crude extract (mg) 1 35℃ 6.0 1h 183.42 2 35℃ 6.5 2h 267.53 3 35℃ 7.0 3h 271.95 4 35℃ 7.5 4h 223.47 5 40℃ 6.0 2h 267.58 6 40℃ 6.5 1h 233.71 7 40℃ 7.0 4h 292.76 8 40℃ 7.5 3h 248.91 9 45℃ 6.0 3h 252.62 10 45℃ 6.5 4h 263.93 11 45℃ 7.0 1h 226.73 12 45℃ 7.5 2h 235.28 13 50℃ 6.0 4h 229.66 14 50℃ 6.5 3h 253.79 15 50℃ 7.0 2h 218.14 16 50℃ 7.5 1h 205.68 As can be seen from Table 3 above, the crude extract yield is higher when the temperature is 35-45℃, the pH value is 6.5-7.0, and the reaction time is 2-4h. Example 1

[0026] A method for extracting and purifying highly active hirudin based on a combined enzymatic hydrolysis-chromatography approach, wherein the extraction and purification method is as follows: S1, Sample pretreatment: Live leeches were starved for one week (the purpose of which was to expel the contents of the leech's intestines), and the leeches were anesthetized with low temperature and then quickly dissected to obtain salivary gland tissue from their heads.

[0027] The salivary gland tissue of the head was rinsed with pre-cooled phosphate buffer (PBS, 0.01M, pH 6.5) and drained. The salivary gland tissue of the head was then placed in an ultra-low temperature freezer at -80°C for rapid freezing and then vacuum freeze-dried. The freeze-dried tissue was then ultra-finely pulverized to obtain salivary gland tissue powder.

[0028] S2, Extraction: Suspend salivary gland tissue powder in 10-15 times its volume of PBS extraction solution (PBS concentration is 0.02M, solution pH is 6.8). Add a complex enzyme to the suspension for gentle enzymatic hydrolysis, wherein the complex enzyme consists of 0.5%-1.5% (w / w) neutral protease, 0.2%-0.8% (w / w) bromelain, and 0.1%-0.5% (w / w) hyaluronidase. The specific hydrolysis conditions are: temperature 35-40℃, reaction pH 6.5-7.0, reaction time 2-4 hours, and slow stirring at 50-100 rpm. After the enzymatic hydrolysis is completed, the system is quickly heated to 85°C and held for 10 minutes to inactivate the enzyme activity. Then, it is immediately cooled in an ice bath and centrifuged at 4°C and 10,000 rpm for 20 minutes. The supernatant is collected, which is the crude extract. S3, repeated purification: S31, preliminary enrichment with macroporous adsorption resin: S311, Resin Pretreatment: HPD-100 macroporous adsorption resin is soaked in 95% ethanol for 24 hours, with stirring 3-4 times during the period. The resin effluent is rinsed with deionized water until no ethanol is found. Then, it is soaked in 5% hydrochloric acid solution for 2 hours and rinsed with deionized water until neutral. Finally, it is soaked in 5% sodium hydroxide solution for 2 hours and rinsed with deionized water until neutral to obtain HPD-100 macroporous adsorption resin with a water content of 45-55%. S312, the HPD-100 macroporous adsorption resin from step S311 is placed into an elution column to obtain an HPD-100 macroporous adsorption resin column. The crude extract is loaded into the HPD-100 macroporous adsorption resin column. After loading, it is washed with 2-3 column volumes of deionized water and eluted with 20%-30% ethanol aqueous solution. The elution peak is collected to obtain the first purified solution. The purpose of this step is to enrich hirudin and remove most water-soluble impurities such as sugars and pigments. S32, purified by cation exchange chromatography: S321, Wash the CM Sepharose Fast Flow cation exchange column with equilibration buffer until the pH and conductivity of the effluent are close to those of the equilibration buffer; Dialyze the first purified solution with equilibration buffer until the difference between the conductivity of the first purified solution and the equilibration buffer is ≤0.5 mS / cm; S322, The first purification solution from step S321 is loaded onto a CM Sepharose Fast Flow cation exchange column. After loading, the column is washed with equilibration buffer until the baseline stabilizes. Then, a linear NaCl gradient elution is performed (0→0.5M NaCl, dissolved in equilibration buffer). The elution peaks in the 0.3-0.4M NaCl range are collected to obtain the second purification solution, thereby removing unbound impurities. The equilibration buffer is a 0.02M acetate-ammonium acetate buffer with a pH of 5.0. S33, affinity chromatography for high purification: The second purified solution is loaded onto an affinity column coupled with thrombin. Hirudin will specifically bind to the column to achieve thrombin affinity chromatography. After thorough washing with equilibration buffer, elution is performed with glycine-hydrochloric acid buffer at pH 2.5-3.0. The collected solution is immediately neutralized with Tris base to maintain the activity of hirudin. S4, Concentration and Freeze-drying: The final purified solution is concentrated and desalted using an ultrafiltration tube with a molecular weight cutoff of 3 kDa, and then freeze-dried under vacuum to obtain a white flocculent or powdery high-purity hirudin product. Example 2

[0029] A method for extracting and purifying highly active hirudin based on a combined enzymatic hydrolysis-chromatography approach, wherein the extraction and purification method is as follows: S1, Sample pretreatment: 100g of live leeches were starved for one week (the purpose of which was to expel the contents of the leech's intestines), and the leeches were anesthetized with low temperature and then quickly dissected to obtain the salivary gland tissue of their heads.

[0030] The salivary gland tissue of the head was rinsed with pre-cooled phosphate buffer (PBS, 0.01M, pH 6.5) and drained. The salivary gland tissue of the head was then placed in an ultra-low temperature freezer at -80°C for rapid freezing and then vacuum freeze-dried. The freeze-dried tissue was then ultra-finely pulverized to obtain 10.03g of salivary gland tissue powder.

[0031] S2, Extraction: The salivary gland tissue powder was suspended in 10 times its volume of PBS extraction solution (PBS concentration was 0.02M, pH value was 6.8). A complex enzyme was added to the suspension for gentle enzymatic hydrolysis, wherein the complex enzyme consisted of 1.5% (w / w) neutral protease, 0.8% (w / w) bromelain, and 0.5% (w / w) hyaluronidase. The specific hydrolysis conditions were: temperature 40℃, reaction pH 6.5, reaction time 3 hours, and slow stirring at 50 rpm. After the enzymatic hydrolysis is completed, the system is quickly heated to 85°C and held for 10 minutes to inactivate the enzyme activity. Then, it is immediately cooled in an ice bath and centrifuged at 4°C and 10,000 rpm for 20 minutes. The supernatant is collected, which is the crude extract. S3, repeated purification: S31, preliminary enrichment with macroporous adsorption resin: S311, Resin Pretreatment: HPD-100 macroporous adsorption resin is soaked in 95% ethanol for 24 hours, with stirring 3-4 times during the period. The resin effluent is rinsed with deionized water until no ethanol is found. Then, it is soaked in 5% hydrochloric acid solution for 2 hours and rinsed with deionized water until neutral. Finally, it is soaked in 5% sodium hydroxide solution for 2 hours and rinsed with deionized water until neutral to obtain HPD-100 macroporous adsorption resin with a water content of 45-55%. S312, the HPD-100 macroporous adsorption resin from step S311 is placed into an elution column to obtain an HPD-100 macroporous adsorption resin column. The crude extract is loaded into the HPD-100 macroporous adsorption resin column. After loading, it is washed with 3 column volumes of deionized water and eluted with 25% ethanol aqueous solution. The elution peak is collected to obtain the first purified solution. The purpose of this step is to enrich hirudin and remove most water-soluble impurities such as sugars and pigments. S32, purified by cation exchange chromatography: S321, Wash the CM Sepharose Fast Flow cation exchange column with equilibration buffer until the pH and conductivity of the effluent are close to those of the equilibration buffer; Dialyze the first purified solution with equilibration buffer until the difference between the conductivity of the first purified solution and the equilibration buffer is ≤0.5 mS / cm; S322, The first purification solution from step S321 is loaded onto a CM Sepharose Fast Flow cation exchange column. After loading, the column is washed with equilibration buffer until the baseline stabilizes. Then, a linear NaCl gradient elution is performed (0→0.5M NaCl, dissolved in equilibration buffer). The elution peaks in the 0.3-0.4M NaCl range are collected to obtain the second purification solution, thereby removing unbound impurities. The equilibration buffer is a 0.02M acetate-ammonium acetate buffer with a pH of 5.0. S33, affinity chromatography for high purification: The second purified solution is loaded onto an affinity column coupled with thrombin. Hirudin will specifically bind to the column to achieve thrombin affinity chromatography. After thorough washing with equilibration buffer, elution is performed with glycine-hydrochloric acid buffer at pH 3.0. The collected solution is immediately neutralized with Tris base to maintain the activity of hirudin. S4, Concentration and Freeze-drying: The final purified solution is concentrated and desalted using an ultrafiltration tube with a molecular weight cutoff of 3 kDa, and then freeze-dried under vacuum to obtain a white flocculent or powdery high-purity hirudin product.

[0032] Comparative Example 1 A method for extracting and purifying highly active hirudin based on a combined enzymatic hydrolysis-chromatography approach, wherein the extraction and purification method is as follows: S1, Sample pretreatment: 100g of live leeches were starved for one week, and after being anesthetized at low temperature, the leeches were quickly dissected to obtain salivary gland tissue from their heads.

[0033] The salivary gland tissue of the head was rinsed with pre-cooled phosphate buffer (PBS, 0.01M, pH 6.5) and drained. The salivary gland tissue of the head was then placed in an ultra-low temperature freezer at -80°C for rapid freezing and then vacuum freeze-dried. The freeze-dried tissue was then ultra-finely pulverized to obtain 9.96g of salivary gland tissue powder.

[0034] S2, Extraction: The salivary gland tissue powder was suspended in 10 times its volume of PBS extraction solution (PBS concentration was 0.02M, pH value was 6.8). A complex enzyme was added to the suspension for gentle enzymatic hydrolysis, wherein the complex enzyme consisted of 1.5% (w / w) neutral protease, 0.8% (w / w) bromelain, and 0.5% (w / w) hyaluronidase. The specific hydrolysis conditions were: temperature 40℃, reaction pH 6.5, reaction time 3 hours, and slow stirring at 50 rpm. After the enzymatic hydrolysis is completed, the system is quickly heated to 85°C and held for 10 minutes to inactivate the enzyme activity. Then, it is immediately cooled in an ice bath and centrifuged at 4°C and 10,000 rpm for 20 minutes. The supernatant is collected, which is the crude extract. S3, preliminary enrichment with macroporous adsorption resin: S31, Resin pretreatment: HPD-100 macroporous adsorption resin is soaked in 95% ethanol for 24 hours, with stirring 3-4 times during the period. The resin effluent is rinsed with deionized water to remove ethanol. Then, it is soaked in 5% hydrochloric acid solution for 2 hours and rinsed with deionized water until neutral. Finally, it is soaked in 5% sodium hydroxide solution for 2 hours and rinsed with deionized water until neutral to obtain HPD-100 macroporous adsorption resin with a water content of 45-55%. S32, the HPD-100 macroporous adsorption resin from step S311 is placed into an elution column to obtain an HPD-100 macroporous adsorption resin column. The crude extract is loaded into the HPD-100 macroporous adsorption resin column. After loading, it is rinsed with 3 column volumes of deionized water and eluted with 25% ethanol aqueous solution. The elution peak is collected to obtain the purified solution. S4, Concentration and Freeze-drying: The obtained purified solution is concentrated and desalted using an ultrafiltration tube with a molecular weight cutoff of 3kDa, and then freeze-dried under vacuum to obtain the product.

[0035] Comparative Example 2 A method for extracting and purifying highly active hirudin based on a combined enzymatic hydrolysis-chromatography approach, wherein the extraction and purification method is as follows: S1, Sample pretreatment: 100g of live leeches were starved for one week, and after being anesthetized at low temperature, the leeches were quickly dissected to obtain salivary gland tissue from their heads.

[0036] The salivary gland tissue of the head was rinsed with pre-cooled phosphate buffer (PBS, 0.01M, pH 6.5) and drained. The salivary gland tissue of the head was then placed in an ultra-low temperature freezer at -80°C for rapid freezing and then vacuum freeze-dried. The freeze-dried tissue was then ultra-finely pulverized to obtain 10.35g of salivary gland tissue powder.

[0037] S2, Extraction: The salivary gland tissue powder was suspended in 10 times its volume of PBS extraction solution (PBS concentration was 0.02M, solution pH was 6.8). A complex enzyme was added to the suspension for gentle enzymatic hydrolysis. The complex enzyme consisted of 1.5% (w / w) neutral protease, 0.8% (w / w) bromelain, and 0.5% (w / w) hyaluronidase. The specific hydrolysis conditions were: temperature 40℃, reaction pH 6.5, reaction time 3 hours, and slow stirring at 50 rpm. After the enzymatic hydrolysis is completed, the system is quickly heated to 85°C and held for 10 minutes to inactivate the enzyme activity. Then, it is immediately cooled in an ice bath and centrifuged at 4°C and 10,000 rpm for 20 minutes. The supernatant is collected, which is the crude extract. S3, cation exchange chromatography purification: S31, wash the CM Sepharose Fast Flow cation exchange column with equilibration buffer until the pH and conductivity of the effluent are close to those of the equilibration buffer; dialyze the first purified solution with equilibration buffer until the difference between the conductivity of the crude extract and the equilibration buffer is ≤0.5 mS / cm; S32, the crude extract from step S321 is loaded onto a CM Sepharose Fast Flow cation exchange column. After loading, the column is washed with equilibration buffer until the baseline stabilizes. Then, a linear NaCl gradient elution is performed (0→0.5M NaCl, dissolved in equilibration buffer). The elution peaks in the 0.3-0.4M NaCl range are collected to obtain the purified solution, thereby removing unbound impurities. The equilibration buffer is a 0.02M acetate-ammonium acetate buffer with a pH of 5.0. S4, Concentration and Freeze-drying: The obtained purified solution was concentrated and desalted using an ultrafiltration tube with a molecular weight cutoff of 3 kDa, and then freeze-dried under vacuum to obtain hirudin product.

[0038] Comparative Example 3 A method for extracting and purifying highly active hirudin based on a combined enzymatic hydrolysis-chromatography approach, wherein the extraction and purification method is as follows: S1, Sample pretreatment: 100g of live leeches were starved for one week, and after being anesthetized at low temperature, the leeches were quickly dissected to obtain salivary gland tissue from their heads.

[0039] The salivary gland tissue of the head was rinsed with pre-cooled phosphate buffer (PBS, 0.01M, pH 6.5) and drained. The salivary gland tissue of the head was then placed in an ultra-low temperature freezer at -80°C for rapid freezing and then vacuum freeze-dried. The freeze-dried tissue was then ultra-finely pulverized to obtain 9.84g of salivary gland tissue powder.

[0040] S2, Extraction: The salivary gland tissue powder was suspended in 10 times its volume of PBS extraction solution (PBS concentration was 0.02M, solution pH was 6.8). A complex enzyme was added to the suspension for gentle enzymatic hydrolysis. The complex enzyme consisted of 1.5% (w / w) neutral protease, 0.8% (w / w) bromelain, and 0.5% (w / w) hyaluronidase. The specific hydrolysis conditions were: temperature 40℃, reaction pH 6.5, reaction time 3 hours, and slow stirring at 50 rpm. After the enzymatic hydrolysis is completed, the system is quickly heated to 85°C and held for 10 minutes to inactivate the enzyme activity. Then, it is immediately cooled in an ice bath and centrifuged at 4°C and 10,000 rpm for 20 minutes. The supernatant is collected, which is the crude extract. S3, affinity chromatography for high purification: The crude extract is loaded onto an affinity column coupled with thrombin. Hirudin will specifically bind to the column to achieve thrombin affinity chromatography. After thorough washing with equilibration buffer, elution is performed with glycine-hydrochloric acid buffer at pH 3.0. The collected solution is immediately neutralized with Tris base to maintain the activity of hirudin and obtain the purified solution. S4, Concentration and Freeze-drying: The purified solution is concentrated and desalted using an ultrafiltration tube with a molecular weight cutoff of 3 kDa, and then freeze-dried under vacuum to obtain a product containing hirudin.

[0041] Comparative Example 4 A method for extracting and purifying highly active hirudin based on a combined enzymatic hydrolysis-chromatography approach, wherein the extraction and purification method is as follows: S1, Sample pretreatment: 100g of live leeches were starved for one week, and after being anesthetized at low temperature, the leeches were quickly dissected to obtain salivary gland tissue from their heads.

[0042] The salivary gland tissue of the head was rinsed with pre-cooled phosphate buffer (PBS, 0.01M, pH 6.5) and drained. The salivary gland tissue of the head was then placed in an ultra-low temperature freezer at -80°C for rapid freezing and then vacuum freeze-dried. The freeze-dried tissue was then ultra-finely pulverized to obtain 9.91g of salivary gland tissue powder.

[0043] S2, Extraction: The salivary gland tissue powder was suspended in 10 times its volume of PBS extraction solution (PBS concentration was 0.02M, pH value was 6.8). A complex enzyme was added to the suspension for gentle enzymatic hydrolysis, wherein the complex enzyme consisted of 1.5% (w / w) neutral protease, 0.8% (w / w) bromelain, and 0.5% (w / w) hyaluronidase. The specific hydrolysis conditions were: temperature 40℃, reaction pH 6.5, reaction time 3 hours, and slow stirring at 50 rpm. After the enzymatic hydrolysis is completed, the system is quickly heated to 85°C and held for 10 minutes to inactivate the enzyme activity. Then, it is immediately cooled in an ice bath and centrifuged at 4°C and 10,000 rpm for 20 minutes. The supernatant is collected, which is the crude extract. S3, preliminary enrichment with macroporous adsorption resin: S31, Resin pretreatment: AB-8 type macroporous adsorption resin is soaked in 95% ethanol for 24 hours, with stirring 3-4 times during the period. The resin effluent is rinsed with deionized water to remove ethanol. Then, it is soaked in 5% hydrochloric acid solution for 2 hours and rinsed with deionized water until neutral. Finally, it is soaked in 5% sodium hydroxide solution for 2 hours and rinsed with deionized water until neutral to obtain AB-8 type macroporous adsorption resin with a water content of 45-55%. S32, the AB-8 type macroporous adsorption resin from step S311 is placed into an elution column to obtain an AB-8 type macroporous adsorption resin column. The crude extract is loaded into the AB-8 type macroporous adsorption resin column. After loading, it is rinsed with 3 column volumes of deionized water and eluted with 25% ethanol aqueous solution. The elution peak is collected to obtain the purified solution. S4, Concentration and Freeze-drying: The obtained purified solution is concentrated and desalted using an ultrafiltration tube with a molecular weight cutoff of 3kDa, and then freeze-dried under vacuum to obtain a product containing hirudin.

[0044] Comparative Example 5 A method for extracting and purifying highly active hirudin based on a combined enzymatic hydrolysis-chromatography approach, wherein the extraction and purification method is as follows: S1, Sample pretreatment: 100g of live leeches were starved for one week (the purpose of which was to expel the contents of the leech's intestines), and the leeches were anesthetized with low temperature and then quickly dissected to obtain the salivary gland tissue of their heads.

[0045] The salivary gland tissue of the head was rinsed with pre-cooled phosphate-buffered saline (PBS, 0.01M, pH 6.5) and drained. The salivary gland tissue of the head was then placed in an ultra-low temperature freezer at -80°C for rapid freezing and then vacuum freeze-dried. The freeze-dried tissue was then ultra-finely pulverized to obtain 10.21g of salivary gland tissue powder.

[0046] S2, Extraction: The salivary gland tissue powder was suspended in 10 times its volume of PBS extraction solution (PBS concentration was 0.02M, pH value was 6.8). A complex enzyme was added to the suspension for gentle enzymatic hydrolysis, wherein the complex enzyme consisted of 1.5% (w / w) neutral protease, 0.8% (w / w) bromelain, and 0.5% (w / w) hyaluronidase. The specific hydrolysis conditions were: temperature 40℃, reaction pH 6.5, reaction time 3 hours, and slow stirring at 50 rpm. After the enzymatic hydrolysis is completed, the system is quickly heated to 85°C and held for 10 minutes to inactivate the enzyme activity. Then, it is immediately cooled in an ice bath and centrifuged at 4°C and 10,000 rpm for 20 minutes. The supernatant is collected, which is the crude extract. S3, repeated purification: S31, preliminary enrichment with macroporous adsorption resin: S311, Resin pretreatment: AB-8 type macroporous adsorption resin is soaked in 95% ethanol for 24 hours, with stirring 3-4 times during the period. The resin effluent is rinsed with deionized water to remove ethanol. Then, it is soaked in 5% hydrochloric acid solution for 2 hours and rinsed with deionized water until neutral. Finally, it is soaked in 5% sodium hydroxide solution for 2 hours and rinsed with deionized water until neutral to obtain AB-8 type macroporous adsorption resin with a water content of 45-55%. S312, the AB-8 macroporous adsorption resin from step S311 is placed into an elution column to obtain an AB-8 macroporous adsorption resin column. The crude extract is loaded into the AB-8 macroporous adsorption resin column. After loading, it is washed with 3 column volumes of deionized water and eluted with 25% ethanol aqueous solution. The elution peak is collected to obtain the first purified solution. The purpose of this step is to enrich hirudin and remove most water-soluble impurities such as sugars and pigments. S32, purified by cation exchange chromatography: S321, Wash the SP-Sephadex C25 cation exchange column with equilibration buffer until the pH and conductivity of the effluent are close to those of the equilibration buffer; Dialyze the first purified solution with equilibration buffer until the difference between the conductivity of the first purified solution and the equilibration buffer is ≤0.5 mS / cm; S322, The first purification solution from step S321 is loaded onto an SP-Sephadex C25 cation exchange column. After loading, the column is washed with equilibration buffer until the baseline stabilizes. Then, a linear NaCl gradient elution is performed (0→0.5M NaCl, dissolved in equilibration buffer). The elution peaks in the 0.3-0.4M NaCl range are collected to obtain the second purification solution, thereby removing unbound impurities. The equilibration buffer is a 0.02M acetate-ammonium acetate buffer with a pH of 5.0. S33, affinity chromatography for high purification: The second purified solution is loaded onto an affinity column coupled with thrombin. Hirudin will specifically bind to the column to achieve thrombin affinity chromatography. After thorough washing with equilibration buffer, elution is performed with glycine-hydrochloric acid buffer at pH 3.0. The collected solution is immediately neutralized with Tris base to maintain the activity of hirudin. S4, Concentration and Freeze-drying: The final purified solution is concentrated and desalted using an ultrafiltration tube with a molecular weight cutoff of 3 kDa, and then freeze-dried under vacuum to obtain a product containing hirudin.

[0047] Table 4. Extraction products, hirudin purity, and specific activity of each embodiment. Crude extract (mg) Product (mg) Hirudin purity (%) Specific activity (ATU / mg) Example 2 369.45 85.79 96.20 15800 Comparative Example 1 365.53 241.25 34.21 6823 Comparative Example 2 372.69 305.61 27.00 8242 Comparative Example 3 358.82 167.72 50.18 9167 Comparative Example 4 263.81 258.59 32.27 6150 Comparative Example 5 273.17 92.64 78.68 12391 As can be seen from Table 4, comparing Comparative Example 1 and Comparative Example 4, it can be seen that the purity of hirudin obtained by purification using HPD-100 macroporous adsorption resin is higher than that of hirudin obtained by purification using AB-8 macroporous adsorption resin; comparing Example 2 and Comparative Example 5, it can be seen that the hirudin obtained by purification method of the present invention has better purity and higher specific activity.

[0048] This invention can be implemented in various ways and is not limited to the embodiments described. Those skilled in the art will understand that the invention can be implemented in other specific ways without changing the technical concept or essential features. Therefore, it should be understood that the embodiments described above are exemplary and not intended to limit the invention.

Claims

1. A method for extracting and purifying highly active hirudin based on a combined enzymatic hydrolysis-chromatography approach, characterized in that, The extraction and purification methods include: S1, Sample pretreatment: Dissect the leech and take the salivary gland tissue from its head. Freeze-dry and pulverize the salivary gland tissue to obtain salivary gland tissue powder. S2, Extraction: The salivary gland tissue powder was extracted by enzymatic hydrolysis with a compound enzyme to obtain a crude extract; S3, repeated purification: S31, Preliminary enrichment with macroporous adsorption resin: The crude extract is loaded into an HPD-100 macroporous adsorption resin column. After loading, it is washed with 2-3 column volumes of deionized water and eluted with 20%-30% ethanol aqueous solution. The elution peak is collected to obtain the first purified solution. S32, Cation exchange chromatography purification: The first purified solution is loaded onto a CM Sepharose Fast Flow cation exchange column. After loading, the column is washed with equilibration buffer until the baseline is stable. Then, the column is eluted with a linear NaCl gradient and the elution peaks in the 0.3-0.4M NaCl range are collected to obtain the second purified solution. S33, affinity chromatography for high purification: The second purification solution is loaded onto an affinity column coupled with thrombin, washed thoroughly with equilibration buffer, eluted with glycine-hydrochloric acid buffer, and the collection solution is neutralized with Tris base to obtain the purified product. S4, the purified product is concentrated and freeze-dried to obtain a high-purity hirudin product.

2. The method for extraction and purification of highly active hirudin based on combined enzymatic hydrolysis and chromatography as described in claim 1, characterized in that, In step S1, the salivary gland tissue from the head of the leech is dissected, and the salivary gland tissue is freeze-dried and pulverized to obtain salivary gland tissue powder, as follows: (1) Live leeches were starved for one week, and after being anesthetized at low temperature, the leeches were quickly dissected to obtain salivary gland tissue from their heads; (2) After rinsing the head salivary gland tissue with pre-cooled phosphate buffer and draining it, the head salivary gland tissue was placed in an ultra-low temperature freezer at -80℃ for quick freezing and then vacuum freeze-drying. The freeze-dried tissue was then ultra-finely pulverized to obtain salivary gland tissue powder.

3. The extraction and purification method of highly active hirudin based on combined enzymatic hydrolysis and chromatography as described in claim 2, characterized in that, The phosphate buffer is PBS, the concentration of which is 0.01M and the pH of the solution is 6.

5.

4. The method for extraction and purification of highly active hirudin based on combined enzymatic hydrolysis and chromatography as described in claim 1, characterized in that, In step S2, the step of using a complex enzyme to extract crude extract from the salivary gland tissue powder is wherein, by weight percentage, the complex enzyme consists of 0.5%-1.5% neutral protease, 0.2%-0.8% bromelain, and 0.1%-0.5% hyaluronidase.

5. The method for extraction and purification of highly active hirudin based on combined enzymatic hydrolysis and chromatography as described in claim 4, characterized in that, The enzymatic hydrolysis conditions for extracting the salivary gland tissue powder using the compound enzyme are: temperature 35-45℃, pH 6.5-7.0, reaction time 2-4h, and slow stirring at 50-100rpm.

6. The method for extraction and purification of highly active hirudin based on combined enzymatic hydrolysis and chromatography as described in claim 5, characterized in that, After enzymatic hydrolysis, the solution is rapidly heated to 80-85℃ and held for 10-13 minutes to inactivate the enzyme. Then, it is immediately cooled in an ice bath and centrifuged at 4-6℃ and 8000-10000 rpm for 18-22 minutes. The supernatant is collected as the crude extract.

7. The method for extraction and purification of highly active hirudin based on combined enzymatic hydrolysis and chromatography as described in claim 1, characterized in that, In step S32, the equilibration buffer is an acetate-ammonium acetate buffer with a concentration of 0.02M and a pH value of 5.0-6.

0.

8. The method for extraction and purification of highly active hirudin based on combined enzymatic hydrolysis and chromatography as described in claim 1, characterized in that, In step S31, the preparation method of the HPD-100 macroporous adsorption resin column is as follows: S311, HPD-100 macroporous adsorption resin is soaked in 95% ethanol for 24 hours, stirred 3-4 times during the period, and the resin effluent is rinsed with deionized water to remove ethanol. Then it is soaked in 5% hydrochloric acid solution for 2 hours and rinsed with deionized water until neutral. Finally, it is soaked in 5% sodium hydroxide solution for 2 hours and rinsed with deionized water until neutral to obtain HPD-100 macroporous adsorption resin with a water content of 45-55%. S312, the HPD-100 macroporous adsorption resin from step S311 is placed into an elution column to obtain an HPD-100 macroporous adsorption resin column.

9. The method for extraction and purification of highly active hirudin based on combined enzymatic hydrolysis and chromatography as described in claim 1, characterized in that, In step S4, the process of concentrating and freeze-drying the purified product to obtain a high-purity hirudin product specifically involves: concentrating and desalting the purified product using an ultrafiltration tube with a molecular weight cutoff of 3 kDa, followed by vacuum freeze-drying.