Process method for preparing heparin based on enzyme method

Abstract

The invention belongs to the technical field of heparin, and particularly relates to a process method for preparing heparin based on an enzyme method. The process method comprises the following steps: (1) pretreating heparin; (2) carrying out crosslinking on the Phe-Leu-Asn-Gln-Asp and the sepharose, and carrying out cross-linking on the obtained product and the sepharose; adding the cross-linked gel into a sodium phosphate buffer solution with the pH value of 7.2-7.5, swelling, and absorbing the supernatant; (3) dissolving the pretreated heparin in a buffer solution with the volume 1-1.2 times that of the pretreated heparin, and absorbing the supernatant; (4) after the active center site of the heparin is adsorbed on the pentapeptide of the gel, centrifuging and taking the precipitate; (5) collecting trapped fluid; (6) carrying out alcohol precipitation and impurity removal on the trapped fluid, and drying and weighing the obtained precipitate; and (7) adding 10-12 times of 1 mol / L NaCl solution into the precipitate for dissolving, collecting filtrate, and freeze-drying the filtrate to obtain the heparin prepared based on the enzyme method. The heparin product prepared by the method is good in clarity, free of opalescence and colorless.

Description

Technical Field

[0001] The invention belongs to the technical field of heparin, and in particular relates to a process for preparing heparin based on an enzymatic method. Background Art

[0002] Heparin is a widely used anticoagulant drug with strong anticoagulant and antithrombotic effects. It is mainly extracted from animal tissues, especially the small intestinal mucosa of pigs and the lungs of cattle. As an important anticoagulant drug, heparin has a wide range of clinical applications, especially in the prevention and treatment of thrombotic diseases.

[0003] Low molecular weight heparin is obtained by chemical degradation, bio-enzyme degradation or physical degradation of heparin. Due to the structural complexity of the raw material heparin and the uncertain impurities in heparin, the quality of low molecular weight heparin made from heparin varies greatly.

[0004] Heparin may encounter a variety of problems during the preparation process, such as poor product clarity, opalescence, yellow or even dark brown-red color, and high absorbance. These problems are usually related to factors such as impurities in the production process, incomplete enzymatic hydrolysis, and by-product generation. 1. Poor product clarity, protein and other macromolecular impurities: The original heparin extract may contain macromolecular impurities such as proteins and polysaccharides that have not been completely removed. These impurities will cause the solution to be turbid and reduce clarity. If the enzymatic hydrolysis step is not thorough, incompletely degraded macromolecules will remain in the solution, resulting in a decrease in clarity. If the filtration step is insufficient and fails to effectively remove tiny particles and insoluble matter, the clarity of the final product will also be poor. 2. The finished product has opalescence. Even after filtration, there may still be some tiny particles or colloidal particles, which will cause the solution to have opalescence. Some enzymatic hydrolysis byproducts may be colloidal or fine particles, which are easily suspended in the solution to form opalescence. If some components are not completely dissolved, the solution will also have opalescence. 3. The color is yellow or even dark brown-red. The original heparin extract may contain naturally occurring pigments (such as carotenoids, polyphenols) or other colored compounds, which are not easily removed during the enzymatic hydrolysis process. Some enzymatic byproducts may be colored, especially when the enzymatic conditions are inappropriate or the enzymatic hydrolysis is incomplete, these byproducts will accumulate and affect the color. 4. High absorbance. As mentioned earlier, the presence of colored impurities will lead to increased absorbance. If large molecular impurities are not completely removed, these impurities usually have a higher absorbance, especially in the ultraviolet region (such as the absorption peak of protein at 280nm).

[0005] By-product generation: Some by-products may be generated during the enzymatic hydrolysis process, and these by-products may also have a high absorbance.

[0006] Therefore, there is an urgent need for a process for preparing heparin based on an enzymatic method. Summary of the invention

[0007] The purpose of the present invention is to provide a process for preparing heparin based on an enzymatic method.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] A process for preparing heparin based on an enzymatic method comprises the following steps:

[0010] (1) enzymatically hydrolyzing commercially available heparin, and after the enzymatic hydrolysis, centrifuging at 3500-3700 rpm to obtain a precipitate to obtain pretreated heparin;

[0011] (2) Phe-Leu-Asn-Gln-Asp and sepharose are cross-linked; after cross-linking, sodium phosphate buffer (pH 7.2-7.5) is added to the gel, and the supernatant is removed after swelling;

[0012] (3) Pre-treated heparin was dissolved in 1-1.2 times the volume of buffer, and 1-1.2 mg of gel was loaded on the sample at a ratio of 3 mg of heparin. After loading, the sample was stirred for 5-10 minutes, allowed to stand for 20-30 minutes, and the supernatant was removed by aspiration;

[0013] (4) After the active center of heparin is adsorbed on the pentapeptide of the gel, 2-3 mg of heparin is added to 2 mU of heparinase II, stirred and hydrolyzed in a water bath at 35-40 °C for 3-4 h, and the precipitate is collected by centrifugation;

[0014] (5) adding 20-22 times the amount of 1 mol / L NaCl solution to the precipitate to dissolve it, and after it is completely dissolved, performing tangential flow circulation ultrafiltration with an ultrafiltration membrane, and collecting the retentate;

[0015] (6) removing impurities from the retentate by alcohol precipitation, and drying and weighing the resulting precipitate;

[0016] (7) adding 10-12 times the amount of 1 mol / L NaCl solution to the precipitate for dissolution, filtering through a sterilization membrane after dissolution, collecting the filtrate, and adjusting the pH value of the filtrate to 6.5-7.2; freeze-drying the filtrate to obtain heparin prepared by the enzymatic method.

[0017] The present invention ensures that the final product has good clarity, no opalescence, is colorless and has extremely low absorbance through a series of fine operation steps. First, an affinity gel that specifically adsorbs heparin is prepared, and the pentapeptide can specifically recognize and bind to the active center site of heparin, thereby achieving efficient separation and purification. The ultrafiltration step effectively removes most of the small molecule impurities and by-products, so that the heparin concentration in the final retentate is higher and the impurities are less, thereby significantly improving the clarity. The alcohol precipitation step further removes the remaining small molecule impurities and soluble by-products, so that the final product is purer and has higher clarity.

[0018] Furthermore, the enzymatic hydrolysis method is: firstly add the modified composite protease to the heparin, enzymatic hydrolysis at 38-40°C for 1-2 hours, filter, continue to add heparinase II, and enzymatic hydrolysis at 38-40°C for 3-4 hours.

[0019] Furthermore, 2 mU of modified composite protease was added to 1-5 mg of heparin.

[0020] Furthermore, 2 mU of heparinase II was added according to 1-5 mg of heparin.

[0021] The enzymatic hydrolysis method of the present invention can significantly improve the clarity of heparin, reduce the absorbance and improve its color through a series of carefully designed steps. The modified composite protease can specifically degrade proteins and other macromolecular organic impurities. Enzymatic hydrolysis at 38-40°C for 1-2 hours ensures sufficient time for the enzyme to fully act on these impurities and decompose them into smaller peptides or amino acids, which are easier to filter and remove. The enzymatic hydrolysis method optimizes the molecular structure of heparin, removes some inactive parts, avoids unnecessary chemical reactions, and reduces the generation of colored byproducts.

[0022] Furthermore, the preparation method of the modified composite protease comprises the following steps:

[0023] (1) mixing acrylamide solution and N,N'-methylenebisacrylamide solution evenly, adding Tris-HCl buffer, adding ammonium persulfate initiator to the mixed solution, quickly adding 2-3 drops of tetramethylethylenediamine, pouring the mixed solution into a mold, and covering the top with a glass plate to ensure a flat surface, polymerizing at room temperature for 14-18 hours, cutting the polymerized polyacrylamide gel into small particles, washing with buffer 1-3 times to remove unbound substances, and obtaining small particles of polyacrylamide gel;

[0024] (2) dissolving fig protease and bromelain in a weight ratio of 1:(1.2-1.4) in Tris-HCl buffer, mixing with small granular polyacrylamide gel, stirring with a magnetic stirrer at a stirring speed of 120-150 rpm / min, adsorption time of 2-4 hours, using a low-speed centrifuge at a centrifugal speed of about 1000-2000 rpm / min, centrifugation time of 5-10 minutes, and then freeze-drying and storing at -20°C to -15°C to obtain a modified composite protease.

[0025] Furthermore, the specific process of alcohol precipitation is as follows: after the microporous filtration is completed, 1.0-1.2 times the volume of 94-96% ethanol is added to the filtrate, and the mixture is stirred and allowed to stand for 13-15 hours.

[0026] The present invention can significantly improve the stability and activity of enzymes by fixing ficin and bromelain on polyacrylamide gel. These enzymes can more effectively degrade proteins and other macromolecular impurities, and reduce the presence of these impurities in the solution. Reduce colored impurities: Proteins and other macromolecular impurities usually have high absorbance, causing the solution color to become darker. By efficiently degrading these impurities, the presence of colored compounds is reduced, thereby reducing the overall absorbance of the solution. The immobilized enzyme can better decompose macromolecular impurities during the enzymolysis process, making it easier to filter and remove them, reducing tiny particles and insolubles in the solution, and making the final product clearer and free of opalescence. By fixing the enzyme on the polyacrylamide gel, the enzyme molecules are not easy to fall off from the carrier, thereby improving the stability and recyclability of the enzyme.

[0027] Furthermore, after the complete dissolution, tangential flow circulation ultrafiltration is performed for 6-8 hours using an ultrafiltration membrane with a molecular weight cutoff of 3000-5000Da, and the retentate is collected.

[0028] Furthermore, the cross-linked gel is added with 10-12 times volume of 50-52 mM sodium phosphate buffer.

[0029] Furthermore, after the dissolution is completed, the solution is filtered through a 0.1 μm sterilization membrane and the filtrate is collected.

[0030] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0031] 1. The enzymatic hydrolysis method of the present invention can significantly improve the clarity, reduce the absorbance and improve the color of heparin through a series of carefully designed steps. The modified composite protease can specifically degrade proteins and other macromolecular organic impurities. If these impurities are retained in the final product, the solution will become turbid (reduced clarity), increase absorbance, and may cause color changes (such as yellow or brown). Enzymatic hydrolysis at 38-40°C for 1-2 hours ensures sufficient time for the enzyme to fully act on these impurities and decompose them into smaller peptides or amino acids, which are easier to be filtered and removed. The enzymatic hydrolysis method optimizes the molecular structure of heparin, removes some inactive parts, avoids unnecessary chemical reactions, and reduces the generation of colored by-products.

[0032] 2. The enzymatic hydrolysis method of the present invention can significantly improve the clarity of heparin, reduce the absorbance and improve its color through a series of carefully designed steps. The modified composite protease can specifically degrade proteins and other macromolecular organic impurities. Enzymatic hydrolysis at 38-40°C for 1-2 hours ensures sufficient time for the enzyme to fully act on these impurities and decompose them into smaller peptides or amino acids, which are easier to filter and remove. The enzymatic hydrolysis method optimizes the molecular structure of heparin, removes some inactive parts, avoids unnecessary chemical reactions, and reduces the generation of colored byproducts. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1

[0035] This embodiment provides a process for preparing heparin based on an enzymatic method, comprising the following steps:

[0036] (1) enzymatically hydrolyzing commercially available heparin, and after the enzymatic hydrolysis, centrifuging at 3600 rpm to obtain a precipitate to obtain pretreated heparin;

[0037] (2) Phe-Leu-Asn-Gln-Asp and sepharose are cross-linked; after cross-linking, sodium phosphate buffer (pH 7.3) is added to the gel, and the supernatant is removed after swelling;

[0038] (3) Pre-treated heparin was dissolved in 1.1 times the volume of buffer, and 1.1 mg of gel was loaded on the sample at a ratio of 3 mg of heparin. After loading, the sample was stirred for 6 minutes, allowed to stand for 25 minutes, and the supernatant was removed by aspiration;

[0039] (4) After the active center of heparin is adsorbed on the pentapeptide of the gel, 2.5 mg of heparin is added to 2 mU of heparinase II, stirred and hydrolyzed in a water bath at 37°C for 3.5 h, and the precipitate is collected by centrifugation;

[0040] (5) adding 21 times the amount of 1 mol / L NaCl solution to the precipitate to dissolve it, and after it is completely dissolved, performing tangential flow circulation ultrafiltration with an ultrafiltration membrane to collect the retentate;

[0041] (6) removing impurities from the retentate by alcohol precipitation, and drying and weighing the resulting precipitate;

[0042] (7) adding 11 times the amount of 1 mol / L NaCl solution to the precipitate for dissolution, filtering through a sterilization membrane after dissolution, collecting the filtrate, and adjusting the pH value of the filtrate to 7; freeze-drying the filtrate to obtain heparin prepared by the enzymatic method.

[0043] The enzymatic hydrolysis method is: first add the modified composite protease to the heparin, enzymatic hydrolyze at 40°C for 1 hour, filter, continue to add heparinase II, and enzymatic hydrolyze at 40°C for 3 hours.

[0044] 2 mU of modified composite protease was added per 5 mg of heparin.

[0045] 2 mU of heparinase II was added per 5 mg of heparin.

[0046] The preparation method of the modified composite protease comprises the following steps:

[0047] (1) Mixing acrylamide solution and N,N'-methylenebisacrylamide solution evenly, adding Tris-HCl buffer, adding ammonium persulfate initiator to the mixed solution, quickly adding 3 drops of tetramethylethylenediamine, pouring the mixed solution into a mold, and covering the top with a glass plate to ensure a flat surface, polymerizing at room temperature for 14 hours, cutting the polymerized polyacrylamide gel into small particles, washing with buffer 3 times to remove unbound substances, and obtaining small particles of polyacrylamide gel;

[0048] (2) Dissolve fig protease and bromelain in a weight ratio of 1:1.2 in Tris-HCl buffer, mix with small granular polyacrylamide gel, stir with a magnetic stirrer at a stirring speed of 150 rpm / min, adsorb for 4 hours, use a low-speed centrifuge at a centrifugal speed of about 2000 rpm / min, centrifuge for 10 minutes, and then freeze-dry and store at -20°C to -15°C to obtain a modified composite protease.

[0049] The specific process of alcohol precipitation is as follows: after the microporous filtration is completed, 1.2 times the volume of 94% ethanol is added to the filtrate, and the mixture is stirred and allowed to stand for 15 hours.

[0050] After complete dissolution, tangential flow circulation ultrafiltration was performed for 8 hours using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and the retentate was collected.

[0051] After cross-linking, 12 volumes of 50 mM sodium phosphate buffer were added to the gel.

[0052] After the dissolution is completed, the solution is filtered through a 0.1 μm sterilization membrane and the filtrate is collected.

[0053] Example 2

[0054] This embodiment provides a process for preparing heparin based on an enzymatic method, comprising the following steps:

[0055] (1) enzymatically hydrolyzing commercially available heparin, and after the enzymatic hydrolysis, centrifuging at 3700 rpm to obtain a precipitate to obtain pretreated heparin;

[0056] (2) Phe-Leu-Asn-Gln-Asp and sepharose are cross-linked; after cross-linking, sodium phosphate buffer (pH 7.2) is added to the gel, and the supernatant is removed after swelling;

[0057] (3) Pre-treated heparin was dissolved in 1.2 times the volume of buffer, and 1 mg of gel was added to 3 mg of heparin. After loading, the mixture was stirred for 5 min, allowed to stand for 30 min, and the supernatant was removed by aspiration.

[0058] (4) After the active center of heparin is adsorbed on the pentapeptide of the gel, 2 mg of heparin is added to 2 mU of heparinase II, stirred and hydrolyzed in a water bath at 40 °C for 4 h, and the precipitate is collected by centrifugation;

[0059] (5) adding 20 times the amount of 1 mol / L NaCl solution to the precipitate to dissolve it, and after it is completely dissolved, performing tangential flow circulation ultrafiltration with an ultrafiltration membrane to collect the retentate;

[0060] (6) removing impurities from the retentate by alcohol precipitation, and drying and weighing the resulting precipitate;

[0061] (7) adding 12 times the amount of 1 mol / L NaCl solution to the precipitate for dissolution, filtering through a sterilization membrane after dissolution, collecting the filtrate, and adjusting the pH value of the filtrate to 6.5; freeze-drying the filtrate to obtain heparin prepared by the enzymatic method.

[0062] The enzymatic hydrolysis method is: first add the modified composite protease to the heparin, enzymatic hydrolyze at 40°C for 2 hours, filter, continue to add heparinase II, and enzymatic hydrolyze at 40°C for 3 hours.

[0063] 2 mU of modified composite protease was added per 5 mg of heparin.

[0064] 2 mU of heparinase II was added per 5 mg of heparin.

[0065] The preparation method of the modified composite protease comprises the following steps:

[0066] (1) Mixing acrylamide solution and N,N'-methylenebisacrylamide solution evenly, adding Tris-HCl buffer, adding ammonium persulfate initiator to the mixed solution, quickly adding 2 drops of tetramethylethylenediamine, pouring the mixed solution into a mold, and covering the top with a glass plate to ensure a smooth surface, polymerizing at room temperature for 18 hours, cutting the polymerized polyacrylamide gel into small particles, washing once with buffer to remove unbound substances, and obtaining small particles of polyacrylamide gel;

[0067] (2) Dissolve fig protease and bromelain in a weight ratio of 1:1.3 in Tris-HCl buffer, mix with small granular polyacrylamide gel, stir with a magnetic stirrer at a stirring speed of 150 rpm / min, adsorb for 2 hours, use a low-speed centrifuge at a centrifugal speed of about 2000 rpm / min, centrifuge for 5 minutes, and then freeze-dry and store at -20°C to -15°C to obtain a modified composite protease.

[0068] The specific process of alcohol precipitation is as follows: after the microporous filtration is completed, 1.2 times the volume of 94% ethanol is added to the filtrate, and the mixture is stirred and allowed to stand for 13 hours.

[0069] After complete dissolution, tangential flow circulation ultrafiltration was performed for 6 hours using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and the retentate was collected.

[0070] After cross-linking, 12 volumes of 50 mM sodium phosphate buffer were added to the gel.

[0071] After the dissolution is completed, the solution is filtered through a 0.1 μm sterilization membrane and the filtrate is collected.

[0072] Example 3

[0073] This embodiment provides a process for preparing heparin based on an enzymatic method, comprising the following steps:

[0074] (1) enzymatically hydrolyzing commercially available heparin, and after the enzymatic hydrolysis, centrifuging at 3500 rpm to obtain a precipitate to obtain pretreated heparin;

[0075] (2) Phe-Leu-Asn-Gln-Asp and sepharose are cross-linked; after cross-linking, sodium phosphate buffer (pH 7.2) is added to the gel, and the supernatant is removed after swelling;

[0076] (3) Pre-treated heparin was dissolved in 1 volume of buffer, and 1 mg of gel was added to 3 mg of heparin. After loading, the mixture was stirred for 10 min, allowed to stand for 20 min, and the supernatant was removed by aspiration.

[0077] (4) After the active center of heparin is adsorbed on the pentapeptide of the gel, 2 mU of heparinase II is added to 3 mg of heparin, stirred and hydrolyzed in a water bath at 35°C for 4 h, and the precipitate is collected by centrifugation;

[0078] (5) adding 20 times the amount of 1 mol / L NaCl solution to the precipitate to dissolve it, and after it is completely dissolved, performing tangential flow circulation ultrafiltration with an ultrafiltration membrane to collect the retentate;

[0079] (6) removing impurities from the retentate by alcohol precipitation, and drying and weighing the resulting precipitate;

[0080] (7) adding 10 times the amount of 1 mol / L NaCl solution to the precipitate for dissolution, filtering through a sterilization membrane after dissolution, collecting the filtrate, and adjusting the pH value of the filtrate to 6.5; freeze-drying the filtrate to obtain heparin prepared by the enzymatic method.

[0081] The enzymatic hydrolysis method is: first add the modified composite protease to the heparin, enzymatic hydrolyze at 38°C for 1 hour, filter, continue to add heparinase II, and enzymatic hydrolyze at 38°C for 3 hours.

[0082] 2 mU of modified composite protease was added per 5 mg of heparin.

[0083] 2 mU of heparinase II was added per 5 mg of heparin.

[0084] The preparation method of the modified composite protease comprises the following steps:

[0085] (1) Mixing acrylamide solution and N,N'-methylenebisacrylamide solution evenly, adding Tris-HCl buffer, adding ammonium persulfate initiator to the mixed solution, quickly adding 3 drops of tetramethylethylenediamine, pouring the mixed solution into a mold, and covering the top with a glass plate to ensure a flat surface, polymerizing at room temperature for 14 hours, cutting the polymerized polyacrylamide gel into small particles, washing with buffer 3 times to remove unbound substances, and obtaining small particles of polyacrylamide gel;

[0086] (2) Dissolve fig protease and bromelain in a weight ratio of 1:1.2 in Tris-HCl buffer, mix with small granular polyacrylamide gel, stir with a magnetic stirrer at a stirring speed of 120 rpm / min, adsorb for 4 hours, use a low-speed centrifuge at a centrifugal speed of about 1000 rpm / min, centrifuge for 10 minutes, and then freeze-dry and store at -20°C to -15°C to obtain a modified composite protease.

[0087] The specific process of alcohol precipitation is as follows: after the microporous filtration is completed, 1.2 times the volume of 96% ethanol is added to the filtrate, and the mixture is stirred and allowed to stand for 15 hours.

[0088] After complete dissolution, tangential flow circulation ultrafiltration was performed for 8 hours using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, and the retentate was collected.

[0089] After cross-linking, 10 volumes of 52 mM sodium phosphate buffer were added to the gel.

[0090] After the dissolution is completed, filter through a 0.1 μm sterilization membrane and collect the filtrate.

[0091] Comparative Example 1

[0092] The difference between this comparative example and Example 1 is that the enzymatic hydrolysis method is: adding heparinase II to heparin and enzymatic hydrolyzing at 40° C. for 4 h.

[0093] Comparative Example 2

[0094] The difference between this comparative example and Example 1 is that ficin and bromelain in a weight ratio of 1:1 are dissolved in Tris-HCl buffer.

[0095] Comparative Example 3

[0096] The difference between this comparative example and Example 1 is that the ficin is replaced by neutral protease.

[0097] Comparative Example 4

[0098] The difference between this comparative example and Example 1 is that the modified composite protease is replaced by the composite protease.

[0099] Performance Testing

[0100] The clarity, opalescence and absorbance of the heparin obtained in Examples 1-3 and Comparative Examples 1-4 were tested. The test results are shown in Table 1.

[0101] Table 1 Performance test results

[0102]

[0103] As can be seen from Table 1, the heparin of the present invention has good clarity, no opalescence, is colorless, and has extremely low absorbance.

[0104] By comparing Example 1 with Comparative Examples 1-3, it can be seen that the enzymatic hydrolysis method of the present invention can significantly improve the clarity of heparin and reduce the absorbance, and at the same time improve the color of heparin.

[0105] By comparing Example 1 with Comparative Example 4, it can be seen that the present invention reduces the absorbance and makes heparin opalescent by modifying the composite protease. At the same time, after the composite protease is modified, it can be reused after filtration after enzymolysis.

[0106] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A process for preparing heparin based on an enzymatic method, characterized in that: The following steps are involved: (1) enzymatically hydrolyzing commercially available heparin, and after the enzymatic hydrolysis, centrifuging at 3500-3700 rpm to obtain a precipitate to obtain pretreated heparin; (2) Phe-Leu-Asn-Gln-Asp and sepharose are cross-linked; after cross-linking, sodium phosphate buffer (pH 7.2-7.5) is added to the gel, and the supernatant is removed after swelling; (3) Pre-treated heparin was dissolved in 1-1.2 times the volume of buffer, and 1-1.2 mg of gel was loaded on the sample at a ratio of 3 mg of heparin. After loading, the sample was stirred for 5-10 minutes, allowed to stand for 20-30 minutes, and the supernatant was removed by aspiration; (4) After the active center of heparin is adsorbed on the pentapeptide of the gel, 2-3 mg of heparin is added to 2 mU of heparinase II, stirred and hydrolyzed in a water bath at 35-40 °C for 3-4 h, and the precipitate is collected by centrifugation; (5) adding 20-22 times the amount of 1 mol / L NaCl solution to the precipitate to dissolve it, and after it is completely dissolved, performing tangential flow circulation ultrafiltration with an ultrafiltration membrane to collect the retentate; (6) removing impurities from the retentate by alcohol precipitation, and drying and weighing the resulting precipitate; (7) adding 10-12 times the amount of 1 mol / L NaCl solution to the precipitate for dissolution, filtering through a sterilization membrane after dissolution, collecting the filtrate, and adjusting the pH value of the filtrate to 6.5-7.2; The filtrate is freeze-dried to obtain heparin prepared by the enzymatic method.

2. The process for preparing heparin based on the enzymatic method according to claim 1, characterized in that: The enzymatic hydrolysis method is: first add the modified composite protease to the heparin, enzymatic hydrolyze at 38-40°C for 1-2 hours, filter, continue to add heparinase II, and enzymatic hydrolyze at 38-40°C for 3-4 hours.

3. The process for preparing heparin based on the enzymatic method according to claim 2, characterized in that: Add 2mU of modified composite protease based on 1-5mg heparin.

4. The process for preparing heparin based on the enzymatic method according to claim 2, characterized in that: Add 2 mU heparinase II per 1-5 mg heparin.

5. The process for preparing heparin based on the enzymatic method according to claim 1, characterized in that: After cross-linking, the gel was added with 10-12 times volume of 50-52 mM sodium phosphate buffer.

6. The process for preparing heparin based on the enzymatic method according to claim 2, characterized in that: The preparation method of the modified composite protease comprises the following steps: (1) mixing acrylamide solution and N,N'-methylenebisacrylamide solution evenly, adding Tris-HCl buffer, adding ammonium persulfate initiator to the mixed solution, quickly adding 2-3 drops of tetramethylethylenediamine, pouring the mixed solution into a mold, and covering the top with a glass plate to ensure a flat surface, polymerizing at room temperature for 14-18 hours, cutting the polymerized polyacrylamide gel into small particles, washing with buffer 1-3 times to remove unbound substances, and obtaining small particles of polyacrylamide gel; (2) Dissolve the ficin and bromelain in Tris-HCl buffer, mix with small granular polyacrylamide gel, stir with a magnetic stirrer at a stirring speed of 120-150 rpm / min, adsorb for 2-4 hours, use a low-speed centrifuge at a centrifugal speed of about 1000-2000 rpm / min, centrifuge for 5-10 minutes, freeze-dry and store at -20°C to -15°C to obtain a modified composite protease.

7. The process for preparing heparin based on the enzymatic method according to claim 6, characterized in that: Ficin and bromelain in a weight ratio of 1: (1.2-1.4) were dissolved in Tris-HCl buffer.

8. The process for preparing heparin based on the enzymatic method according to claim 1, characterized in that: The specific process of alcohol precipitation is as follows: after microporous filtration is completed, 1.0-1.2 times the volume of 94-96% ethanol is added to the filtrate, and the mixture is stirred and allowed to stand for 13-15 hours.

9. The process for preparing heparin based on the enzymatic method according to claim 1, characterized in that: After complete dissolution, tangential flow circulation ultrafiltration is performed for 6-8 hours using an ultrafiltration membrane with a molecular weight cutoff of 3000-5000Da, and the retentate is collected.

10. The process for preparing heparin based on the enzymatic method according to claim 1, characterized in that: After the dissolution is completed, the solution is filtered through a 0.1 μm sterilization membrane and the filtrate is collected.

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