A method for preparing low molecular heparan sulfate by enzyme
Low molecular weight heparan sulfate was prepared by enzymatic method. Heparinase loaded on modified carbon nanotubes was used to cleave heparan sulfate under mild conditions, which solved the problems of low stability and low yield of chemical cleavage method and achieved efficient preparation of low molecular weight heparan sulfate.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HUANGCHUAN PENGSHENG ANIMAL PROD CO LTD
- Filing Date
- 2026-02-24
- Publication Date
- 2026-05-29
AI Technical Summary
Existing chemical pyrolysis methods for preparing low-molecular-weight heparan sulfate involve violent reactions that easily damage sulfate groups, leading to reduced stability and activity, as well as low yield.
Low molecular weight heparan sulfate was prepared by enzymatic method. Heparinase was loaded onto modified carbon nanotubes and lysed under mild conditions. The combination of carbon nanotube modification and heparinase improved the yield and maintained product stability.
Heparan sulfate was successfully cleaved into low-molecular-weight heparan sulfate under mild conditions, improving the yield. Furthermore, the modified carbon nanotubes can be reused, enhancing the enzymatic hydrolysis efficiency.
Abstract
Description
Technical Field
[0001] This invention belongs to the field of heparin extraction technology, specifically relating to an enzymatic method for preparing low molecular weight heparan sulfate. Background Technology
[0002] Heparan sulfate (HS), a member of the glycosaminoglycan (GAG) family, is a special carbohydrate molecule widely distributed in tissues and an important component of the extracellular matrix (ECM). It often binds to proteins to form proteoglycans, playing a vital role in regulating key life processes such as development, angiogenesis, tumor metastasis, and viral infection. In recent years, scientists have made breakthroughs in the artificial synthesis of HS, beginning to explore its potential in stem cell regulation and the treatment of certain diseases (such as fibrolamellar hepatocellular carcinoma). These studies have opened new hopes for medical innovation and disease treatment. Compared to unfractionated heparin, heparan sulfate has a lower degree of sulfate formation and weaker anticoagulant activity, making it important for the prevention and treatment of thrombosis and ulcerative colitis. The oral antithrombotic drug sulodexide is already on the market, one of the main components of which is heparan sulfate extracted from heparin production byproducts. Low molecular weight heparan sulfate is a smaller molecular weight fragment obtained from heparan sulfate through fractionation or degradation. Due to its smaller molecular weight and lower degree of sulfation, it exhibits superior absorption and pharmacokinetic properties in vivo. Currently, methods for preparing low molecular weight heparan sulfate include chemical cleavage and enzymatic hydrolysis. Chemical cleavage methods primarily involve β-elimination, nitrite degradation, hydrogen peroxide degradation, acid degradation, and radiation degradation. These methods involve relatively vigorous reactions that easily lead to the removal of sulfate groups from heparan sulfate, thereby compromising its stability and activity. Furthermore, the yield of low molecular weight heparan sulfate obtained using these methods is low; therefore, existing technologies require further improvement. Summary of the Invention
[0003] The purpose of this invention is to provide a mild and high-yield enzymatic method for preparing low-molecular-weight heparan sulfate.
[0004] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing low molecular weight heparan sulfate by enzymatic method comprises the following steps: (1) dissolving heparan sulfate in deionized water, adjusting the pH to 8-8.5 with NaOH solution at 20-25℃, adding modified carbon nanotubes loaded with heparinase, and then enzymatically hydrolyzing at 35℃-45℃ for 3.0-3.5h with stirring to obtain an enzymatic hydrolysate; (2) detecting the enzymatic hydrolysate in step (1) online, and when the specific absorbance measured at a wavelength of 230nm is 1.3-1.5, rapidly heating the enzymatic hydrolysate to 95-98℃, and terminating the reaction after 0.5-1.0h; (3) cooling the enzymatic hydrolysate after the reaction to 20-30℃, adding anhydrous ethanol and stirring for 1-2h, then centrifuging to collect the precipitate, adding deionized water, filtering with a 0.1-0.22μm filter membrane, and then freeze-drying under vacuum to obtain low molecular weight heparan sulfate.
[0005] Furthermore, the method for modifying carbon nanotubes comprises the following steps: adding anhydrous ethanol and anhydrous ferric chloride to carbon nanotubes, stirring at 55-65℃ for 1-2 hours, centrifuging to remove the supernatant, vacuum drying at 30℃-35℃ for 10-14 hours, then adding ethylene glycol, precipitating for 2-3 hours, centrifuging to collect the precipitate, and heating to 550-650℃ under a nitrogen atmosphere for 1-2 hours to obtain modified carbon nanotubes.
[0006] Furthermore, the modified carbon nanotubes loaded with heparinase are prepared by the following steps: after adding the modified carbon nanotubes to deionized water for the first time, heparinase is added at 35-45℃, the mixture is stirred evenly, centrifuged and the precipitate is collected, then washed with deionized water for the second time, the precipitate is collected and dried at 35-45℃.
[0007] Furthermore, the heparinase is at least one of heparinase I, heparinase II, and heparinase III.
[0008] Furthermore, in step (1), the mass-to-volume ratio of heparan sulfate, deionized water, and modified carbon nanotubes loaded with heparinase is 100-200g: 1000-2000ml: 80-150g; and the mass percentage of NaOH solution is 30%-40%.
[0009] Furthermore, in step (3), the volume ratio of the enzymatic hydrolysate, anhydrous ethanol, and deionized water is: 1000-2000ml∶400-600ml∶200-300ml.
[0010] Furthermore, the potency of the heparan sulfate is 60-70 IU / mg.
[0011] Furthermore, in the method for modifying carbon nanotubes, the mass-to-volume ratio of carbon nanotubes, anhydrous ethanol, anhydrous ferric chloride, and ethylene glycol is 80-150g : 200-300ml : 100-200ml : 200-300ml.
[0012] Furthermore, in the preparation method of the modified carbon nanotubes loaded with heparinase, the mass-volume ratio of the modified carbon nanotubes, the first deionized water, the heparinase, and the second deionized water is: 80-150g: 200-300ml: 20-50g: 100-200ml.
[0013] This invention employs an enzymatic process to degrade heparan sulfate into the desired low-molecular-weight heparan sulfate under mild conditions. Furthermore, this invention utilizes modified carbon nanotubes loaded with heparinase. The carbon nanotubes, modified with iron ions, possess the ability to cleave heparan sulfate. The modified carbon nanotube-loaded heparinase can simultaneously cleave large-molecule heparan sulfate into low-molecular-weight heparan sulfate during enzymatic hydrolysis, improving the yield of low-molecular-weight heparan sulfate. Moreover, the modified carbon nanotubes loaded with heparinase can be reused after filtration through a filter membrane, further enhancing their utilization rate. Detailed Implementation Example
[0014] A method for preparing low molecular weight heparan sulfate by enzymatic method comprises the following steps: (1) Dissolving heparan sulfate sample in deionized water, adjusting pH to 8 with NaOH solution at 20°C, adding modified carbon nanotubes loaded with heparinase, and then enzymatically hydrolyzing at 35°C for 3.0 h while stirring to obtain enzymatic hydrolysate; (2) Detecting the enzymatic hydrolysate in step (1) online, when the specific absorbance measured at 230 nm wavelength is 1.3, rapidly raising the temperature of the enzymatic hydrolysate to 95°C, and terminating the reaction after 0.5 h; (3) Cooling the enzymatic hydrolysate after the reaction to 20°C, adding anhydrous ethanol and stirring for 1 h, then centrifuging to obtain the precipitate, adding deionized water, filtering with a 0.1 μm filter membrane, and then freeze-drying under vacuum to obtain low molecular weight heparan sulfate.
[0015] The method for modifying carbon nanotubes comprises the following steps: adding anhydrous ethanol and anhydrous ferric chloride to carbon nanotubes, stirring at 55°C for 1 hour, centrifuging to remove the supernatant, vacuum drying at 30°C for 10 hours, then adding ethylene glycol, precipitating for 2 hours, centrifuging to collect the precipitate, heating to 550°C under a nitrogen atmosphere, and reacting for 1 hour to obtain modified carbon nanotubes.
[0016] The modified carbon nanotubes loaded with heparinase are prepared by the following steps: after adding the modified carbon nanotubes to deionized water for the first time, heparinase is added at 35°C, the mixture is stirred evenly, centrifuged and the precipitate is collected, then washed with deionized water for the second time, the precipitate is collected and dried at 35°C.
[0017] The heparinase mentioned is heparinase I.
[0018] In step (1), the mass-to-volume ratio of heparan sulfate, deionized water, and modified carbon nanotubes loaded with heparinase is 100g:1000ml:80g; the mass percentage of NaOH solution is 30%.
[0019] In step (3), the volume ratio of the enzymatic hydrolysate, anhydrous ethanol and deionized water is 1000ml: 400ml: 200ml.
[0020] The potency of the heparan sulfate is 60 IU / mg.
[0021] In the carbon nanotube modification method, the mass-volume ratio of carbon nanotubes, anhydrous ethanol, anhydrous ferric chloride, and ethylene glycol is 80g: 200ml: 100ml: 200ml.
[0022] In the preparation method of the modified carbon nanotubes loaded with heparinase, the mass-volume ratio of the modified carbon nanotubes, the first deionized water, the heparinase, and the second deionized water is 80g: 200ml: 20g: 100ml.
[0023] The yield of low molecular weight heparan sulfate obtained by the above method was 77%. Example
[0024] A method for preparing low molecular weight heparan sulfate by enzymatic method comprises the following steps: (1) Heparan sulfate is dissolved in deionized water, the pH is adjusted to 8.3 with NaOH solution at 22°C, modified carbon nanotubes loaded with heparinase are added, and then the enzyme is hydrolyzed at 40°C for 3.2 h with stirring to obtain the enzymatic hydrolysate; (2) The enzymatic hydrolysate in step (1) is detected online. When the specific absorbance measured at a wavelength of 230 nm is 1.4, the enzymatic hydrolysate is rapidly heated to 97°C and the reaction is terminated after 0.8 h; (3) The enzymatic hydrolysate after the reaction is completed is cooled to 25°C, anhydrous ethanol is added and stirred for 1.5 h, then the precipitate is centrifuged and deionized water is added. After filtration with a 0.2 μm filter membrane, the precipitate is freeze-dried under vacuum to obtain low molecular weight heparan sulfate.
[0025] The method for modifying carbon nanotubes comprises the following steps: adding anhydrous ethanol and anhydrous ferric chloride to carbon nanotubes, stirring at 60°C for 1.5 h, centrifuging to remove the supernatant, vacuum drying at 32°C for 12 h, then adding ethylene glycol, precipitating for 2.5 h, centrifuging to collect the precipitate, heating to 600°C under a nitrogen atmosphere, and reacting for 1.5 h to obtain modified carbon nanotubes.
[0026] The modified carbon nanotubes loaded with heparinase are prepared by the following steps: after adding the modified carbon nanotubes to the first deionized water, heparinase is added at 40°C, the mixture is stirred evenly, centrifuged and the precipitate is collected, then washed with a second deionized water, the precipitate is collected and dried at 40°C.
[0027] The heparinase mentioned is heparinase II.
[0028] In step (1), the mass-to-volume ratio of heparan sulfate, deionized water, and modified carbon nanotubes loaded with heparinase is 150g:1500ml:100g; the mass percentage of NaOH solution is 35%.
[0029] In step (3), the volume ratio of the enzymatic hydrolysate, anhydrous ethanol and deionized water is 1500ml: 500ml: 250ml.
[0030] The potency of the heparan sulfate is 65 IU / mg.
[0031] In the method for modifying carbon nanotubes, the mass-volume ratio of carbon nanotubes, anhydrous ethanol, anhydrous ferric chloride, and ethylene glycol is 100g:250ml:150ml:250ml.
[0032] In the preparation method of the modified carbon nanotubes loaded with heparinase, the mass-volume ratio of the modified carbon nanotubes, the first deionized water, the heparinase, and the second deionized water is 100g: 250ml: 40g: 150ml.
[0033] The yield of low molecular weight heparan sulfate obtained by the above method was 72%. Example
[0034] A method for preparing low molecular weight heparan sulfate by enzymatic method comprises the following steps: (1) Heparan sulfate is dissolved in deionized water, the pH is adjusted to 8.5 with NaOH solution at 25°C, modified carbon nanotubes loaded with heparinase are added, and then the enzyme is hydrolyzed at 45°C for 3.5 h with stirring to obtain the enzymatic hydrolysate; (2) The enzymatic hydrolysate in step (1) is detected online. When the specific absorbance measured at 230 nm wavelength is 1.5, the enzymatic hydrolysate is rapidly heated to 98°C and the reaction is terminated after 0.5-1.0 h; (3) The enzymatic hydrolysate after the reaction is completed is cooled to 30°C, anhydrous ethanol is added and stirred for 2 h, then the precipitate is centrifuged and deionized, then deionized water is added, and the mixture is filtered through a 0.22 μm filter membrane and then freeze-dried under vacuum to obtain low molecular weight heparan sulfate.
[0035] The method for modifying carbon nanotubes comprises the following steps: adding anhydrous ethanol and anhydrous ferric chloride to carbon nanotubes, stirring at 65°C for 2 hours, centrifuging to remove the supernatant, vacuum drying at 35°C for 14 hours, then adding ethylene glycol, precipitating for 3 hours, centrifuging to collect the precipitate, heating to 650°C under a nitrogen atmosphere, and reacting for 2 hours to obtain modified carbon nanotubes.
[0036] The modified carbon nanotubes loaded with heparinase are prepared by the following steps: after adding the modified carbon nanotubes to the first deionized water, heparinase is added at 45°C, the mixture is stirred evenly, centrifuged and the precipitate is collected, then washed with a second deionized water, the precipitate is collected and dried at 45°C.
[0037] The heparinase mentioned is heparinase III.
[0038] In step (1), the mass-to-volume ratio of heparan sulfate, deionized water, and modified carbon nanotubes loaded with heparinase is 200g:2000ml:150g; the mass percentage of NaOH solution is 40%.
[0039] In step (3), the volume ratio of the enzymatic hydrolysate, anhydrous ethanol, and deionized water is 2000ml: 600ml: 300ml.
[0040] The potency of the heparan sulfate is 70 IU / mg.
[0041] In the method for modifying carbon nanotubes, the mass-volume ratio of carbon nanotubes, anhydrous ethanol, anhydrous ferric chloride, and ethylene glycol is 150g: 300ml: 200ml: 300ml.
[0042] In the preparation method of the modified carbon nanotubes loaded with heparinase, the mass-volume ratio of the modified carbon nanotubes, the first deionized water, the heparinase, and the second deionized water is 150g:300ml:50g:200ml.
[0043] The yield of low molecular weight heparan sulfate obtained by the above method was 71%.
Claims
1. A method for preparing low molecular weight heparan sulfate using an enzymatic process, characterized in that, The process consists of the following steps: (1) Dissolve heparan sulfate in deionized water, adjust the pH to 8-8.5 with NaOH solution at 20-25℃, add modified carbon nanotubes loaded with heparinase, and then enzymatically hydrolyze at 35℃-45℃ for 3.0-3.5h while stirring to obtain the enzymatic hydrolysate; (2) Detect the enzymatic hydrolysate in step (1) online. When the specific absorbance measured at 230nm wavelength is 1.3-1.5, quickly raise the temperature of the enzymatic hydrolysate to 95-98℃ and terminate the reaction after 0.5-1.0h; (3) Cool the enzymatic hydrolysate after the reaction to 20-30℃, add anhydrous ethanol and stir for 1-2h, then centrifuge to collect the precipitate, add deionized water, filter with a 0.1-0.22μm filter membrane, and freeze-dry under vacuum to obtain low molecular weight heparan sulfate.
2. The method for preparing low molecular weight heparan sulfate by enzymatic method according to claim 1, characterized in that, The method for modifying carbon nanotubes comprises the following steps: adding anhydrous ethanol and anhydrous ferric chloride to carbon nanotubes, stirring at 55-65℃ for 1-2 hours, centrifuging to remove the supernatant, vacuum drying at 30℃-35℃ for 10-14 hours, then adding ethylene glycol, precipitating for 2-3 hours, centrifuging to collect the precipitate, heating to 550-650℃ under a nitrogen atmosphere, and reacting for 1-2 hours to obtain modified carbon nanotubes.
3. The method for preparing low molecular weight heparan sulfate by enzymatic method according to claim 1, characterized in that, The modified carbon nanotubes loaded with heparinase are prepared by the following steps: after adding the modified carbon nanotubes to deionized water for the first time, heparinase is added at 35-45℃, the mixture is stirred evenly, centrifuged and the precipitate is collected, then washed with deionized water for the second time, the precipitate is collected and dried at 35-45℃.
4. The method for preparing low molecular weight heparan sulfate by enzymatic method according to claim 1, characterized in that, The heparinase mentioned is at least one of heparinase I, heparinase II, and heparinase III.
5. The method for preparing low molecular weight heparan sulfate by enzymatic method according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of heparan sulfate, deionized water, and modified carbon nanotubes loaded with heparinase is 100-200g: 1000-2000ml: 80-150g; the mass percentage of NaOH solution is 30%-40%.
6. The method for preparing low molecular weight heparan sulfate by enzymatic method according to claim 1, characterized in that, In step (3), the volume ratio of the enzymatic hydrolysate, anhydrous ethanol and deionized water is 1000-2000ml: 400-600ml: 200-300ml.
7. The method for preparing low molecular weight heparan sulfate by enzymatic method according to claim 1, characterized in that, The potency of the heparan sulfate is 60-70 IU / mg.
8. The method for preparing low molecular weight heparan sulfate by enzymatic method according to claim 1, characterized in that, In the method for modifying carbon nanotubes, the mass-to-volume ratio of carbon nanotubes, anhydrous ethanol, anhydrous ferric chloride, and ethylene glycol is 80-150g: 200-300ml: 100-200ml: 200-300ml.
9. The method for preparing low molecular weight heparan sulfate by enzymatic method according to claim 1, characterized in that, In the preparation method of the modified carbon nanotubes loaded with heparinase, the mass-volume ratio of the modified carbon nanotubes, the first deionized water, the heparinase, and the second deionized water is 80-150g: 200-300ml: 20-50g: 100-200ml.