Method for preparing heparin sodium through dual-enzymolysis method

Abstract

The invention belongs to the field of biochemical industries, and particularly relates to a method for preparing heparin sodium through a dual-enzymolysis method. The method for preparing the heparin sodium through the dual-enzymolysis method includes the following steps that heparin sodium in heparin sodium-protein complex is dissolved and separated through the dual-enzymolysis method, then the heparin sodium is completely extracted out through the steps of temperature raising, resin adsorption, elution, alcohol precipitation, drying and the like, and the heparin sodium yield is raised. By means of dual-enzymolysis, the salt concentration can be lowered appropriately, pollution can be alleviated, and steam consumption and warm water consumption are lowered. Pig pancreas can be activated and used for large-scale production, and the cost of the pig pancreas is about 100 yuan for every 6 tons of reaction liquid; the cost of the pig pancreas and the cost of the steam can be counteracted.

Description

(1) Technical field

[0001] The invention belongs to the field of biochemical industry, in particular to a method for preparing heparin sodium by double enzymatic hydrolysis. (2) Background technology

[0002] Heparin is a mucopolysaccharide that widely exists in mammalian tissues, mainly exists in mast cells, has anticoagulant effect, and is widely used in the treatment of surgery, cerebral thrombosis, myocardial infarction, etc. The molecular weight of heparin is about 3000-37500. Research on commercial heparin shows that there are at least 21 molecular entities. Heparin sodium is the sodium salt of heparin. As a natural anticoagulant substance, heparin sodium has been valued by countries all over the world. It is also one of the main export drugs in my country. With the deepening of research, it has been found that heparin sodium not only has anticoagulant, antithrombotic and blood lipid regulation effects, but also has anti-inflammatory, anti-allergic, anti-viral, anti-c...

Examples

Embodiment 1

[0019] A method for preparing heparin sodium by a double enzymatic hydrolysis method is realized through the following steps:

[0020] (1) Enzymolysis: Take 500kg of small intestinal mucosa obtained from the porcine heparin workshop, add special saline for casings until the salinity of the feed solution is 3.5%, put it in an enzymolysis tank with water, adjust the pH to 7.8, add 100g of pancreatic enzyme, and heat When the temperature rises to 51°C, keep warm for 1 hour, filter through an 80-mesh membrane, collect the filtrate, add 125 g of 2709 alkaline protease, and keep warm for 1 hour.

[0021] (2) Heating: after step (1), heat until the temperature rises to 63°C, keep warm for 5 minutes, filter through an 80-mesh membrane, and collect the filtrate.

[0022] (3) Resin adsorption: After step (2), add the obtained filtrate into the adsorption tank, stir, cool down to 55°C, add 500g resin, keep warm at 55°C, stir and adsorb for 8 hours, filter, and release the waste liquid. ...

Embodiment 2

[0027] A method for preparing heparin sodium by a double enzymatic hydrolysis method is realized through the following steps:

[0028] (1) Enzymolysis: Take 600kg of small intestinal mucosa obtained from the porcine heparin workshop, add special salt for casings until the salinity of the feed solution is 3.0%, put it in an enzymolysis tank with water, adjust the pH to 7.5, add 120g of pancreatin, and heat When the temperature rises to 45°C, keep warm for 1 hour, filter through an 80-mesh membrane, collect the filtrate, add 300 g of 2709 alkaline protease, and keep warm for 1 hour.

[0029] (2) Heating: after step (1), heat until the temperature rises to 60°C, keep warm for 5 minutes, filter through an 80-mesh membrane, and collect the filtrate.

[0030] (3) Resin adsorption: after step (2), add the obtained filtrate into the adsorption tank, stir, cool down to 55°C, add 650g resin, keep warm at 55°C, stir and adsorb for 8 hours, filter, and release the waste liquid.

[0031] ...

Embodiment 3

[0035] A method for preparing heparin sodium by a double enzymatic hydrolysis method is realized through the following steps:

[0036] (1) Enzymolysis: Take 800kg of small intestinal mucosa obtained from the porcine heparin workshop, add special salt for casings until the salinity of the feed solution is 4.0%, put it in an enzymolysis tank with water, adjust the pH to 8, add 160g of pancreatic enzyme, and heat to temperature Raise to 55°C, keep warm for 1 hour, filter through an 80-mesh membrane, collect the filtrate, add 300 g of 2709 alkaline protease, and keep warm for 1 hour.

[0037] (2) Heating: after step (1), heat until the temperature rises to 65°C, keep warm for 5 minutes, filter through an 80-mesh membrane, and collect the filtrate.

[0038] (3) Resin adsorption: After step (2), add the obtained filtrate into the adsorption tank, stir, cool down to 55°C, add 1200g resin, keep warm at 55°C, stir and adsorb for 8 hours, filter, and release the waste liquid.

[0039] ...

(1) Technical field

[0001] The invention belongs to the field of biochemical industry, in particular to a method for preparing heparin sodium by double enzymatic hydrolysis. (2) Background technology

[0002] Heparin is a mucopolysaccharide that widely exists in mammalian tissues, mainly exists in mast cells, has anticoagulant effect, and is widely used in the treatment of surgery, cerebral thrombosis, myocardial infarction, etc. The molecular weight of heparin is about 3000-37500. Research on commercial heparin shows that there are at least 21 molecular entities. Heparin sodium is the sodium salt of heparin. As a natural anticoagulant substance, heparin sodium has been valued by countries all over the world. It is also one of the main export drugs in my country. With the deepening of research, it has been found that heparin sodium not only has anticoagulant, antithrombotic and blood lipid regulation effects, but also has anti-inflammatory, anti-allergic, anti-viral, anti-c...

Examples

Embodiment 1

[0019] A method for preparing heparin sodium by a double enzymatic hydrolysis method is realized through the following steps:

[0020] (1) Enzymolysis: Take 500kg of small intestinal mucosa obtained from the porcine heparin workshop, add special saline for casings until the salinity of the feed solution is 3.5%, put it in an enzymolysis tank with water, adjust the pH to 7.8, add 100g of pancreatic enzyme, and heat When the temperature rises to 51°C, keep warm for 1 hour, filter through an 80-mesh membrane, collect the filtrate, add 125 g of 2709 alkaline protease, and keep warm for 1 hour.

[0021] (2) Heating: after step (1), heat until the temperature rises to 63°C, keep warm for 5 minutes, filter through an 80-mesh membrane, and collect the filtrate.

[0022] (3) Resin adsorption: After step (2), add the obtained filtrate into the adsorption tank, stir, cool down to 55°C, add 500g resin, keep warm at 55°C, stir and adsorb for 8 hours, filter, and release the waste liquid. ...

Embodiment 2

[0027] A method for preparing heparin sodium by a double enzymatic hydrolysis method is realized through the following steps:

[0028] (1) Enzymolysis: Take 600kg of small intestinal mucosa obtained from the porcine heparin workshop, add special salt for casings until the salinity of the feed solution is 3.0%, put it in an enzymolysis tank with water, adjust the pH to 7.5, add 120g of pancreatin, and heat When the temperature rises to 45°C, keep warm for 1 hour, filter through an 80-mesh membrane, collect the filtrate, add 300 g of 2709 alkaline protease, and keep warm for 1 hour.

[0029] (2) Heating: after step (1), heat until the temperature rises to 60°C, keep warm for 5 minutes, filter through an 80-mesh membrane, and collect the filtrate.

[0030] (3) Resin adsorption: after step (2), add the obtained filtrate into the adsorption tank, stir, cool down to 55°C, add 650g resin, keep warm at 55°C, stir and adsorb for 8 hours, filter, and release the waste liquid.

[0031] ...

Embodiment 3

[0035] A method for preparing heparin sodium by a double enzymatic hydrolysis method is realized through the following steps:

[0036] (1) Enzymolysis: Take 800kg of small intestinal mucosa obtained from the porcine heparin workshop, add special salt for casings until the salinity of the feed solution is 4.0%, put it in an enzymolysis tank with water, adjust the pH to 8, add 160g of pancreatic enzyme, and heat to temperature Raise to 55°C, keep warm for 1 hour, filter through an 80-mesh membrane, collect the filtrate, add 300 g of 2709 alkaline protease, and keep warm for 1 hour.

[0037] (2) Heating: after step (1), heat until the temperature rises to 65°C, keep warm for 5 minutes, filter through an 80-mesh membrane, and collect the filtrate.

[0038] (3) Resin adsorption: After step (2), add the obtained filtrate into the adsorption tank, stir, cool down to 55°C, add 1200g resin, keep warm at 55°C, stir and adsorb for 8 hours, filter, and release the waste liquid.

[0039] ...