CN113528488B - Method for preparing high-quality crude hyaluronidase product by stirring and adsorbing resin - Google Patents

Method for preparing high-quality crude hyaluronidase product by stirring and adsorbing resin Download PDF

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CN113528488B
CN113528488B CN202110943295.6A CN202110943295A CN113528488B CN 113528488 B CN113528488 B CN 113528488B CN 202110943295 A CN202110943295 A CN 202110943295A CN 113528488 B CN113528488 B CN 113528488B
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stirring
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resin
hyaluronidase
mixing
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CN113528488A (en
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李海星
林芝雨
潘豪
刘翻
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Nanchang University
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    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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Abstract

The invention provides a method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin. The extraction process adopts a hydrogen ionization filter membrane, the filter membrane prepared by treating the weak acid type modified solution is weak in acid, the filter membrane conforms to the existing environment of the hyaluronidase, the activity of the hyaluronidase cannot be influenced by long-time filtration, and the content of bacterial endotoxin is effectively reduced. Cation exchange resin with good stability and high adsorption efficiency is used for extraction, the titer of the prepared hyaluronidase reaches more than 500IU/mg, the specific activity is more than 800 IU/mg.pr, and the bacterial endotoxin is as low as 0.103 EU. Compared with a precipitation extraction method, the method has the advantages that the potency of the hyaluronidase crude product is greatly improved by using a resin adsorption process, high-quality raw materials are provided for preparing products with higher purity in the subsequent process, and in addition, the potency and the use safety of the hyaluronidase are further guaranteed by using the impurity adsorbent.

Description

Method for preparing high-quality crude hyaluronidase product by stirring and adsorbing resin
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for preparing a high-quality crude hyaluronidase product by stirring and adsorbing resin.
Background
The hyaluronidase is an enzyme extracted from mammalian testis and capable of hydrolyzing mucopolysaccharide, and is a white or yellowish powder, which is soluble in water and insoluble in ethanol, acetone or diethyl ether. No odor, good stability, no loss of activity after continuous heating at 42 deg.C for 60min, and 80% of activity after continuous heating at 100 deg.C for 5 min. It is relatively easy to inactivate in low concentration aqueous solution, and can be protected from inactivation by gelatin or acacia. Fe2+ and Cu2+ ions have reversible inhibition effect on enzyme. Pb2+, Hg2+, Ni 2+, and the like have no obvious influence on the enzyme activity. The inhibition of hyaluronidase by chondroitin sulfate B (dermatan), heparan sulfate, keratan sulfate, sodium heparin and high concentrations of hyaluronic acid can be reversed in the presence of sodium chloride (0.15mol/L) or protamine sulfate.
The traditional extraction method of the hyaluronidase is a fractional precipitation extraction method, and the method has the defects that the extracted hyaluronidase has low effective value, general specific activity and pyrogen risk. In addition, there is a problem that the extraction rate is low. Therefore, how to prepare high-quality hyaluronidase becomes the focus of research of numerous biochemical enterprises and college research institutes. Based on the method, the invention provides a method for preparing a high-quality crude hyaluronidase product by resin stirring and adsorption.
Disclosure of Invention
The first object of the present invention: the method for preparing the high-quality crude hyaluronidase by resin stirring adsorption is provided, the titer of the hyaluronidase prepared by the method is more than 500IU/mg, the specific activity is more than 800 IU/mg.pr, the extraction rate can be more than 14%, and the method has obvious advantages compared with the traditional ammonium sulfate fractional precipitation extraction method.
Another object of the invention is: provides a preparation method of cation exchange resin in the method for preparing high-quality crude hyaluronidase by resin stirring and adsorption.
The invention has three other purposes: provides a preparation method of a hydrogen ionization filter membrane in the method for preparing high-quality crude hyaluronidase by resin stirring and adsorption.
The fourth object of the present invention: provides a preparation method of an impurity adsorbent in the method for preparing high-quality crude hyaluronidase by stirring and adsorbing resin.
The invention is realized by the following technical scheme: a method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin comprises the following steps:
a. mincing tissues and grinding into slurry: taking 40-60 Kg of sheep testis, peeling, cutting tissues into fine blocks, grinding into pulp by using a grinder, and repeatedly grinding into pulp for three times;
b. stirring and extracting: adding purified water with the weight being 2-4 times of the weight of the testis tissue, stirring, adjusting the pH value of the solution to 3.0-3.5 by using cold acetic acid, and fully homogenizing the testis tissue; stirring and extracting for 4-8 hours, then passing through a hydrogen ionization filter membrane, controlling the filtering time to be 8-14 hours, removing the precipitate, and collecting the filtrate;
c. primary ammonium sulfate precipitation for impurity removal: gradually stirring and adding solid ammonium sulfate until the final concentration is 25-35%, precipitating, standing for 6-8 hours, siphoning, taking supernatant, and removing precipitate;
d. secondary treatment of impurities: mixing the supernatant with 0.5-1.5% impurity adsorbent, stirring, standing for 1-3 hr, and filtering to obtain filtrate;
e. adsorption by cation exchange resin: taking the filtrate obtained in the step d, adjusting the pH value to 5.0-5.5, stirring, adding 3-7 KG of cation exchange resin, stirring and adsorbing for 1-3 hours at 50-60 r/min, and maintaining the pH value in the adsorption process at 5.0-5.5; standing for 20-40min after adsorption, siphoning supernatant, collecting resin adsorbed at the bottom by using a Buchner funnel, washing for 8-10 times by using a sodium acetate solution with the concentration of 40-60L, PH being 5.5 and 0.1M, removing part of impurities, stirring and eluting for 1-3 hours by using a sodium acetate solution with the concentration of 40-60L, PH 5.5.5 and 0.1M and sodium chloride solution with the concentration of 0.1M at the speed of 50-60 r/min, standing for 20-40min, siphoning to collect supernatant containing a hyaluronidase product, recovering bottom resin, recycling the resin, and eluting the supernatant for later use;
f. secondary ammonium sulfate precipitation product: and taking the resin elution supernatant, gradually stirring and adding solid ammonium sulfate until the final concentration is 82-88%, precipitating, standing for 6-8 hours, siphoning, carrying out suction filtration, taking the precipitate, and drying at 40-50 ℃ to obtain the ammonium sulfate.
The preparation method of the hydrogen ionization filter membrane comprises the following steps of raw materials, by weight, 20-40 parts of a casting membrane liquid main material, 1-3 parts of a plasticizer, 80-100 parts of an organic solvent and 3-7 parts of a pore-forming component; the method comprises the following specific steps:
(1) and preparing a casting solution: mixing the main material of the casting solution with an organic solvent, stirring until the main material is completely dissolved, heating to 40-50 ℃, adding the rest raw materials, stirring at a low speed of 300-500 r/min for 20-30 min, transferring to a defoaming machine for defoaming, and cooling to normal temperature to obtain the casting solution;
(2) and preparing a filter membrane: preparing on plate glass in a blade coating mode, wherein the thickness of a wet film after blade coating is 200-300 um, naturally drying for 3-5 min at normal temperature, immediately immersing in water for phase conversion, immersing for 30-60min, taking out, and drying at 40-60 ℃ to obtain the hydrogen ionization filter membrane.
The main material of the casting film liquid is one or a mixture of more than two of polyvinylidene fluoride, polyvinyl chloride, polyether sulfone and polysulfone.
The plasticizer is acetyl tributyl citrate;
the organic solvent is prepared from the following components in a mass ratio of 1-3: 1, dipropylene glycol dimethyl ether and methyl pyrrolidone.
The preparation method of the pore-forming component comprises the following steps of mixing 5-9 parts of polyethylene glycol, 7-11 parts of polyvinylpyrrolidone, 3-7 parts of toluene and 15-25 parts of an acidification modification solution by weight, and specifically, mixing the polyethylene glycol, the polyvinylpyrrolidone and the toluene, slowly adding the mixture into the acidification modification solution within 1-2h while stirring, wherein the stirring speed is 100-200rpm, standing for 3-5h after the addition is finished, filtering, and roasting and activating filter residues at 35-55 ℃ for 0.5-1.5h to obtain the pore-forming component.
Wherein the acidification modification component comprises 2-6 parts of 2- (2-methoxycarbonyl ethylamine) methyl benzoate and K2HPO by weight parts of raw materials 4 5-9 parts of sodium ethylene diamine tetracetate 7-11 parts of water 80-100 parts of K2HPO 4 Mixing with water for dissolution, adding 2- (2-methoxycarbonylethylamine) methyl benzoate and sodium ethylene diamine tetracetate, stirring and mixing at the speed of 500rpm for 300 times, standing for 30min, and then mixing with dimethylbenzyl ortho ester butyrate according to the mass ratio of 3-7: 1, and the product is the acidification modification component.
The impurity adsorbent is modified sepiolite, and the preparation method comprises the following steps of taking 10-16 parts by weight of sepiolite and 2-6 parts by weight of modified additive as raw materials, grinding and crushing the sepiolite into 200 meshes, dispersing the sepiolite into water with the particle size of 8-12 times, and stirring at the rotating speed of 50-90rpm to obtain sepiolite dispersion liquid for later use; mixing the modified additive with 3-5 times of water, stirring for 5-15min to form a chitosan-gelatin-cellulose crosslinked mixture, then combining with the sepiolite dispersion liquid, heating to 50-70 ℃, dropwise adding acetic acid, continuously stirring for 10-20h to form gel, instantly cooling and freeze-drying by adopting a liquid nitrogen refrigeration mode when the gel is hot to form a porous substance, and crushing to 100-mesh particles to obtain the product.
The modifying additive is prepared from the following components in a mass ratio of 5-9: 3-5: 1-3, mixing the chitosan, the cellulose and the gelatin with an enzyme solution in an amount which is 10-30 times the mass of the chitosan, the cellulose and the gelatin, and performing enzymolysis for 1-2 hours at 40-50 ℃ and 60-80Mpa, wherein the pH of the enzyme solution is adjusted to 3.5-4.5 by hydrochloric acid, and the mass ratio is 3-9: 5-11: 65-85 of chitosanase, cellulase and water;
the cellulose is extracted from agricultural and forestry residues including rice straw, waste straw and bagasse.
The preparation method of the cation exchange resin comprises the following steps of introducing 10-20 parts by weight of a main material, 6-10 parts by weight of a modifier and 1-3 parts by weight of an auxiliary material of the cation exchange resin into a reaction tank, mixing the main material and the auxiliary material, heating to 40-60 ℃, and keeping for 25-45 min for later use; and (2) mixing a modifier with water according to a mass ratio of 5: 1, adding the mixture into a reaction tank, heating to 70-90 ℃, uniformly stirring at a rotating speed of 800-1000 rpm, preserving heat for 1-2 hours, transferring the mixture into a shaping mold, preparing microspheres with the thickness of 0.5-1 mm, and cooling to obtain the microsphere.
The main material of the cation exchange resin is macroporous weak-acidic acrylic cation exchange resin;
the modifier is prepared from the following components in a mass ratio of 1: 1-5 of a pretreatment mixture of sodium alginate and chitin, wherein the preparation method comprises the following steps: mixing the sodium alginate, the chitin and the dispersion liquid according to the mass volume ratio of 1 kg: 2-2.8L, mechanically stirring at 400-600 rpm for 15-25 min, filtering, freezing the filter residue in a freezer at-50 to-70 ℃ for 24-48 h, taking out the filter residue, transferring to an oven at 200 ℃ for heat preservation for 2h, and cooling to normal temperature.
The preparation method of the dispersion comprises the following steps: adding 3-5 parts of heptadecafluorodecyltrimethoxysilane into 10-16 parts of ethanol, and stirring and mixing to obtain a product a; adding 5-7 parts of 1, 3-bis (4-hydroxybutyl) tetramethyldisiloxane into 15-25 parts of diethyl ether, stirring and mixing to obtain a product b, combining the product a and the product b to obtain a product c, heating the product c to 60 ℃ at 1-3 ℃/min, adding 4-8 parts of dimethylbenzene at the dropping speed of 30mL/min, rotationally stirring at the rotating speed of 400rpm of 200-4 ℃, and standing for 3-4h to obtain the product.
The auxiliary material is zeolite powder, and the particle size of the zeolite powder is 20-50 um.
The invention has the advantages that:
(1) compared with the traditional ammonium sulfate fractional precipitation extraction method, the prepared hyaluronidase has the advantages that the titer of the crude hyaluronidase is greatly improved by using a resin adsorption process, and high-quality raw materials are provided for preparing products with higher purity in the subsequent process.
(2) The hydrogen ionization filter membrane is adopted in the extraction process, the filter membrane prepared by treating the weak acid type pore-forming component is weak acid, the existence environment of the hyaluronidase is met, and the activity of the hyaluronidase cannot be influenced by long-time filtration.
(3) Aiming at the defects that the stability of the conventional macroporous weakly acidic acrylic cation exchange resin is general and the adsorption efficiency needs to be improved, the inventor proposes that the macroporous weakly acidic acrylic cation exchange resin is modified by using a three-dimensional reticular modifier and assisting with microparticle zeolite powder, and the internal structure of the macroporous weakly acidic acrylic cation exchange resin is slowly expanded through mild pretreatment at the low temperature of 40-60 ℃, so that the bearing capacity and the binding force of the modifier and auxiliary materials are increased, and the adsorption efficiency and the stability of the resin are greatly improved.
(4) According to the invention, the impurity adsorbent is adopted for treatment between the primary precipitation and the secondary precipitation, the sepiolite is modified by the modified additive subjected to short-time enzymolysis treatment, and the modified additive with adsorption capacity is effectively combined with the sepiolite to form freeze-dried gel by utilizing the characteristic that the sepiolite is soft when meeting water, so that the adsorption capacity of the impurity adsorbent is greatly improved, in addition, a certain effect on removing heavy metals can be achieved, the titer of a hyaluronidase product is improved, and the use risk is reduced.
In order to make the method for preparing high-quality crude hyaluronidase by resin stirring adsorption more clear, the invention is further described with reference to the following embodiments.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
Example 1
A method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin comprises the following steps:
a. mincing tissues and grinding into slurry: taking 50Kg of sheep testis, peeling, cutting the tissue into fine blocks, grinding into pulp by a mincing machine, and repeatedly grinding into pulp for three times;
b. stirring and extracting: adding purified water with the weight of 3 times of the testis tissue, stirring, adjusting the pH value of the solution to 3.3 by using cold acetic acid, and fully homogenizing the testis tissue; stirring and extracting for 6 hours, then passing through a hydrogen ionization filter membrane, controlling the filtering time to be 11 hours, removing the precipitate, and collecting the filtrate;
c. primary ammonium sulfate precipitation for impurity removal: gradually stirring, adding solid ammonium sulfate to a final concentration of 30% for precipitation, standing for 7 hr, siphoning, collecting supernatant, and removing precipitate;
d. secondary treatment of impurities: mixing the supernatant with 1% impurity adsorbent, stirring, standing for 2 hr, and filtering to obtain filtrate;
e. adsorption by cation exchange resin: taking the filtrate obtained in the step d, adjusting the pH value to 5.3, stirring and adding 5KG cation exchange resin, stirring and adsorbing for 2 hours at 55r/min, and maintaining the pH value unchanged in the adsorption process; standing for 30min after adsorption, siphoning supernatant, collecting resin adsorbed at the bottom by using a Buchner funnel, washing for 9 times by using a 50L, PH sodium acetate solution with the concentration of 5.5 and 0.1M to remove partial impurities, stirring and eluting for 2 hours at 55r/min by using a 50L, PH 5.5.5 and 0.1M sodium acetate-1.0M sodium chloride solution, standing for 30min, siphoning to collect supernatant containing a hyaluronidase product, recovering bottom resin, recycling the resin for reuse, and eluting the resin for later use;
f. secondary ammonium sulfate precipitation product: collecting the resin elution supernatant, gradually stirring, adding solid ammonium sulfate to a final concentration of 85%, precipitating, standing for 7 hr, siphoning, vacuum filtering, collecting precipitate, and oven drying at 45 deg.C.
The preparation method of the hydrogen ionization filter membrane comprises the following steps of calculating raw materials by weight part of 30 parts of casting membrane liquid main material, 2 parts of plasticizer, 90 parts of organic solvent and 5 parts of pore-forming component; the method comprises the following specific steps:
(1) and preparing a casting solution: mixing the main material of the casting solution with an organic solvent, stirring until the main material is completely dissolved, heating to 45 ℃, adding the rest raw materials, stirring at a low speed of 400r/min for 25min, transferring to a defoaming machine for defoaming, and cooling to normal temperature to obtain the casting solution;
(2) and (3) preparing a filter membrane: preparing on plate glass by adopting a blade coating mode, wherein the thickness of a wet film after blade coating is 250um, naturally drying for 4min at normal temperature, immediately soaking in water for phase conversion, soaking for 45min, taking out, and drying at 50 ℃ to obtain the hydrogen ionization filter membrane.
The main material of the casting film liquid is polyvinylidene fluoride.
The plasticizer is acetyl tributyl citrate;
the organic solvent is prepared from the following components in a mass ratio of 2: 1, dipropylene glycol dimethyl ether and methyl pyrrolidone.
The preparation method of the pore-forming component comprises the following steps of mixing 7 parts of polyethylene glycol, 9 parts of polyvinylpyrrolidone, 5 parts of toluene and 20 parts of an acidification modification solution by weight, specifically, mixing the polyethylene glycol, the polyvinylpyrrolidone and the toluene, slowly adding the mixture into the acidification modification solution within 1.5h while stirring at a stirring speed of 150rpm, standing for 4h after the addition, filtering, and baking and activating filter residues for 1h at 45 ℃.
Wherein the acidification modification component comprises 4 parts of 2- (2-methoxycarbonyl ethylamine) methyl benzoate and K2HPO by weight parts of raw materials 4 7 parts of sodium ethylene diamine tetracetate 9 parts of water 90 parts, adding K2HPO 4 Mixing with water for dissolving, adding 2- (2-methoxycarbonylethylamine) methyl benzoate and sodium ethylene diamine tetracetate, stirring and mixing at 400rpm, standing for 30min, and then mixing with dimethylbenzyl ortho ester butyrate according to the mass ratio of 5: 1, and the product is the acidification modification component.
The impurity adsorbent is modified sepiolite, and the preparation method comprises the following steps of calculating the raw materials by weight part to include 13 parts of sepiolite and 4 parts of modified additive, grinding and crushing the sepiolite into 200 meshes, dispersing the crushed sepiolite in 10 times of water, and stirring at a rotating speed of 70pm to obtain sepiolite dispersion liquid for later use; mixing the modified additive with 4 times of water, stirring for 10min to form a chitosan-gelatin-cellulose crosslinked mixture, then mixing with the sepiolite dispersion liquid, heating to 60 ℃, dropwise adding acetic acid, continuously stirring for 15h to form gel, instantly cooling and freeze-drying by adopting a liquid nitrogen refrigeration mode when the solution is hot to form a porous substance, and crushing to 100-mesh particles to obtain the product.
The modifying additive is prepared from the following components in percentage by mass: 4: 2, uniformly mixing the chitosan, the cellulose and the gelatin with 20 times of enzyme solution by mass, and carrying out enzymolysis for 1.5 hours at 45 ℃ and 70Mpa, wherein the enzyme solution is prepared by adjusting the pH value to 4 by hydrochloric acid according to the mass ratio of 6: 8: 75 of chitosanase, cellulase and water,
the cellulose is extracted from straw.
The preparation method of the cation exchange resin comprises the following steps of introducing 15 parts by weight of a main material, 8 parts by weight of a modifier and 2 parts by weight of an auxiliary material of the cation exchange resin into a reaction tank, mixing the main material and the auxiliary material, heating to 50 ℃, and keeping for 35min for later use; and (2) mixing the modifier with water according to a mass ratio of 5: 1, adding the mixture into a reaction tank, heating to 80 ℃, uniformly stirring at the rotating speed of 900rpm, preserving heat for 1.5 hours, transferring the mixture into a shaping mold, preparing microspheres with the thickness of 0.8mm, and cooling to obtain the microsphere.
The main material of the cation exchange resin is macroporous weak-acid acrylic cation exchange resin;
the modifier is prepared from the following components in a mass ratio of 1: 3, the preparation method of the pretreatment mixture of sodium alginate and chitin comprises the following steps: mixing the sodium alginate, the chitin and the dispersion liquid according to the mass-volume ratio of 1 kg: mixing at a ratio of 2.4L, mechanically stirring at 500rpm for 20min, filtering, freezing the residue in-60 deg.C refrigerator for 36h, taking out the residue, transferring into oven at 175 deg.C, maintaining for 2h, and cooling to room temperature.
The preparation method of the dispersion comprises the following steps: adding 4 parts of heptadecafluorodecyltrimethoxysilane into 13 parts of ethanol, and stirring and mixing to obtain a product a; adding 6 parts of 1, 3-bis (4-hydroxybutyl) tetramethyldisiloxane into 20 parts of diethyl ether, stirring and mixing to obtain a product b, combining the product a and the product b to obtain a product c, heating to 60 ℃ at the speed of 2 ℃/min, adding 6 parts of dimethylbenzene at the dropping speed of 30mL/min, rotating and stirring at the rotating speed of 300rpm for 30min, and standing for 3.5h to obtain the product.
The auxiliary material is zeolite powder, and the particle size of the auxiliary material is 30 um.
Example 2
A method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin comprises the following steps:
a. mincing tissues and grinding into slurry: taking 40Kg of sheep testis, peeling, cutting the tissue into fine blocks, grinding into pulp by a mincing machine, and repeatedly grinding into pulp for three times;
b. stirring and extracting: adding purified water 2 times the weight of the testis tissue, stirring, adjusting the pH value of the solution to 3.0 with cold acetic acid, and fully homogenizing the testis tissue; stirring and extracting for 4 hours, then passing through a hydrogen ionization filter membrane, controlling the filtering time for 8 hours, removing the precipitate, and collecting the filtrate;
c. primary ammonium sulfate precipitation for impurity removal: gradually stirring, adding solid ammonium sulfate to a final concentration of 25%, precipitating, standing for 6 hr, siphoning, collecting supernatant, and removing precipitate;
d. secondary treatment of impurities: mixing the supernatant with 1.5% impurity adsorbent, stirring, standing for 3 hr, and filtering to obtain filtrate;
e. adsorption by cation exchange resin: taking the filtrate obtained in the step d, adjusting the pH value to 5.5, stirring and adding 7KG cation exchange resin, stirring and adsorbing for 3 hours at 60r/min, and maintaining the pH value in the adsorption process at 5.5; standing for 40min after adsorption, siphoning supernatant, collecting resin adsorbed at the bottom by using a Buchner funnel, washing for 10 times by using a sodium acetate solution with the concentration of 60L, PH being 5.5 and 0.1M, removing partial impurities, finally stirring and eluting for 3 hours by using a sodium acetate solution with the concentration of 60L, PH 5.5.5 and 0.1M-1.0M, standing for 40min, siphoning and collecting supernatant containing a hyaluronidase product, recovering bottom resin, recycling the resin after regeneration, and eluting the supernatant by using the resin for later use;
f. secondary ammonium sulfate precipitation product: collecting the resin elution supernatant, gradually stirring, adding solid ammonium sulfate to a final concentration of 88%, precipitating, standing for 8 hr, siphoning, vacuum filtering, collecting precipitate, and oven drying at 50 deg.C.
The preparation method of the hydrogen ionization filter membrane comprises the following steps of calculating raw materials by weight parts of 20 parts of a casting membrane liquid main material, 1 part of a plasticizer, 80 parts of an organic solvent and 3 parts of a pore-forming component; the method comprises the following specific steps:
(1) and preparing a casting solution: mixing the main material of the casting solution with an organic solvent, stirring until the main material is completely dissolved, heating to 40 ℃, adding the rest raw materials, stirring at a low speed of 300r/min for 20min, transferring to a defoaming machine for defoaming, and cooling to normal temperature to obtain the casting solution;
(2) and (3) preparing a filter membrane: preparing on plate glass by adopting a blade coating mode, wherein the thickness of a wet film after blade coating is 200um, naturally drying for 3min at normal temperature, immediately soaking in water for phase conversion, soaking for 30min, taking out, and drying at 40 ℃ to obtain the hydrogen ionization filter membrane.
The main material of the casting solution is polyvinyl chloride.
The plasticizer is acetyl tributyl citrate;
the organic solvent is prepared from the following components in a mass ratio of 1: 1, dipropylene glycol dimethyl ether and methyl pyrrolidone.
The preparation method of the pore-forming component comprises the following steps of mixing 9 parts of polyethylene glycol, 11 parts of polyvinylpyrrolidone, 7 parts of toluene and 25 parts of an acidification modification solution by weight, and specifically, mixing the polyethylene glycol, the polyvinylpyrrolidone and the toluene, slowly adding the mixture into the acidification modification solution within 2 hours while stirring at a stirring speed of 200rpm, standing for 5 hours after the addition, filtering, and baking and activating filter residues at 55 ℃ for 1.5 hours to obtain the pore-forming component.
Wherein the acidification modification component comprises 2 parts of 2- (2-methoxycarbonylethylamine) methyl benzoate and K2HPO by weight parts of raw materials 4 5 parts of sodium ethylene diamine tetracetate 11 parts of water 100 parts, adding K2HPO 4 Mixing with water for dissolving, adding 2- (2-methoxycarbonylethylamine) methyl benzoate and sodium ethylene diamine tetracetate, stirring and mixing at 300rpm, standing for 30min, and then mixing with dimethylbenzyl ortho ester butyrate according to the mass ratio of 3: 1, and the product is the acidification modification component.
The impurity adsorbent is modified sepiolite, and the preparation method comprises the following steps of calculating raw materials by weight part including 10 parts of sepiolite and 2 parts of modified additive, grinding and crushing the sepiolite into 200 meshes, dispersing the crushed sepiolite in 8 times of water, and stirring at a rotating speed of 50rpm to obtain sepiolite dispersion liquid for later use; mixing the modified additive with 3 times of water, stirring for 5min to form a chitosan-gelatin-cellulose crosslinked mixture, then mixing with the sepiolite dispersion liquid, heating to 50 ℃, dropwise adding acetic acid, continuously stirring for 10h to form gel, instantly cooling and freeze-drying by adopting a liquid nitrogen refrigeration mode when the solution is hot to form a porous substance, and crushing to 100-mesh particles to obtain the product.
The modifying additive is 9: 5: 3, mixing the chitosan, the cellulose and the gelatin with an enzyme solution in an amount which is 30 times that of the chitosan, the cellulose and the gelatin, and performing enzymolysis for 2 hours at 50 ℃ and 80Mpa, wherein the enzyme solution is prepared by adjusting the pH value to 4.5 with hydrochloric acid according to a mass ratio of 9: 11: 85 chitosanase, cellulase and water,
the cellulose is extracted from waste straw.
The preparation method of the cation exchange resin comprises the following steps of introducing the main material and the auxiliary material into a reaction tank for blending, heating to 40 ℃, and keeping for 25min for later use, wherein the raw materials comprise 10 parts by weight of the main material, 6 parts by weight of the modifier and 1 part by weight of the auxiliary material; and (2) mixing the modifier with water according to a mass ratio of 5: 1, adding the mixture into a reaction tank, heating to 70 ℃, uniformly stirring at the rotating speed of 800rpm, preserving heat for 1h, transferring the mixture into a shaping mold, preparing microspheres with the thickness of 0.5mm, and cooling to obtain the microsphere.
The main material of the cation exchange resin is macroporous weak-acidic acrylic cation exchange resin;
the modifier is prepared from the following components in a mass ratio of 1: 5, the preparation method of the pretreatment mixture of sodium alginate and chitin comprises the following steps: mixing the sodium alginate, the chitin and the dispersion liquid according to the mass volume ratio of 1 kg: mixing at a ratio of 2.8L, mechanically stirring at 600rpm for 25min, filtering, freezing the residue in a refrigerator at-70 deg.C for 48 hr, taking out the residue, transferring into an oven at 150 deg.C, maintaining the temperature for 2 hr, and cooling to room temperature.
The preparation method of the dispersion comprises the following steps: adding 3 parts of heptadecafluorodecyltrimethoxysilane into 10 parts of ethanol, and stirring and mixing to obtain a product a; adding 5 parts of 1, 3-bis (4-hydroxybutyl) tetramethyldisiloxane into 15 parts of diethyl ether, stirring and mixing to obtain a product b, combining the product a and the product b to obtain a product c, heating to 60 ℃ at 3 ℃/min, adding 8 parts of dimethylbenzene at a dropping speed of 30mL/min, rotating and stirring at a rotating speed of 400rpm for 40min, and standing for 4h to obtain the final product.
The auxiliary material is zeolite powder, and the particle size of the auxiliary material is 50 um.
Example 3
A method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin comprises the following steps:
a. mincing tissues and grinding into slurry: taking 60Kg of sheep testis, peeling, cutting the tissue into fine blocks, grinding into pulp by a mincing machine, and repeatedly grinding into pulp for three times;
b. stirring and extracting: adding purified water with the weight of 4 times of the testis tissue, stirring, adjusting the pH value of the solution to 3.5 by using cold acetic acid, and fully homogenizing the testis tissue; stirring and extracting for 8 hours, then passing through a hydrogen ionization filter membrane, controlling the filtration time for 14 hours, removing the precipitate, and collecting the filtrate;
c. primary ammonium sulfate precipitation for impurity removal: gradually stirring, adding solid ammonium sulfate to a final concentration of 35% for precipitation, standing for 8 hr, siphoning, collecting supernatant, and removing precipitate;
d. secondary treatment of impurities: mixing the supernatant with 0.5 wt% impurity adsorbent, stirring, standing for 1 hr, filtering, and collecting filtrate;
e. adsorption by cation exchange resin: taking the filtrate obtained in the step d, adjusting the pH value to 5.0, stirring and adding 3KG cation exchange resin, stirring and adsorbing for 1 hour at the speed of 50r/min, and maintaining the pH value in the adsorption process at 5.0; standing for 20min after adsorption, siphoning supernatant, collecting resin adsorbed at the bottom by using a Buchner funnel, washing for 8 times by using a sodium acetate solution with the concentration of 40L, PH being 5.5 and 0.1M, removing partial impurities, finally stirring and eluting for 1 hour at the speed of 50r/min by using a sodium acetate solution with the concentration of 40L, PH 5.5.5 and 0.1M and sodium chloride solution with the concentration of 1.0M, standing for 20min, siphoning and collecting supernatant containing a hyaluronidase product, recovering bottom resin, recycling the resin after regeneration, and eluting the supernatant by using the resin for later use;
f. secondary ammonium sulfate precipitation product: collecting the resin elution supernatant, gradually stirring, adding solid ammonium sulfate to a final concentration of 82%, precipitating, standing for 6 hr, siphoning, vacuum filtering, collecting precipitate, and oven drying at 40 deg.C.
The preparation method of the hydrogen ionization filter membrane comprises the following steps of calculating raw materials by weight part to include 40 parts of casting membrane liquid main material, 3 parts of plasticizer, 100 parts of organic solvent and 7 parts of pore-forming component; the method comprises the following specific steps:
(1) and preparing a casting solution: mixing the main material of the casting solution with an organic solvent, stirring until the main material is completely dissolved, heating to 50 ℃, adding the rest raw materials, stirring at a low speed of 500r/min for 30min, transferring to a defoaming machine for defoaming, and cooling to normal temperature to obtain the casting solution;
(2) and preparing a filter membrane: preparing on plate glass by adopting a blade coating mode, wherein the thickness of a wet film after blade coating is 300um, naturally drying for 5min at normal temperature, immediately soaking in water for phase conversion, soaking for 60min, taking out, and drying at 60 ℃ to obtain the hydrogen ionization filter membrane.
The casting film liquid is mainly polyether sulfone.
The plasticizer is acetyl tributyl citrate;
the organic solvent is prepared from the following components in a mass ratio of 3: 1, dipropylene glycol dimethyl ether and methyl pyrrolidone.
The preparation method of the pore-forming component comprises the following raw materials of, by weight, 5 parts of polyethylene glycol, 7 parts of polyvinylpyrrolidone, 3 parts of toluene and 15 parts of an acidified modified solution, and specifically, the polyethylene glycol, the polyvinylpyrrolidone and the toluene are mixed, slowly added into the acidified modified solution within 1 hour while stirring at a stirring speed of 100rpm, and then are kept stand for 3 hours after the addition, filtered, and the filter residue is baked and activated at 35 ℃ for 0.5 hour, so that the pore-forming component is obtained.
Wherein the acidification modification component comprises 6 parts of 2- (2-methoxycarbonyl ethylamine) methyl benzoate and K2HPO by weight part of raw materials 4 9 parts of sodium ethylene diamine tetracetate 7 parts of water 80 parts, adding K2HPO 4 Mixing with water, dissolving, adding 2- (2-methoxycarbonylethylamine) methyl benzoate and sodium ethylene diamine tetracetate, stirring at 500rpm, standing for 30min, and mixing with waterThe mass ratio of the butyric acid dimethylbenzyl ortho ester is 7: 1, and the product is the acidification modification component.
The impurity adsorbent is modified sepiolite, and the preparation method comprises the following steps of calculating raw materials by weight parts including 16 parts of sepiolite and 6 parts of modified additive, grinding and crushing the sepiolite into 200 meshes, dispersing the crushed sepiolite into water of 12 times, and stirring at a rotating speed of 90rpm to obtain sepiolite dispersion liquid for later use; mixing the modified additive with 5 times of water, stirring for 15min to form a chitosan-gelatin-cellulose crosslinked mixture, then mixing with the sepiolite dispersion liquid, heating to 70 ℃, dropwise adding acetic acid, continuously stirring for 20h to form gel, instantly cooling and freeze-drying in a liquid nitrogen refrigeration mode when the gel is hot to form a porous substance, and crushing to 100-mesh particles to obtain the porous substance.
The modifying additive is prepared from the following components in percentage by mass: 3: 1, mixing chitosan, cellulose and gelatin with an enzyme solution in an amount which is 10 times that of the chitosan, the cellulose and the gelatin, and performing enzymolysis for 1 hour at 40 ℃ and 60Mpa, wherein the enzyme solution is prepared by adjusting the pH value to 3.5 by hydrochloric acid, and the mass ratio of the enzyme solution is 3: 5: 65 chitosanase, cellulase and water,
the cellulose is extracted from bagasse.
The preparation method of the cation exchange resin comprises the following steps of introducing 20 parts by weight of a main material, 10 parts by weight of a modifier and 3 parts by weight of an auxiliary material of the cation exchange resin into a reaction tank, mixing the main material and the auxiliary material, heating to 60 ℃, and keeping for 45min for later use; and (2) mixing the modifier with water according to a mass ratio of 5: 1, adding the mixture into a reaction tank, heating to 90 ℃, uniformly stirring at the rotating speed of 1000rpm, preserving heat for 2 hours, transferring the mixture into a shaping mold, preparing microspheres with the diameter of 1mm, and cooling to obtain the microsphere.
The main material of the cation exchange resin is macroporous weak-acidic acrylic cation exchange resin;
the modifier is prepared from the following components in a mass ratio of 1: 1, the preparation method of the pretreatment mixture of sodium alginate and chitin comprises the following steps: mixing the sodium alginate, the chitin and the dispersion liquid according to the mass-volume ratio of 1 kg: mixing at a ratio of 2L, mechanically stirring at 400rpm for 15min, filtering, freezing the residue in a freezer at-50 deg.C for 24h, taking out the residue, transferring into an oven at 200 deg.C, maintaining the temperature for 2h, and cooling to room temperature.
The preparation method of the dispersion liquid comprises the following steps: adding 5 parts of heptadecafluorodecyltrimethoxysilane into 16 parts of ethanol, and stirring and mixing to obtain a product a; adding 7 parts of 1, 3-bis (4-hydroxybutyl) tetramethyldisiloxane into 15 parts of diethyl ether, stirring and mixing to obtain a product b, combining the product a and the product b to obtain a product c, heating to 60 ℃ at 1 ℃/min, adding 4 parts of dimethylbenzene at a dropping speed of 30mL/min, rotating and stirring at 200rpm for 20min, and standing for 3h to obtain the product.
The auxiliary material is zeolite powder, and the particle size of the auxiliary material is 20 um.
Example 4
A method for preparing a high-quality crude product of hyaluronidase by resin stirring and adsorption, wherein the preparation raw materials of a hydrogen ionization filter membrane comprise 30 parts of a casting membrane liquid main material, 2 parts of a plasticizer, 90 parts of an organic solvent and 2 parts of a pore-forming component by weight;
the rest is the same as example 1.
Example 5
A method for preparing a high-quality crude product of hyaluronidase by resin stirring and adsorption, wherein the preparation raw materials of a hydrogen ionization filter membrane comprise 30 parts of a casting membrane liquid main material, 2 parts of a plasticizer, 90 parts of an organic solvent and 8 parts of a pore-forming component by weight;
the rest is the same as example 1.
Example 6
A method for preparing a high-quality crude product of hyaluronidase by resin stirring and adsorption is disclosed, wherein raw materials for preparing a hydrogen ionization filter membrane comprise 30 parts of a main material of a membrane casting liquid, 2 parts of a plasticizer, 90 parts of an organic solvent and 5 parts of a pore-forming component by weight;
the pore-forming component comprises 7 parts by weight of polyethylene glycol, 9 parts by weight of polyvinylpyrrolidone, 5 parts by weight of toluene and 14 parts by weight of acidified modified solution.
The rest is the same as example 1.
Example 7
A method for preparing a high-quality crude product of hyaluronidase by resin stirring and adsorption, wherein the preparation raw materials of a hydrogen ionization filter membrane comprise 30 parts of a casting membrane liquid main material, 2 parts of a plasticizer, 90 parts of an organic solvent and 5 parts of a pore-forming component by weight;
the pore-forming component comprises 7 parts by weight of polyethylene glycol, 9 parts by weight of polyvinylpyrrolidone, 5 parts by weight of toluene and 26 parts by weight of acidified modified solution.
The rest is the same as example 1.
Example 8
A method for preparing a high-quality crude product of hyaluronidase by resin stirring and adsorption, wherein the preparation raw materials of a hydrogen ionization filter membrane comprise 30 parts of a casting membrane liquid main material, 2 parts of a plasticizer, 90 parts of an organic solvent and 5 parts of a pore-forming component by weight;
the preparation raw materials of the pore-forming component comprise 7 parts by weight of polyethylene glycol, 9 parts by weight of polyvinylpyrrolidone and 5 parts by weight of toluene.
The rest is the same as example 1.
Example 9
A method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin is disclosed, wherein the preparation method of the hydrogen ionization filter membrane comprises the following steps of calculating raw materials by weight part of 30 parts of a casting membrane liquid main material, 2 parts of a plasticizer, 90 parts of an organic solvent and 5 parts of a pore-forming component; the method comprises the following specific steps:
(1) and preparing a casting solution: mixing the main material of the casting solution with an organic solvent, stirring until the main material is completely dissolved, heating to 45 ℃, adding the rest raw materials, stirring at a low speed of 400r/min for 25min, transferring to a defoaming machine for defoaming, and cooling to normal temperature to obtain the casting solution;
(2) and preparing a filter membrane: preparing on plate glass by adopting a blade coating mode, wherein the thickness of a wet film after blade coating is 250um, naturally drying for 4min at normal temperature, immediately soaking in water for phase conversion, soaking for 45min, taking out, transferring, soaking in an acidification modification solution for 15s, and drying at 50 ℃ to obtain the hydrogen ionization filter membrane.
The rest is the same as example 1.
Example 10
A method for preparing a high-quality crude hyaluronidase product by resin stirring adsorption is disclosed, wherein an acidification modification solution is prepared by mixing a raw material of 5: 1, a K2HPO4 solution and a dimethylbenzyl ortho butyrate mixed solution;
wherein the acidification modification component comprises K2HPO in parts by weight as raw material 4 7 parts of water, 90 parts of K2HPO 4 Mixing with water for dissolving, standing for 30min, and mixing with dimethylbenzyl ortho ester butyrate according to a mass ratio of 5: 1, and the product is the acidification modification component.
The rest is the same as example 1.
Example 11
A method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin, wherein the secondary treatment of impurities in the step d comprises the following steps: mixing the supernatant with 0.4% impurity adsorbent, stirring, standing for 2 hr, and filtering to obtain filtrate;
the rest is the same as example 1.
Example 12
A method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin, wherein the secondary treatment of impurities in the step d comprises the following steps: mixing the supernatant with 1.6% impurity adsorbent, stirring, standing for 2 hr, and filtering to obtain filtrate;
the rest is the same as example 1.
Example 13
A method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin, wherein an impurity adsorbent is modified sepiolite, and the preparation raw materials comprise 13 parts of sepiolite and 1 part of modified additive by weight;
the rest is the same as example 1.
Example 14
A method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin, wherein an impurity adsorbent is modified sepiolite, and the preparation raw materials comprise 13 parts of sepiolite and 7 parts of modified additive by weight;
the rest is the same as example 1.
Example 15
A method for preparing high-quality crude product of hyaluronidase by stirring and adsorbing resin is provided, wherein the impurity adsorbent is sepiolite;
the rest is the same as example 1.
Example 16
A method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin, wherein the raw materials for preparing an impurity adsorbent comprise 13 parts of sepiolite and 4 parts of modified additive by weight;
the modifying additive is prepared from the following components in percentage by mass: 4: 2 chitosan, cellulose, gelatin mixture;
the rest is the same as example 1.
Example 17
A method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin, wherein the raw materials for preparing an impurity adsorbent comprise 13 parts of sepiolite and 4 parts of modified additive by weight;
the modifying additive is prepared from the following components in percentage by mass: 4: 2, mixing the chitosan, the cellulose and the gelatin with an enzyme solution in an amount which is 20 times that of the chitosan, the cellulose and the gelatin, and performing enzymolysis for 1.5 hours at 45 ℃ and 70Mpa, wherein the enzyme solution is prepared by mixing the chitosan, the cellulose and the gelatin in a mass ratio of 6: 8: 75 of chitosanase, cellulase and water,
the rest is the same as example 1.
Example 18
A method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin, wherein the raw materials for preparing an impurity adsorbent comprise 13 parts of sepiolite and 4 parts of modified additive by weight;
the modifying additive is 7: 4: 2, uniformly mixing the chitosan, the cellulose and the gelatin with 20 times of enzyme solution by mass, and carrying out enzymolysis for 1.5 hours at 45 ℃ and 70Mpa, wherein the enzyme solution is prepared by adjusting the pH value to 2.5 by hydrochloric acid according to the mass ratio of 6: 8: 75 of chitosanase, cellulase and water;
the rest is the same as example 1.
Example 19
A method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin, wherein the raw materials for preparing an impurity adsorbent comprise 13 parts of sepiolite and 4 parts of modified additive by weight;
the modifying additive is prepared from the following components in percentage by mass: 4: 2, uniformly mixing the chitosan, the cellulose and the gelatin with 20 times of enzyme solution by mass, and carrying out enzymolysis for 1.5 hours at 45 ℃ and 70Mpa, wherein the enzyme solution is prepared by adjusting the pH value to 5.0 by hydrochloric acid according to the mass ratio of 6: 8: 75 of chitosanase, cellulase and water;
the rest is the same as example 1.
Example 20
A method for preparing a high-quality crude product of hyaluronidase by resin stirring adsorption, wherein in the step e, in the cation exchange resin adsorption, the addition amount of the cation exchange resin is 2 kg;
the rest is the same as example 1.
Example 21
A method for preparing a high-quality crude product of hyaluronidase by resin stirring adsorption, wherein in the step e, in the cation exchange resin adsorption, the addition amount of the cation exchange resin is 8 kg;
the rest is the same as example 1.
Example 22
A method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin, wherein raw materials for preparing cation exchange resin comprise 15 parts of a main material of the cation exchange resin, 5 parts of a modifier and 2 parts of an auxiliary material by weight;
the rest is the same as example 1.
Example 23
A method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin, wherein the raw materials for preparing cation exchange resin comprise 15 parts of a main material of the cation exchange resin, 11 parts of a modifier and 2 parts of an auxiliary material by weight;
the rest is the same as example 1.
Example 24
A method for preparing high-quality crude product of hyaluronidase by resin stirring adsorption comprises selecting macroporous weakly acidic acrylic cation exchange resin as cation exchange resin, and performing unmodified treatment;
the rest is the same as example 1.
Example 25
A method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin is disclosed, wherein raw materials for preparing cation exchange resin comprise, by weight, 15 parts of a main material of the cation exchange resin, 8 parts of a modifier and 2 parts of an auxiliary material.
The modifier is prepared from the following components in a mass ratio of 1: 3 sodium alginate and chitin; the modifier is used without pretreatment.
The rest is the same as example 1.
Example 26
A method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin is disclosed, wherein raw materials for preparing cation exchange resin comprise, by weight, 15 parts of a main material of the cation exchange resin, 8 parts of a modifier and 2 parts of an auxiliary material.
The modifier is prepared from the following components in a mass ratio of 1: 3, the preparation method of the pretreatment mixture of sodium alginate and chitin comprises the following steps: mixing the sodium alginate, the chitin and the dispersion liquid according to the mass-volume ratio of 1 kg: mixing at a ratio of 2.4L, mechanically stirring at 500rpm for 20min, filtering, freezing the residue in a freezer at-60 deg.C for 36h, taking out the residue, transferring into an oven at 175 deg.C, maintaining the temperature for 2h, and cooling to room temperature.
The raw materials of the dispersion liquid comprise 13 parts of ethanol, 20 parts of diethyl ether and 6 parts of xylene by weight.
The rest is the same as example 1.
Test of
The quality of the hyaluronidase prepared by the method of each embodiment is detected, and the method is specifically as follows:
Figure 716656DEST_PATH_IMAGE001
from the above table results, it can be seen that:
1. the methods of the examples are preferred to those of examples 1, 2 and 3, particularly preferred to those of example 1;
2. compared with examples 4-10 and example 1, the hydrogen ionization filter membrane of the present invention has a significant effect on the primary extraction effect, especially affecting the extraction rate and bacterial endotoxin, the pore-forming components of the hydrogen ionization filter membranes of examples 4 and 5 are less or more, the acidification modification solution in the pore-forming components of the hydrogen ionization filter membranes of examples 6 and 7 is less or more, the acidification modification solution is not used for the pore-forming component of the hydrogen ionization filter membrane of example 8, the filter membrane is used for membrane formation in example 9, and then the filter membrane is soaked in the acidification modification solution for hydrogen ionization treatment, the ordinary weak acid salt K2HPO4 is used for the acidification modification solution of example 10, and the results are all inferior to those of example 1;
3. compared with examples 11 to 19 and example 1, the use of the impurity adsorbent improves the titer of the hyaluronidase to a certain extent, the impurity adsorbents of examples 11 and 12 are used in a small or large amount, the raw material modification additives for preparing the impurity adsorbents of examples 13 and 14 are too small or too much, the impurity adsorbent of example 15 is common sepiolite and is not subjected to the modification treatment of the invention, the modification additive of example 16 is not subjected to the enzymolysis treatment, the enzyme solution is not subjected to the acid treatment during the enzymolysis treatment of the modification additive of example 17, the pH of the enzyme solution is not in the technical requirement range during the enzymolysis treatment of the modification additives of examples 18 and 19, and the change of the formula or the process results in inferior effect to that of example 1;
4. comparative examples 20 to 26 and example 1, the preparation formulation and process of cation exchange resin were changed and compared, the amount of cation exchange resin used in examples 20 and 21 was not within the range of technical requirements, the preparation formulation of examples 22 and 23 was not within the range of technical requirements, the cation exchange resin in example 24 was a macroporous weakly acidic acrylic cation exchange resin which was not subjected to the modification treatment of the present invention, the modifier in example 25 was not pretreated before use, the dispersion liquid in the pretreatment step before use of the modifier in example 26 was a common organic solvent dispersion liquid, heptadecafluorodecyltrimethoxysilane and 1, 3-bis (4-hydroxybutyl) tetramethyldisiloxane were not introduced; the preparation formulas and the processes of the above embodiments are changed, so that the extraction effect of the cation exchange resin on the hyaluronidase is obviously inferior to that of the embodiment 1, the titer and the specific activity of the hyaluronidase are influenced, and the extraction rate is also influenced to a certain extent.

Claims (8)

1. A method for preparing a high-quality crude product of hyaluronidase by stirring and adsorbing resin is characterized by comprising the following steps: the method comprises the following steps:
a. mincing tissues and grinding into slurry: taking 40-60 Kg of sheep testis, peeling, cutting tissues into fine blocks, grinding into pulp by using a mincing machine, and repeatedly grinding the pulp for three times;
b. stirring and extracting: adding purified water with the weight being 2-4 times of that of the testis tissue, stirring, adjusting the pH value of the solution to 3.0-3.5 by using cold acetic acid, and fully homogenizing the testis tissue; stirring and extracting for 4-8 hours, then passing through a hydrogen ionization filter membrane, controlling the filtering time to be 8-14 hours, removing the precipitate, and collecting the filtrate;
c. primary ammonium sulfate precipitation for impurity removal: gradually stirring and adding solid ammonium sulfate until the final concentration is 25-35%, precipitating, standing for 6-8 hours, siphoning, taking supernatant, and removing precipitate;
d. secondary treatment of impurities: mixing the supernatant with 0.5-1.5% impurity adsorbent, stirring, standing for 1-3 hr, and filtering to obtain filtrate;
e. adsorption by cation exchange resin: taking the filtrate obtained in the step d, adjusting the pH value to 5.0-5.5, stirring, adding 3-7 KG of cation exchange resin, stirring and adsorbing for 1-3 hours at 50-60 r/min, and maintaining the pH value in the adsorption process at 5.0-5.5; standing for 20-40min after adsorption, siphoning supernatant, collecting resin adsorbed at the bottom by using a Buchner funnel, washing for 8-10 times by using 40-60L, pH as 5.5 and 0.1M sodium acetate solution, removing partial impurities, stirring and eluting for 1-3 hours by using 40-60L, pH5.5 and 0.1M sodium acetate-1.0M sodium chloride solution at 50-60 r/min, standing for 20-40min, siphoning and collecting supernatant containing a hyaluronidase product, recovering bottom resin, recycling the resin for reuse, and eluting the supernatant by using the resin for later use;
f. secondary ammonium sulfate precipitation product: taking the resin elution supernatant, gradually stirring and adding solid ammonium sulfate until the final concentration is 82-88%, precipitating, standing for 6-8 hours, siphoning, filtering, taking the precipitate, and drying at 40-50 ℃ to obtain the ammonium sulfate-containing water-soluble organic acid;
the preparation method of the hydrogen ionization filter membrane comprises the following steps of preparing 20-40 parts by weight of a casting membrane liquid main material, 1-3 parts by weight of a plasticizer, 80-100 parts by weight of an organic solvent and 3-7 parts by weight of a pore-forming component; the method comprises the following specific steps:
(1) and preparing a casting solution: mixing the main material of the casting solution with an organic solvent, stirring until the main material is completely dissolved, heating to 40-50 ℃, adding the rest raw materials, stirring at a low speed of 300-500 r/min for 20-30 min, transferring to a defoaming machine for defoaming, and cooling to normal temperature to obtain the casting solution;
(2) and preparing a filter membrane: preparing on plate glass in a blade coating mode, wherein the thickness of a wet film after blade coating is 200-300 um, naturally drying for 3-5 min at normal temperature, immediately soaking in water for phase conversion, soaking for 30-60min, taking out, and drying at 40-60 ℃ to obtain the hydrogen ionization filter membrane;
the main material of the casting film liquid is one or a mixture of more than two of polyvinylidene fluoride, polyvinyl chloride, polyether sulfone and polysulfone; the plasticizer is acetyl tributyl citrate; the organic solvent is prepared from the following components in a mass ratio of 1-3: 1, dipropylene glycol dimethyl ether and methyl pyrrolidone.
2. The method for preparing high-quality crude hyaluronidase by resin stirring adsorption according to claim 1, wherein: the preparation method of the pore-forming component comprises the following steps of mixing 5-9 parts of polyethylene glycol, 7-11 parts of polyvinylpyrrolidone, 3-7 parts of toluene and 15-25 parts of an acidification modification solution by weight, specifically, blending the polyethylene glycol, the polyvinylpyrrolidone and the toluene, slowly adding the mixture into the acidification modification solution within 1-2h while stirring, wherein the stirring speed is 100-200rpm, standing for 3-5h after the addition is finished, filtering, and baking and activating filter residues at 35-55 ℃ for 0.5-1.5h to obtain the pore-forming component; wherein the acidification modification solution is taken as a raw material and comprises 2-6 parts of 2- (2-methoxycarbonylethylamine) methyl benzoate and K 2 HPO 4 5-9 parts of sodium ethylene diamine tetracetate 7-11 parts of water 80-100 parts of 2 HPO 4 Mixing with water for dissolving, adding 2- (2-methoxycarbonylethylamine) methyl benzoate and sodium ethylene diamine tetracetate, stirring and mixing at the speed of 300-500rpm, standing for 30min, and then mixing with dimethylbenzyl orthobutyrate according to the mass ratio of 3-7: 1, and obtaining the acidified modified solution.
3. The method for preparing high-quality crude hyaluronidase by resin stirring adsorption according to claim 1, wherein: the impurity adsorbent is modified sepiolite, and the preparation method comprises the following steps of taking 10-16 parts by weight of sepiolite and 2-6 parts by weight of modified additive as raw materials, grinding and crushing the sepiolite into 200 meshes, dispersing the sepiolite into water with the particle size of 8-12 times, and stirring at the rotating speed of 50-90rpm to obtain sepiolite dispersion liquid for later use; mixing the modified additive with 3-5 times of water, stirring for 5-15min to form a chitosan-gelatin-cellulose crosslinked mixture, then combining with the sepiolite dispersion liquid, heating to 50-70 ℃, dropwise adding acetic acid, continuously stirring for 10-20h to form gel, instantly cooling and freeze-drying by adopting a liquid nitrogen refrigeration mode when the gel is hot to form a porous substance, and crushing to 100-mesh particles to obtain the product.
4. The method for preparing high-quality crude hyaluronidase by resin stirring adsorption as claimed in claim 3, wherein: the modifying additive is prepared from the following components in a mass ratio of 5-9: 3-5: 1-3, mixing the chitosan, the cellulose and the gelatin with an enzyme solution in an amount which is 10-30 times the mass of the chitosan, the cellulose and the gelatin, and performing enzymolysis for 1-2 hours at 40-50 ℃ and 60-80Mpa, wherein the enzyme solution is prepared by adjusting the pH value to 3.5-4.5 with hydrochloric acid according to a mass ratio of 3-9: 5-11: 65-85 of chitosanase, cellulase and water; the cellulose is extracted from agricultural and forestry residues including rice straw, waste straw and bagasse.
5. The method for preparing high-quality crude hyaluronidase by resin stirring adsorption according to claim 1, wherein: the preparation method of the cation exchange resin comprises the following steps of introducing 10-20 parts by weight of a main material, 6-10 parts by weight of a modifier and 1-3 parts by weight of an auxiliary material of the cation exchange resin into a reaction tank, mixing the main material and the auxiliary material, heating to 40-60 ℃, and keeping for 25-45 min for later use; and (2) mixing the modifier with water according to a mass ratio of 5: 1, adding the mixture into a reaction tank, heating to 70-90 ℃, uniformly stirring at the rotating speed of 800-1000 rpm, preserving heat for 1-2 hours, transferring the mixture into a shaping mold, preparing microspheres with the particle size of 0.5-1 mm, and cooling to obtain the zeolite microsphere, wherein the auxiliary material is zeolite powder, and the particle size of the zeolite powder is 20-50 microns.
6. The method for preparing high-quality crude hyaluronidase by resin stirring adsorption according to claim 5, wherein: the main material of the cation exchange resin is macroporous weak-acid acrylic cation exchange resin.
7. The method for preparing high-quality crude hyaluronidase by resin stirring adsorption according to claim 5, wherein: the modifier is a mixture of 1: 1-5 of a pretreatment mixture of sodium alginate and chitin, wherein the preparation method comprises the following steps: mixing the sodium alginate, the chitin and the dispersion liquid according to the mass-volume ratio of 1 kg: 2-2.8L, mechanically stirring at the rotating speed of 400-600 rpm for 15-25 min, filtering, freezing the filter residue in a refrigerator at the temperature of-50-70 ℃ for 24-48 h, taking out the filter residue, transferring the filter residue to an oven at the temperature of 150-200 ℃, preserving heat for 2h, and cooling to the normal temperature.
8. The method for preparing high-quality crude hyaluronidase by resin agitation and adsorption according to claim 7, wherein: the preparation method of the dispersion comprises the following steps: adding 3-5 parts of heptadecafluorodecyltrimethoxysilane into 10-16 parts of ethanol, and stirring and mixing to obtain a product a; adding 5-7 parts of 1, 3-bis (4-hydroxybutyl) tetramethyldisiloxane into 15-25 parts of diethyl ether, stirring and mixing to obtain a product b, combining the product a and the product b to obtain a product c, heating the product c to 60 ℃ at 1-3 ℃/min, adding 4-8 parts of dimethylbenzene at the dropping speed of 30mL/min, rotationally stirring at the rotating speed of 400rpm of 200-4 ℃, and standing for 3-4h to obtain the product.
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101597296A (en) * 2008-06-02 2009-12-09 中国科学院过程工程研究所 A kind of novel method of efficiently extracting and producing artemisinin by ionic liquid
WO2010031876A1 (en) * 2008-09-22 2010-03-25 Universiteit Gent Carboxyethylated cyclodextrin polysulfates useful as medicaments
CN103060288A (en) * 2012-12-30 2013-04-24 青岛九龙生物医药有限公司 Method for extracting hyaluronidase from pig testis
CN103060291A (en) * 2012-12-28 2013-04-24 青岛九龙生物医药有限公司 Extraction method for hyaluronidase
CN103421053A (en) * 2013-08-08 2013-12-04 南京标科生物科技有限公司 Extraction method for pomegranate foline
CN103725659A (en) * 2013-11-24 2014-04-16 青岛康原药业有限公司 Method for extracting hyaluronidase from bovine testes
CN106119225A (en) * 2016-06-30 2016-11-16 宁波市江东林清环保科技有限公司 A kind of preparation method of hyaluronidase
CN113135982A (en) * 2021-04-12 2021-07-20 内蒙古农业大学 Method for decoloring sunflower seed protein and decolored sunflower seed protein
WO2022031894A1 (en) * 2020-08-07 2022-02-10 Gilead Sciences, Inc. Prodrugs of phosphonamide nucleotide analogues and their pharmaceutical use

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4920162A (en) * 1988-12-22 1990-04-24 Clark Peter D Electrocoating composition of a polyamine self-condensed epoxy adduct and coatings produced thereby
CN200960075Y (en) * 2006-05-17 2007-10-17 王宝根 Photohydroionization sterilizing toilet utensils composition device
CN103103170B (en) * 2013-02-06 2014-10-15 甘肃天森药业有限公司 Production process for cow or sheep hyaluronidase
CN103451166A (en) * 2013-08-31 2013-12-18 青岛康原药业有限公司 Method for extracting hyaluronidase from pig testicle
CN104531635A (en) * 2014-12-23 2015-04-22 青岛康原药业有限公司 Method for extracting hyaluronidase crude product
CN104498452A (en) * 2014-12-23 2015-04-08 青岛康原药业有限公司 Method for extracting hyaluronidase crude product by low-temperature ethanol method
CN107312015A (en) * 2017-06-21 2017-11-03 禹州市天源生物科技有限公司 A kind of technique of efficiently extracting and producing artemisinin by ionic liquid
CN107640874A (en) * 2017-11-14 2018-01-30 安徽名创新材料科技有限公司 The technique that a kind of MBR is combined processing dyeing waste water with membrane separation process
CN111019985B (en) * 2019-12-27 2022-02-15 华熙生物科技股份有限公司 Method for preparing low molecular weight or oligomeric hyaluronic acid or salt thereof by enzymolysis
CN113774044B (en) * 2021-08-26 2023-05-12 南昌市万华生化制品有限公司 Preparation process of high-purity hyaluronidase
CN114250212A (en) * 2021-12-14 2022-03-29 华熙生物科技股份有限公司 Hyaluronidase freeze-dried preparation and preparation method thereof

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101597296A (en) * 2008-06-02 2009-12-09 中国科学院过程工程研究所 A kind of novel method of efficiently extracting and producing artemisinin by ionic liquid
WO2010031876A1 (en) * 2008-09-22 2010-03-25 Universiteit Gent Carboxyethylated cyclodextrin polysulfates useful as medicaments
CN103060291A (en) * 2012-12-28 2013-04-24 青岛九龙生物医药有限公司 Extraction method for hyaluronidase
CN103060288A (en) * 2012-12-30 2013-04-24 青岛九龙生物医药有限公司 Method for extracting hyaluronidase from pig testis
CN103421053A (en) * 2013-08-08 2013-12-04 南京标科生物科技有限公司 Extraction method for pomegranate foline
CN103725659A (en) * 2013-11-24 2014-04-16 青岛康原药业有限公司 Method for extracting hyaluronidase from bovine testes
CN106119225A (en) * 2016-06-30 2016-11-16 宁波市江东林清环保科技有限公司 A kind of preparation method of hyaluronidase
WO2022031894A1 (en) * 2020-08-07 2022-02-10 Gilead Sciences, Inc. Prodrugs of phosphonamide nucleotide analogues and their pharmaceutical use
CN113135982A (en) * 2021-04-12 2021-07-20 内蒙古农业大学 Method for decoloring sunflower seed protein and decolored sunflower seed protein

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
肝素钠粗品清洁生产方案评估;侍爱秋;《环境科学与管理》;20071215(第12期);全文 *

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