CN117721099A - Preparation method and application of chymotrypsin - Google Patents
Preparation method and application of chymotrypsin Download PDFInfo
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- CN117721099A CN117721099A CN202311741836.2A CN202311741836A CN117721099A CN 117721099 A CN117721099 A CN 117721099A CN 202311741836 A CN202311741836 A CN 202311741836A CN 117721099 A CN117721099 A CN 117721099A
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- 108090000317 Chymotrypsin Proteins 0.000 title claims abstract description 135
- 229960002376 chymotrypsin Drugs 0.000 title claims abstract description 135
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 111
- 239000000243 solution Substances 0.000 claims abstract description 87
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- 229940088598 enzyme Drugs 0.000 claims abstract description 39
- 239000002244 precipitate Substances 0.000 claims abstract description 39
- 238000003756 stirring Methods 0.000 claims abstract description 39
- 239000007853 buffer solution Substances 0.000 claims abstract description 37
- 239000011734 sodium Substances 0.000 claims abstract description 32
- 229910052708 sodium Inorganic materials 0.000 claims abstract description 32
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims abstract description 30
- 238000000605 extraction Methods 0.000 claims abstract description 29
- 238000002386 leaching Methods 0.000 claims abstract description 29
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- 238000001035 drying Methods 0.000 claims abstract description 25
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
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- 229920002684 Sepharose Polymers 0.000 claims description 2
- 239000004480 active ingredient Substances 0.000 claims description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 2
- 238000000108 ultra-filtration Methods 0.000 abstract description 14
- 229920002873 Polyethylenimine Polymers 0.000 abstract description 6
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- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 abstract description 3
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- 230000000052 comparative effect Effects 0.000 description 26
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- 239000004721 Polyphenylene oxide Substances 0.000 description 10
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- 238000002835 absorbance Methods 0.000 description 9
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- 238000000926 separation method Methods 0.000 description 7
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- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
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- HVBSAKJJOYLTQU-UHFFFAOYSA-N 4-aminobenzenesulfonic acid Chemical compound NC1=CC=C(S(O)(=O)=O)C=C1 HVBSAKJJOYLTQU-UHFFFAOYSA-N 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
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- 229940024606 amino acid Drugs 0.000 description 2
- 125000000129 anionic group Chemical group 0.000 description 2
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- 239000012588 trypsin Substances 0.000 description 2
- 238000002137 ultrasound extraction Methods 0.000 description 2
- LZKGFGLOQNSMBS-UHFFFAOYSA-N 4,5,6-trichlorotriazine Chemical compound ClC1=NN=NC(Cl)=C1Cl LZKGFGLOQNSMBS-UHFFFAOYSA-N 0.000 description 1
- 206010063560 Excessive granulation tissue Diseases 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
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- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 108090000083 Serine Endopeptidases Proteins 0.000 description 1
- 102000003667 Serine Endopeptidases Human genes 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 108010027597 alpha-chymotrypsin Proteins 0.000 description 1
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- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- SKAWDTAMLOJQNK-LBPRGKRZSA-N ethyl N-acetyl-L-tyrosinate Chemical compound CCOC(=O)[C@@H](NC(C)=O)CC1=CC=C(O)C=C1 SKAWDTAMLOJQNK-LBPRGKRZSA-N 0.000 description 1
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- 238000011031 large-scale manufacturing process Methods 0.000 description 1
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 210000001819 pancreatic juice Anatomy 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
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- PVGBHEUCHKGFQP-UHFFFAOYSA-N sodium;n-[5-amino-2-(4-aminophenyl)sulfonylphenyl]sulfonylacetamide Chemical compound [Na+].CC(=O)NS(=O)(=O)C1=CC(N)=CC=C1S(=O)(=O)C1=CC=C(N)C=C1 PVGBHEUCHKGFQP-UHFFFAOYSA-N 0.000 description 1
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- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a preparation method and application of chymotrypsin, which relate to the technical field of biological pharmacy and sequentially carry out raw material pretreatment, leaching, extraction, activation, ultrafiltration, fine extraction, purification and drying and crushing, wherein sodium polyvinylsulfonate is added into chymotrypsin crude enzyme liquid during fine extraction, and insoluble polymers are collected after stirring and centrifugation; adding acetic acid buffer solution into insoluble polymer, stirring, adding polyetherimide solution, fully dissociating, freeze centrifuging, filtering, and collecting precipitate; the sodium polyvinylsulfonate has negatively charged sulfonic acid group capable of forming electrostatic interaction with positively charged histidine residue of chymotrypsin to form polymeric precipitate, and the presence of low concentration polyethylenimine can destroy coulomb force action between the two to dissociate the two mutually, i.e. the prepared chymotrypsin has higher quality through the synergistic action between the added sodium polyvinylsulfonate and polyethylenimine.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to a preparation method and application of chymotrypsin.
Background
Chymotrypsin, also known as chymotrypsin, is biosynthesized in the pancreas in the form of chymotrypsinogen and is secreted out with pancreatic juice. In the small intestine, chymotrypsinogen is converted to active alpha-chymotrypsin by trypsin and chymotrypsin, which is a serine endopeptidase. Chymotrypsin acts mainly on peptide bonds formed by amino acids such as aromatic amino acids (tyrosine, tryptophan, phenylalanine).
The use of chymotrypsin has focused mainly on medical aspects: chymotrypsin can decompose fibrin coagulum at inflammation part, promote dissolving and decomposing blood clot, purulent secretion and necrotic tissue, thereby purifying wound surface, promoting granulation tissue regeneration, and promoting wound healing. At home and abroad, clinical local application of chymotrypsin in ophthalmology and dermatology has been confirmed, and the chymotrypsin is mainly used for ophthalmology operation in domestic clinic, can also be used for wounds or local inflammation so as to reduce local secretion and edema, and in recent years, the clinical application range of chymotrypsin is also gradually widened along with continuous development of clinical pharmacology research.
At present, the main problems in chymotrypsin extraction are low product quality (the requirements of pharmacopoeia are not met, the enzyme activity is low), long production period, low yield and the like.
Chinese patent CN104531650a discloses a method for purifying chymotrypsin by affinity chromatography and sectional elution and a pharmaceutical composition containing chymotrypsin, the method comprises the following steps: (1) multiple crystallization; (2) activation; (3) salting out; (4) ultrafiltration; (5) pyrogen treatment; (6) dialysis; (7) affinity chromatography; (8) lyophilizing to prepare chymotrypsin. However, this method has a problem of complicated process and serious loss of enzyme activity.
Chinese patent CN104419695B discloses a preparation method of chymotrypsinogen bionic affinity material and purification method of chymotrypsin, comprising the following steps: (1) After the basic chromatography medium reacts with trichlorotriazine for activation, the basic chromatography medium reacts with L-valine and sulfanilic acid respectively to synthesize a bionic affinity separation material; (2) Purifying chymotrypsinogen by using the prepared bionic affinity separation material, allowing a sample containing chymotrypsinogen to flow through the affinity chromatography column, adsorbing chymotrypsinogen on the affinity chromatography column, flushing the affinity chromatography column, removing the foreign proteins which are not adsorbed on the affinity chromatography column, and then changing buffer conditions to wash out the bound chymotrypsinogen. (3) activation of chymotrypsinogen with trypsin. The artificially synthesized small molecular compound ligand has the advantages of large multivalent price, large medium usage amount, low purification column recycling rate and short service life, and is not suitable for large-scale production.
Disclosure of Invention
Based on the problems existing in the background technology, the invention aims to provide a preparation method and application of chymotrypsin, which utilize ultrasonic extraction and combining ultrafiltration, column chromatography and other processes, and simultaneously use sodium polyethylene sulfonate for polymerization, so that polyetherimide is used for degrading and refining the chymotrypsin, wherein the extraction rate of the chymotrypsin reaches 70% -75%, and the activity of the prepared chymotrypsin reaches 1659U/mg.
The invention is realized by the following technical scheme:
in a first aspect, the present application provides a method for preparing chymotrypsin comprising the steps of:
step 1: pretreating raw materials, washing pancreas with isotonic physiological saline, and mincing;
step 2: leaching, namely adding tris (hydroxymethyl) aminomethane hydrochloride into minced pancreas, and stirring and homogenizing to obtain a chymotrypsin leaching solution;
step 3: extracting, freezing and centrifuging the extracted leaching solution, repeatedly dissolving, extracting and centrifuging the obtained precipitate by using the leaching solution, and collecting all supernatant;
step 4: activating, namely adding calcium chloride and pancreatin powder into the collected supernatant, adjusting the pH value by using sodium bicarbonate, and fully stirring to obtain an activated enzyme solution;
step 5: ultrafiltering, ultrafiltering with ultrafilter tube, concentrating the dialysate to obtain chymotrypsin crude enzyme solution;
step 6: fine extracting, namely adding sodium polyvinylsulfonate into the chymotrypsin crude enzyme liquid, stirring, centrifuging, and collecting insoluble polymers; adding acetic acid buffer solution into insoluble polymer, stirring, adding polyetherimide solution, fully dissociating, freeze centrifuging, filtering, and collecting precipitate;
step 7: purifying, adding acetic acid buffer solution into the precipitate, loading and adsorbing by using a chromatographic column, eluting, and collecting eluent containing chymotrypsin activity peaks;
step 8: drying and crushing, freezing the collected eluent, drying in vacuum, crushing and sieving to obtain chymotrypsin.
According to the preparation method of chymotrypsin, the anionic polymer-sodium polyvinylsulfonate is added in the fine extraction process, the negative charged sulfonic acid group of sodium polyvinylsulfonate can be used for generating an interaction electrostatic interaction with the positively charged histidine residue of chymotrypsin to form a polymeric precipitate, and the presence of low-concentration polyethyleneimine can destroy the coulomb force action between the two to enable the two to be dissociated from each other, namely, the prepared chymotrypsin has higher quality through the synergistic action between the added sodium polyvinylsulfonate and polyethyleneimine.
The invention has the advantages of improving the adsorption capacity to protein, obviously improving the separation effect of protein, along with mild separation condition, no damage to protein activity, high separation effect and large column capacity.
The integral preparation method of the invention utilizes the polyelectrolyte polymeric protein with low concentration and combines the chromatographic column purification method, thereby achieving the effects of simplifying the process and producing high-quality chymotrypsin with low cost and meeting the domestic and foreign market demands.
Further, the concentration of the tris (hydroxymethyl) aminomethane hydrochloride added in the step 2 is 2.8mmol/L to 4.2mmol/L.
Further, the extraction temperature in the step 3 is controlled to be 4-5 ℃.
Further, the dosage of the calcium chloride added in the step 4 is 1% -1.5%, and the dosage of the pancreatin powder added is 3-5%.
Further, in the step 4, sodium bicarbonate is added to adjust the pH to 7.0-8.0, and the temperature is maintained at 20-30 ℃ and fully stirred.
Further, the concentration of the sodium polyvinyl sulfonate added in the step 6 is 0.015mol/L to 0.02mol/L.
Further, the concentration of the acetic acid buffer solution added in the step 6 was 0.01mol/L.
Further, the concentration of the polyetherimide solution added in the step 6 is 4.0E-06mol/L to 4.5E-06mol/L.
Further, in step 7, a packed phenyl-sepharose column was used.
In a second aspect, the present application provides a pharmaceutical composition comprising chymotrypsin prepared using the preparation process described above as an active ingredient.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) According to the preparation method of chymotrypsin, the anionic polymer-sodium polyvinylsulfonate is added in the fine extraction process, the negative charged sulfonic acid group of sodium polyvinylsulfonate can be used for generating an interaction electrostatic interaction with the positively charged histidine residue of chymotrypsin to form a polymeric precipitate, and the presence of low-concentration polyethyleneimine can destroy the coulomb force action between the two to enable the two to be dissociated from each other, namely, the prepared chymotrypsin has higher quality through the synergistic action between the added sodium polyvinylsulfonate and polyethyleneimine;
(2) The invention has the advantages of mild separation condition, no damage to protein activity, high separation effect and large column capacity;
(3) The integral preparation method of the invention utilizes polyelectrolyte polymeric protein with low concentration and combines a chromatographic column purification method, thereby achieving the effects of simplifying the process and producing high-quality chymotrypsin with low cost and meeting the domestic and foreign market demands;
(4) The invention utilizes ultrasonic extraction and combines the technologies of ultrafiltration, column chromatography and the like, and simultaneously uses sodium polyethylene sulfonate for polymerization, and polyetherimide is used for degrading and refining chymotrypsin, wherein the extraction rate of the chymotrypsin reaches 70-75%, and the activity of the chymotrypsin prepared by the method reaches 1659U/mg.
Drawings
In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the drawings that are needed in the examples will be briefly described below, it being understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope, and that other related drawings may be obtained from these drawings without inventive effort for a person skilled in the art. In the drawings:
FIG. 1 is a graph showing the relationship between polyethylene sodium sulfate polymerase at different concentrations in examples and comparative examples;
FIG. 2 is a graph showing the relationship between polyetherimide degradation chymotrypsin and sodium polyvinylsulfonate polymers at various concentrations in examples and comparative examples;
FIG. 3 is an SEM image of the chymotrypsin prepared in example 1 of the present invention;
FIG. 4 is an SEM image of the chymotrypsin prepared in comparative example 1.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The embodiment provides a preparation method of chymotrypsin, which comprises the following specific steps:
s1: pretreating raw materials, collecting pig pancreas 100g, removing fat connective tissue, adding 1L isotonic physiological saline (0.9% NaCl), washing, removing impurity dirt on pancreas surface, and flaking and mincing.
S2: leaching, adding 3mmol/L of 250mLTris-HCl into minced pancreas, and fully stirring and homogenizing at 5 ℃ for 1 hour to obtain chymotrypsin leaching solution.
S3: extracting, namely extracting the extract in a 700w power ultrasonic wave instrument at the temperature of 5 ℃ for 7 minutes, freezing and centrifuging (4000 r/min,10 minutes and 5 ℃) after full extraction, repeatedly dissolving and extracting the obtained precipitate with the extract and centrifuging twice, and collecting all supernatant.
S4: activating, adding 1.5% calcium chloride (w/v) and 3%o pancreatin powder (w/v) into the collected supernatant, adjusting pH value to 7.5 by using sodium bicarbonate, maintaining the temperature at 25 ℃ and fully stirring for 110 minutes, and activating enzyme solution.
S5: ultrafiltering with ultrafiltration tube with cut-off molecular weight of 10KD (30 min, 4deg.C), and concentrating the dialysate to appropriate volume to obtain chymotrypsin crude enzyme solution.
S6: fine extraction, namely slowly adding 10ml of 0.015mol/L polyvinyl sodium sulfonate into the chymotrypsin crude enzyme solution, maintaining the pH value to be 7.5, fully and uniformly stirring, standing overnight, centrifuging, removing the supernatant, and collecting insoluble polymer; 100ml of an acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L is added into the insoluble polymer, 10ml of a 4.0E-06mol/L polyether imine solution is added after the mixture is slowly and evenly stirred, the solution is fully dissociated, frozen and centrifuged (12000 r/min,15 min), filtered, and the precipitate is collected.
S7: purifying, adding 100ml of acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L and 0.5mol/LnaCl into the precipitate, loading the mixture into a column, selecting a phenyl-agarose gel chromatographic column (1.6 cm x 15cm,30 ml) for loading and adsorbing, eluting with 0.5mol/L and 0.01mol/L of acetic acid buffer solution with the pH value of 5.0, controlling the flow rate to be 1ml/min, collecting eluent containing chymotrypsin activity peaks, and preserving at the temperature of minus 20 ℃.
S8: drying and crushing, freezing the collected eluent, drying in vacuum, crushing and sieving to obtain the chymotrypsin product.
Example 2
Based on example 1, this example provides a method for preparing chymotrypsin, and compared with example 1, the concentration of sodium polyvinylsulfonate added in step S6 of this implementation is 0.02mol/L, and other technical characteristics are exactly the same as example 1. The specific method comprises the following steps:
s1: pretreating raw materials, collecting pig pancreas 100g, removing fat connective tissue, adding 1L isotonic physiological saline (0.9% NaCl), washing, removing impurity dirt on pancreas surface, and flaking and mincing.
S2: leaching, adding 3mmol/L of 250mLTris-HCl into minced pancreas, and fully stirring and homogenizing at 5 ℃ for 1 hour to obtain chymotrypsin leaching solution.
S3: extracting, namely extracting the extract in a 700w power ultrasonic wave instrument at the temperature of 5 ℃ for 7 minutes, freezing and centrifuging (4000 r/min,10 minutes and 5 ℃) after full extraction, repeatedly dissolving and extracting the obtained precipitate with the extract and centrifuging twice, and collecting all supernatant.
S4: activating, adding 1.5% calcium chloride (w/v) and 3%o pancreatin powder (w/v) into the collected supernatant, adjusting pH value to 7.5 by using sodium bicarbonate, maintaining the temperature at 25 ℃ and fully stirring for 110 minutes, and activating enzyme solution.
S5: ultrafiltering with ultrafiltration tube with cut-off molecular weight of 10KD (30 min, 4deg.C), and concentrating the dialysate to appropriate volume to obtain chymotrypsin crude enzyme solution.
S6: fine extracting, slowly adding 10ml of 0.02mol/L polyvinyl sodium sulfonate into the chymotrypsin crude enzyme liquid, maintaining the pH value to be 7.5, fully and uniformly stirring, standing overnight, centrifuging, removing the supernatant, and collecting insoluble polymer; 100ml of an acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L is added into the insoluble polymer, 10ml of a 4.0E-06mol/L polyether imine solution is added after the mixture is slowly and evenly stirred, the solution is fully dissociated, frozen and centrifuged (12000 r/min,15 min), filtered, and the precipitate is collected.
S7: purifying, adding 100ml of acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L and 0.5mol/LnaCl into the precipitate, loading the mixture into a column, selecting a phenyl-agarose gel chromatographic column (1.6 cm x 15cm,30 ml) for loading and adsorbing, eluting with 0.5mol/L and 0.01mol/L of acetic acid buffer solution with the pH value of 5.0, controlling the flow rate to be 1ml/min, collecting eluent containing chymotrypsin activity peaks, and preserving at the temperature of minus 20 ℃.
S8: drying and crushing, freezing the collected eluent, drying in vacuum, crushing and sieving to obtain the chymotrypsin product.
Example 3
Based on example 1, this example provides a method for preparing chymotrypsin, and compared with example 1, the concentration of sodium polyvinylsulfonate added in step S6 of this implementation is 0.025mol/L, and other technical characteristics are exactly the same as those of example 1. The specific method comprises the following steps:
s1: pretreating raw materials, collecting pig pancreas 100g, removing fat connective tissue, adding 1L isotonic physiological saline (0.9% NaCl), washing, removing impurity dirt on pancreas surface, and flaking and mincing.
S2: leaching, adding 3mmol/L of 250mLTris-HCl into minced pancreas, and fully stirring and homogenizing at 5 ℃ for 1 hour to obtain chymotrypsin leaching solution.
S3: extracting, namely extracting the extract in a 700w power ultrasonic wave instrument at the temperature of 5 ℃ for 7 minutes, freezing and centrifuging (4000 r/min,10 minutes and 5 ℃) after full extraction, repeatedly dissolving and extracting the obtained precipitate with the extract and centrifuging twice, and collecting all supernatant.
S4: activating, adding 1.5% calcium chloride (w/v) and 3%o pancreatin powder (w/v) into the collected supernatant, adjusting pH value to 7.5 by using sodium bicarbonate, maintaining the temperature at 25 ℃ and fully stirring for 110 minutes, and activating enzyme solution.
S5: ultrafiltering with ultrafiltration tube with cut-off molecular weight of 10KD (30 min, 4deg.C), and concentrating the dialysate to appropriate volume to obtain chymotrypsin crude enzyme solution.
S6: fine extracting, slowly adding 10ml of 0.025mol/L polyvinyl sodium sulfonate into the chymotrypsin crude enzyme liquid, maintaining the pH value to be 7.5, fully and uniformly stirring, standing overnight, centrifuging, removing the supernatant, and collecting insoluble polymer; 100ml of an acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L is added into the insoluble polymer, 10ml of a 4.0E-06mol/L polyether imine solution is added after the mixture is slowly and evenly stirred, the solution is fully dissociated, frozen and centrifuged (12000 r/min,15 min), filtered, and the precipitate is collected.
S7: purifying, adding 100ml of acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L and 0.5mol/LnaCl into the precipitate, loading the mixture into a column, selecting a phenyl-agarose gel chromatographic column (1.6 cm x 15cm,30 ml) for loading and adsorbing, eluting with 0.5mol/L and 0.01mol/L of acetic acid buffer solution with the pH value of 5.0, controlling the flow rate to be 1ml/min, collecting eluent containing chymotrypsin activity peaks, and preserving at the temperature of minus 20 ℃.
S8: drying and crushing, freezing the collected eluent, drying in vacuum, crushing and sieving to obtain the chymotrypsin product.
Example 4
Based on example 1, this example provides a method for preparing chymotrypsin, and compared with example 1, the concentration of sodium polyvinylsulfonate added in step S6 of this implementation is 0.03mol/L, and other technical characteristics are exactly the same as those of example 1. The specific method comprises the following steps:
s1: pretreating raw materials, collecting pig pancreas 100g, removing fat connective tissue, adding 1L isotonic physiological saline (0.9% NaCl), washing, removing impurity dirt on pancreas surface, and flaking and mincing.
S2: leaching, adding 3mmol/L of 250mLTris-HCl into minced pancreas, and fully stirring and homogenizing at 5 ℃ for 1 hour to obtain chymotrypsin leaching solution.
S3: extracting, namely extracting the extract in a 700w power ultrasonic wave instrument at the temperature of 5 ℃ for 7 minutes, freezing and centrifuging (4000 r/min,10 minutes and 5 ℃) after full extraction, repeatedly dissolving and extracting the obtained precipitate with the extract and centrifuging twice, and collecting all supernatant.
S4: activating, adding 1.5% calcium chloride (w/v) and 3%o pancreatin powder (w/v) into the collected supernatant, adjusting pH value to 7.5 by using sodium bicarbonate, maintaining the temperature at 25 ℃ and fully stirring for 110 minutes, and activating enzyme solution.
S5: ultrafiltering with ultrafiltration tube with cut-off molecular weight of 10KD (30 min, 4deg.C), and concentrating the dialysate to appropriate volume to obtain chymotrypsin crude enzyme solution.
S6: fine extraction, namely slowly adding 10ml of 0.03mol/L polyvinyl sodium sulfonate into the chymotrypsin crude enzyme liquid, maintaining the pH value to be 7.5, fully and uniformly stirring, standing overnight, centrifuging, removing the supernatant, and collecting insoluble polymer; 100ml of an acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L is added into the insoluble polymer, 10ml of a 4.0E-06mol/L polyether imine solution is added after the mixture is slowly and evenly stirred, the solution is fully dissociated, frozen and centrifuged (12000 r/min,15 min), filtered, and the precipitate is collected.
S7: purifying, adding 100ml of acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L and 0.5mol/LnaCl into the precipitate, loading the mixture into a column, selecting a phenyl-agarose gel chromatographic column (1.6 cm x 15cm,30 ml) for loading and adsorbing, eluting with 0.5mol/L and 0.01mol/L of acetic acid buffer solution with the pH value of 5.0, controlling the flow rate to be 1ml/min, collecting eluent containing chymotrypsin activity peaks, and preserving at the temperature of minus 20 ℃.
S8: drying and crushing, freezing the collected eluent, drying in vacuum, crushing and sieving to obtain the chymotrypsin product.
Comparative example 1
The comparative example provides a preparation method of chymotrypsin, and compared with the example 1, the comparative example is not added with polyethylene sodium sulfonate, and other technical characteristics are exactly the same as the example 1, and the specific method is as follows:
s1: pretreating raw materials, collecting pig pancreas 100g, removing fat connective tissue, adding 1L isotonic physiological saline (0.9% NaCl), washing, removing impurity dirt on pancreas surface, and flaking and mincing.
S2: leaching, adding 3mmol/L of 250mLTris-HCl into minced pancreas, and fully stirring and homogenizing at 5 ℃ for 1 hour to obtain chymotrypsin leaching solution.
S3: extracting, namely extracting the extract in a 700w power ultrasonic wave instrument at the temperature of 5 ℃ for 7 minutes, freezing and centrifuging (4000 r/min,10 minutes and 5 ℃) after full extraction, repeatedly dissolving and extracting the obtained precipitate with the extract and centrifuging twice, and collecting all supernatant.
S4: activating, adding 1.5% calcium chloride (w/v) and 3%o pancreatin powder (w/v) into the collected supernatant, adjusting pH value to 7.5 by using sodium bicarbonate, maintaining the temperature at 25 ℃ and fully stirring for 110 minutes, and activating enzyme solution.
S5: ultrafiltering with ultrafiltration tube with cut-off molecular weight of 10KD (30 min, 4deg.C), and concentrating the dialysate to appropriate volume to obtain chymotrypsin crude enzyme solution.
S6: fine extraction, namely centrifuging chymotrypsin crude enzyme liquid, discarding supernatant, and collecting insoluble polymer; 100ml of an acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L is added into the insoluble polymer, 10ml of a 4.0E-06mol/L polyether imine solution is added after the mixture is slowly and evenly stirred, the solution is fully dissociated, frozen and centrifuged (12000 r/min,15 min), filtered, and the precipitate is collected.
S7: purifying, adding 100ml of acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L and 0.5mol/LnaCl into the precipitate, loading the mixture into a column, selecting a phenyl-agarose gel chromatographic column (1.6 cm x 15cm,30 ml) for loading and adsorbing, eluting with 0.5mol/L and 0.01mol/L of acetic acid buffer solution with the pH value of 5.0, controlling the flow rate to be 1ml/min, collecting eluent containing chymotrypsin activity peaks, and preserving at the temperature of minus 20 ℃.
S8: drying and crushing, freezing the collected eluent, drying in vacuum, crushing and sieving to obtain the chymotrypsin product.
As shown in FIG. 3, an SEM image of chymotrypsin prepared by the method of example 1 and an SEM image of chymotrypsin prepared by the method of comparative example 1 are shown in FIG. 4.
As can be seen from fig. 3 and 4, the chymotrypsin has a loose structure and a smooth surface, and sodium polyvinylsulfonate is added to interact with chymotrypsin, so that small granular substances can be adsorbed together, and the internal structure of the polymer is more compact.
Comparative example 2
This comparative example provides a method for preparing chymotrypsin, which is different from example 1 in that the concentration of sodium polyvinylsulfonate added in step S6 of this comparative example is 0.005mol/L, and other technical features are exactly the same as in example 1. The specific method comprises the following steps:
s1: pretreating raw materials, collecting pig pancreas 100g, removing fat connective tissue, adding 1L isotonic physiological saline (0.9% NaCl), washing, removing impurity dirt on pancreas surface, and flaking and mincing.
S2: leaching, adding 3mmol/L of 250mLTris-HCl into minced pancreas, and fully stirring and homogenizing at 5 ℃ for 1 hour to obtain chymotrypsin leaching solution.
S3: extracting, namely extracting the extract in a 700w power ultrasonic wave instrument at the temperature of 5 ℃ for 7 minutes, freezing and centrifuging (4000 r/min,10 minutes and 5 ℃) after full extraction, repeatedly dissolving and extracting the obtained precipitate with the extract and centrifuging twice, and collecting all supernatant.
S4: activating, adding 1.5% calcium chloride (w/v) and 3%o pancreatin powder (w/v) into the collected supernatant, adjusting pH value to 7.5 by using sodium bicarbonate, maintaining the temperature at 25 ℃ and fully stirring for 110 minutes, and activating enzyme solution.
S5: ultrafiltering with ultrafiltration tube with cut-off molecular weight of 10KD (30 min, 4deg.C), and concentrating the dialysate to appropriate volume to obtain chymotrypsin crude enzyme solution.
S6: fine extracting, slowly adding 10ml of 0.005mol/L polyvinyl sodium sulfonate into the chymotrypsin crude enzyme solution, maintaining the pH value to be 7.5, fully and uniformly stirring, standing overnight, centrifuging, removing the supernatant, and collecting insoluble polymer; 100ml of an acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L is added into the insoluble polymer, 10ml of a 4.0E-06mol/L polyether imine solution is added after the mixture is slowly and evenly stirred, the solution is fully dissociated, frozen and centrifuged (12000 r/min,15 min), filtered, and the precipitate is collected.
S7: purifying, adding 100ml of acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L and 0.5mol/LnaCl into the precipitate, loading the mixture into a column, selecting a phenyl-agarose gel chromatographic column (1.6 cm x 15cm,30 ml) for loading and adsorbing, eluting with 0.5mol/L and 0.01mol/L of acetic acid buffer solution with the pH value of 5.0, controlling the flow rate to be 1ml/min, collecting eluent containing chymotrypsin activity peaks, and preserving at the temperature of minus 20 ℃.
S8: drying and crushing, freezing the collected eluent, drying in vacuum, crushing and sieving to obtain the chymotrypsin product.
Comparative example 3
This comparative example provides a method for preparing chymotrypsin, which is different from example 1 in that the concentration of sodium polyvinylsulfonate added in step S6 of this comparative example is 0.01mol/L, and other technical features are exactly the same as in example 1. The specific method comprises the following steps:
s1: pretreating raw materials, collecting pig pancreas 100g, removing fat connective tissue, adding 1L isotonic physiological saline (0.9% NaCl), washing, removing impurity dirt on pancreas surface, and flaking and mincing.
S2: leaching, adding 3mmol/L of 250mLTris-HCl into minced pancreas, and fully stirring and homogenizing at 5 ℃ for 1 hour to obtain chymotrypsin leaching solution.
S3: extracting, namely extracting the extract in a 700w power ultrasonic wave instrument at the temperature of 5 ℃ for 7 minutes, freezing and centrifuging (4000 r/min,10 minutes and 5 ℃) after full extraction, repeatedly dissolving and extracting the obtained precipitate with the extract and centrifuging twice, and collecting all supernatant.
S4: activating, adding 1.5% calcium chloride (w/v) and 3%o pancreatin powder (w/v) into the collected supernatant, adjusting pH value to 7.5 by using sodium bicarbonate, maintaining the temperature at 25 ℃ and fully stirring for 110 minutes, and activating enzyme solution.
S5: ultrafiltering with ultrafiltration tube with cut-off molecular weight of 10KD (30 min, 4deg.C), and concentrating the dialysate to appropriate volume to obtain chymotrypsin crude enzyme solution.
S6: fine extracting, slowly adding 10ml of 0.01mol/L polyvinyl sodium sulfonate into the chymotrypsin crude enzyme liquid, maintaining the pH value to be 7.5, fully and uniformly stirring, standing overnight, centrifuging, removing the supernatant, and collecting insoluble polymer; 100ml of an acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L is added into the insoluble polymer, 10ml of a 4.0E-06mol/L polyether imine solution is added after the mixture is slowly and evenly stirred, the solution is fully dissociated, frozen and centrifuged (12000 r/min,15 min), filtered, and the precipitate is collected.
S7: purifying, adding 100ml of acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L and 0.5mol/LnaCl into the precipitate, loading the mixture into a column, selecting a phenyl-agarose gel chromatographic column (1.6 cm x 15cm,30 ml) for loading and adsorbing, eluting with 0.5mol/L and 0.01mol/L of acetic acid buffer solution with the pH value of 5.0, controlling the flow rate to be 1ml/min, collecting eluent containing chymotrypsin activity peaks, and preserving at the temperature of minus 20 ℃.
S8: drying and crushing, freezing the collected eluent, drying in vacuum, crushing and sieving to obtain the chymotrypsin product.
Example 5
Based on example 1, this example provides a method for preparing chymotrypsin, which differs from example 1 in that the concentration of the polyetherimide solution added in this example is 4.5E-06mol/L, and other technical features are exactly the same as in example 1. The specific method comprises the following steps:
s1: pretreating raw materials, collecting pig pancreas 100g, removing fat connective tissue, adding 1L isotonic physiological saline (0.9% NaCl), washing, removing impurity dirt on pancreas surface, and flaking and mincing.
S2: leaching, adding 3mmol/L of 250mLTris-HCl into minced pancreas, and fully stirring and homogenizing at 5 ℃ for 1 hour to obtain chymotrypsin leaching solution.
S3: extracting, namely extracting the extract in a 700w power ultrasonic wave instrument at the temperature of 5 ℃ for 7 minutes, freezing and centrifuging (4000 r/min,10 minutes and 5 ℃) after full extraction, repeatedly dissolving and extracting the obtained precipitate with the extract and centrifuging twice, and collecting all supernatant.
S4: activating, adding 1.5% calcium chloride (w/v) and 3%o pancreatin powder (w/v) into the collected supernatant, adjusting pH value to 7.5 by using sodium bicarbonate, maintaining the temperature at 25 ℃ and fully stirring for 110 minutes, and activating enzyme solution.
S5: ultrafiltering with ultrafiltration tube with cut-off molecular weight of 10KD (30 min, 4deg.C), and concentrating the dialysate to appropriate volume to obtain chymotrypsin crude enzyme solution.
S6: fine extraction, namely slowly adding 10ml of 0.015mol/L polyvinyl sodium sulfonate into the chymotrypsin crude enzyme solution, maintaining the pH value to be 7.5, fully and uniformly stirring, standing overnight, centrifuging, removing the supernatant, and collecting insoluble polymer; 100ml of an acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L is added into the insoluble polymer, 10ml of a 4.0E-06mol/L polyether imine solution is added after the mixture is slowly and evenly stirred, the solution is fully dissociated, frozen and centrifuged (12000 r/min,15 min), filtered, and the precipitate is collected.
S7: purifying, adding 100ml of acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L and 0.5mol/LnaCl into the precipitate, loading the mixture into a column, selecting a phenyl-agarose gel chromatographic column (1.6 cm x 15cm,30 ml) for loading and adsorbing, eluting with 0.5mol/L and 0.01mol/L of acetic acid buffer solution with the pH value of 5.0, controlling the flow rate to be 1ml/min, collecting eluent containing chymotrypsin activity peaks, and preserving at the temperature of minus 20 ℃.
S8: drying and crushing, freezing the collected eluent, drying in vacuum, crushing and sieving to obtain the chymotrypsin product.
Comparative example 4
This comparative example provides a method for preparing chymotrypsin, and the concentration of the polyetherimide solution added in this comparative example is 2.5E-06mol/L compared with example 1, and other technical characteristics are exactly the same as example 1. The specific method comprises the following steps:
s1: pretreating raw materials, collecting pig pancreas 100g, removing fat connective tissue, adding 1L isotonic physiological saline (0.9% NaCl), washing, removing impurity dirt on pancreas surface, and flaking and mincing.
S2: leaching, adding 3mmol/L of 250mLTris-HCl into minced pancreas, and fully stirring and homogenizing at 5 ℃ for 1 hour to obtain chymotrypsin leaching solution.
S3: extracting, namely extracting the extract in a 700w power ultrasonic wave instrument at the temperature of 5 ℃ for 7 minutes, freezing and centrifuging (4000 r/min,10 minutes and 5 ℃) after full extraction, repeatedly dissolving and extracting the obtained precipitate with the extract and centrifuging twice, and collecting all supernatant.
S4: activating, adding 1.5% calcium chloride (w/v) and 3%o pancreatin powder (w/v) into the collected supernatant, adjusting pH value to 7.5 by using sodium bicarbonate, maintaining the temperature at 25 ℃ and fully stirring for 110 minutes, and activating enzyme solution.
S5: ultrafiltering with ultrafiltration tube with cut-off molecular weight of 10KD (30 min, 4deg.C), and concentrating the dialysate to appropriate volume to obtain chymotrypsin crude enzyme solution.
S6: fine extraction, namely slowly adding 10ml of 0.015mol/L polyvinyl sodium sulfonate into the chymotrypsin crude enzyme solution, maintaining the pH value to be 7.5, fully and uniformly stirring, standing overnight, centrifuging, removing the supernatant, and collecting insoluble polymer; 100ml of an acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L is added into the insoluble polymer, 10ml of a 2.5E-06mol/L polyether imine solution is added after the mixture is slowly and evenly stirred, the solution is fully dissociated, frozen and centrifuged (12000 r/min,15 min), filtered, and the precipitate is collected.
S7: purifying, adding 100ml of acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L and 0.5mol/LnaCl into the precipitate, loading the mixture into a column, selecting a phenyl-agarose gel chromatographic column (1.6 cm x 15cm,30 ml) for loading and adsorbing, eluting with 0.5mol/L and 0.01mol/L of acetic acid buffer solution with the pH value of 5.0, controlling the flow rate to be 1ml/min, collecting eluent containing chymotrypsin activity peaks, and preserving at the temperature of minus 20 ℃.
S8: drying and crushing, freezing the collected eluent, drying in vacuum, crushing and sieving to obtain the chymotrypsin product.
Comparative example 5
This comparative example provides a method for preparing chymotrypsin, and the concentration of the polyetherimide solution added in this comparative example is 6.0E-06mol/L compared with example 1, and other technical characteristics are exactly the same as example 1. The specific method comprises the following steps:
s1: pretreating raw materials, collecting pig pancreas 100g, removing fat connective tissue, adding 1L isotonic physiological saline (0.9% NaCl), washing, removing impurity dirt on pancreas surface, and flaking and mincing.
S2: leaching, adding 3mmol/L of 250mLTris-HCl into minced pancreas, and fully stirring and homogenizing at 5 ℃ for 1 hour to obtain chymotrypsin leaching solution.
S3: extracting, namely extracting the extract in a 700w power ultrasonic wave instrument at the temperature of 5 ℃ for 7 minutes, freezing and centrifuging (4000 r/min,10 minutes and 5 ℃) after full extraction, repeatedly dissolving and extracting the obtained precipitate with the extract and centrifuging twice, and collecting all supernatant.
S4: activating, adding 1.5% calcium chloride (w/v) and 3%o pancreatin powder (w/v) into the collected supernatant, adjusting pH value to 7.5 by using sodium bicarbonate, maintaining the temperature at 25 ℃ and fully stirring for 110 minutes, and activating enzyme solution.
S5: ultrafiltering with ultrafiltration tube with cut-off molecular weight of 10KD (30 min, 4deg.C), and concentrating the dialysate to appropriate volume to obtain chymotrypsin crude enzyme solution.
S6: fine extraction, namely slowly adding 10ml of 0.015mol/L polyvinyl sodium sulfonate into the chymotrypsin crude enzyme solution, maintaining the pH value to be 7.5, fully and uniformly stirring, standing overnight, centrifuging, removing the supernatant, and collecting insoluble polymer; 100ml of acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L is added into insoluble polymer, 10ml of 6.0E-06mol/L polyether imine solution is added after being slowly and evenly stirred, the solution is fully dissociated, frozen and centrifuged (12000 r/min,15 min), filtered, and sediment is collected.
S7: purifying, adding 100ml of acetic acid buffer solution with the pH value of 5.0 and 0.01mol/L and 0.5mol/LnaCl into the precipitate, loading the mixture into a column, selecting a phenyl-agarose gel chromatographic column (1.6 cm x 15cm,30 ml) for loading and adsorbing, eluting with 0.5mol/L and 0.01mol/L of acetic acid buffer solution with the pH value of 5.0, controlling the flow rate to be 1ml/min, collecting eluent containing chymotrypsin activity peaks, and preserving at the temperature of minus 20 ℃.
S8: drying and crushing, freezing the collected eluent, drying in vacuum, crushing and sieving to obtain the chymotrypsin product.
The activity of chymotrypsin prepared by the preparation methods of examples 1 to 5 and comparative examples 1 to 5 was examined, and the results of the examination are shown in fig. 1 and 2.
As is clear from FIG. 1, the chymotrypsin activity gradually increases and then decreases when the concentration of the sodium polyethylene sulfate increases, and the chymotrypsin activity is highest when the concentration is 0.015 to 0.025 mol/L.
As is clear from FIG. 2, since the concentration of the polyetherimide solution increases gradually and decreases as the concentration increases to the maximum, the concentration of the polyetherimide solution is selected to be 3.8E-06mol/L to 4.5E-06mol/L, and the chymotrypsin activity is the highest.
Chymotrypsin potency assay
1. Preparation of substrate solution N-acetyl-L-tyrosine ethyl ester 23.7mg is put into a 100ml measuring flask, phosphate buffer solution (38.9 ml of 0.067mol/L potassium dihydrogen phosphate solution and 61.1ml of 0.067mol/L disodium hydrogen phosphate solution are mixed, pH value is 7.0) is added, the mixture is warmed to dissolve, cooled and diluted to scale, and shaking is carried out uniformly. Frozen for preservation, but should not be frozen and thawed repeatedly.
2. Preparation of sample solution A proper amount of the sample is precisely weighed, and 0.0012mol/L hydrochloric acid solution is added for dissolution and quantitative dilution to prepare a solution containing 12-16 chymotrypsin units in each 1 ml.
3. Measurement method 0.0012mol/L hydrochloric acid solution 0.2ml and substrate solution 3.0ml were taken, and absorbance was measured at 25 ℃ + -0.5 ℃ and adjusted to 0.200 at 237nm wavelength according to ultraviolet-visible spectrophotometry (appendix IVA of second edition pharmacopoeia 2010). And then 0.2ml of the sample solution and 3.0ml of the substrate solution are taken, immediately timed and uniformly shaken, the absorbance is read every 30 seconds for 5 minutes (repeated), the change rate of the absorbance is constant, and the constant time is not less than 3 minutes. If the rate of change cannot be kept constant, it can be measured separately with a lower concentration. The absorbance change rate per 30 seconds should be controlled to 0.008 to 0.012, plotted on the ordinate of absorbance and the abscissa of time, and the absorbance at the straight line portion within 3 minutes is taken, and calculated as follows.
Wherein P is the potency per 1mg chymotrypsin, unit; a2 is absorbance starting on a straight line; a1 is absorbance terminated on a straight line; t is the time of A2 to A1 readings, minutes; w is the amount of the test sample contained in the measurement solution and mg;0.0075 is the absorbance change of 0.0075 per minute under the above conditions, i.e., equivalent to 1 chymotrypsin unit. The results of the detection are shown in the following table.
| Scheme for the production of a semiconductor device | Chymotrypsin potency (u/mg) | Extraction yield (%) |
| Example 1 | 1551 | 75.1 |
| Example 2 | 1659 | 73.4 |
| Example 3 | 1598 | 70.4 |
| Example 4 | 1480 | 70.2 |
| Example 5 | 1579 | 70.3 |
| Comparative example 1 | 1086 | 54.8 |
| Comparative example 2 | 1124 | 58.2 |
| Comparative example 3 | 235 | 9.8 |
| Comparative example 4 | 768 | 42.8 |
| Comparative example 5 | 926 | 47.5 |
From the test data in the above table, the chymotrypsin titer and the extraction rate in the examples are higher than those in the comparative examples, which indicates that the chymotrypsin prepared by the preparation method of the present application has significantly improved titer and extraction rate.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (10)
1. A method for preparing chymotrypsin, comprising the steps of:
step 1: pretreating raw materials, washing pancreas with isotonic physiological saline, and mincing;
step 2: leaching, namely adding tris (hydroxymethyl) aminomethane hydrochloride into minced pancreas, and stirring and homogenizing to obtain a chymotrypsin leaching solution;
step 3: extracting, freezing and centrifuging the extracted leaching solution, repeatedly dissolving, extracting and centrifuging the obtained precipitate by using the leaching solution, and collecting all supernatant;
step 4: activating, namely adding calcium chloride and pancreatin powder into the collected supernatant, adjusting the pH value by using sodium bicarbonate, and fully stirring to obtain an activated enzyme solution;
step 5: ultrafiltering, ultrafiltering with ultrafilter tube, concentrating the dialysate to obtain chymotrypsin crude enzyme solution;
step 6: fine extracting, namely adding sodium polyvinylsulfonate into the chymotrypsin crude enzyme liquid, stirring, centrifuging, and collecting insoluble polymers; adding acetic acid buffer solution into insoluble polymer, stirring, adding polyetherimide solution, fully dissociating, freeze centrifuging, filtering, and collecting precipitate;
step 7: purifying, adding acetic acid buffer solution into the precipitate, loading and adsorbing by using a chromatographic column, eluting, and collecting eluent containing chymotrypsin activity peaks;
step 8: drying and crushing, freezing the collected eluent, drying in vacuum, crushing and sieving to obtain chymotrypsin.
2. The method for producing chymotrypsin according to claim 1, wherein the concentration of the tris hydrochloride added in the step 2 is 2.8mmol/L to 4.2mmol/L.
3. The method for preparing chymotrypsin according to claim 1, wherein the extraction temperature in the step 3 is controlled to be 4-5 ℃.
4. The method for preparing chymotrypsin according to claim 1, wherein the amount of calcium chloride added in the step 4 is 1% -1.5%, and the amount of pancreatin powder added is 3-5%.
5. The method for preparing chymotrypsin according to claim 1, wherein sodium bicarbonate is added in the step 4 to adjust the pH to 7.0-8.0, and the mixture is fully stirred at 20-30 ℃.
6. The method for preparing chymotrypsin according to claim 1, wherein the concentration of the sodium polyvinylsulfonate added in the step 6 is 0.015mol/L to 0.02mol/L.
7. The method for preparing chymotrypsin according to claim 1, wherein the concentration of the acetic acid buffer solution added in the step 6 is 0.01mol/L.
8. The method for producing chymotrypsin according to claim 1, wherein the concentration of the polyetherimide solution added in the step 6 is 4.0E-06mol/L to 4.5E-06mol/L.
9. The method according to claim 1, wherein a built-in phenyl-sepharose column is used in step 7.
10. A pharmaceutical composition comprising, as an active ingredient, chymotrypsin prepared by the preparation method according to any one of claims 1 to 9.
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2023
- 2023-12-18 CN CN202311741836.2A patent/CN117721099A/en active Pending
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