CN109929022A - A kind of preparation method of catfish protamine sulfate - Google Patents
A kind of preparation method of catfish protamine sulfate Download PDFInfo
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- CN109929022A CN109929022A CN201711358481.3A CN201711358481A CN109929022A CN 109929022 A CN109929022 A CN 109929022A CN 201711358481 A CN201711358481 A CN 201711358481A CN 109929022 A CN109929022 A CN 109929022A
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- protamine sulfate
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- sulfuric acid
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Abstract
The invention discloses a kind of preparation method of catfish protamine sulfate, this method passes through following processing step: pretreatment, sulfuric acid solution extraction, low temperature precipitation, gel permeation chromatography, dehydration, freeze-drying, the extraction purification protamine sulfate from catfish.The present invention continuously improves the production technology of protamine sulfate, especially carries out experimental study repeatedly to each technological parameter, continues to optimize, finally establish a set of more scientific scale production process.Preparation method of the present invention, product quality is high, and the heparin anticoagulant effect that the resulting every mg of protamine sulfate is neutralized is up to 150 units, and content is up to 95% or more, 95% or more chromatographic purity, other indices meet Chinese Pharmacopoeia, United States Pharmacopeia, European Pharmacopoeia standard.
Description
Technical field
The present invention relates to field of biotechnology, relate in particular to a kind of preparation method of catfish protamine sulfate.
Background technique
Nucleoprotamine is a kind of polycation peptide, is primarily present in the spermary tissue of birds, fish and mammal.
It is a kind of alkaline protein, is combined together with DNA, is existed in the form of nucleprotamine.The molecular weight of nucleoprotamine compared with
Small, molecular weight is made of mainly 5.5 between 13.0kDa 30-50 amino acid.Nucleoprotamine has good stability,
Heating is not easy to solidify, and isoelectric point is between 12-13.At present successfully from various fishs, fresh-water fishes and mammal at
Nucleoprotamine is extracted in ripe spermary tissue.
Substitution since McClean in 1937 etc. reports nucleoprotamine with bacteriostatic activity, as chemical preservative
Product occur gradually over food additives ranks.Nowadays, nucleoprotamine is more and more widely used in the food industry.With
The advantages that chemical synthesis preservative is compared, and nucleoprotamine is with high security, antiseptic property is good, thermal stability is high, and milt
Albumen also has very high trophism and functionality, and energy blood pressure lowering helps breathing, promotees digestion, inhibiting cancer, antithrombotic, strengthens liver function
Can, inhibit blood clotting etc..In medical domain, nucleoprotamine is also widely applied, it can postpone or prevent insulin
Release, or as heparin antidote etc..
The extraction of nucleoprotamine at present, from solvent using upper, substantially with sulfuric acid or hydrochloric acid for main extractant,
Purified using citrate or organic solvent as scarvenger, also have in sulfuric acid or hydrochloric acid Extract do not have to organic solvent and
It with Quadrafos, is precipitated out nucleoprotamine with phos-phate forms, is then dissolved in the sulfuric acid of high concentration in, subdivision
Solution is Protamine.In addition, also can be used organic solvent (acetone, ethyl alcohol, chloroform etc.) be dehydrated repeatedly, degreasing,
Extracting, washing nucleoprotamine, are aided with the reagents such as sodium sulphate, ammonium hydroxide, hydrogen peroxide, EDTA disodium salt therebetween.
But the protamine sulfate foreign protein containing non-functional that the above method extracts is more, and pharmacopoeia of each country is to sulfuric acid milt egg
It all has higher requirements in terms of white purity, especially United States Pharmacopeia is molten in the content of protamine sulfate, biological value, residual
Agent, chromatographic purity, element impurity, iron content, methyl mercury content etc. are all made that requirement.Therefore, how to nucleoprotamine
It is purified, becomes urgent problem to be solved in current nucleoprotamine purifying.
Summary of the invention
The method that the present invention provides a kind of to extract protamine sulfate from catfish, mainly solves milt in the prior art
The low problem of purity of protein.
In order to achieve the above-mentioned object of the invention, the invention discloses a kind of purifying process of protamine sulfate, including it is following
Step:
(1) it pre-processes: the catfish fish sperm of freezing being taken to thaw, be (1~5) in the mass ratio of the fish sperm and purified water: 1 ratio adds
Enter cold water and be placed in tissue mashing machine to smash to pieces, obtains slurries with four layers of filtered through gauze；
(2) sulfuric acid solution: being added 0.3%~1.0% sulfuric acid solution in Xiang Shangshu slurries, slurries and volume ratio are 1:(5~15),
Acidolysis 1 ~ 3h, 5100 х g centrifugation 20 ~ 30 minutes；Supernatant is collected, adjusts pH to 8 ~ 10,5100 х with 30% ~ 40%NaOH solution
G is centrifuged 20 ~ 30 minutes；
(3) low temperature be precipitated: collect supernatant, 0 ~ 4 DEG C, stand 12 ~ 16h, incline supernatant liquor, to lower sediment thing by 1:(3 ~
10) 50 ~ 70 DEG C of water dissolution is added in the ratio of (w/v)；
(4) gel permeation chromatography: being splined on Sephadex G-25 gel column for above-mentioned solution, contains 0.01 with pH 7.0~9.0
The Tris-HCl buffer of~0.20mol/L KCl balances, and Ultraviolet Detector is detected and recorded, and elutes enzymatic activity peak；
(5) be dehydrated: solid dehydrated alcohol is dehydrated twice, and centrifugation discards supernatant liquid, leaves and takes sediment；
(6) it is lyophilized: above-mentioned sediment being packed into disk into freeze dryer, is lyophilized up to protamine sulfate
Preparation method of the present invention, product purity is high, and the heparin anticoagulant that every mg protamine sulfate is neutralized acts on high
Up to 150 units, content is up to 95% or more, and 95% or more chromatographic purity, other indices meet Chinese Pharmacopoeia, U.S.'s medicine
Allusion quotation, European Pharmacopoeia standard meet the requirement to high-purity sulfuric acid nucleoprotamine, improve economic benefit.
Specific embodiment
The present invention is further explained in the light of specific embodiments, without limiting in any way.
Embodiment 1
(1) it pre-processes: the catfish fish sperm 2kg of freezing being taken to thaw, addition 1kg cold water, which is placed in tissue mashing machine, to be smashed to pieces, with four
Layer filtered through gauze obtains slurries；
(2) sulfuric acid solution: being added 0.5% sulfuric acid solution in Xiang Shangshu slurries, slurries and volume ratio are 1:7, acidolysis 2h,
5100 х g are centrifuged 20 minutes；Supernatant is collected, adjusts pH to 8 with 30%NaOH solution, 5100 х g are centrifuged 20 minutes；
(3) low temperature is precipitated: collecting supernatant, 0 DEG C, stands 12h, incline supernatant liquor, presses 1:5(w/v to lower sediment thing)
60 DEG C of water dissolution is added in ratio；
(4) gel permeation chromatography: being splined on Sephadex G-25 gel column for above-mentioned solution, contains 0.05mol/ with pH 8.0
The Tris-HCl buffer of L KCl balances, and Ultraviolet Detector is detected and recorded, and elutes enzymatic activity peak；
(5) be dehydrated: solid dehydrated alcohol is dehydrated twice, and centrifugation discards supernatant liquid, leaves and takes sediment；
(6) it is lyophilized: above-mentioned sediment being packed into disk into freeze dryer, is lyophilized up to protamine sulfate.
The heparin anticoagulant effect that the every mg of obtained protamine sulfate is neutralized is 156 units, content 96.4%, color
Spectral purity is 95.7%.
Embodiment 2
(1) it pre-processes: the catfish fish sperm 2kg of freezing being taken to thaw, addition 0.5kg cold water, which is placed in tissue mashing machine, to be smashed to pieces, is used
Four layers of filtered through gauze obtain slurries；
(2) sulfuric acid solution: being added 0.9% sulfuric acid solution in Xiang Shangshu slurries, slurries and volume ratio are 1:5, acidolysis 3h, and 5100
х g is centrifuged 30 minutes；Supernatant is collected, adjusts pH to 8 with 35%NaOH solution, 5100 х g are centrifuged 30 minutes；
(3) low temperature is precipitated: collecting supernatant, 2 DEG C, stands 16h, incline supernatant liquor, presses 1:7(w/v to lower sediment thing)
65 DEG C of water dissolution is added in ratio；
(4) gel permeation chromatography: being splined on Sephadex G-25 gel column for above-mentioned solution, contains 0.10mol/ with pH 8.3
The Tris-HCl buffer of L KCl balances, and Ultraviolet Detector is detected and recorded, and elutes enzymatic activity peak；
(5) be dehydrated: solid dehydrated alcohol is dehydrated twice, and centrifugation discards supernatant liquid, leaves and takes sediment；
(6) it is lyophilized: above-mentioned sediment being packed into disk into freeze dryer, is lyophilized up to protamine sulfate.
The heparin anticoagulant effect that the every mg of obtained protamine sulfate is neutralized is 164 units, content 95.6%, color
Spectral purity is 95.5%.
Embodiment 3
(1) it pre-processes: the catfish fish sperm 2kg of freezing being taken to thaw, addition 0.5kg cold water, which is placed in tissue mashing machine, to be smashed to pieces, is used
Four layers of filtered through gauze obtain slurries；
(2) sulfuric acid solution: being added 0.4% sulfuric acid solution in Xiang Shangshu slurries, slurries and volume ratio are 1:12, acidolysis 2.5h,
5100 х g are centrifuged 25 minutes；Supernatant is collected, adjusts pH to 9.5 with 50%NaOH solution, 5100 х g are centrifuged 25 minutes；
(3) low temperature is precipitated: collecting supernatant, 4 DEG C, stands 16h, incline supernatant liquor, presses 1:10(w/v to lower sediment thing)
55 DEG C of water dissolution is added in ratio；
(4) gel permeation chromatography: being splined on Sephadex G-25 gel column for above-mentioned solution, contains 0.15mol/L with pH 9.5
The Tris-HCl buffer of KCl balances, and Ultraviolet Detector is detected and recorded, and elutes enzymatic activity peak；
(5) be dehydrated: solid dehydrated alcohol is dehydrated twice, and centrifugation discards supernatant liquid, leaves and takes sediment；
(6) it is lyophilized: above-mentioned sediment being packed into disk into freeze dryer, is lyophilized up to protamine sulfate.
The heparin anticoagulant effect that the every mg of obtained protamine sulfate is neutralized is 152 units, content 96.8%, color
Spectral purity is 97.2%
The above described is only a preferred embodiment of the present invention, be not that the invention has other forms of limitations, it is any ripe
Know the equivalent reality that professional and technical personnel was changed or be modified as equivalent variations possibly also with the technology contents of the disclosure above
Apply example.But without departing from the technical solutions of the present invention, to the above embodiments according to the technical essence of the invention
Any simple modification, equivalent variations and remodeling, still fall within the protection scope of technical solution of the present invention.
Claims (5)
1. a kind of preparation method of catfish protamine sulfate, which comprises the steps of:
Pretreatment: taking the catfish fish sperm of freezing to thaw, and addition cold water, which is placed in tissue mashing machine, to be smashed to pieces, with four layers of filtered through gauze
Obtain slurries；
Sulfuric acid solution: sulfuric acid solution, acidolysis, 5100 х g centrifugation are added in Xiang Shangshu slurries；Supernatant is collected, NaOH solution is used
Adjust pH, 5100 х g centrifugation；
Low temperature is precipitated: collecting supernatant, stands, incline supernatant liquor, is dissolved in water into lower sediment thing；
Gel permeation chromatography: being splined on Sephadex G-25 gel column for above-mentioned concentrate, slow with the Tris-HCl containing KCl
Fliud flushing balance, Ultraviolet Detector are detected and are recorded, and elute enzymatic activity peak；
Dehydration: solid dehydrated alcohol is dehydrated twice, and centrifugation discards supernatant liquid, leaves and takes sediment；
Freeze-drying: the sediment of step (5) is packed into disk into freeze dryer, is lyophilized up to protamine sulfate.
2. the method as described in claim 1, which is characterized in that in step (1), the water is purified water, the fish sperm
Mass ratio with the water is (1~5): 1.
3. the method as described in claim 1, which is characterized in that in step (2), the sulfuric acid solution concentration be 0.3%~
1.0%, the volume ratio of the slurries and the sulfuric acid solution is 1:(5~15), the acidolysis time is 1-3h；The hydrogen-oxygen
Change sodium solution concentration is 30%-40%, and the pH is 8 ~ 10, and the centrifugation time is 20-30 minutes.
4. the method as described in claim 1, which is characterized in that in step (3), the dwell temperature is 0-4 DEG C, when standing
Between be 12-16h;The water is purified water, and the coolant-temperature gage is 50-70 DEG C, and the ratio of the sediment and water is 1:
(3-10) (w/v).
5. the method as described in claim 1, which is characterized in that equilibrium liquid described in step (4) is that pH 7.0~9.0 contains
The Tris-HCl buffer of 0.01~0.20mol/L KCl.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116179638A (en) * | 2023-04-28 | 2023-05-30 | 瑞吉明(山东)生物科技有限公司 | Preparation method of protamine polypeptide and product |
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2017
- 2017-12-17 CN CN201711358481.3A patent/CN109929022A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116179638A (en) * | 2023-04-28 | 2023-05-30 | 瑞吉明(山东)生物科技有限公司 | Preparation method of protamine polypeptide and product |
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