CN100351373C - Process for preparing artemia hatching enzyme by using artemia hatching liquid - Google Patents
Process for preparing artemia hatching enzyme by using artemia hatching liquid Download PDFInfo
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- CN100351373C CN100351373C CNB2005100434641A CN200510043464A CN100351373C CN 100351373 C CN100351373 C CN 100351373C CN B2005100434641 A CNB2005100434641 A CN B2005100434641A CN 200510043464 A CN200510043464 A CN 200510043464A CN 100351373 C CN100351373 C CN 100351373C
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The present invention relates to a process for preparing artemia hatching enzymes by artemia hatching liquid. The process comprises the steps: artemia ova are firstly cultured in artificial sea water, and hatching liquid, namely filtrate is collected by a filter screen when more than 90% of artemias are hatched; then, ammonium sulfate is added to the hatching liquid to be stirred until the saturation of the ammonium ulfate is between 50 and 70%; the mixture is then placed into a refrigerator with 4 DEG C to precipitate over night for centrifugal precipitation; buffer solutions completely dissolve the precipitates, and distilled water is dialyzed and desalted in a dialysis bag under 4 DEG C; the dialysate is frozen and dried until a sample reach a constant weight, so that the coarse products of the artemia hatching enzymes are obtained; the coarse products are purified through anion exchange column chromatography and gel filtration column chromatography to obtain the pure products of the artemia hatching enzymes. By the adoption of a freezing and drying technology, the present invention has the advantages of simple and reasonable technique, full unitization for waste artemia hatching liquid as raw materials, good solubility for the made-up artemia hatching enzymes, low cost and long-term storage, and can be applied to improving the hatchability of artemia strains with low hatchability and lowering the breeding cost of aquaculture industry in sea water.
Description
Technical field
The invention belongs to the marine biotechnology field, relate to a kind of method of producing halogen worm hatching enzyme with halogen worm artemia hatching solution.
Background technology
It is very important animality living bait during aquatic product sprout is produced that the halogen worm just incubates nauplius larva, contain higher protein (50%～60%), a certain amount of fat and higher unsaturated fatty acid, minerals and vitamins etc. in the artemia imago body, be the agreeable to the taste bait of many aquatic animal seed post-larvas and Shui nationality pet, the halogen worm post-larva of throwing something and feeding is very favourable to the body colour that improves the crab seedling rearing surviving rate, improves Shui nationality pet.At present, in the world 85% marine products economic animal with the halogen worm as fresh and alive open-mouthed bait.According to the comprehensive test of relevant expert to 14 indexs such as growth characteristics, cultural method, market value and market potential of 24 kinds of main aquaculture kinds such as halogen worm, salmon, prawn, scallop, the feasibility assessment of halogen worm aquaculture is only second to salmon and occupies second.
But its application in aquatic fry growing and aquaculture also can meet the demands far from.This mainly is because adult quality instability, and the hatching rate of some halogen worm strain is too low and influenced its using value.In China and some countries of USSR (Union of Soviet Socialist Republics), the reserves of low hatchability artemia cysts (hatching rate of some strain halogen worm has only 30-50%) are considerable at present.If can with this utilization of resources on the aquatic fry growing of some kind, partly or entirely substitute expensive high quality artemia cysts, will alleviate the contradiction of artemia cysts anxiety to a certain extent, reduce seedling cost, avoid the waste of resource.But also there is not correlation technique can solve low this problem of halogen worm hatching rate at present.And halogen worm hatching enzyme will help to improve the hatching rate of halogen worm, solve the quality problem of low hatchability artemia cysts.In addition, the research of hatching enzyme is to the control hatching rate of animal and hatching process and then control its seedling and form rate and also have crucial guiding value.And then the control of hatching rate can be the protection of endangered species, the aquaculture of useful animals and the researchs such as quantity control of pest again a technological line that broad prospect of application is arranged is provided.
Summary of the invention
The present invention seeks to overcoming the deficiency of existing halogen worm hatching technique, a kind of method of producing halogen worm hatching enzyme with halogen worm artemia hatching solution is provided.
The method or the technology of extraction halogen worm hatching enzyme of the present invention: earlier artemia cysts is incubated in the seawater, treats that the halogen worm more than 90% is finished when hatching, collect filtrate-artemia hatching solution through filter screen; Then ammonium sulfate is added in it, and stir that to make the saturation ratio of ammonium sulfate be 50-70%; Put into 4 ℃ of refrigerator overnight precipitations again, and then centrifugation, and after will precipitating fully dissolving with damping fluid, under 4 ℃, in dialysis tubing, carry out dialysis desalting again with distilled water, above-mentioned dialyzate is carried out lyophilize, reach constant weight to sample, pack after dry powder is taken out promptly obtain halogen worm hatching enzyme crude product.
On this basis, for halogen worm hatching enzyme is carried out more deep research, the present invention has done to be further purified to halogen worm hatching enzyme crude product: be splined on anion exchange chromatography after above-mentioned halogen worm hatching enzyme crude product is dissolved with damping fluid, carry out gradient elution, the sample that wash-out goes out is received the sample device automatically with sample and is collected, and chooses the highest elution samples of enzymic activity value as the anion exchange chromatography sample; Again above-mentioned anion exchange chromatography sample is concentrated with concentrated centrifuge tube, be further purified through gel-filtration column.Choose the highest elution samples of enzymic activity value and be the pure product of halogen worm hatching enzyme.For the pure product of prolonged preservation halogen worm hatching enzyme, the pure product of halogen worm hatching enzyme of above-mentioned solution state are carried out lyophilize, taking-up is packed promptly obtain the pure product of halogen worm hatching enzyme.
The present invention is to be the extraction and purification that material carries out hatching enzyme with high hatchability strain halogen worm, and its method and detailed step are as follows:
1. the collection of the cultivation of artemia cysts and artemia hatching solution:
For the flow process that simplifies the operation, artemia cysts is incubated in the nature seawater usually in the batch production running.Consider and contain a certain amount of calcium ions and magnesium ions in the natural sea-water, easily with follow-up ammonium sulfate generation insoluble precipitation, so adopt artificial seawater to cultivate artemia cysts in the present invention.The prescription of artificial seawater is dissolved in the distilled water of 1000ml for 30g sodium-chlor.The seawater of manually preparation is at room temperature added artemia cysts with after aerator pump inflation half an hour, under 31 ℃, the condition of illumination, cultivate artemia cysts.The embryo who is cultured to more than 90% finishes when hatching (cultivating 24h approximately), with filter screen artificial seawater is filtered, and the filtrate that obtains is artemia hatching solution.
2. the preparation of halogen worm hatching enzyme crude product:
To add solid ammonium sulfate in the above-mentioned artemia hatching solution, the saturation ratio that makes ammonium sulfate is 50-70%, and stirred solution raises with the concentration of avoiding local ammonium sulfate in the solution and causes protein denaturation when adding ammonium sulfate.Then, this liquid is placed on 4 ℃ of refrigerator overnight precipitations, uses refrigerated centrifuge again with the centrifugal 30min of 5000g (4 ℃).Supernatant liquor in the centrifuge tube is inclined to falling gently, and (20mmol/L Tris-HCl pH7.5) fully after the dissolving, dialyses 12h with desalination with distilled water to the precipitation of centrifuge tube bottom in dialysis tubing under 4 ℃ with damping fluid; The dialyzate that obtains is at first used the freeze drier lyophilize at-20 ℃ after freezing again, and freezing conditions is: vacuum tightness reaches 80Pa, carries out freeze-drying, reaches constant weight to sample.Be halogen worm hatching enzyme crude product with packing after the dry powder taking-up.Halogen worm hatching enzyme crude product promptly can be applied to aquaculture to improve the hatching rate of low hatchability strain halogen worm.
But halogen worm hatching enzyme crude product can not satisfy the requirement of amino acid sequence analysis research contents.Therefore, the present invention has carried out further purifying to the crude product of halogen worm hatching enzyme, so that further provide the basis for the further investigation of halogen worm hatching enzyme.Following steps have been carried out purifying on the basis of thick enzyme.
3. anion exchange chromatography:
Halogen worm hatching enzyme crude product is splined on anion exchange chromatography with after the damping fluid dissolving, respectively with contain 0,0.1,0.2,0.3,0.4, the damping fluid (pH7.5) of the 20mmol/L Tris-HCl of 0.5mol/L NaCl carries out gradient elution, the sample that wash-out goes out is received the sample device automatically with sample and is collected, be determined at the enzymic activity of each elution samples at wavelength 280nm place with ultraviolet spectrophotometer, the size of the photoabsorption that the size of this enzymic activity causes with the enzymatic lysis casein (being called for short 0D280) is represented.The concrete operations step is: get 20 μ l casein solution (5mg/ml) in the 1ml centrifuge tube, add 20 μ l elution samples, behind the reaction 30min, add 500 μ l trichoroacetic acid(TCA)s with termination reaction in 35 ℃ of water-baths.4 ℃ leave standstill 30min after, with refrigerated centrifuge in 4 ℃, the centrifugal 30min of 5000g.The sucking-off supernatant liquor is measured the 0D280 value of each elution samples with ultraviolet spectrophotometer, and this is worth the experimental value of elution samples for this reason; Control group only substitutes each above-mentioned elution samples with the above-mentioned damping fluid of 50 μ l, measure the 0D280 value of control group again, and this value is control value.The enzymic activity of each elution samples is represented with " experimental value-control value "; Order with elution samples is an X-coordinate, is that ordinate zou is drawn the enzymic activity curve with the enzymic activity value of each elution samples, chooses the enzymic activity value is the highest on the enzymic activity curve elution samples as the anion exchange chromatography sample.
4. gel filtration chromatography:
Again above-mentioned anion exchange chromatography sample is concentrated with concentrated centrifuge tube, be further purified through gel-filtration column.Behind spissated anion exchange chromatography sample upper prop, (pH7.5) carries out wash-out with the 20mmol/LTris-HCl damping fluid, the component that wash-out goes out is received the sample device automatically with sample and is collected, and is determined at the enzymic activity of each elution samples of 280nm place again with ultraviolet spectrophotometer.Concrete operations are identical with step described in the anion exchange chromatography.Choose that the highest elution samples of enzymic activity value is the pure product of halogen worm hatching enzyme on the enzymic activity curve.
For with the pure product prolonged preservation of halogen worm hatching enzyme, the pure product of halogen worm hatching enzyme of above-mentioned solution state are carried out lyophilize, the lyophilize condition: temperature drops to-54 ℃, vacuum tightness and reaches 80Pa, carry out lyophilize, reach constant weight to sample, take out the pure product of halogen worm hatching enzyme that are that pack.
Above-mentioned selected halogen worm strain requires the scope of its hatching rate at 70%-90%; The hatching rate of halogen worm strain is high more, and the yield of halogen worm hatching enzyme is high more.
In order to further specify above method, with the halogen worm strain (hatching rate is more than 90%) in Bohai Sea Gulf, the place of production as embodiment:
The seawater of manually preparation at room temperature with the artemia cysts that adds Bohai Sea Gulf, the 50g place of production after aerator pump inflation half an hour, is cultivated artemia cysts under 31 ℃, the condition of illumination.The embryo who is cultured to more than 90% finishes when hatching, and uses the strainer filtering artificial seawater, and the filtrate that obtains is artemia hatching solution;
Add solid ammonium sulfate in the above-mentioned artemia hatching solution, the whole saturation ratio that makes ammonium sulfate is 67%, then, this liquid is placed on 4 ℃ of refrigerator overnight precipitations, uses refrigerated centrifuge with the centrifugal 30min of 5000g (4 ℃), collecting precipitation again; With damping fluid (20mmol/L Tris-HCl, pH7.5) behind the complete dissolution precipitation, under 4 ℃, in dialysis tubing, dialyse 12h with desalination with distilled water, the dialyzate that obtains is at first used the freeze drier lyophilize at-20 ℃ after freezing again, freezing conditions is: vacuum tightness reaches 80Pa, carry out freeze-drying, reach constant weight to sample.Be halogen worm hatching enzyme crude product with packing after the dry powder taking-up.
Halogen worm hatching enzyme crude product is splined on the DEAE-sepharose Fast Flow (anion exchange chromatography of 1cm * 20cm) after with the dissolving of the 20mmol/L Tris-HCl damping fluid of pH7.5, with contain 0,0.1,0.2,0.3,0.4, the damping fluid (pH7.5) of the 20mmol/L of 0.5mol/L NaCl carries out gradient elution, elution speed 20ml/h, the collected volume of each elution samples is 2ml.Be determined at the enzymic activity of each elution samples of 280nm place with ultraviolet spectrophotometer.Draw the enzymic activity curve at last, choose the enzymic activity value is the highest on the enzymic activity curve elution samples as the anion exchange chromatography sample.
The ion exchange column sample is splined on Sephacryl gel-filtration column (1cm * 50cm) be further purified after concentrating; Carry out wash-out with 20mmol/L Tris-HCl damping fluid (pH7.5), flow velocity is 15ml/h, and the collected volume of each elution samples is 2ml.Be determined at the enzymic activity at 280nm place with ultraviolet spectrophotometer.Draw and to choose the enzymic activity value is the highest on the enzymic activity curve elution samples behind the enzymic activity curve as the gel filtration chromatography sample.The lyophilize of gel-filtration column sample, lyophilize condition: when vacuum tightness reaches 80Pa, carry out lyophilize, reach constant weight to sample, take out the pure product of halogen worm hatching enzyme that are that pack.
Because the present invention has adopted the higher halogen worm strain of hatching rate to prepare hatching enzyme, improved the output of enzyme, and the homology of hatching enzyme is very high between different strain halogen worm, the hatching enzyme for preparing from high hatchability halogen worm strain can be applied to low hatchability halogen worm strain fully.Cultivate the halogen worm with artificial seawater again in the method, the precipitation of having avoided heavy metal ion such as calcium magnesium in the natural sea-water and vitriol to produce, while has also been got rid of in the natural sea-water other not clear components to the active influence of hatching enzyme.Other proteinic kinds and content are all few in the artemia hatching solution of Huo Deing on this basis, have therefore simplified the leaching process of hatching enzyme.Present method has adopted freeze drying process again, makes whole extraction process simple specification of the present invention, and is workable, the yield height, and can prolonged preservation; The halogen worm hatching enzyme that obtains by the present invention can improve the hatching rate of low-quality halogen worm greatly, makes full use of artemia resource, reduces the seedling cost of mariculture industry significantly, alleviates the nervous situation of halogen worm supply in the production demand.
Claims (6)
1, a kind of method of producing halogen worm hatching enzyme with halogen worm artemia hatching solution is characterized in that earlier artemia cysts being incubated in the seawater, treats that the halogen worm more than 90% is finished when hatching, and collects filtrate--artemia hatching solution through filter screen; Then ammonium sulfate is added in it, and stir that to make the saturation ratio of ammonium sulfate be 50-70%; Put into 4 ℃ of refrigerator overnight precipitations again, and then centrifugation, and after will precipitating fully dissolving with damping fluid, under 4 ℃, in dialysis tubing, carry out dialysis desalting again with distilled water, above-mentioned dialyzate is carried out lyophilize, reach constant weight to sample, pack after dry powder is taken out and be halogen worm hatching enzyme crude product.
2, a kind of method of producing halogen worm hatching enzyme as claimed in claim 1 with halogen worm artemia hatching solution, it is characterized in that above-mentioned halogen worm hatching enzyme crude product is splined on anion exchange chromatography with after the damping fluid dissolving, carry out gradient elution, the sample that wash-out goes out is received the sample device automatically with sample and is collected, choose the highest elution samples of enzymic activity value as the anion exchange chromatography sample, again above-mentioned anion exchange chromatography sample is concentrated with concentrated centrifuge tube, be further purified through gel-filtration column, choose the highest elution samples of enzymic activity value and be the pure product of halogen worm hatching enzyme.
3, a kind of method of producing halogen worm hatching enzyme with halogen worm artemia hatching solution as claimed in claim 2 is characterized in that the above-mentioned pure product of halogen worm hatching enzyme are carried out lyophilize, take out pack obtain can prolonged preservation the pure product of halogen worm hatching enzyme.
4, a kind of method of producing halogen worm hatching enzyme with halogen worm artemia hatching solution as claimed in claim 1 is characterized in that described seawater is an artificial seawater, and its NaCl concentration is 30 ‰.
5, a kind of method of producing halogen worm hatching enzyme with halogen worm artemia hatching solution as claimed in claim 1 is characterized in that the damping fluid that is adopted is 20mmol/L Tris-HCl, and pH 7.5.
6, a kind of method of producing halogen worm hatching enzyme with halogen worm artemia hatching solution as claimed in claim 3 is characterized in that the lyophilize condition: temperature drops to-54 ℃, vacuum tightness and reaches 80Pa, carries out lyophilize to sample and reaches constant weight.
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| CNB2005100434641A CN100351373C (en) | 2005-05-05 | 2005-05-05 | Process for preparing artemia hatching enzyme by using artemia hatching liquid |
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| CNB2005100434641A CN100351373C (en) | 2005-05-05 | 2005-05-05 | Process for preparing artemia hatching enzyme by using artemia hatching liquid |
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| CN1699561A CN1699561A (en) | 2005-11-23 |
| CN100351373C true CN100351373C (en) | 2007-11-28 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105815276A (en) * | 2015-01-09 | 2016-08-03 | 天津丰年水产养殖有限公司 | Special culturing salt for increasing hatchability of brine shrimp eggs |
| CN107019667A (en) * | 2017-05-07 | 2017-08-08 | 辽宁石油化工大学 | The preparation method of artemia ovum hatching liquid extract |
| CN107114606A (en) * | 2017-05-07 | 2017-09-01 | 辽宁石油化工大学 | A kind of method for improving artemia ovum hatching synchronism |
| CN111034656A (en) * | 2019-11-24 | 2020-04-21 | 南通科技职业学院 | Preparation method for preparing hatching enzyme solution based on medaka hatching fluid |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001050880A2 (en) * | 2000-01-14 | 2001-07-19 | Baldur Hjaltason | Cultivation of dha-rich prey organisms for aquatic species |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001050880A2 (en) * | 2000-01-14 | 2001-07-19 | Baldur Hjaltason | Cultivation of dha-rich prey organisms for aquatic species |
Non-Patent Citations (4)
| Title |
|---|
| 《生物化学》(下册) 王镜岩，朱圣庚，pp300.302和pp337.339，高等教育出版社. 2004 * |
| 卤水Na/Mg比值对卤虫发育生长的影响 张福.海湖盐与化工，第32卷第2期 2003 * |
| 卤虫(Artemia salina)孵化腺细胞分化时相的免疫细胞化学研究(简报) 李凌，樊廷俊.实验生物学报，第37卷第2期 2004 * |
| 囊尾蚴膜联蛋白32在大肠杆菌中的表达及纯化 郭瀛军，吴丹.生物工程学报，第17卷第5期 2001 * |
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