CN103966175A - Extraction separation technology of marine organism source SOD (superoxide dismutase) - Google Patents

Extraction separation technology of marine organism source SOD (superoxide dismutase) Download PDF

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Publication number
CN103966175A
CN103966175A CN201410187808.5A CN201410187808A CN103966175A CN 103966175 A CN103966175 A CN 103966175A CN 201410187808 A CN201410187808 A CN 201410187808A CN 103966175 A CN103966175 A CN 103966175A
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sod
extraction
extracting solution
marine organisms
separation process
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孙继鹏
易瑞灶
洪碧红
黄文文
陈伟珠
晋文慧
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Third Institute of Oceanography SOA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
    • C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
    • C12Y115/01001—Superoxide dismutase (1.15.1.1)

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The invention relates to an extraction separation technology of marine organism source SOD (superoxide dismutase). The extraction separation technology comprises the following steps: (1) adding a buffer solution to fish viscera of marine aquatic processing byproducts through homogenate treatment in proportion, adjusting the pH of an extracting solution to be 6.0-7.4, controlling the extraction temperature, and stirring for 2-6h for extraction; (2) heating the extracting solution to remove hybrid protein; (3) cooling the extracting solution and then centrifuging to remove sediments, removing macromolecules of supernate through a multi-stage super-filtration, micro-filtration and nano-filtration multi-element film integrated purification system for separation, purification and concentration so as to obtain an SOD crude product; (4) separating and purifying the SOD crude product through combination of dextrangel G-100 and DEAE-FF chromatography, and then carrying out drying treatment so as to obtain a high-purity marine organism source SOD product. The conventional application of a large amount of organic solvent acetone and inorganic salt ammonium sulfate when the SOD is prepared from animal blood is changed, so that the influence to the activity and safety of SOD enzyme caused by the added reagents is reduced, and the waste for the reagents and subsequent removal process are reduced.

Description

A kind of marine organisms source superoxide-dismutase extraction and separation process
Technical field
The present invention relates to the high-valued development technique field of fishery products, espespecially from marine organisms source, extract and separate superoxide-dismutase industrialization extraction and separation process.
Background technology
Superoxide radical is acknowledged as human body cell " killer ", generation and the evolution of a lot of serious diseases of the mankind are participated in, such as the existence of O2-in human body can directly cause the diseases such as growth of cancer cells, radiation injury, human senility, rheumatoid arthritis, pulmonary emphysema, senile cataract, Senile osteoarthritis, in addition as heart trouble, diabetes, lupus erythematosus, spread very fast acquired immune deficiency syndrome (AIDS) even in recent years, also all having substantial connection with superoxide radical, is the chief-criminal who causes the serious disease such as aging, tumour.And superoxide-dismutase (Superoxide dismutase, SOD) be the key defence line of prevention oxygen toxicity in organism, be " jinx " of natural superoxide radical, its function is to remove O2-, is also the antioxidant reductase of unique removing O2-in organism.SOD has anti-inflammatory, antiviral, radioprotective, the anti-ageing effect of waiting for a long time, and in the research in the field such as radiotherapy prevention and Human Longevity of virus disease, autoimmune disorder, cardiovascular disorder, Radiation sickness, cancer and cancer, also oneself has breakthrough.Be widely used at aspects such as medical science, makeup, food supplements at present.
SOD rule of origin aspect is main still taking animal tissuess such as pig ox blood, livers as main so far, has set up the several different methods of separation and purification SOD from animal erythrocyte.In the extraction purge process of traditional SOD, used respectively a large amount of organic solvent-acetones and inorganic salt ammonium sulfate, these reagent add the activity that not only affects enzyme, and for the waste of reagent and follow-up removal technique, all improved its industry cost.And itself carries various bacteriophagees etc. animal, just inevitably easily there are some cross infections in this, the phenomenons such as anaphylaxis, particularly foot and mouth disease, mad cow disease and spreading by synzoic pernicious transmissible disease, people have produced query for the security of extracting SOD isoreactivity composition from pig, ox, the risk of production animal source blood products strengthens, resist taking animal blood and internal organ as the superoxide-dismutase of waste many countries and regions, makes superoxide-dismutase very in short supply in the international market.
Rational SOD source is enriched in selection, simplifies purification step, reduces costs, and the SOD that obtains higher specific activity produces SOD key issue.Follow China's aquaculture adjustment of agricultural stracture, fish processing industry is also progressively developed, but the product that cures fish can produce a large amount of tankage simultaneously, accounts for and cures fish 30%～50%, if effectively do not processed, not only contaminate environment, also causes ample resources waste.In fish viscera, contain abundant biological enzyme, wherein SOD activity is higher, if the resource using cheap and abundant fish viscera as industrial production SOD, simplify purification procedures, preparation high added value, the wide SOD product of market outlook, realize utilization of waste material, turn waste into wealth, just seem rather important.
Summary of the invention
Technical problem to be solved by this invention is taking marine fishery processing byproduct fish viscera as raw material, a kind of high purity marine organisms source superoxide-dismutase extraction and separation process is provided, can obtains safer efficient marine organisms source superoxide radical scavenger product by this technique.
For achieving the above object, solution of the present invention is:
A kind of marine organisms source superoxide-dismutase extraction and separation process, its step comprises:
Step 1, through the marine fishery processing byproduct fish viscera of homogenized, add in proportion buffered soln, regulate extracting solution pH to 6.0～7.4, control extract temperature, stir extract 2～6 hours;
Step 2, extracting solution are removed foreign protein through heat treated, directly enter next step operation;
After step 3, extracting solution are cooling, through centrifugal removal throw out, supernatant liquor removes macromole, separating-purifying and concentrated by multi-ultrafiltration, micro-filtration, the integrated purification system of nanofiltration multicomponent membrane, obtains SOD crude product;
Step 4, SOD crude product, through sephadex g-100 and DEAE-FF chromatography united separation and purification, by drying treatment, obtain high purity marine organisms source SOD product.
Kilogram in described step 1, through the mass volume ratio of homogenate marine fishery processing byproduct fish viscera and buffered soln: rising, is 1:2～1:10.
In described step 1, the Tris-HCL that the buffered soln of employing is 0.05～0.3mol/L or phosphate buffer solution.
In described step 1, the buffered soln of employing adopts extracting solution pH to 6.0～7.4 in hydrochloric acid and sodium hydroxide control leaching process.
In described step 1, extract temperature and be controlled at below 60 DEG C.
In described step 1, add copper, two kinds of salt of zinc in buffered soln, copper ion concentration is controlled at 0.2～20.0mmol/L, and zinc ion concentration is controlled at 1～20.0mmol/L.
Copper zinc salt used in described step 1 can be vitriol, hydrochloride or acetate.
In described step 2, extracting solution is by adding heat abstraction part foreign protein, and Heating temperature is 60 DEG C, and the time is controlled at 30～120 minutes.
Adopt after such scheme, the high purity marine organisms source SOD vigor that the present invention extracts can reach 15000U/mg albumen, and iso-electric point is 4.45～4.50, contain two subunits, formed molecular weight ranges by 17 seed amino acids: 34960～37600Da, lives stable at pH 5.0～9.0 scope endoenzymes.SOD extraction process disclosed by the invention has changed organic solvent-acetones a large amount of while in the past preparing SOD with animal blood and the use of inorganic salt ammonium sulfate, not only reduce the impact on SOD enzymic activity and security causing because adding these reagent, and reduced for the waste of reagent and follow-up removal technique, all reduce its industry cost.
The present invention sets up marine organisms source SOD industrialized technology, prepare and compared at present because produce the wide SOD product of high added value, market outlook of query with terrestrial animal blood extraction process, reduce and produce environmental pollution and the wasting of resources that a large amount of by products bring because of the product that cures fish simultaneously, realize utilization of waste material, turn waste into wealth.
Brief description of the drawings
The suitableeest extraction mass volume ratio of Fig. 1 marine organisms source SOD, kilogram: rise;
Fig. 2 marine organisms the suitableeest extraction time of source SOD;
The suitableeest extraction of Fig. 3 marine organisms source SOD pH;
The suitableeest Extraction buffer concentration of Fig. 4 marine organisms source SOD;
Fig. 5 marine organisms source SOD is at Superdex 200(10/300) reservation collection of illustrative plates;
The SDS-page(12% of Fig. 6 marine organisms source SOD).
Embodiment
The present invention has disclosed a kind of marine organisms source superoxide-dismutase extraction and separation process, and its step comprises:
Step 1, through the marine fishery processing byproduct fish viscera of homogenized, add in proportion buffered soln, regulate extracting solution pH to 6.0～7.4, control extract temperature, stir extract 2～6 hours; Kilogram through the mass volume ratio of homogenate marine fishery processing byproduct fish viscera and buffered soln: rising, is 1:2～1:10, but consider energy consumption in actual industrial production, should control ratio below 1:5; Adopt Tris-HCL or phosphate buffer solution that buffered soln is 0.05～0.3mol/L, also can replace with the sodium-chlor of a great deal of for reducing extraction cost, with extracting solution pH to 6.0～7.4 in hydrochloric acid and sodium hydroxide control leaching process; Unaffected for ensureing SOD vigor, extract temperature and be controlled at below 60 DEG C; For improving the stability of SOD in leaching process, remove part foreign protein simultaneously, in buffered soln, add copper, two kinds of salt of zinc, copper ion concentration is controlled at 0.2～20.0mmol/L, zinc ion concentration is controlled at 1～20.0mmol/L, and copper zinc salt used can be vitriol, hydrochloride, acetate etc.;
Step 2, extracting solution are removed foreign protein through heat treated, directly enter next step operation; Extracting solution can be by adding heat abstraction part foreign protein, Heating temperature is 60 DEG C, time is controlled at 30～120 minutes, in leaching process in a small amount, 80 DEG C are heated 10～20 minutes, also part that can be removed foreign protein, considers the control that is difficult to carry out at short notice heating and cooling in actual industrial production for tonne extracting solution, and Heating temperature should be controlled at 60 DEG C;
After step 3, extracting solution are cooling, through centrifugal removal throw out, supernatant liquor removes macromole, separating-purifying and concentrated by multi-ultrafiltration, micro-filtration, the integrated purification system of nanofiltration multicomponent membrane, obtains SOD crude product;
Step 4, SOD crude product, through sephadex g-100 and DEAE-FF chromatography united separation and purification, by drying treatment, obtain high purity marine organisms source SOD product.
By concrete case study on implementation, the present invention is explained below, advantage and disadvantage of the present invention will be more clear along with description.But these case study on implementation are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of invention technical scheme and form be modified or be replaced, but these amendments and replacement all fall into protection scope of the present invention.
Embodiment mono-:
By the quick homogenate of marine fishery processing byproduct fish viscera, take homogenate raw material 50Kg, be 1:4 by the mass volume ratio of raw material and buffered soln, drop into 200L 0.05 mol/L Tris-HCL damping fluid, in damping fluid, contain the copper sulfate of 2.0mmol/L and the zinc sulfate of 20mmol/L, with hydrochloric acid and sodium hydroxide control extracting solution pH to 6.4, 40 DEG C are stirred extraction 4 hours, extracting solution is removed foreign protein for 60 minutes through 60 DEG C of heating, after cooling, remove throw out by continuously streamed whizzer, first centrifugate filter clarification by plate and frame microfiltration membrane, remove macromolecular cpd simultaneously, again by ultrafiltration, the integrated purification system separating-purifying of the multicomponent membranes such as nanofiltration and concentrated, obtain SOD crude product.SOD crude product, through sephadex g-100 and DEAE-FF chromatography united separation and purification, by drying treatment, obtains high purity marine organisms source SOD product.
Embodiment bis-:
By the quick homogenate of marine fishery processing byproduct fish viscera, take homogenate raw material 50Kg, be 1:3 by the mass volume ratio of raw material and buffered soln, drop into 150L 0.2 mol/L phosphoric acid buffer, in damping fluid, contain the copper sulfate of 8.0mmol/L and the zinc sulfate of 10mmol/L, with hydrochloric acid and sodium hydroxide control extracting solution pH to 7.0, 10 DEG C are stirred extraction 6 hours, extracting solution is removed foreign protein for 120 minutes through 60 DEG C of heating, after cooling, remove throw out by continuously streamed whizzer, first centrifugate filter clarification by plate and frame microfiltration membrane, remove macromolecular cpd simultaneously, again respectively by molecular weight cut-off 500KDa, 50KDa and 5KDa carry out separating-purifying and concentrated, obtain SOD crude product.SOD crude product, through sephadex g-100 and DEAE-FF chromatography united separation and purification, is collected SOD active ingredient, by drying treatment, obtains high purity marine organisms source SOD product.
Embodiment tri-:
By the quick homogenate of marine fishery processing byproduct fish viscera, take homogenate raw material 50Kg, be 1:5 by the mass volume ratio of raw material and buffered soln, drop into 250L 0.2 mol/L sodium chloride solution, in extracting solution, contain the copper sulfate of 1.2mmol/L and the zinc sulfate of 16mmol/L, with hydrochloric acid and sodium hydroxide control extracting solution pH to 6.4, 25 DEG C are stirred extraction 5 hours, extracting solution is removed foreign protein for 80 minutes through 60 DEG C of heating, after cooling, remove throw out by continuously streamed whizzer, first centrifugate filter clarification by plate and frame microfiltration membrane, remove macromolecular cpd simultaneously, carry out separating-purifying and concentrated by molecular weight cut-off 100KDa and 10KDa respectively again, obtain SOD crude product.SOD crude product, through sephadex g-100 and DEAE-FF chromatography united separation and purification, is collected SOD active ingredient, by drying treatment, obtains high purity marine organisms source SOD product.
Embodiment tetra-:
By the quick homogenate of marine fishery processing byproduct fish viscera, take homogenate raw material 50Kg, be 1:4 by the mass volume ratio of raw material and buffered soln, drop into 200L 0.05 mol/L Tris-HCL damping fluid (including 0.05 mol/L sodium chloride solution), in extracting solution, contain the copper sulfate of 4mmol/L and the zinc sulfate of 20mmol/L, with hydrochloric acid and sodium hydroxide control extracting solution pH to 6.8, 40 DEG C are stirred extraction 4 hours, extracting solution is removed foreign protein for 80 minutes through 60 DEG C of heating, after cooling, remove throw out by continuously streamed whizzer, first centrifugate filter clarification by plate and frame microfiltration membrane, remove macromolecular cpd simultaneously, carry out separating-purifying and concentrated by molecular weight cut-off 50KDa and 5KDa respectively again, obtain SOD crude product.SOD crude product, through sephadex g-100 and DEAE-FF chromatography united separation and purification, is collected SOD active ingredient, by drying treatment, obtains high purity marine organisms source SOD product.
Embodiment five:
By the quick homogenate of marine fishery processing byproduct fish viscera, take homogenate raw material 50Kg, be 1:10 by the mass volume ratio of raw material and buffered soln, drop into 500L 0.15mol/L sodium chloride solution, in extracting solution, contain the copper sulfate of 8mmol/L and the zinc sulfate of 6mmol/L, with hydrochloric acid and sodium hydroxide control extracting solution pH to 6.4, 45 DEG C are stirred extraction 2 hours, extracting solution is removed foreign protein for 80 minutes through 60 DEG C of heating, after cooling, remove throw out by continuously streamed whizzer, first centrifugate filter clarification by plate and frame microfiltration membrane, remove macromolecular cpd simultaneously, carry out separating-purifying and concentrated by molecular weight cut-off 100KDa and 10KDa respectively again, obtain SOD crude product.SOD crude product, through sephadex g-100 and DEAE-FF chromatography united separation and purification, is collected SOD active ingredient, by drying treatment, obtains high purity marine organisms source SOD product.
The present invention enriches marine fishery processing byproduct fish viscera as raw material taking cheapness, simplifies purification procedures, and the extraction purifies and separates technique of a kind of high purity marine organisms source SOD is provided.SOD extraction process disclosed by the invention has changed organic solvent-acetones a large amount of while in the past preparing SOD with animal blood and the use of inorganic salt ammonium sulfate, not only reduce the impact on SOD enzymic activity and security causing because adding these reagent, and reduced for the waste of reagent and follow-up removal technique, all reduce its industry cost.Compare at present and query SOD product because producing with terrestrial animal blood extraction process, by this technique can obtain high added value, market outlook are wide, safer efficient marine organisms source superoxide radical scavenger SOD product, reduce and produce environmental pollution and the wasting of resources that a large amount of by products bring because of the product that cures fish simultaneously, realize utilization of waste material, turn waste into wealth.
It should be noted that above-mentioned case study on implementation is only to better case study on implementation of the present invention; do not have the protection scope of the present invention of restriction; being equal to replacement or substituting of having done on the basis of technique scheme all belongs to protection scope of the present invention, and protection scope of the present invention is as the criterion with claims.

Claims (8)

1. a marine organisms source superoxide-dismutase extraction and separation process, its step comprises:
Step 1, through the marine fishery processing byproduct fish viscera of homogenized, add in proportion buffered soln, regulate extracting solution pH to 6.0～7.4, control extract temperature, stir extract 2～6 hours;
Step 2, extracting solution are removed foreign protein through heat treated, directly enter next step operation;
After step 3, extracting solution are cooling, through centrifugal removal throw out, supernatant liquor removes macromole, separating-purifying and concentrated by multi-ultrafiltration, micro-filtration, the integrated purification system of nanofiltration multicomponent membrane, obtains SOD crude product;
Step 4, SOD crude product, through sephadex g-100 and DEAE-FF chromatography united separation and purification, by drying treatment, obtain high purity marine organisms source SOD product.
Kilogram 2. marine organisms as claimed in claim 1 source superoxide-dismutase extraction and separation process, is characterized in that: in described step 1, through the mass volume ratio of homogenate marine fishery processing byproduct fish viscera and buffered soln: rising, is 1:2～1:10.
3. marine organisms as claimed in claim 1 or 2 source superoxide-dismutase extraction and separation process, is characterized in that: in described step 1, and the Tris-HCL that the buffered soln of employing is 0.05～0.3mol/L or phosphate buffer solution.
4. marine organisms as claimed in claim 1 or 2 source superoxide-dismutase extraction and separation process, is characterized in that: in described step 1, the buffered soln of employing adopts extracting solution pH to 6.0～7.4 in hydrochloric acid and sodium hydroxide control leaching process.
5. marine organisms as claimed in claim 1 source superoxide-dismutase extraction and separation process, is characterized in that: in described step 1, extract temperature and be controlled at below 60 DEG C.
6. marine organisms as claimed in claim 1 or 2 source superoxide-dismutase extraction and separation process, it is characterized in that: in described step 1, in buffered soln, add copper, two kinds of salt of zinc, copper ion concentration is controlled at 0.2～20.0mmol/L, and zinc ion concentration is controlled at 1～20.0mmol/L.
7. marine organisms as claimed in claim 6 source superoxide-dismutase extraction and separation process, is characterized in that: copper zinc salt used in described step 1 can be vitriol, hydrochloride or acetate.
8. marine organisms as claimed in claim 1 source superoxide-dismutase extraction and separation process, is characterized in that: in described step 2, extracting solution is by adding heat abstraction part foreign protein, and Heating temperature is 60 DEG C, and the time is controlled at 30～120 minutes.
CN201410187808.5A 2014-05-06 2014-05-06 Extraction separation technology of marine organism source SOD (superoxide dismutase) Pending CN103966175A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104946611A (en) * 2015-07-15 2015-09-30 北京中科紫鑫科技有限责任公司 Purification method of ATP sulfurylase
CN107213029A (en) * 2017-05-22 2017-09-29 大连大学 A kind of moisturizing toner for adding low-temperature superoxide dismutase and preparation method thereof
CN109628418A (en) * 2019-01-04 2019-04-16 广东海洋大学 A kind of marine organisms source superoxide dismutase extraction and separation process

Citations (1)

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CN1821394A (en) * 2006-02-28 2006-08-23 中国海洋大学 Method for extracting SOD emzyme from tilapia mossambica viscus

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CN1821394A (en) * 2006-02-28 2006-08-23 中国海洋大学 Method for extracting SOD emzyme from tilapia mossambica viscus

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104946611A (en) * 2015-07-15 2015-09-30 北京中科紫鑫科技有限责任公司 Purification method of ATP sulfurylase
CN104946611B (en) * 2015-07-15 2016-05-25 北京中科紫鑫科技有限责任公司 A kind of purification process of ATP sulfurylase
CN107213029A (en) * 2017-05-22 2017-09-29 大连大学 A kind of moisturizing toner for adding low-temperature superoxide dismutase and preparation method thereof
CN109628418A (en) * 2019-01-04 2019-04-16 广东海洋大学 A kind of marine organisms source superoxide dismutase extraction and separation process

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