CN106987565A - A kind of marine low temperature superoxide dismutase extraction and separation process - Google Patents
A kind of marine low temperature superoxide dismutase extraction and separation process Download PDFInfo
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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
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- 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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Abstract
The present invention relates to the high-valued development technique field of marine organisms product, particularly a kind of marine low temperature superoxide dismutase extraction and separation process.The present invention extracts low-temperature superoxide dismutase using marine bacteria, and specific method is：The centrifugation of marine bacteria broken wall is obtained into crude enzyme liquid, through ammonium sulfate precipitation, is concentrated by ultrafiltration, the column chromatographies of Sephadex G 100, spray drying finally give the low-temperature superoxide dismutase of outward appearance micro-strip pale bluish green, its enzyme activity 576.83U/mg.The inventive method is fermented by raw material of microorganism, with short production cycle, simple to operate, is easy to storage and transport, and production cost is low, is difficult to go mouldy and is damaged by worms, and security and stability are high, and product yield is high, it is easy to large-scale production, with wide application prospect.
Description
Technical field
The present invention relates to the high-valued development technique field of marine organisms product, more particularly to a kind of marine low temperature super oxygen
Thing mutase extraction and separation process.
Background technology
Superoxide dismutase (superoxide dismutase, ECl.15.1.1, abbreviation SOD) is that a class is widely present
In the metalloenzyme in organism, ultra-oxygen anion free radical can be catalyzed and hydrogen ion reacts to form hydrogen peroxide and molecular oxygen,
So as to remove the oxygen radical in organism, play and protect organisms from injury effect.Mccord and Fridovich in 1969
It was found that the enzymes biocatalysis is active and is named as superoxide dismutase.It is different by the metal species of its combination, it can be classified as
Three classes：One class is glaucous Cu, and Zn-SOD, relative molecular weight about 32000, the cytosol and leaf for being primarily present in eukaryotic is green
It is current most study, a most deep class in body；Equations of The Second Kind is the Mn of aubergine, Fe-SOD, relative molecular weight about 40000,
It is primarily present in prokaryote and mitochondrial matrix；3rd class is filemot Fe-SOD, and relative molecular weight is about
38700, it is primarily present in prokaryotic and some plants.
SOD is medically mainly used in adjuvant radiotherapy and chemotherapy as biological enzyme formulation, the protection of the organ such as kidney, liver, heart
And transplanting, eliminate radiation-induced side effect, and the probe as some diseases etc.；Food industry as additive application in
Beverage and beer etc..In recent years SOD be widely used in treatment oxygen poisoning, it is cataract of old people, diabetes, angiocardiopathy, each
Plant a variety of diseases such as inflammation.SOD is always domestic and international study hotspot in decades, initialization phase focus mostly in from animal blood or
SOD is extracted in tissue, the research for producing SOD to mid-term stage microbial fermentation starts to start to walk and increasingly increase, up to the present
Micro-organisms SOD already turns into the main body of research.But mostly medium and high temperature SOD, optimum temperature typically more than 45 DEG C,
And the physiological temp of human body is generally 36.5~37 DEG C, the medium and high temperature SOD for causing microbial fermentation to produce can not give full play to work
With there is therapeutic effect difference or without effect.Low temperature SOD optimum temperatures are generally 35~40 DEG C, relatively human body
Physiological temp, it is obvious applied to therapeutic effect.But low temperature SOD industrialization, large-scale production and application have not been reported.Mirror
In animal blood source difficulty and the limitation of the medium and high temperature SOD optimum temperatures of microbial fermentation production, microorganism is utilized
The low temperature SOD of fermenting and producing has very big application advantage.Low temperature SOD at a temperature of natural environment there is high enzymatic activity and height to urge
Change efficiency, and to thermo-responsive, can lose the vigor of cold-adapted enzyme by gentle heat treatment, if SOD is applied into food
Industry so as to greatly shorten the time of processing procedure and save costliness expense is heated or cooled, and do not influence product quality, this
It will be helpful to low temperature SOD popularization and use.The heating of cumbersome extraction process and medium and high temperature enzyme can fundamentally be broken away from, it is cold
But equipment and flow, energy consumption and production cost are reduced while yield and enzymatic activity is improved.In view of low temperature SOD is natural and raw
Advantage under the conditions of reason, low temperature SOD possesses in terms of health care, food, cosmetics to be more widely applied prospect and opens
Send out potentiality.
The content of the invention
It is an object of the invention to provide a kind of extraction and separation process of marine low temperature superoxide dismutase, using microorganism as
Raw material is fermented, with short production cycle, simple to operate, is easy to storage and transport, and production cost is low, is difficult to go mouldy and is damaged by worms,
Security and stability are high, and product yield is high, it is easy to large-scale production, with wide application prospect.
The present invention is realized by the following method there is provided a kind of extraction and separation process of marine low temperature superoxide dismutase,
Utilize marine bacteria thalline extraction purification low-temperature superoxide dismutase.
The extraction and separation process process of described marine low temperature superoxide dismutase is：Marine bacteria broken wall is centrifuged
To crude enzyme liquid, through ammonium sulfate precipitation, it is concentrated by ultrafiltration, Sephadex G-100 column chromatographies, spray drying obtain outward appearance micro-strip light blue
Green, enzyme activity 576.83U/mg low-temperature superoxide dismutase.
The extraction and separation process of above-mentioned marine low temperature superoxide dismutase, the preparation method of the crude enzyme liquid is：It is logical
Sterile working is crossed, by the extra large sample inoculation activated into liquid fermentation medium, in 20 DEG C, 160r/min fermentation 48h, hair
Yeast-like fungi liquid is in 4 DEG C, and 6 000r/min centrifugation 30min obtain wet thallus, 4 DEG C, 6 000r/min centrifugation 30min, wet thallus phosphorus
Acid buffer dissolves, in broken 30min under 480W ultrasonic wave ice baths (broken 5s, be spaced 5s), after crushing, 4 DEG C, 12000r/min
15min is centrifuged, supernatant is crude enzyme liquid, and aforesaid liquid fermentative medium formula percentage is：Glucose 2.0, yeast extract 0.5,
Chen Haishui is prepared, and pH is natural.
The extraction and separation process of above-mentioned marine low temperature superoxide dismutase, the method for the ammonium sulfate precipitation is：System
Standby 6 parts of 25ml crude enzyme liquids, add the cold ammonium sulfate of solid thereto respectively, its saturated solution concentration is distinguished 20%, 30%,
40%th, 50%, 60%, 70%, 80%, 90%, 4 DEG C of 16h that saltout, pH7.8 phosphate buffers dissolving protein precipitation, determine low temperature
Superoxide dismutase activity highest ammonium sulfate precipitation condition.
The cold ammonium sulfate is that ammonium sulfate is cooled into -20 DEG C.
The extraction and separation process of above-mentioned marine low temperature superoxide dismutase, the ultrafiltration concentration method is：It will saltout
Completion sample is transferred to the molecular cut off 10KDa super filter tubes by pretreatment, and 4 DEG C, 6000r/min, ultrafiltration 30min are removed
SO42-With part small molecular protein, liquid-transfering gun carefully takes out concentrate, and 4 DEG C, preservation is standby, is partially soluble in phosphate buffer, surveys
Determine low-temperature superoxide dismutase enzyme activity, calculate protein recovery.
The extraction and separation process of above-mentioned marine low temperature superoxide dismutase, the Sephadex G-100 column chromatography sides
Method is：Sephadex G-100 gel columns are balanced with pH7.8 phosphate buffers, after after baseline balance, take enzyme liquid after 2ml ultrafiltration to add
Enter the equilibrated Sephadex G-100 gel columns of pH7.8 phosphate buffers (Ф 1.6cm × 60cm), regulation sets automatic collect
Device, often pipe collection 2ml or so.According to the eluting peak on display, it is determined that enzyme containing SOD is respectively managed, collect and determine its enzyme activity, close
And respectively managed containing enzyme activity, 4 DEG C, preservation is standby.
The extraction and separation process of above-mentioned marine low temperature superoxide dismutase, the parameter of the spray drying be set into
110 DEG C of temperature of mouth, outlet temperature are 60 DEG C, pressure position 2kgf/cm2, flow rate of liquid be 5mL/min.
It is extra large in the extraction and separation process of above-mentioned marine low temperature superoxide dismutase, the protective agent in the spray drying
Algae sugar, sucrose, the consumption of sodium alginate are 2%-5%, and the consumption of soluble starch is 6%-8%, protectant total consumption control
System is within 10%.
The extraction and separation process of above-mentioned marine low temperature superoxide dismutase, the marine bacteria is glutinous false alternately single for sea
Born of the same parents bacterium, extra large Pseudoalteromonas, coral Pseudoalteromonas of dwelling.
Brief description of the drawings
Fig. 1 is that marine low temperature superoxide dismutase of the present invention isolates and purifies result；
Fig. 2 is marine low temperature superoxide dismutase polyacrylamide gel electrophoresis result of the present invention；
Fig. 3 is marine low temperature superoxide dismutase enzyme type qualification result of the present invention.
Embodiment
Specification is described further with reference to embodiment.
Embodiment 1
A kind of extraction and separation process of described marine low temperature superoxide dismutase, comprises the following steps：
1. the preparation of crude enzyme liquid：The glutinous Pseudoalteromonas Pseudoalteromonas in 1L marine bacterias sea
Mariniglutinosa (numberings：1.8476, DSMZ of institute of microbiology of the Chinese Academy of Sciences) zymotic fluid obtains 50 grams of yeast
Bacterium bacterium mud, with distillation water washing 2-3 time, 4 DEG C, 6 000r/min centrifuge 30min, obtain wet thallus, 4 DEG C, 6 000r/min from
Heart 30min, wet thallus phosphate buffer dissolves, and in crushing 30min (broken 5s, be spaced 5s) under 480W ultrasonic wave ice baths, breaks
After broken, 4 DEG C, 12 000r/min centrifugation 15min, supernatant is crude enzyme liquid, and aforesaid liquid fermentative medium formula percentage is：
Glucose 2.0, yeast extract 0.5, Chen Haishui is prepared, and pH is natural.
2. ammonium sulfate precipitation：6 parts of 25ml crude enzyme liquids are prepared, the cold ammonium sulfate of solid is added thereto respectively, makes its saturation molten
Liquid concentration difference 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 4 DEG C of 16h that saltout, pH7.8 phosphate buffers
Protein precipitation is dissolved, the active highest ammonium sulfate precipitation conditions of SOD are determined.
It 3. is concentrated by ultrafiltration：It will saltout and complete sample and be transferred to molecular cut off 10KDa super filter tubes by pretreatment, 4 DEG C,
6000r/min, ultrafiltration 30min, remove SO4 2-With part small molecular protein, liquid-transfering gun carefully takes out concentrate, and 4 DEG C, preservation is standby
With being partially soluble in phosphate buffer, determine low-temperature superoxide dismutase enzyme activity, calculate protein recovery.
4.Sephadex G-100 column chromatographies：Sephadex G-100 gel columns are balanced with pH7.8 phosphate buffers, base is treated
After line balance, enzyme liquid after 2ml ultrafiltration is taken to add the equilibrated Sephadex G-100 gel columns (Ф of pH7.8 phosphate buffers
1.6cm × 60cm), regulation sets automatic collector, and often pipe collects 2ml or so.Through determining the protein quantity and low temperature superoxides
Mutase enzyme assay, the high eluent of the activity containing low-temperature superoxide dismutase is merged, you can obtain the low of high vigor
Warm superoxide dismutase enzyme liquid 20mL, total enzyme activity is 576.83U/mg.Finally super oxygen is identified with poly- propionamide gel electrophoresis
The enzyme purity of thing mutase, is as a result shown in Fig. 2；Copper-zinc superoxide dismutase is accredited as through enzyme type, Fig. 3 is as a result seen.
5. spray drying：It is 60 DEG C, pressure position 2kgf/cm that its parameter, which is set to 110 DEG C of inlet temperature, outlet temperature,2, liquid
Rate of flow of fluid is 5mL/min.Trehalose, sucrose, the consumption of sodium alginate are 2%-5% in protective agent in spray drying, solvable
Property starch consumption be 6%-8%, protectant total consumption control is within 10%.
Embodiment 2
A kind of extraction and separation process of described marine low temperature superoxide dismutase, comprises the following steps：
1. the preparation of crude enzyme liquid：1L marine bacterias sea Pseudoalteromonas Pseudoalteromonas marina (numberings：
1.10151, DSMZ of institute of microbiology of the Chinese Academy of Sciences) zymotic fluid obtains 50 grams of saccharomycete bacterium muds, with distillation water washing
2-3 times, 4 DEG C, 6 000r/min centrifugation 30min obtain wet thallus, 4 DEG C, 6 000r/min centrifugation 30min, wet thallus phosphoric acid
Buffer solution.In broken 30min under 480W ultrasonic wave ice baths (broken 5s, be spaced 5s).After broken, 4 DEG C, 12 000r/min
15min is centrifuged, supernatant is crude enzyme liquid, and aforesaid liquid fermentative medium formula percentage is：Glucose 2.0, yeast extract 0.5,
Chen Haishui is prepared, and pH is natural.
2. ammonium sulfate precipitation：6 parts of 25ml crude enzyme liquids are prepared, the cold ammonium sulfate of solid is added thereto respectively, makes its saturation molten
Liquid concentration difference 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 4 DEG C of 16h that saltout, pH7.8 phosphate buffers
Protein precipitation is dissolved, the active highest ammonium sulfate precipitation condition of low-temperature superoxide dismutase is determined.
It 3. is concentrated by ultrafiltration：It will saltout and complete sample and be transferred to (molecular cut off 10KDa) super filter tube by pretreatment, 4 DEG C,
6000r/min, ultrafiltration 30min, remove SO4 2-With part small molecular protein, liquid-transfering gun carefully takes out concentrate, and 4 DEG C, preservation is standby
With being partially soluble in phosphate buffer, determine low-temperature superoxide dismutase enzyme activity, calculate protein recovery.
4.Sephadex G-100 column chromatographies：Sephadex G-100 gel columns are balanced with pH7.8 phosphate buffers, base is treated
After line balance, enzyme liquid after 2ml ultrafiltration is taken to add the equilibrated Sephadex G-100 gel columns (Ф of pH7.8 phosphate buffers
1.6cm × 60cm), regulation sets automatic collector, and often pipe collects 2ml or so.Through determining the protein quantity and low temperature superoxides
Mutase enzyme assay, the high eluent of the activity containing low-temperature superoxide dismutase is merged, you can obtain the low of high vigor
Warm superoxide dismutase enzyme liquid 20mL, total enzyme activity is 576.83U/mg.Finally super oxygen is identified with poly- propionamide gel electrophoresis
The enzyme purity of thing mutase, is as a result shown in Fig. 2；Copper-zinc superoxide dismutase is accredited as through enzyme type, Fig. 3 is as a result seen.
5. spray drying：It is 60 DEG C, pressure position 2kgf/cm that its parameter, which is set to 110 DEG C of inlet temperature, outlet temperature,2, liquid
Rate of flow of fluid is 5mL/min.Trehalose, sucrose, the consumption of sodium alginate are 2%-5% in protective agent in spray drying, solvable
Property starch consumption be 6%-8%, protectant total consumption control is within 10%.
Embodiment 3
A kind of extraction and separation process of described marine low temperature superoxide dismutase, comprises the following steps：
1. the preparation of crude enzyme liquid：1L marine bacterias dwell coral Pseudoalteromonas Pseudoalteromonas
Paragorgicola (numberings：1.10153, DSMZ of institute of microbiology of the Chinese Academy of Sciences) zymotic fluid obtains 50 grams of yeast
Bacterium bacterium mud, with distillation water washing 2-3 time, 4 DEG C, 6 000r/min centrifuge 30min, obtain wet thallus, 4 DEG C, 6 000r/min from
Heart 30min, wet thallus phosphate buffer dissolves.In broken 30min under 480W ultrasonic wave ice baths (broken 5s, be spaced 5s).It is broken
After broken, 4 DEG C, 12 000r/min centrifugation 15min, supernatant is crude enzyme liquid, and aforesaid liquid fermentative medium formula percentage is：
Glucose 2.0, yeast extract 0.5, Chen Haishui is prepared, and pH is natural.
2. ammonium sulfate precipitation：6 parts of 25ml crude enzyme liquids are prepared, the cold ammonium sulfate of solid is added thereto respectively, makes its saturation molten
Liquid concentration difference 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 4 DEG C of 16h that saltout, pH7.8 phosphate buffers
Protein precipitation is dissolved, the active highest ammonium sulfate precipitation condition of low-temperature superoxide dismutase is determined.
It 3. is concentrated by ultrafiltration：It will saltout and complete sample and be transferred to (molecular cut off 10KDa) super filter tube by pretreatment, 4 DEG C,
6000r/min, ultrafiltration 30min, remove SO4 2-With part small molecular protein, liquid-transfering gun carefully takes out concentrate, and 4 DEG C, preservation is standby
With being partially soluble in phosphate buffer, determine low-temperature superoxide dismutase enzyme activity, calculate protein recovery.
4.Sephadex G-100 column chromatographies：Sephadex G-100 gel columns are balanced with pH7.8 phosphate buffers, base is treated
After line balance, enzyme liquid after 2ml ultrafiltration is taken to add the equilibrated Sephadex G-100 gel columns (Ф of pH7.8 phosphate buffers
1.6cm × 60cm), regulation sets automatic collector, and often pipe collects 2ml or so.Through determining the protein quantity and low temperature superoxides
Mutase enzyme assay, the high eluent of the activity containing low-temperature superoxide dismutase is merged, you can obtain the low of high vigor
Warm superoxide dismutase enzyme liquid 20mL, total enzyme activity is 576.83U/mg.Finally super oxygen is identified with poly- propionamide gel electrophoresis
The enzyme purity of thing mutase, is as a result shown in Fig. 2；Copper-zinc superoxide dismutase is accredited as through enzyme type, Fig. 3 is as a result seen.
5. spray drying：It is 60 DEG C, pressure position 2kgf/cm that its parameter, which is set to 110 DEG C of inlet temperature, outlet temperature,2, liquid
Rate of flow of fluid is 5mL/min.Trehalose, sucrose, the consumption of sodium alginate are 2%-5% in protective agent in spray drying, solvable
Property starch consumption be 6%-8%, protectant total consumption control is within 10%.
Embodiment 4
The measure of low-temperature superoxide dismutase activity
0.1mol/L Tris-HCl buffer solutions, distilled water, low temperature superoxides discrimination are added in test tube according to table 1 respectively
Change enzyme enzyme liquid, 10mmol/L HCl, in after 20 DEG C of constant temperature 20min, 20 DEG C of preheated neighbours are added in sample cell and control tube
Benzenetriol (pyrogallol addition is on the basis of mouse thymus cells speed is 0.07/min), shakes up rapidly, immediately with shifting
Liquid rifle moves into sample in quartz cuvette, determines a light absorption value per 30s at wavelength 325nm, 3min is surveyed altogether, figure is as a result seen
1。
The assay NBT photoreduction of table 1 determines low temperature SOD sample-adding tables
V1：Reaction solution cumulative volume, mL；V2：Determination sample volume, mL；n：Sample diluting liquid multiple；
ODA：Mouse thymus cells speed；ODB：Sample OD value changes speed
Embodiment 5
Protein content determination standard curve
Using Bradford methods, using bovine serum albumin as standard protein, standard protein curve determination enzyme liquid protein is drawn
Content.
1. standard BSA solution
2. dye reagent：100mg Coomassie brilliant G-250s, 50ml ethanol (95%), phosphoric acid (85%) is diluted with water to
1L, is filtered standby.
3. according to the form below adds corresponding reagent, and 5min determines light absorption value at 595nm.
The protein concentration standard curve of table 2
Above content is to combine specific preferred embodiment further description made for the present invention, it is impossible to assert
The specific implementation of the present invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of not departing from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the present invention's
Protection domain.
Claims (9)
1. a kind of marine low temperature superoxide dismutase extraction and separation process, it is characterised in that：Utilize marine bacteria extraction purification
Low-temperature superoxide dismutase, specific method is：The centrifugation of marine bacteria broken wall is obtained into crude enzyme liquid, through ammonium sulfate precipitation, ultrafiltration
Concentration, Sephadex G-100 column chromatographies, spray drying, finally gives low-temperature superoxide dismutase.
2. a kind of marine low temperature superoxide dismutase extraction and separation process according to claim 1, it is characterised in that：Institute
The preparation method for stating crude enzyme liquid is：Sterile working, the marine bacteria strain of activation is inoculated into liquid fermentation medium and obtains wet bacterium
Body, after ultrasonication, centrifugation supernatant is crude enzyme liquid, and the liquid fermentation medium formula percentage is：Glucose 2.0, ferment
Female cream 0.5, Chen Haishui is prepared, and pH is natural.
3. a kind of marine low temperature superoxide dismutase extraction and separation process according to claim 1, it is characterised in that：Institute
The method for stating ammonium sulfate precipitation is：Added into crude enzyme liquid saturated concentration be respectively 20%, 30%, 40%, 50%, 60%,
70%th, 80%, 90% cold ammonium sulfate precipitation 16h, pH7.8 phosphate buffers dissolving protein precipitation, determines low temperature superoxides
Dismutase activity highest ammonium sulfate precipitation condition.
4. a kind of marine low temperature superoxide dismutase extraction and separation process according to claim 3, it is characterised in that：Institute
The cold ammonium sulfate stated is that ammonium sulfate is cooled into -20 DEG C.
5. a kind of marine low temperature superoxide dismutase extraction and separation process according to claim 1, it is characterised in that：Institute
The method for stating ultrafiltration concentration is：It will saltout and complete the super filter tube that sample is transferred to the molecular cut off 10KDa by pretreatment, remove
SO4 2-With part small molecular protein, concentrate is taken out, 4 DEG C, preservation is standby, be partially soluble in phosphate buffer, determine low temperature super oxygen
Compound mutase enzyme activity, calculates protein recovery.
6. a kind of marine low temperature superoxide dismutase extraction and separation process according to claim 1, it is characterised in that：Institute
Stating Sephadex G-100 column chromatography methods is：Purify enzyme liquid with Sephadex G-100 gel columns, collect and determine its enzyme activity
Power, merging is respectively managed containing enzyme activity, and 4 DEG C, preservation is standby.
7. a kind of marine low temperature superoxide dismutase extraction and separation process according to claim 1, it is characterised in that：Spray
Protective agent during mist is dried is one kind in trehalose, sucrose, sodium alginate, soluble starch or wherein several combinations.
8. a kind of marine low temperature superoxide dismutase extraction and separation process according to claim 7, it is characterised in that：Institute
It is 2%-5% to state protective agent trehalose, sucrose, the consumption of sodium alginate, and the consumption of soluble starch is 6%-8%, protective agent
Total consumption control within 10%.
9. a kind of marine low temperature superoxide dismutase extraction and separation process according to claim 1-8 any one, its
It is characterised by：The marine bacteria is the glutinous Pseudoalteromonas in sea, extra large Pseudoalteromonas, coral Pseudoalteromonas of dwelling.
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Cited By (1)
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| CN107865423A (en) * | 2017-11-10 | 2018-04-03 | 大连大学 | A kind of SOD oral liquids of anti-enteron aisle oxidation |
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Application publication date: 20170728 |