CN104593339A - Bio-active enzyme and extraction process thereof - Google Patents

Bio-active enzyme and extraction process thereof Download PDF

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CN104593339A
CN104593339A CN201410851247.4A CN201410851247A CN104593339A CN 104593339 A CN104593339 A CN 104593339A CN 201410851247 A CN201410851247 A CN 201410851247A CN 104593339 A CN104593339 A CN 104593339A
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gained
solution
ammonium sulfate
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elutriant
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陈�光
高云龙
盛美田
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AESTHETIC TECHNOLOGY (BEIJING) Co Ltd
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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.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01006—Catalase (1.11.1.6)

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Abstract

The invention relates to a bio-active enzyme and an extraction process thereof. Particularly, the bio-active enzyme provided by the invention is extracted from a red blood cell. In a preparation process of the bio-active enzyme, the washed red blood cell and a citrate buffer solution are mixed, the red blood cell is smashed, and centrifuging is carried out to take a liquid supernatant, thereby obtaining the sediment by an ammonium sulfate precipitation method. According to the invention, use of the common dialysis or organic solvent precipitation method in the prior art is avoided, a Sephade*G-25 sephadex column and a metal chelate affinity column are selected to purify the bio-active enzyme, a phosphate buffer solution with a specific pH value is selected to be used as an eluant according to the characteristics of the bio-active enzyme, and the high-purity bio-active enzyme is successfully obtained. The extraction process of the bio-active enzyme provided by the invention is simple in operation, safe and environmentally friendly. After being tested, the bio-active enzyme provided by the invention is high in purity and good in antioxidant activity.

Description

A kind of bioactive enzyme and extraction process thereof
Technical field
The present invention relates to and there is bioactive material, be specifically related to a kind of bioactive enzyme and extraction process thereof.
Background technology
At present, domestic and international bioactive enzyme majority derives from animal blood, liver, has rich in natural resources in China, and prior art has report for the method extracting bioactive enzyme from blood or blood related raw material more." purifying of pig hemase and some character research thereof " (Pharmaceutical Biotechnology that Zhang Erxian etc. deliver, 2nd phase 5-8 in 1994) disclose the purification process of pig hemase, it adopts CM-23 and sephadex G-100 chromatography and ammonium sulfate salting-out process purifying pig hemase.In addition, patent documentation CN102690794A discloses one and prepares catalatic method, the method is raw material with fresh beef liver, homogenate after being shredded, and adopts the techniques such as ultrasonic extraction, ion exchange chromatography, gel permeation chromatography, lyophilize to produce high enzyme and lives through hydrogen oxide enzyme product.
But the technology of traditional extracting and developing bioactive enzyme, exists that length consuming time, efficiency are low, the defect that activity is not high and contaminative is strong of enzyme.
Summary of the invention
The present invention is intended to the defect overcoming prior art existence, provides a kind of and has the active enzyme of good biological.
The invention provides a kind of bioactive enzyme, described bioactive enzyme is prepared from by the method comprised the following steps:
1) erythrocyte after cleaning mix with water, with flash extracter extraction, in gained extracting solution, add chloroform and 95% alcohol mixed solvent extract, get supernatant liquor, obtain crude enzyme liquid;
2) in step 1) add ammonium sulfate in gained crude enzyme liquid, leave standstill, collected after centrifugation precipitates; To be precipitated and dissolved in 0.01 ~ 0.02mol/L phosphate buffered saline buffer of pH7.0 ~ 7.4, gained solution is added the sephadex column Sephadex G-25 top balanced, wash-out is carried out with phosphate buffered saline buffer described in this step, collect containing protein and the elutriant of not liquid containing ammonium sulfate, concentrated, obtain concentrated solution;
3) in step 2) add NaCl in gained concentrated solution, obtain the solution that NaCl concentration is 0.4 ~ 0.6mol/L, gained solution is added the affine capital end of the metal-chelating balanced, carry out wash-out with 0.015 ~ 0.025mol/L phosphate buffered saline buffer of pH 5.8 ~ 6.2, collect the elutriant containing protein;
4) enrichment step 3) gained elutriant, lyophilize, obtains bioactive enzyme.
Be mammiferous erythrocyte as the erythrocyte of raw material in the present invention, the acquisition methods of erythrocyte is biological field ordinary method.
Step 1 of the present invention) in, the concentration of described citrate buffer solution is preferably 0.02mol/L, and pH value is preferably 5.0; The volume ratio of erythrocyte and described citrate buffer solution is 1:0.5 ~ 2, is preferably 1:1; The device of described broken erythrocyte is preferably flash extracter, described flash extracter is the Conventional flash extractor of this area for extracting, also Other Instruments or the method replacement of crushing effect described in the application can be realized by this area, its operating parameters is: extract under 3000 ~ 8000rpm condition, each extraction 20 ~ 45s, extract 2 ~ 5 times altogether, every minor tick 1 ~ 2min, operating parameters is preferably extraction under 5000rpm condition, extracts 30s at every turn, extracts 3 times, every minor tick 2min altogether; Described centrifugal be specially 3000 ~ 3200rpm condition under centrifugal 5 ~ 15min, be preferably under 3000rpm condition centrifugal 15min.
Step 2 of the present invention) described in crude enzyme liquid, add ammonium sulfate thus enough bioactive enzyme fully to be precipitated, the final concentration adding ammonium sulfate is 0.3 ~ 0.5g/ml, after adding ammonium sulfate, 20 ~ 40min should be left standstill, at 0 ~ 4 DEG C, centrifugal 10 ~ 20min under 7500 ~ 8500rpm condition, separation of supernatant, collecting precipitation; In gained supernatant liquor, add 0.7 ~ 0.9g/ml ammonium sulfate, leave standstill 20 ~ 40min, at 0 ~ 4 DEG C, centrifugal 25 ~ 35min under 8500 ~ 9500rpm condition, collecting precipitation; The precipitation of twice being collected gained merges.
After ammonium sulfate precipitation, the present invention does not use conventional dialysis process, but adopts gel chromatography removing ammonium sulfate, and the lengthy and tedious process avoiding conventional dialysis desalination method and the target product loss that may cause.Concrete, described phosphate buffered saline buffer is potassium primary phosphate-dipotassium hydrogen phosphate damping fluid; The concentration of damping fluid is preferably 0.015mol/L, and pH value is preferably 7.2; Step 3) gained precipitation be dissolved in phosphate buffered saline buffer with mass volume ratio 1:1 ~ 5, the mass unit herein precipitated is g, and the unit of phosphate buffered saline buffer volume is ml; Sephadex column selects Sephadex G-25, and the balance of gel column and the wash-out of moving phase are routine operation; Described concentrated preferably polyoxyethylene glycol method of enrichment, described polyoxyethylene glycol is preferably PEG4000, and enrichment step is routine operation.In order to differentiate whether ammonium sulfate is eliminated totally, the present invention selects the ammonium sulfate in Nessler's reagent qualification elutriant, and concrete steps are: get 1 elutriant, drop in white colorimetric disk hole, add 1 Nessler's reagent mixing, if occur, brown color precipitates, and shows in elutriant containing ammonium sulfate.
Simultaneously, step 2 of the present invention) should protein be contained in the elutriant collected, specifically, in elutriant, the discrimination method of protein is specially: get 1 elutriant, drop in black colorimetric disk, add 1 20% sulphosalicylic acid mixing, if there is white flock precipitate, show in elutriant containing protein.
Step 3 of the present invention) purifying is carried out to biological activity enzyme solution, and adopt and step 2) identical method identify in solution whether also have protein, when can't detect protein, stopping collection.Specifically, this step carries out protein purification by metal affinity chromatography method, and the metal ion of described metal-chelating is affine post institute chelating is selected from chelating metal ion conventional in the metal chelate affinity chromatographies such as copper, zinc, iron, nickel ion; Described phosphate buffered saline buffer is potassium primary phosphate-dipotassium hydrogen phosphate damping fluid; The concentration of damping fluid is preferably 0.02mol/L, and pH value is preferably 6.0; The discrimination method of protein and step 3 in elutriant) identical; Described concentrated preferably polyoxyethylene glycol method of enrichment, described polyoxyethylene glycol is preferably PEG4000, and enrichment step is routine operation.
As most preferably scheme of the present invention, described bioactive enzyme is prepared from by the method comprised the following steps:
1) erythrocyte after cleaning is mixed with the 0.02mol/L citrate buffer solution of pH5.0 mix with volume ratio 1:1, with the broken erythrocyte of flash extracter, its operating parameters is: extract under 5000rpm condition, extract 30s at every turn, extract 3 times altogether, every minor tick 2min; Under 3000rpm condition, centrifugal 15min, gets supernatant liquor, obtains crude enzyme liquid;
2) in step 1) in gained crude enzyme liquid, add 0.4g/ml ammonium sulfate, leave standstill 30min, at 0 DEG C, centrifugal 15min under 8000rpm condition, separation of supernatant, collecting precipitation; Ammonium sulfate is added in gained supernatant liquor with mass volume ratio 0.8:1, leaves standstill 30min, at 0 DEG C, centrifugal 30min under 9000rpm condition, collecting precipitation; The precipitation of twice being collected gained merges; Precipitation is dissolved in the 0.015mol/L potassium primary phosphate-dipotassium hydrogen phosphate damping fluid of pH7.2 with mass volume ratio 1:5, gained solution is added the sephadex column Sephadex G-25 top balanced, wash-out is carried out with phosphate buffered saline buffer described in this step, collect containing protein and the elutriant of not liquid containing ammonium sulfate, concentrate with PEG4000, obtain concentrated solution;
3) in step 2) add NaCl in gained concentrated solution, obtain the solution that NaCl concentration is 0.5mol/L, gained solution is added the nickel ion chelating affinity column top balanced, carry out wash-out with pH6.0,0.02mol/L potassium primary phosphate-dipotassium hydrogen phosphate damping fluid, collect the elutriant containing protein;
4) step 3 is got) gained elutriant, concentrate with PEG4000, lyophilize, obtains bioactive enzyme.
In order to ensure the activity of the bioactive enzyme prepared, the service temperature of each step of the present invention is all preferably 0 ~ 4 DEG C.
Through method qualifications such as high performance liquid chromatography, the main component of bioactive enzyme provided by the invention is catalase, and catalatic content is more than 90%.
The present invention protects described bioactive enzyme preparing the application in makeup further.
Compared with prior art, the extraction process that the invention provides bioactive enzyme is easy and simple to handle, safety, environmental protection, and bioactive enzyme productive rate is high, purity is high, activity is good, is suitable for industrialization scale operation.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.Erythrocyte in each embodiment of the application conveniently acquisition method is collected in the pig propagated artificially.
In various embodiments of the present invention, in elutriant, the discrimination method of ammonium sulfate is specially: get 1 elutriant, drops in white colorimetric disk hole, and add 1 Nessler's reagent mixing, if occur, brown color precipitates, and shows in elutriant containing ammonium sulfate; In elutriant, the discrimination method of protein is specially: get 1 elutriant, drops in black colorimetric disk, adds 1 20% sulphosalicylic acid mixing, if there is white flock precipitate, shows in elutriant containing protein.
Embodiment 1
Bioactive enzyme is prepared from by following steps:
1) the 0.02mol/L citrate buffer solution 100ml of erythrocyte 100ml and pH5.0 after cleaning is mixed, with the broken erythrocyte of flash extracter, its operating parameters is: extract under 5000rpm condition, extract 30s at every turn, extract 3 times altogether, every minor tick 2min; Under 3000rpm condition, centrifugal 15min, gets supernatant liquor, obtains crude enzyme liquid 170ml;
2) in step 1) in gained crude enzyme liquid, add 68g ammonium sulfate and dissolve, leave standstill 30min, at 0 DEG C, centrifugal 15min under 8000rpm condition, separation of supernatant, collecting precipitation; In gained supernatant liquor, add 65g ammonium sulfate, leave standstill 30min, at 4 DEG C, centrifugal 30min under 9000rpm condition, collecting precipitation; The precipitation of twice being collected gained merges, and must precipitate 4.41g; To be precipitated and dissolved in pH7.2,0.015mol/L potassium primary phosphate-dipotassium hydrogen phosphate damping fluid 15ml, gained solution is added the sephadex column Sephadex G-25 top balanced, wash-out is carried out with phosphate buffered saline buffer described in this step, collect containing protein and the elutriant of not liquid containing ammonium sulfate, concentrate with PEG4000, obtain concentrated solution 7.2ml;
3) in step 2) add 0.21gNaCl in gained concentrated solution and dissolve, gained solution is added the nickel ion chelating affinity column top balanced, carry out wash-out with pH6.0,0.02mol/L potassium primary phosphate-dipotassium hydrogen phosphate damping fluid, collect the elutriant containing protein;
4) with PEG4000 enrichment step 3) gained concentrated solution, lyophilize, obtains white powder 39.1mg.
Detect through proteins gel electrophoresis SDS-PAGE, products obtained therefrom molecular weight is identical with commercially available catalase standard substance, proves that product is catalase.
Embodiment 2
Bioactive enzyme is prepared from by following steps:
1) the 0.025mol/L citrate buffer solution 200ml of erythrocyte 100ml and pH4.8 after cleaning is mixed, with the broken erythrocyte of flash extracter, its operating parameters is: extract under 8000rpm condition, extract 20s at every turn, extract 2 times altogether, every minor tick 2min; Under 3200rpm condition, centrifugal 5min, gets supernatant liquor, obtains crude enzyme liquid 265ml;
2) in step 1) in gained crude enzyme liquid, add 132.5g ammonium sulfate and dissolve, leave standstill 40min, at 4 DEG C, centrifugal 10min under 8500rpm condition, separation of supernatant, collecting precipitation; In gained supernatant liquor, add 135g ammonium sulfate, leave standstill 40min, at 4 DEG C, centrifugal 25min under 9500rpm condition, collecting precipitation; The precipitation of twice being collected gained merges, and must precipitate 4.02g; To be precipitated and dissolved in pH6.9,0.02mol/L potassium primary phosphate-dipotassium hydrogen phosphate damping fluid 20.1ml, gained solution is added the sephadex column Sephadex G-25 top balanced, wash-out is carried out with phosphate buffered saline buffer described in this step, collect containing protein and the elutriant of not liquid containing ammonium sulfate, concentrate with PEG4000, obtain concentrated solution 10ml;
3) in step 2) add 0.3gNaCl in gained concentrated solution and dissolve, gained solution is added the chelating copper ions affinity column top balanced, carry out wash-out with pH6.2,0.015mol/L potassium primary phosphate-dipotassium hydrogen phosphate damping fluid, collect the elutriant containing protein;
4) with PEG4000 enrichment step 3) gained elutriant, lyophilize, obtains white powder 36.2mg.
Embodiment 3
Bioactive enzyme is prepared from by following steps:
1) the 0.015mol/L citrate buffer solution 50ml of erythrocyte 100ml and pH5.2 after cleaning is mixed, with the broken erythrocyte of flash extracter, its operating parameters is: extract under 3000rpm condition, extract 45s at every turn, extract 5 times altogether, every minor tick 1min; Under 3200rpm condition, centrifugal 15min, gets supernatant liquor, obtains crude enzyme liquid 139ml;
2) in step 1) in gained crude enzyme liquid, add 41.7g ammonium sulfate and dissolve, leave standstill 20min, at 0 DEG C, centrifugal 20min under 7500rpm condition, separation of supernatant, collecting precipitation; In gained supernatant liquor, add 50g ammonium sulfate, leave standstill 20min, at 4 DEG C, centrifugal 35min under 8500rpm condition, collecting precipitation; The precipitation of twice being collected gained merges, and must precipitate 3.88g; To be precipitated and dissolved in pH6.7,0.01mol/L potassium primary phosphate-dipotassium hydrogen phosphate damping fluid 4ml, gained solution is added the sephadex column Sephadex G-25 top balanced, wash-out is carried out with phosphate buffered saline buffer described in this step, collect containing protein and the elutriant of not liquid containing ammonium sulfate, concentrate with PEG4000, obtain concentrated solution 5ml;
3) in step 2) add 0.73gNaCl in gained concentrated solution and dissolve, gained solution is added the Zinc Ions Chelated affinity column top balanced, carry out wash-out with pH5.8,0.025mol/L potassium primary phosphate-dipotassium hydrogen phosphate damping fluid, collect the elutriant containing protein;
4) with PEG4000 enrichment step 3) gained elutriant, lyophilize, obtains white powder 34.8mg.
Comparative example 1
Compared with embodiment 1, difference is only, described step 2) replace in order to lower operation:
2) in step 1) in gained crude enzyme liquid, add 68g ammonium sulfate and dissolve, leave standstill 30min, at 0 DEG C, centrifugal 15min under 8000rpm condition, separation of supernatant, collecting precipitation; In gained supernatant liquor, add 65g ammonium sulfate, leave standstill 30min, at 4 DEG C, centrifugal 30min under 9000rpm condition, collecting precipitation; The precipitation of twice being collected gained merges, and must precipitate 4.41g; To be precipitated and dissolved in pH7.2,0.015mol/L potassium primary phosphate-dipotassium hydrogen phosphate damping fluid 15ml, be placed in dialysis tubing, conventionally ammonium sulfate is removed in dialysis, a dialysis buffer liquid is changed every 5 hours, after 50 hours, not containing ammonium sulfate in solution, concentrate with PEG4000, obtain concentrated solution.
Products therefrom is white powder 30.23mg.
Comparative example 2
Compared with embodiment 1, difference is only, step 3) described phosphate buffered saline buffer step 2) described phosphate buffered saline buffer replacement.
Products therefrom is white powder 34.69mg.
Experimental example: bioactive enzyme detects
1, sample is detected: commercially available catalase, as standard substance, detects embodiment 1 ~ 3 and comparative example 1,2 products therefrom.
2, purity detecting: adopt reversed-phased high performace liquid chromatographic, conveniently operate, reference standard product go out peak situation, determine the target absorption peak of sample, adopt peak area method counting yield purity.The present invention for stationary phase, with the acetonitrile-water containing 0.3% trifluoroacetic acid for moving phase, detects the purity of bioactive enzyme with C18 post.
3, Enzyme assay:, measure H at 240nm wavelength 2o 2by the decreasing value of absorbancy during bioactive enzyme catalytic decomposition; Enzyme activity unit is defined as 1min every milligram protein makes the decline enzyme amount of 0.001 unit of optical density(OD) be 1 enzyme activity unit under 240nm wavelength, and the activity of enzyme represents by specific activity (U/mg).
4, the purity of bioactive enzyme and the detected result of enzymic activity are in table 1.
Table 1: bioactive enzyme detected result
Sample Purity (%) Specific activity (U/mg)
Embodiment 1 92.33 13302.2
Embodiment 2 91.96 13256.9
Embodiment 3 90.26 13203.9
Comparative example 1 88.56 12987.6
Comparative example 2 63.16 11036.9
Standard substance -- 13249.0
From above detected result, the purity of bioactive enzyme provided by the invention is all greater than 88%, is better than comparative example 1 and is significantly better than comparative example 2; Bioactive enzyme specific activity provided by the invention is all not less than the specific activity of standard substance, is better than comparative example 1, and is significantly better than comparative example 2.
Through more known, the bulk properties of the product that the embodiment of the present invention 1 provides is best.
Although above with general explanation, embodiment and test, the present invention is described in detail, and on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims (7)

1. a bioactive enzyme, is characterized in that, described bioactive enzyme is prepared from by the method comprised the following steps:
1) by clean after erythrocyte mix with 0.015 ~ 0.025mol/L citrate buffer solution of pH4.8 ~ 5.2, broken erythrocyte, centrifuging and taking supernatant liquor, obtains crude enzyme liquid;
2) in step 1) add ammonium sulfate in gained crude enzyme liquid, leave standstill, centrifugal, collecting precipitation; To be precipitated and dissolved in 0.01 ~ 0.02mol/L phosphate buffered saline buffer of pH7.0 ~ 7.4, gained solution is added the sephadex column Sephadex G-25 top balanced, wash-out is carried out with phosphate buffered saline buffer described in this step, collect containing protein and the elutriant of not liquid containing ammonium sulfate, concentrated, obtain concentrated solution;
3) in step 2) add NaCl in gained concentrated solution, obtain the solution that NaCl concentration is 0.4 ~ 0.6mol/L, gained solution is added the affine capital end of the metal-chelating balanced, carry out wash-out with 0.015 ~ 0.025mol/L phosphate buffered saline buffer of pH 5.8 ~ 6.2, collect the elutriant containing protein;
4) by step 3) gained elutriant concentrate, lyophilize, obtains bioactive enzyme.
2. bioactive enzyme according to claim 1, is characterized in that, step 1) described damping fluid is the citric acid solution of concentration 0.02mol/L, pH5.0.
3. bioactive enzyme according to claim 1, is characterized in that, step 1) device of described broken erythrocyte is flash extracter, its operating parameters is: extract under 3000 ~ 8000rpm condition, each extraction 20 ~ 45s, extracts 2 ~ 5 times altogether, every minor tick 1 ~ 2min.
4. bioactive enzyme according to claim 1, it is characterized in that, described step 2) described in add ammonium sulfate, leave standstill, to be centrifugally specially: ammonium sulfate is dissolved in step 1 with mass volume ratio 0.3 ~ 0.5:1) in gained crude enzyme liquid, leave standstill 20 ~ 40min, at 0 ~ 4 DEG C, centrifugal 10 ~ 20min under 7500 ~ 8500rpm condition, separation of supernatant, collecting precipitation; Ammonium sulfate is added in gained supernatant liquor with mass volume ratio 0.7 ~ 0.9:1, leaves standstill 20 ~ 40min, at 0 ~ 4 DEG C, centrifugal 25 ~ 35min under 8500 ~ 9500rpm condition, collecting precipitation; The precipitation of twice being collected gained merges.
5. bioactive enzyme according to claim 1, is characterized in that, described bioactive enzyme is prepared from by the method comprised the following steps:
1) erythrocyte after cleaning is mixed with 0.015 ~ 0.025mol/L citrate buffer solution of pH4.8 ~ 5.2 mix with volume ratio 1:0.5 ~ 2, with the broken erythrocyte of flash extracter, the operating parameters of flash extracter is: extract under 3000 ~ 8000rpm condition, each extraction 20 ~ 45s, extract 2 ~ 5 times altogether, every minor tick 1 ~ 2min; Under 3000 ~ 3200rpm condition, centrifugal 5 ~ 15min, gets supernatant liquor, obtains crude enzyme liquid;
2) in step 1) in gained crude enzyme liquid, add 0.3 ~ 0.5g/ml ammonium sulfate, leave standstill 20 ~ 40min, at 0 ~ 4 DEG C, centrifugal 10 ~ 20min under 7500 ~ 8500rpm condition, separation of supernatant, collecting precipitation; Ammonium sulfate is added in gained supernatant liquor with mass volume ratio 0.7 ~ 0.9:1, leaves standstill 20 ~ 40min, at 0 ~ 4 DEG C, centrifugal 25 ~ 35min under 8500 ~ 9500rpm condition, collecting precipitation; The precipitation of twice being collected gained merges; Again precipitation is dissolved in the 0.01 ~ 0.02mol/L potassium primary phosphate-dipotassium hydrogen phosphate damping fluid of pH7.0 ~ 7.4 with mass volume ratio 1:1 ~ 5, gained solution is added the sephadex column Sephadex G-25 top balanced, wash-out is carried out with phosphate buffered saline buffer described in this step, collect containing protein and the elutriant of not liquid containing ammonium sulfate, concentrate with PEG4000, obtain concentrated solution;
3) in step 2) add NaCl in gained concentrated solution, obtain the solution that NaCl concentration is 0.4 ~ 0.6mol/L, gained solution is added the affine capital end of the metal-chelating balanced, carry out wash-out with the 0.015 ~ 0.025mol/L potassium primary phosphate-dipotassium hydrogen phosphate damping fluid of pH value 5.8 ~ 6.2, collect the elutriant containing protein;
4) with PEG4000 enrichment step 3) gained elutriant, lyophilize, obtains bioactive enzyme.
6. bioactive enzyme according to claim 1, is characterized in that, described bioactive enzyme is prepared from by the method comprised the following steps:
1) erythrocyte after cleaning is mixed with the 0.02mol/L citrate buffer solution of pH5.0 mix with volume ratio 1:1, with the broken erythrocyte of flash extracter, the operating parameters of flash extracter is: extract under 5000rpm condition, each extraction 30s, extract 3 times altogether, every minor tick 2min; Under 3000rpm condition, centrifugal 15min, gets supernatant liquor, obtains crude enzyme liquid;
2) in step 1) in gained crude enzyme liquid, add 0.4g/ml ammonium sulfate, leave standstill 30min, at 0 DEG C, centrifugal 15min under 8000rpm condition, separation of supernatant, collecting precipitation; Ammonium sulfate is added in gained supernatant liquor with mass volume ratio 0.8:1, leaves standstill 30min, at 0 DEG C, centrifugal 30min under 9000rpm condition, collecting precipitation; The precipitation of twice being collected gained merges; Precipitation is dissolved in the 0.015mol/L potassium primary phosphate-dipotassium hydrogen phosphate damping fluid of pH7.2 with mass volume ratio 1:5, gained solution is added the sephadex column Sephadex G-25 top balanced, wash-out is carried out with phosphate buffered saline buffer described in this step, collect containing protein and the elutriant of not liquid containing ammonium sulfate, concentrate with PEG4000, obtain concentrated solution;
3) in step 2) add NaCl in gained concentrated solution, obtain the solution that NaCl concentration is 0.5mol/L, gained solution is added the nickel ion chelating affinity column top balanced, carry out wash-out with pH6.0,0.02mol/L potassium primary phosphate-dipotassium hydrogen phosphate damping fluid, collect the elutriant containing protein;
4) with PEG4000 enrichment step 3) gained elutriant, lyophilize, obtains bioactive enzyme.
7. bioactive enzyme described in claim 1 ~ 6 any one is preparing the application in makeup.
CN201410851247.4A 2014-12-31 2014-12-31 Bio-active enzyme and extraction process thereof Pending CN104593339A (en)

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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
CN117089531A (en) * 2023-10-07 2023-11-21 佰东靶向(深圳)生物科技有限公司 Preparation method of ganoderma lucidum extraction active enzyme

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