CN108892702A - A kind of isolation and purification method of glomalin related soil albumen - Google Patents
A kind of isolation and purification method of glomalin related soil albumen Download PDFInfo
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- CN108892702A CN108892702A CN201810597831.XA CN201810597831A CN108892702A CN 108892702 A CN108892702 A CN 108892702A CN 201810597831 A CN201810597831 A CN 201810597831A CN 108892702 A CN108892702 A CN 108892702A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
- C07K1/303—Extraction; Separation; Purification by precipitation by salting out
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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Abstract
The invention discloses a kind of isolation and purification methods of glomalin related soil albumen, this method according to the characteristic of glomalin related soil albumen it is comprehensive used it is sour sink, saltout, ultrafiltration, chromatography, the techniques such as chromatogram purification and activated carbon adsorption, eliminate the interference of humic acid and other impurity in albumen, the purity of glomalin related soil albumen greatly improved, the advantages such as this method also has a product yield high, and production cost is low.
Description
Technical field
The present invention relates to the preparation methods of protein, the especially isolation and purification method of glomalin related soil albumen.
Background technique
Glomalin related soil albumen (GRSP) is a kind of mycetogenetic glycoprotein substance of arbuscular mycorrhiza (AM), property
Matter is stablized, and not soluble in water, high temperature resistant plays an important role in soil agreegate is formed, and can be improved the stabilization of soil agreegate
Property.As a kind of organic matter, GRSP can provide a large amount of carbon sources for soil, can adsorb the heavy metal in fixing soil, counterweight
Metallic pollution geobiont repair plays an important role.The discovery humic acid of existing research at present is the main interfering substance in GRSP,
The presence of humic acid has seriously affected GRSP property and function.The purifying of GRSP is mainly passed through at simple physics at present
Reason carries out preliminary purification, can partially remove the interference of humic acid, obtained purity of protein is very low, affects further answering for GRSP
With, therefore the GRSP for how obtaining high-purity is current urgent problem.
Summary of the invention
The object of the present invention is to provide a kind of isolation and purification methods of high-purity glomalin related soil albumen.
Above-mentioned purpose is achieved through the following technical solutions：
A kind of isolation and purification method of glomalin related soil albumen, includes the following steps：
1) it extracts：Pedotheque is subjected to high temperature extraction with the sodium citrate solution of 10-100mM, collects extracting solution；
2) acid is heavy：It is adjusted with acid the pH to 2-2.5 of soil extract, precipitating is collected by centrifugation；
3) it saltouts：Precipitating is dissolved with sodium hydroxide solution, ammonium sulfate is then added into solution, makes the saturation of ammonium sulfate
Degree reaches 50-80%, and precipitating is collected by centrifugation after saltouing；
4) ultrafiltration：Precipitating is dissolved with sodium hydroxide solution, the ultrafiltration membrance filter for being 30-100kDa with molecular cut off,
It obtains that liquid is concentrated by ultrafiltration；
5) it chromatographs：ConA affinity column on liquid will be concentrated by ultrafiltration, first eluted with equilibration buffer, then with containing 0.1-
The equilibration buffer of 0.2M agglutinin specific sugar elutes and collects eluent, and the equilibration buffer is 5-15mM Tris-
HCl, 0.5-2mM MgCl2, 0.5-2mM CaCl2, 0.1-0.2M NaCl, pH 7-8；
6) chromatogram purification：It by molecular volume exclusion chromatography column on eluent, is eluted with mobile phase, ultraviolet detection analyzes
Peak situation simultaneously collects eluent, and the mobile phase is 0.01-0.05M NaH2PO4Solution；
7) active carbon decoloring：Active carbon is added into eluent to decolourize, is freeze-dried after filtering, obtains high-purity sacculus
Mycin related soil albumen.
Preferably, the concentration of the sodium citrate solution is 50mM.
Preferably, it is adjusted with acid the pH to 2.3 of soil extract, the acid is dilute hydrochloric acid.
Preferably, the concentration of the sodium hydroxide solution is 0.05-0.2M.
Preferably, the saturation degree of the ammonium sulfate is 70%.
Preferably, the equilibration buffer is 10mM Tris-HCl, 1mM MgCl2, 1mM CaCl2, 0.15M NaCl, pH
It is 8.
Preferably, the agglutinin specific sugar is Alpha-Methyl-D-MANNOSE, and content is 0.15M.
Preferably, the Detection wavelength of the ultraviolet detection is 280nm
Preferably, the mobile phase is 0.02M NaH2PO4Solution.
Saltouing is the method for more common protein precipitation, its biggest advantage is will not to make protein denaturation, when to albumen
When inorganic salts are added in matter solution, protein surface hydrated sheath can rupture and since the change surface charge of ionic strength can be big
Amount neutralizes, and more albumen solubility is caused to reduce, makes to assemble and precipitate between protein molecule, and the present invention is by the way that sulfuric acid is added
Ammonium forms ammonium sulfate saturated solution and carries out protein precipitation.Ultrafiltration is that the ultrafiltration of corresponding hole is made according to the difference of molecular weight
Film, protein can be retained when passing through ultrafiltration membrane according to molecular weight, while play the role of concentration.ConA agglutinin is affine layer
Analysis is the Characteristics of glycoproteins in view of GRSP, since ConA agglutinin can adsorb glycoprotein to separate target saccharic composition, is first used
Equilibration buffer elutes nonspecific proteins, then with the elution containing Alpha-Methyl-D-MANNOSE equilibration buffer in conjunction with agglutinin
Glycoprotein.It is purified according to the molecular weight characteristics of GRSP using molecular volume exclusion chromatography, separation principle is molecular weight
Difference, macromolecular first goes out, is eluted after small molecule.Activated carbon adsorption is mainly the effect decolourized, adsorptive hindrance substance
Humic acid reduces impurity interference.
The invention has the advantages that：
The present invention can from soil isolated high-purity glomalin related soil albumen, reduce foreign protein and
The advantages such as the interference of humic acid, the present invention also have a product yield high, and production cost is low.
Specific embodiment
The present invention is described in detail below by specific embodiment.
Embodiment 1
(1) it extracts：The pedotheque for weighing levigate sieving, is added the 20mM sodium citrate solution of 10 times of weight, and high temperature adds
Thermal extraction 1h, is collected by centrifugation extracting solution, repeats the above steps, until extracting solution is colorless and transparent；
(2) acid is heavy：It is slowly added to dilute hydrochloric acid into extracting solution, adjusts pH to 2, precipitating is collected by centrifugation after ice bath；
(3) it saltouts：Precipitating 0.05M NaOH solution is dissolved, ammonium sulfate is then added, reaches the saturation degree of ammonium sulfate
To 80%, precipitating is collected by centrifugation after saltouing；
(4) ultrafiltration：Precipitating after saltouing 0.05M NaOH solution is dissolved, the ultrafiltration for being 30kDa with molecular cut off
Film filtering obtains that liquid is concentrated by ultrafiltration；
(5) it chromatographs：ConA affinity column (filler is ConA Sepharose 4B) is first balanced with buffer, then
Loading first uses equilibration buffer (5mM Tris-HCl, 0.5mM MgCl2, 2mM CaCl2, 0.2M NaCl, pH7.0) and elution, then
It is eluted with containing 0.1M Alpha-Methyl-D-MANNOSE equilibration buffer, collection contains Alpha-Methyl-D-MANNOSE eluent；
(6) chromatogram purification：Molecular volume exclusion chromatography column (SEC s2000, specification on the eluent that step (5) is obtained
For 300 × 7.8mm, 5 μm), with mobile phase (0.01M NaH2PO4Solution) elution, ultraviolet detection wavelength is 280nm, according to appearance
Situation collects eluent；
(7) active carbon decoloring：Active carbon is added in the eluent obtained to step (6) to decolourize, is freezed after filtering dry
It is dry to get.
Embodiment 2
(1) it extracts：The 50mM sodium citrate solution of 8 times of weight is added in the pedotheque for weighing levigate sieving, high-temperature heating
1h is extracted, extracting solution is collected by centrifugation, repeats the above steps, until extracting solution is colorless and transparent；
(2) acid is heavy：It is slowly added to 0.1M dilute hydrochloric acid into extracting solution, adjusts pH to 2.3, precipitating is collected by centrifugation after ice bath；
(3) it saltouts：Precipitating 0.1M NaOH solution is dissolved, ammonium sulfate is then added, reaches the saturation degree of ammonium sulfate
70%, precipitating is collected by centrifugation after saltouing；
(4) ultrafiltration：Precipitating after saltouing 0.1M NaOH solution is dissolved, the ultrafiltration membrane for being 50kDa with molecular cut off
Filtering obtains that liquid is concentrated by ultrafiltration；
(5) it chromatographs：ConA affinity column is first balanced with buffer, then loading, first uses equilibration buffer (10mM
Tris-HCl, 1mM MgCl2, 1mM CaCl2, 0.15M NaCl, pH8.0) elution, then with contain 0.15M Alpha-Methyl-D- sweet dew
The equilibration buffer elution of sugar, collection contain Alpha-Methyl-D-MANNOSE eluent；
(6) chromatogram purification：Molecular volume exclusion chromatography column on the eluent that step (5) is obtained, with mobile phase (0.02M
NaH2PO4Solution) elution, ultraviolet detection wavelength is 280nm, collects eluent according to appearance situation；
(7) active carbon decoloring：Active carbon is added in the eluent obtained to step (6) to decolourize, is freezed after filtering dry
It is dry to get.
Embodiment 3
(1) it extracts：The 70mM sodium citrate solution of 6 times of weight is added in the pedotheque for weighing levigate sieving, high-temperature heating
1h is extracted, extracting solution is collected by centrifugation, repeats the above steps, until extracting solution is colorless and transparent；
(2) acid is heavy：It is slowly added to dilute hydrochloric acid into extracting solution, adjusts pH to 2.5, precipitating is collected by centrifugation after ice bath；
(3) it saltouts：Precipitating 0.2M NaOH solution is dissolved, ammonium sulfate is then added, reaches the saturation degree of ammonium sulfate
60%, precipitating is collected by centrifugation after saltouing；
(4) ultrafiltration：Precipitating after saltouing 0.2M NaOH solution is dissolved, the ultrafiltration for being 100kDa with molecular cut off
Film filtering obtains that liquid is concentrated by ultrafiltration；
(5) it chromatographs：ConA affinity column is first balanced with buffer, then loading, first uses equilibration buffer (15mM
Tris-HCl, 2mM MgCl2, 0.5mM CaCl2, 0.1M NaCl, pH7.5) elution, then with contain 0.2M Alpha-Methyl-D- sweet dew
The equilibration buffer elution of sugar, collection contain Alpha-Methyl-D-MANNOSE eluent；
(6) chromatogram purification：Molecular volume exclusion chromatography column on the eluent that step (5) is obtained, with mobile phase (0.05M
NaH2PO4Solution) elution, ultraviolet detection wavelength is 280nm, collects eluent according to appearance situation；
(7) active carbon decoloring：Active carbon is added in the eluent obtained to step (5) to decolourize, is freezed after filtering dry
It is dry to get.
It calculates product yield (products weight/pedotheque weight × 100%), detects glomalin related soil in product
The content of albumen, as a result see the table below：
| Yield (%) | Content (%) | |
| Embodiment 1 | 2.4 | 58 |
| Embodiment 2 | 2.8 | 83 |
| Embodiment 3 | 2 | 66 |
As can be seen from the above results, the glomalin related soil albumen of high-purity can be obtained using the present invention.
Claims (9)
1. a kind of isolation and purification method of glomalin related soil albumen, it is characterised in that include the following steps：
1) it extracts：Pedotheque is subjected to high temperature extraction with the sodium citrate solution of 10-100mM, collects extracting solution；
2) acid is heavy：It is adjusted with acid the pH to 2-2.5 of soil extract, precipitating is collected by centrifugation；
3) it saltouts：Precipitating is dissolved with sodium hydroxide solution, ammonium sulfate is then added into solution, reaches the saturation degree of ammonium sulfate
To 50-80%, precipitating is collected by centrifugation after saltouing；
4) ultrafiltration：Precipitating is dissolved with sodium hydroxide solution, the ultrafiltration membrance filter for being 30-100kDa with molecular cut off obtains
Liquid is concentrated by ultrafiltration；
5) it chromatographs：ConA affinity column on liquid will be concentrated by ultrafiltration, first eluted with equilibration buffer, then coagulated with containing 0.1-0.2M
The equilibration buffer for collecting plain specific sugar elutes and collects eluent, and the equilibration buffer is 5-15mM Tris-HCl, 0.5-
2mM MgCl2, 0.5-2mM CaCl2, 0.1-0.2M NaCl, pH 7-8；
6) chromatogram purification：It by molecular volume exclusion chromatography column on eluent, is eluted with mobile phase, ultraviolet detection, analyzes appearance feelings
Condition simultaneously collects eluent, and the mobile phase is 0.01-0.05M NaH2PO4Solution；
7) active carbon decoloring：Active carbon is added into eluent to decolourize, is freeze-dried after filtering, obtains high-purity glomalin
Related soil albumen.
2. the isolation and purification method of glomalin related soil albumen as described in claim 1, it is characterised in that：The lemon
The concentration of acid sodium solution is 50mM.
3. the isolation and purification method of glomalin related soil albumen as described in claim 1, it is characterised in that：It is adjusted with acid
The pH to 2.3 of soil extract, the acid are dilute hydrochloric acid.
4. the isolation and purification method of glomalin related soil albumen as described in claim 1, it is characterised in that：The hydrogen-oxygen
The concentration for changing sodium solution is 0.05-0.2M.
5. the isolation and purification method of glomalin related soil albumen as described in claim 1, it is characterised in that：The sulfuric acid
The saturation degree of ammonium is 70%.
6. the isolation and purification method of glomalin related soil albumen as described in claim 1, it is characterised in that：The balance
Buffer is 10mM Tris-HCl, 1mM MgCl2, 1mM CaCl2, 0.15M NaCl, pH 8.
7. the isolation and purification method of glomalin related soil albumen as described in claim 1, it is characterised in that：The agglutination
Plain specific sugar is Alpha-Methyl-D-MANNOSE, and content is 0.15M.
8. the isolation and purification method of glomalin related soil albumen as described in claim 1, it is characterised in that：It is described ultraviolet
The Detection wavelength of detection is 280nm.
9. the isolation and purification method of glomalin related soil albumen as described in claim 1, it is characterised in that：The flowing
It is mutually 0.02M NaH2PO4Solution.
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2018
- 2018-06-11 CN CN201810597831.XA patent/CN108892702A/en active Pending
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Inventor after: Chen Xiuhua Inventor after: Guo Xuejia Inventor after: Ji Lingling Inventor after: Ma Qilian Inventor after: Tan Wenfeng Inventor before: Chen Xiuhua Inventor before: Guo Xuejia Inventor before: Ji Lingling Inventor before: Ma Qilian |
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Application publication date: 20181127 |