CN103073627B - Method for microwave-assisted reverse micelle separation and purification of kidney bean agglutinin - Google Patents
Method for microwave-assisted reverse micelle separation and purification of kidney bean agglutinin Download PDFInfo
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Abstract
The invention discloses a method for microwave-assisted reverse micelle separation and purification of kidney bean agglutinin, and relates to a separation and purification method of the agglutinin, aiming at solving the problems of low ratio, high cost, more processes and long time for separation and purification of the kidney bean agglutinin. According to the method for the microwave-assisted reverse micelle separation and purification of the kidney bean agglutinin, the agglutinin is directly separated and purified from kidney bean agglutinin water extract; and the method comprises a pre-extraction process and a back extraction process. Under an auxiliary effect of microwave, water-soluble protein in kidney bean is quickly dissolved into the water extract, so that the dissolubility is greatly improved, and the leaching time is shortened. Based on a liquid-liquid extraction principle, the selectivity of extraction is improved, and high-efficiency pre-extraction and back extraction of the kidney bean agglutinin can be realized. By applying a reverse micelle extraction technology, a better separation and purification effect can be achieved with only the pre-extraction and back extraction steps; the method has simple steps and is convenient to operate; and the pre-treatment time can be greatly shortened in combination with microwave assistance so as to save the cost.
Description
Technical field
The present invention relates to a kind of separation purification method of lectin, relate in particular to a kind of directly method of separation and purification kidney bean lectin from water extraction liquid of inverse micelle abstraction technique of using.
Background technology
Lectin (lectin or agglutinin) is that a class has carbohydrate specificity, can be combined with the oligosaccharide structure of the special glycoprotein of cell surface, glycolipid, impels cell agglutination or saccharide complex to precipitate, the glycoprotein in nonimmune source or carbohydrate-binding protein.Lectin is of many uses, can be used for the evaluation of sugary high molecular separation and purification and sugar chain structure in biochemical research; Can single-minded identification cell surface signaling molecule in biological study, for selecting mutant clone, research cell mechanism and cell surface feature and Identifying micro-organisms; In immunology research, can urge cell mitogen, suppress the growth of the malignant cells such as tumour; Medically can identify blood group, diagnosis pathology cell and the tissue different with make single-minded ground of medicine target as carrier molecule.Therefore, lectin is a kind of important research tool in modern scientific research field, and the separation and purification of lectin has very tempting scientific research value and market outlook.
The research of lectin starts from Herman Stillmark in 1888 and chances on Ricin (Ricicn).So far the lectin of having found be take Plant lectins as main, nearly 1000 kinds, be distributed widely in the phyto-group that pulse family, Solanaceae, Euphorbiaceae, grass tree section, Liliaceae and Amaryllidaceae etc. are numerous, wherein abundant with the lectin kind in leguminous plants, existing more than 600 plant at present.China most areas has the advantage that kidney bean is wide in variety, output is high, the lectin high feature that has that content is high, blood coagulation is tired in its beans, and therefore, the separation of kidney bean lectin and purifying research have important realistic meaning.
At present, the separation purification method of lectin carries out according to the traditional separation method of protein substantially, generally first that extract is heavy or saltout through acid, then through negatively charged ion and cationic exchange chromatography repeatedly.In the situation that lectin sugar binding specificity is known, can utilize affinity chromatography to carry out separation and purification.Cheng Yuesheng etc. (1997) utilize the special hydrophobic binding site of lectin, adopt hydrophobic interaction chromatography separation to obtain soybean agglutinin.200510011480.2 by the ammonium sulfate precipitation of different saturation and the ion-exchange chromatography of multistep, obtain a kind of agglutinin protein of astragalus root.200610097429.2 by ultrasound-enhanced acid carry, saltout, the technique means such as thermal treatment, ultrafiltration, affinity chromatography obtains wheat germ agglutinin.201110114904.3 disclose a kind of affinity chromatography filler D-GalN-FF-sepharose4B of purifying soybean agglutinin.201110025266.8 purify and obtain a kind of Malania oleifera lectin by methods such as ammonium sulfate precipitation, ultrafiltration desalination and concentrated, ion exchange chromatographies.But above method also not yet breaks away from that the complex operation, the treatment capacity that exist in lectin separation and purification process are low, loss is large and the weak point of length consuming time (being generally 1～3 day).Therefore, set up that lectin separating and purifying technology is imperative fast and efficiently.
In recent years, inverse micelle abstraction technique is easy and simple to handle with it, cost is low, green, pollution-free, percentage extraction is high, mild condition, solvent can Reusabilities and be difficult for causing protein-denatured advantage and the extensive concern that is subject to various countries researchist.Reverse micelle is the nanoscale molecular aggregate (10-100nm) of specific surfactant molecule spontaneous formation in nonsurfactant system, is generally W/O form.Inverse micelle abstraction technique generally includes front extraction and the two step processes of reextraction.In front extraction process, protein molecule is diffused into water and organic phase interface, by controlling the relevant technological condition of extraction process, at interface formation, contain the reverse micelle system of specific objective protein molecule, the reverse micelle that contains protein spreads and leaves interface in organic phase.In reextraction process, by changing relevant technological condition, the reverse micelle that contains protein bursts apart at water and organic phase interface, and specific objective albumen is from interfacial diffusion to aqueous solution main body.Hou Yufang etc. (2010) and Liu Yanyan etc. (2011) be take red kidney bean fine powder as raw material, utilize inverse micelle abstraction technique to extract phytohemagglutinin under solid-liquid state.But solid-liquid inverse micelle abstraction has the limitation of separation and purification multiplying power low (2～5 times), specific protein poor selectivity, and technology based on liquid-liquid reverse micelle technology extraction kidney bean lectin there is no systematicness report or patent, more without relevant report or the patent of microwave-assisted.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, solve the problem that kidney bean lectin separation and purification multiplying power is low, cost is high, technique is many, the time is grown, provide a kind of high-level efficiency, easy handling, cost is low and the method for the microwave-assisted reverse micelle separation and purification kidney bean lectin of safety and environmental protection.
The method of the microwave-assisted reverse micelle separation and purification kidney bean lectin that the present invention proposes, is direct separation and purification lectin from kidney bean lectin water extraction liquid, comprises front extraction process and reextraction process, and as shown in Figure 1, concrete steps are as follows:
(1) kidney bean lectin water extraction liquid preparation: described kidney bean lectin water extraction liquid is kidney bean abrasive dust, cross 40～100 mesh sieves, be dissolved in 5～50 volumes times aqueous solution, microwave-assisted lixiviate 5～60min at 10～60 ℃, power is 150～800W, lixiviate, by 8000～15000rpm, 4 ℃ of centrifugal 10～20min, is got supernatant liquor;
Described kidney bean kind is the kidney bean kinds such as red kidney bean, red coloured kidney bean, milk coloured kidney bean, black kidney bean, Semen Phaseoli Vulgaris and yellow kidney bean;
The described aqueous solution is distilled water, physiological saline, phosphate buffer soln or Tris-HCl buffered soln, and described buffer concentration is 10～50mM, and described pH value of water solution is 6.0～8.0;
25 ℃ of described lixiviate microwave-assisted condition optimization temperature, power 325W, time 30min.
(2) inverse micellar solution preparation: the component of described inverse micellar solution comprises tensio-active agent, organic solvent and water;
Described tensio-active agent is a kind of in aniorfic surfactant, cationic surfactant or nonionic surface active agent, preferred version is aniorfic surfactant or cationic surfactant, preferred succinic acid 2-the ethylhexyl of described aniorfic surfactant sodium sulfonate (AOT), the preferred cetrimonium bronmide/cetyl trimethyl of described cationic surfactant amine bromine (CTAB);
The concentration that described tensio-active agent is dissolved in organic solvent is 15～300mM;
Described moisture addition is to regulate W
ovalue (reverse micelle water ratio) is foundation 15～35;
Described organic solvent is n-hydro carbons (C
6～C
10), the one or two or more in pentane, hexanaphthene, normal hexane, octane, octane-iso, hexanol and butanols, preferably organic solvent is octane-iso, preferred co-solvents is one or both of hexanol and butanols, it is 5～20 % that preferred co-solvents is added volume ratio;
The preparation steps of described inverse micellar solution is: in proportion tensio-active agent is joined in organic solvent, be stirred to tensio-active agent and dissolve completely, add the water of certain amount, make system W
ovalue is in optimum range, and standing gained clear solution is inverse micellar solution.
(3) front extraction process: regulate kidney bean lectin water extraction liquid pH value and ionic strength, kidney bean lectin water extraction liquid is mixed with described inverse micellar solution, shaking table concussion mixes after certain hour, and the centrifugal layering of 1500～5000rpm, gets upper organic phase;
Described kidney bean lectin water extraction liquid pH value is 4.0～6.0 in aniorfic surfactant reverse micelle system, preferably 5.6; In cationic surfactant reverse micelle system, be 6.0～10.0, preferably 8.0;
Described kidney bean lectin water extraction liquid ionic strength is 20～200mM, the preferred NaCl of salt ion type, preferred 80mM in aniorfic surfactant reverse micelle system, preferred 100mM in aniorfic surfactant reverse micelle system;
Described kidney bean lectin water extraction liquid is that kidney bean lectin water extraction liquid is long-pending: inverse micellar solution volume=1:1～1:50 with inverse micellar solution mixed volume ratio;
Described mixing time is 5～40min.
(4) reextraction process: described upper organic phase is mixed with the back extraction buffered soln with certain ionic strength, shaking table concussion mixes after certain hour, and the centrifugal layering of 1500～5000rpm, takes off a layer water, through desalination, lyophilize, obtain the kidney bean lectin after separation and purification.
Described back extraction buffered soln is preferably phosphoric acid salt buffer solution or Tris-HCl buffered soln in aniorfic surfactant reverse micelle system, and concentration is 10～50mM, and pH value is 6.0～10.0; Described back extraction buffered soln is that You selects Lin Suan Yan – citric acid solution in cationic surfactant reverse micelle system, and concentration is 10～50mM, and pH value is 3.5～5.5;
Described back extraction buffered soln ionic strength is 100～1000mM, the preferred KCl of salt ion type or KBr, preferably 500mM;
Described reextraction buffered soln auxiliary agent is ethanol, propyl alcohol or butanols, preferred butanols, and it is 0～10 % that the buffered soln auxiliary agent of preferably stripping adds volume ratio;
Described upper organic phase is upper strata water with back extraction buffered soln mixed volume ratio: back extraction buffered soln=1:1～1:50;
Described mixing time is 5～40min.
Kidney bean lectin separation and purification efficiency rating of the present invention is:
Compared with prior art, the invention has the advantages that: the present invention, under microwave-assisted effect, makes water-soluble protein in kidney bean dissolve in rapidly water extraction liquid, has greatly improved solubleness, has shortened extraction time.The present invention is based on liquid-liquid extraction principle, by extraction process parameters such as regulation system pH value, ionic strength, phase volume ratio and reaction times, improved the selectivity of extraction, can realize the efficient front extraction of kidney bean lectin and strip.Application inverse micelle abstraction technique only needs front extraction and two steps of stripping can reach good separation and purification effect, and step is simple, easy to operate, can greatly shorten the pre-treatment time with microwave-assisted set, cost-saving.
The present invention has following beneficial effect:
1, the inventive method required time is short, low for equipment requirements, and implementation cost is little, it is fast and convenient to operate, being easy to serialization production and industrialization amplifies, separation and purification and concentrated can simultaneously completing, protein extraction rate is between 20 %～55 %, and kidney bean lectin purifying multiplying power can reach more than 8 times.
2, kidney bean lectin of the present invention is protected by tensio-active agent and water molecules, is difficult for inactivation, and purity is high, and the follow-up simple ion exchange chromatography of a step or the chromatography of only needing can obtain high sterling.
3, the present invention's extraction solvent used all can reuse, cost-saving, reduces environmental pollution, is the biologically active substance extracting method of a kind of green, environmental protection.
4, the prepared kidney bean lectin product of the present invention, can reach the test requirements document of biological level, can be widely used in blood group checking, short cell mitogen, suppress, in the biomedical engineering researchs such as malignant cell growth, to be with a wide range of applications and market potential.
Accompanying drawing explanation
Fig. 1 is the process flow sheet of the inventive method separation and purification kidney bean lectin;
Fig. 2 is SDS-polypropylene amine gel electrophoresis analysis figure of the present invention, wherein: 1 is standard protein molecule Marker; 2 is anionic reverse micelle separation and purification gained kidney bean lectin sample; 3 is cationic reverse micelle separation and purification gained kidney bean lectin sample; 4 is kidney bean lectin water extraction liquid.
Embodiment
Below in conjunction with preferred embodiment, the present invention is further illustrated; but do not limit to so; every technical solution of the present invention is modified or is equal to replacement, and not departing from the spirit and scope of technical solution of the present invention, all should be encompassed in protection scope of the present invention.
Embodiment 1:
A method for aniorfic surfactant microwave-assisted reverse micelle separation and purification kidney bean lectin of the present invention, specifically comprises the following steps:
(1) kidney bean lectin water extraction liquid preparation:
Select high-quality kidney bean abrasive dust, cross 80 mesh sieves, be dissolved in 10 times of (volume) phosphate buffer solns (pH7.2,25mM) 25 ℃ of microwave-assisted lixiviate 30min(325W), 12000rpm, 4 ℃ of centrifugal 20min, get supernatant liquor.
(2) inverse micellar solution preparation:
Aniorfic surfactant succinic acid 2-ethylhexyl sodium sulfonate (AOT) is dissolved in octane-iso completely to the inverse micellar solution that compound concentration is 130mM.Add certain water to reverse micelle system W
ovalue is 25.In the present embodiment, solubility promoter is butanols, and it is 10 % that solubility promoter adds volume ratio.
(3) front extraction process:
Regulate the pH=5.6 of kidney bean lectin water extraction liquid, with NaCl, regulate the ionic strength of kidney bean lectin water extraction liquid to 80mM.Kidney bean lectin water extraction liquid and inverse micellar solution mix with the volume ratio of 1:1, are placed on shaking table the velocity fluctuation mixing 15min with 200rpm.By the centrifugal layering of mixed solution 3000rpm, 15min, get upper organic phase.
(5) reextraction process:
Back extraction buffered soln is phosphate buffer soln (pH8.0,25mM), regulates the ionic strength of back extraction buffered soln to 500mM with KCl.Back extraction buffered soln and upper organic phase are mixed with the volume ratio of 1:1, add reextraction buffered soln auxiliary agent butanols, and adding volume ratio is 5 %.Mixing solutions is placed on shaking table with the velocity fluctuation mixing 15min of 200rpm, and by the centrifugal layering of mixed solution 3000rpm, 15min, lower floor's water is the kidney bean lectin aqueous solution after separation and purification.Through desalination, lyophilize, obtain the kidney bean lectin after separation and purification.
Employing bicinchoninic acid method (BCA method) records the amount of protein, and the kidney bean lectin blood coagulation before and after hemagglutination test records reverse micelle separation and purification is tired.From measurement result, microwave-assisted reverse micelle separation and purification kidney bean lectin effect is fine, and protein extraction rate is 43.5%, and kidney bean lectin separation and purification multiplying power can reach 9.3 left and right.
Embodiment 2:
A method for cationic surfactant microwave-assisted reverse micelle separation and purification kidney bean lectin of the present invention, specifically comprises the following steps:
(1) kidney bean lectin water extraction liquid preparation:
Select high-quality kidney bean abrasive dust, cross 80 mesh sieves, be dissolved in 10 times of (volume) phosphate buffer solns (pH7.2,25mM) 25 ℃ of microwave-assisted lixiviate 30min(325W), 12000rpm, 4 ℃ of centrifugal 20min, get supernatant liquor.
(2) inverse micellar solution preparation:
Cationic surfactant cetrimonium bronmide/cetyl trimethyl amine bromine (CTAB) is dissolved in octane-iso completely to the inverse micellar solution that compound concentration is 150mM.Add certain water to reverse micelle system W
ovalue is 25.In the present embodiment, solubility promoter is butanols, and it is 10 % that solubility promoter adds volume ratio.
(3) front extraction process:
Regulate the pH=8.0 of kidney bean lectin water extraction liquid, with NaCl, regulate the ionic strength of kidney bean lectin water extraction liquid to 100mM.Kidney bean lectin water extraction liquid and inverse micellar solution mix with the volume ratio of 1:1, are placed on shaking table the velocity fluctuation mixing 15min with 200rpm.By the centrifugal layering of mixed solution 3000rpm, 15min, get upper organic phase.
(4) reextraction process:
Back extraction buffered soln is Lin Suan Yan – citric acid solution (pH4.5,25mM), regulates the ionic strength of back extraction buffered soln to 500mM with KBr.Back extraction buffered soln and upper organic phase are mixed with the volume ratio of 1:1, add reextraction buffered soln auxiliary agent butanols, and adding volume ratio is 5%.Mixing solutions is placed on shaking table with the velocity fluctuation mixing 15min of 200rpm, and by the centrifugal layering of mixed solution 3000rpm, 15min, lower floor's water is the kidney bean lectin aqueous solution after separation and purification.Through desalination, lyophilize, obtain the kidney bean lectin after separation and purification.
Employing bicinchoninic acid method (BCA method) records the amount of protein, and the kidney bean lectin blood coagulation before and after hemagglutination test records reverse micelle separation and purification is tired.From measurement result, microwave-assisted reverse micelle separation and purification kidney bean lectin effect is fine, and protein extraction rate is 48.5%, and kidney bean lectin separation and purification multiplying power can reach 8.8 left and right.
According to embodiment 1 and embodiment 2 definite best reverse micelle separation and purification parameters, test, adopt SDS-polyacrylamide gel electrophoresis to analyze (Fig. 2) to protein ingredient known, the kidney bean lectin water extraction of comparing liquid sample (swimming lane 4), the kidney bean lectin sample (swimming lane 2) of aniorfic surfactant microwave-assisted reverse micelle separation and purification and the kidney bean lectin sample (swimming lane 3) of cationic surfactant microwave-assisted reverse micelle separation and purification are single band.
In sum, the method effect of microwave-assisted reverse micelle separation and purification kidney bean lectin involved in the present invention is remarkable.
Claims (7)
1. a method for microwave-assisted reverse micelle separation and purification kidney bean lectin, is characterized in that described method, for direct separation and purification lectin from kidney bean lectin water extraction liquid, comprises front extraction process and reextraction process, and concrete steps are as follows:
(1) kidney bean lectin water extraction liquid preparation: kidney bean abrasive dust, cross 40～100 mesh sieves, be dissolved in 5～50 volumes times aqueous solution, microwave-assisted lixiviate 5～60min at 10～60 ℃, power is 150～800W, and lixiviate, by 8000～15000rpm, 4 ℃ of centrifugal 10～20min, is got supernatant liquor;
(2) inverse micellar solution preparation: tensio-active agent is joined in organic solvent, be stirred to tensio-active agent and dissolve completely, add the water of certain amount, make system W
ovalue is in 15～35 scopes, and standing gained clear solution is inverse micellar solution, and its concentration is 15～300mM; The described aqueous solution is distilled water, physiological saline, phosphate buffer soln or Tris-HCl buffered soln, and pH value of water solution is 6.0～8.0, and buffer concentration is 10～50mM; Described tensio-active agent is aniorfic surfactant, cationic surfactant or nonionic surface active agent; Described organic solvent is a kind of, two or more the mixture in hydro carbons, pentane, hexanaphthene, normal hexane, octane, octane-iso, hexanol and butanols;
(3) front extraction process: regulating kidney bean lectin water extraction liquid pH value is 4.0～10.0, ionic strength is 20～200mM, the ratio that is 1:1～1:50 with inverse micellar solution according to volume ratio by kidney bean lectin water extraction liquid is mixed after 5～40min, the centrifugal layering of 1500～5000rpm, gets upper organic phase;
(4) reextraction process: the ratio that is 1:1～1:50 with the back extraction buffered soln with certain ionic strength according to volume ratio by above-mentioned upper organic phase is mixed after 5～40min, the centrifugal layering of 1500～5000rpm, take off a layer water, through desalination, lyophilize, obtain the kidney bean lectin after separation and purification.
2. the method for microwave-assisted reverse micelle separation and purification kidney bean lectin according to claim 1, is characterized in that described kidney bean kind is red kidney bean, red coloured kidney bean, milk coloured kidney bean, black kidney bean, Semen Phaseoli Vulgaris or yellow kidney bean.
3. the method for microwave-assisted reverse micelle separation and purification kidney bean lectin according to claim 1, is characterized in that in front extraction process, and regulate kidney bean lectin water extraction liquid pH value is 4.0～6.0 in aniorfic surfactant reverse micelle system; In cationic surfactant reverse micelle system, be 6.0～10.0.
4. the method for microwave-assisted reverse micelle separation and purification kidney bean lectin according to claim 1, is characterized in that described back extraction buffered soln is phosphate buffer soln, Tris-HCl buffered soln or Lin Suan Yan – citric acid solution.
5. the method for microwave-assisted reverse micelle separation and purification kidney bean lectin according to claim 4, it is characterized in that described back extraction buffered soln is phosphate buffer soln or Tris-HCl buffered soln in aniorfic surfactant reverse micelle system, concentration is 10～50mM, and pH value is 6.0～10.0; Described back extraction buffered soln is Lin Suan Yan – citric acid solution in cationic surfactant reverse micelle system, and concentration is 10～50mM, and pH value is 3.5～5.5.
6. the method for microwave-assisted reverse micelle separation and purification kidney bean lectin according to claim 4, is characterized in that described back extraction buffered soln ionic strength is 100～1000mM.
7. according to the method for the microwave-assisted reverse micelle separation and purification kidney bean lectin described in claim 4,5 or 6, the auxiliary agent that it is characterized in that described reextraction buffered soln is ethanol, propyl alcohol or butanols, and it is 0～10 % that reextraction buffered soln auxiliary agent adds volume ratio.
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| CN106632042B (en) * | 2016-12-15 | 2019-10-01 | 中国农业科学院麻类研究所 | The method extracted and purify cucoline |
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| CN110755601B (en) * | 2019-10-31 | 2023-06-23 | 云南康洲生物科技有限公司 | Kidney bean lectin plant medicine composition for treating HIV/AIDS immune reconstruction defect and preparation method and application thereof |
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