CN101054407A - Method for separating and purifying hydrophobin - Google Patents
Method for separating and purifying hydrophobin Download PDFInfo
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- CN101054407A CN101054407A CN 200710099646 CN200710099646A CN101054407A CN 101054407 A CN101054407 A CN 101054407A CN 200710099646 CN200710099646 CN 200710099646 CN 200710099646 A CN200710099646 A CN 200710099646A CN 101054407 A CN101054407 A CN 101054407A
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Abstract
The invention discloses hydrophobic protein separation and purification method. The method comprise: soaking solid substrate with hydrophobic particle on surface in hydrophobic protein solution, then taking the substrate out of the hydrophobic protein solution, rinsing, carrying out supersonic oscillation in container filled with water, then taking the substrate out of the solution, obtaining purified hydrophobic protein solution. The principle that hydrophobic protein can adsorb at hydrophobic substance surface, and hydrophobic particle has hetero phase nucleation growth effect to hydrophobic protein can be utilized to achieve the aim for separating and purifying the hydrophobic protein. The inventive method is simply and conveniently operated, and cost is low. The hydrophobic substance surface can be repeatedly used which is a merit for industrial production. The present invention will have a broad prospect in hydrophobic protein separation and purification field.
Description
Technical field
The present invention relates to proteic separating and purifying method, particularly relate to the separating and purifying method of hydrophobin.
Background technology
Epiphyte hydrophobic protein (hydrophobin) is extensively to be present in the small-sized protein (containing 100 amino-acid residues approximately) that the cluster in the filamentous fungus has unusual Physiology and biochemistry character, does not find as yet in other organism.Solvability feature according to hydrophobicity collection of illustrative plates and its component film can be divided into hydrophobin two classes: I type and II type.In order to understand hydrophobin more specifically, scientist has carried out big quantity research to their structure and the characteristic of aspects such as biophysics and biological chemistry.Discover that hydrophobin plays important effect to the growth and the breeding of Mycophyta, comprise absorption, the stability of fungi the surface, the formation of pulp and aerial mycelia, and to the covering protection of fungi structure etc.By the secreted hydrophobin of the mycelia in the water can in water, spread and with the interface of other medium or air on self-assembly takes place, the surface tension of water is declined to a great extent, grow in air thereby make mycelia can break through water surrounding.Wherein, hydrophobin SC3 can make the surface tension of the 50 μ g/mL aqueous solution from maximum 72mj/m
2Be reduced to 24mj/m
2, illustrate that hydrophobin also is good tensio-active agent.Why this protein has strong functions like this, is because they have on hydrophilic-hydrophobic interfaces (for example hydrophobic solid interface of water-air interface, water-oily interface or water and similar Teflon) forms amphipathic film by self-assembly characteristic.
Hydrophobin approximately includes 100 amino-acid residues, thereby forms the natural characteristic that amphipathic film changes the surface at the interface by self-assembly.The hydrophobin film is very firm, can resist 2% SDS down at 100 ℃.Hydrophobin both no cytotoxicity does not have strong immunogenicity yet, and therefore, they not only have very high scientific research value at biological field, have extraordinary application prospect equally at medicine and industrial circle.Hydrophobin can be fixed other protein on solid, and the protein that maybe will dissolve purifies.For example, make the hydrophobin self-assembly on the surface of medical science graft, the biocompatibility that can significantly improve these grafts maybe can prevent morbific bacterial adhesion to the surface as conduit, and promotes the growth of human fibroblast.This hydrophobin can also perhaps be called " adhesive agent " as a kind of media, the molecule of cell, protein and other kind is fixed on hydrophobic solid surface, such as biosensor.Utilize the unreactiveness of assembling back hydrophobin, can predict aspect held stationary enzyme active and can have superiority that the porousness that the film of moulding simultaneously has helps some small molecules and enters transmitter.In field of nanometer technology, hydrophobin through some processing can carry different functional groups, can form the differing molecular figure of nano-precision on the surface, because the club shaped structure of the I type hydrophobin after the assembling approximately has only 10nm wide, but chemical property is highly stable.In addition, hydrophobin has that very high surfactivity can (SC3 approximately be 30mj/m
2), quite similar with traditional bio-surfactant, for example glycolipid class, lipopeptid enzyme/lipoprotein, phosphatide, neutral grease and lipopolysaccharides.Like this, just hydrophobin can be applied to pharmacy industry, to substitute traditional tensio-active agent.
These characteristics of hydrophobin have caused the extensive concern of scientific circles, and present research mainly concentrates on how to realize its suitability for industrialized production and the concrete application facet in each field thereof.But the throughput of hydrophobin is also very limited, aspect the separation purification very big difficulty is being arranged also, thereby is hindering its tempo greatly.Therefore a lot of International Technology workers are devoted to research in this respect always; " Method of purifyinga hydrophobin present in a hydrophobin-containing solution " such as people such as de Vocht proposition; applied for worldwide intellecture property patent protection in calendar year 2001, publication number is WO/2001/057076.Utilize hydrophobin that the absorption of solid substrate is purified to solution, solid substrate is immersed the long-time back of purification solution to be taken out, use strong tensio-active agent will be adsorbed on the hydrophobin dissolving of solid surface again, again hydrophobin is separated purification with tensio-active agent by chromatography and centrifugal separation at last.
China Nankai University magazine " biologically active substance Separation ﹠ Purification " at home rolled up the article of delivering on the 7th phase " separation and purification of the mould hydrophobin HFBI of Rui Shi wood " in 2005 the 31st, reported the extracting and purifying method of hydrophobin HFBI, be to use 1% SDS extracting successively, the KCl post precipitation, can obtain hydrophobin through Berol 532 aqueous two-phase extractions and slightly carry product, and then be further purified by Sephadex G-25 and Q-Sepharose.
Common hydrophobic grouping has alkyl, aromatic base etc. in the material, and it is many more to contain hydrophobic group in the material, and its hydrophobicity will be big more, and such as polyethylene, polystyrene, polypropylene etc. all belong to lyophobic dust.Teflon (Teflon) chemical name is called tetrafluoroethylene, is ethene (C
2H
4) in four hydrogen (H) all be replaced as fluorine (F), become tetrafluoroethylene (C
2F
4), form through polymerization again.Because Teflon is to be made by carbon atom and fluorine atom, and is not hydrogeneous, so hydrophobic performance is strong especially.Characteristics such as that Teflon also has is heat-resisting, low temperature resistant, solidity to corrosion, excellent Abherent and self-lubricity, low frictional factor.
Summary of the invention
The purpose of this invention is to provide a kind of particle that utilizes lyophobic dust the absorption and the heterogeneous nucleation growth effect of hydrophobin separated the method for purification to it.
For solving the problems of the technologies described above, the present invention takes following technical scheme: a kind of separating and purifying method of hydrophobin, be to have the solid substrate of lyophobic dust particle to place hydrophobin solution to be purified to soak surface arrangement earlier, then substrate is taken out from hydrophobin solution, use water rinse, put into the container that fills water again and carry out sonic oscillation, make after the hydrophobin of hydrophobic particles surface adsorption and nucleating growth comes off soluble in water, at last substrate is taken out from solution, obtain purified hydrophobin solution.
In the separating and purifying method of above-mentioned hydrophobin, because hydrophobin can be in all lyophobic dust surface adsorption, thereby the selection of described lyophobic dust is diversified, as Teflon (Teflon) etc.
The size that how much can influence the hydrophobin nucleating growth of the particle of the lyophobic dust of described solid substrate surface arrangement, general particle is many more, the size of hydrophobin growth is big more, particle is few more, and the size of hydrophobin growth is more little, and is not high to the purification efficiency of hydrophobin, then can form the lyophobic dust film but density is too high, the nucleating growth that is unfavorable for hydrophobin, therefore, the lyophobic dust Particle Density that distributes on the described solid substrate is generally 1 * E2-3 * E15/cm
2
The described surface arrangement that is used to separate the purification hydrophobin has the preparation method of the solid substrate of lyophobic dust particle to have a lot, as the physical chemistry etching, and the physical chemistry deposition, chemical corrosion, particle bombardment, physical adsorption adheres to, and applies etc.Consider that particle is in problems such as the size of substrate surface sticking power and roughness, using the ion beam sputtering deposition machine is reasonable method, concrete operations are: use ion beam sputtering deposition machine solid substrate surface bombardment lyophobic dust target to smooth cleaning under vacuum condition, make the particle of lyophobic dust be attached to clean solid substrate surface, and by control to sputtering time, the particle density of the lyophobic dust that solid substrate is distributed is regulated and control, and to make surface arrangement the solid substrate of desired density lyophobic dust particle is arranged.Usually, sputtering time is oversize, forms hydrophobic film easily, be unfavorable for the nucleating growth of hydrophobin, and sputtering time is too short, and then hydrophobic particles density is too little, and is not high to the purification efficiency of hydrophobin.Described sputtering time is generally 1-600s, is preferably 5s.The material of described solid substrate is diversified, all can as quartz, silicon chip or sheet glass etc.
In addition, surface arrangement has the length of the soak time of solid substrate in hydrophobin solution to be purified of lyophobic dust particle also can influence the size of hydrophobin nucleating growth, general soak time is long more, the size of hydrophobin growth is big more, soak time is short more, the size of hydrophobin growth is more little, can adjust soak time according to the concentration of hydrophobin solution and the particle density of lyophobic dust, is generally 0.01-72 hour.
Handle by the sonic oscillation that the hydrophobin sample is carried out different capacity, discovery is not that the big more separation refining effect of power is good more, power is excessive, can promote hydrophobin soluble in water on the lyophobic dust particle, to assemble on the contrary, therefore, the power density of sonic oscillation is generally 0.01-500W/mL (being preferably 1.35-1.45W/mL), and oscillation frequency is 1-300KHz, and duration of oscillation is 0.01-300min.
Hydrophobin with the aforesaid method separation and purification also belongs to protection scope of the present invention.
The invention provides a kind of hydrophobic protein separation and purification method.This method is to utilize hydrophobin to hydrophobin very strong heterogeneous nucleation growth effect to be arranged in the surface adsorption of lyophobic dust and the particle of lyophobic dust, thereby reaches the purpose of hydrophobin being separated purification.Can obtain the higher hydrophobin solution of purity easily with method of the present invention, and this method has simple to operately, conditional request is low, and is with low cost, and the substrate that is distributed with the lyophobic dust particle can be reused, and is easy to carry out the advantage of industrial applications.The present invention will play a significant role in hydrophobic protein separation and purification, have a extensive future.
Below in conjunction with specific embodiment the present invention is described in further details.
Description of drawings
Fig. 1 has the atomic force microscopy image of the quartz base plate of Teflon particle for surface arrangement
Fig. 2 has the quartz base plate of Teflon particle to soak 12 hours atomic force microscopy image and image analysis results after the taking-up in hydrophobin HFBII solution for surface arrangement
Substrate the atomic force microscopy image through the sonic oscillation of different capacity handle after of Fig. 3 for being adsorbed with hydrophobin
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
The separation of embodiment 1, hydrophobin is purified and is detected the influence of the sonic oscillation processing of different capacity to purification effect
One, the surface arrangement that is used to separate the purification hydrophobin has the preparation of the solid substrate of Teflon particle
The polishing quartz base plate of cleaning is put into the ion beam sputtering deposition machine, install the Teflon target, vacuum is evacuated to 1.3E-3pa, take out behind the sputter 5s, obtaining surface arrangement has the solid substrate of Teflon particle, (figure A is of a size of 5 microns to the atomic force microscope observed result as shown in Figure 1, figure B is of a size of 1 micron), the roughness of the quartzy substrate surface of clean polishing is very low, by seeing substrate surface many particles that distributed in the atomic power image behind sputter coating, find that by the test of the surface being carried out water contact angle contact angle becomes big, prove that thus these are exactly the Teflon particle at the point that substrate surface distributes, the Teflon Particle Density is about 8 * E11/cm
2
Two, the separation of hydrophobin is purified and is detected the influence of the sonic oscillation processing of different capacity to purification effect
There is the solid substrate of Teflon particle to place hydrophobin HFBII solution to be purified to soak 12 hours the surface arrangement of step 1 preparation, then substrate is taken out from hydrophobin solution, observation and image analyzed under atomic force microscope, (figure A is an atomic force microscopy image, is of a size of 5 microns as shown in Figure 2 for observation and image analysis result; Figure B is the image analysis result), can see that substrate surface has grown very big hydrophobin aggregate in irregular shape, this be since hydrophobin due to Teflon particle surface absorption and nucleating growth.The height that can be got aggregate by the supporting analysis software of atomic force microscope can reach about 130nm, and diameter can reach about 550nm, illustrates that the growth velocity of hydrophobin is very high.Water to the substrate rinsing that is adsorbed with hydrophobin after, it is put into the container that fills water again carry out sonic oscillation, make the hydrophobin of absorption of Teflon particle and nucleating growth come off back soluble in water, the sonic oscillation power density is made as 0.85W/mL respectively, 1.42W/mL, 1.90W/mL, other condition is the same, frequency is 20-25KHz, duration of oscillation is 15min, after the ultrasonic end substrate is taken out from solution, under atomic force microscope, observe, (figure A is the atomic force microscopy image of vibration metacoxa under 0.85W/mL to observed result as shown in Figure 3, figure B is the atomic force microscopy image of vibration metacoxa under 1.42W/mL, figure C is the atomic force microscopy image of vibration metacoxa under 1.90W/mL, size is 5 microns, the Z axle highly is 15nm), adopt different capacity to carry out the effect of gained after the supersound process and different to sample as seen from Figure 3, remaining hydrophobin is minimum on the substrate of sonic oscillation under 1.42W/mL, big aggregated particles obviously disappears, reached and separated the effect of purifying, explanation is not that the big more effect of ultrasonic power is good more thus, this is because hydrophobin has very high activity, power is excessive can to promote the hydrophobin in the solution to assemble on the Teflon particle on the contrary again, and 1.35-1.45W/mL is best hunting power density.Above-mentioned detected result shows, can obtain the higher hydrophobin of purity with method of the present invention.
The separation of embodiment 2, hydrophobin is purified
One, the surface arrangement that is used to separate the purification hydrophobin has the preparation of the solid substrate of Teflon particle
The glass substrate of cleaning is put into the ion beam sputtering deposition machine, install the Teflon target, vacuum is evacuated to 1.3E-3pa, takes out behind the sputter 300s, obtains the solid substrate that surface arrangement has the Teflon particle.
Two, the separation of hydrophobin is purified
There is the solid substrate of Teflon particle to place hydrophobin HFBII solution to be purified to soak 72 hours the surface arrangement of step 1 preparation, then substrate is taken out from hydrophobin solution, observation and image analyzed under atomic force microscope, can see that substrate surface has grown very big hydrophobin aggregate in irregular shape, this be since hydrophobin due to Teflon particle surface absorption and nucleating growth.The height that can be got aggregate by the supporting analysis software of atomic force microscope can reach about 130nm, and diameter can reach about 550nm, illustrates that the growth velocity of hydrophobin is very high.Then water to the substrate rinsing that is adsorbed with hydrophobin after, it is put into the container that fills water again carry out sonic oscillation, the sonic oscillation power density is made as 0.01W/mL respectively, oscillation frequency is 200KHz, and duration of oscillation is 300min, after the ultrasonic end substrate is taken out from solution, under atomic force microscope, observe, observed result shows that remaining hydrophobin is less on the glass substrate, and big aggregated particles obviously disappears, and has obtained the higher hydrophobin of purity.
The separation of embodiment 3, hydrophobin is purified
One, the surface arrangement that is used to separate the purification hydrophobin has the preparation of the solid substrate of Teflon particle
The glass substrate of cleaning is put into the ion beam sputtering deposition machine, install the Teflon target, vacuum is evacuated to 1.3E-3pa, takes out behind the sputter 600s, obtains the solid substrate that surface arrangement has the Teflon particle.
Two, the separation of hydrophobin is purified
There is the solid substrate of Teflon particle to place hydrophobin HFBII solution to be purified to soak 24 hours the surface arrangement of step 1 preparation, then substrate is taken out from hydrophobin solution, observation and image analyzed under atomic force microscope, can see that substrate surface has grown very big hydrophobin aggregate in irregular shape, this be since hydrophobin due to Teflon particle surface absorption and nucleating growth.The height that can be got aggregate by the supporting analysis software of atomic force microscope can reach about 130nm, and diameter can reach about 550nm, illustrates that the growth velocity of hydrophobin is very high.Then water to the substrate rinsing that is adsorbed with hydrophobin after, it is put into the container that fills water again carry out sonic oscillation, the sonic oscillation power density is made as 300W/mL respectively, oscillation frequency is 100KHz, and duration of oscillation is 60min, after the ultrasonic end substrate is taken out from solution, under atomic force microscope, observe, observed result shows that remaining hydrophobin is less on the glass substrate, and big aggregated particles obviously disappears, and has obtained the higher hydrophobin of purity.
The separation of embodiment 4, hydrophobin is purified
One, the surface arrangement that is used to separate the purification hydrophobin has the preparation of the solid substrate of Teflon particle
The silicon chip substrate of cleaning is put into the ion beam sputtering deposition machine, install the Teflon target, vacuum is evacuated to 1.3E-3pa, takes out behind the sputter 1s, obtains the solid substrate that surface arrangement has the Teflon particle.
Two, the separation of hydrophobin is purified
There is the solid substrate of Teflon particle to place hydrophobin HFBII solution to be purified to soak 1 hour the surface arrangement of step 1 preparation, then substrate is taken out from hydrophobin solution, observation and image analyzed under atomic force microscope, can see that substrate surface has grown very big hydrophobin aggregate in irregular shape, this be since hydrophobin due to Teflon particle surface absorption and nucleating growth.The height that can be got aggregate by the supporting analysis software of atomic force microscope can reach about 130nm, and diameter can reach about 550nm, illustrates that the growth velocity of hydrophobin is very high.Then water to the substrate rinsing that is adsorbed with hydrophobin after, it is put into the container that fills water again carry out sonic oscillation, the sonic oscillation power density is made as 500W/mL respectively, oscillation frequency is 300KHz, and duration of oscillation is 200min, after the ultrasonic end substrate is taken out from solution, under atomic force microscope, observe, observed result shows that remaining hydrophobin is less on the silicon chip substrate, and big aggregated particles obviously disappears, and has obtained the higher hydrophobin of purity.
The separation of embodiment 5, hydrophobin is purified
One, the surface arrangement that is used to separate the purification hydrophobin has the preparation of the solid substrate of Teflon particle
The quartz base plate of cleaning is put into the ion beam sputtering deposition machine, install the Teflon target, vacuum is evacuated to 1.3E-3pa, takes out behind the sputter 50s, obtains the solid substrate that surface arrangement has the Teflon particle.
Two, the separation of hydrophobin is purified
There is the solid substrate of Teflon particle to place hydrophobin HFBII solution to be purified to soak 36 hours the surface arrangement of step 1 preparation, then substrate is taken out from hydrophobin solution, observation and image analyzed under atomic force microscope, can see that substrate surface has grown very big hydrophobin aggregate in irregular shape, this be since hydrophobin due to Teflon particle surface absorption and nucleating growth.The height that can be got aggregate by the supporting analysis software of atomic force microscope can reach about 130nm, and diameter can reach about 550nm, illustrates that the growth velocity of hydrophobin is very high.Then water to the substrate rinsing that is adsorbed with hydrophobin after, it is put into the container that fills water again carry out sonic oscillation, the sonic oscillation power density is made as 1.45W/mL respectively, oscillation frequency is 150KHz, and duration of oscillation is 150min, after the ultrasonic end substrate is taken out from solution, under atomic force microscope, observe, observed result shows that remaining hydrophobin is less on the quartz base plate, and big aggregated particles obviously disappears, and has obtained the higher hydrophobin of purity.
The separation of embodiment 6, hydrophobin is purified
One, the surface arrangement that is used to separate the purification hydrophobin has the preparation of the solid substrate of Teflon particle
The quartz base plate of cleaning is put into the ion beam sputtering deposition machine, install the Teflon target, vacuum is evacuated to 1.3E-3pa, takes out behind the sputter 100s, obtains the solid substrate that surface arrangement has the Teflon particle.
Two, the separation of hydrophobin is purified
There is the solid substrate of Teflon particle to place hydrophobin HFBII solution to be purified to soak 48 hours the surface arrangement of step 1 preparation, then substrate is taken out from hydrophobin solution, observation and image analyzed under atomic force microscope, can see that substrate surface has grown very big hydrophobin aggregate in irregular shape, this be since hydrophobin due to Teflon particle surface absorption and nucleating growth.The height that can be got aggregate by the supporting analysis software of atomic force microscope can reach about 130nm, and diameter can reach about 550nm, illustrates that the growth velocity of hydrophobin is very high.Then water to the substrate rinsing that is adsorbed with hydrophobin after, it is put into the container that fills water again carry out sonic oscillation, the sonic oscillation power density is made as 1.35W/mL respectively, oscillation frequency is 250KHz, and duration of oscillation is 30min, after the ultrasonic end substrate is taken out from solution, under atomic force microscope, observe, observed result shows that remaining hydrophobin is less on the quartz base plate, and big aggregated particles obviously disappears, and has obtained the higher hydrophobin of purity.
Claims (9)
1, the separating and purifying method of hydrophobin, be to have the solid substrate of lyophobic dust particle to place hydrophobin solution to be purified to soak surface arrangement earlier, then substrate is taken out from hydrophobin solution, use water rinse, put into the container that fills water again and carry out sonic oscillation, at last substrate is taken out from solution, obtain purified hydrophobin solution.
2, separating and purifying method according to claim 1 is characterized in that: described lyophobic dust is a Teflon.
3, separating and purifying method according to claim 1 is characterized in that: the Particle Density of the lyophobic dust that distributes on the described solid substrate is 1 * E2-3 * E15/cm
2
4, separating and purifying method according to claim 1, it is characterized in that: the described surface arrangement that is used to separate the purification hydrophobin has the preparation method of the solid substrate of lyophobic dust particle to be: use ion beam sputtering deposition machine solid substrate surface bombardment lyophobic dust target to smooth cleaning under vacuum condition, obtain the solid substrate that surface arrangement has the lyophobic dust particle.
5, separating and purifying method according to claim 4 is characterized in that: described sputtering time is 1-600s.
6, separating and purifying method according to claim 1 is characterized in that: it is 0.01-72 hour that described surface arrangement has the soak time of solid substrate in hydrophobin solution to be purified of lyophobic dust particle.
7, according to each described separating and purifying method of claim 1-6, it is characterized in that: the power density of described sonic oscillation is 0.01-500W/mL, and oscillation frequency is 1-300KHz, and duration of oscillation is 0.01-300min.
8, separating and purifying method according to claim 7 is characterized in that: the power density of described sonic oscillation is 1.35-1.45W/mL.
9, the hydrophobin that obtains with each described method of claim 1-8 through separation and purification.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| USRE44812E1 (en) | 2008-12-16 | 2014-03-18 | Conopco, Inc. | Method for extracting hydrophobin from a solution |
| US9115349B2 (en) | 2008-10-16 | 2015-08-25 | Conopco, Inc. | Hydrophobin solution containing antifoam |
| CN106749683A (en) * | 2015-11-20 | 2017-05-31 | 中国科学院理化技术研究所 | A kind of cellulose nanometer sheet material and preparation method thereof |
-
2007
- 2007-05-25 CN CN 200710099646 patent/CN101054407A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9115349B2 (en) | 2008-10-16 | 2015-08-25 | Conopco, Inc. | Hydrophobin solution containing antifoam |
| USRE44812E1 (en) | 2008-12-16 | 2014-03-18 | Conopco, Inc. | Method for extracting hydrophobin from a solution |
| CN106749683A (en) * | 2015-11-20 | 2017-05-31 | 中国科学院理化技术研究所 | A kind of cellulose nanometer sheet material and preparation method thereof |
| CN106749683B (en) * | 2015-11-20 | 2019-08-20 | 中国科学院理化技术研究所 | A kind of cellulose nanometer sheet material and preparation method thereof |
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