CN101215320B - Method for modifying protein separation action by using ultrasonic enhancement charged ultra-filtration - Google Patents
Method for modifying protein separation action by using ultrasonic enhancement charged ultra-filtration Download PDFInfo
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- CN101215320B CN101215320B CN2007101732789A CN200710173278A CN101215320B CN 101215320 B CN101215320 B CN 101215320B CN 2007101732789 A CN2007101732789 A CN 2007101732789A CN 200710173278 A CN200710173278 A CN 200710173278A CN 101215320 B CN101215320 B CN 101215320B
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Abstract
The invention discloses a process which improves protein separation by adopting ultrasonic enhanced charging hyper-filtration, which comprises changing a membrane into a charging hyper-filtration membrane through adopting charging modifying property technology, deciding environment conditions of separating protein solution according to the molecular weight size and isoelectric point of protein which will be separated and the electric quantity of the charging hyper-filtration membrane, increasing the separation selective of protein and the yield of product protein through utilizing the electrostatic interaction between the charging hyper-filtration membrane and protein, arranging the charging hyper-filtration membrane which is prepared on a hyper-filtration device, and loading ultrasonic wave on the hyper-filtration device. The invention can effectively combine each advantage of increasing membrane flux through ultrasonic enhanced and increasing membrane selective through charging hyper-filtration membrane, can simultaneously increase the separation speed and yield of protein, and effectively solves the two problems of low selective and pollution of membrane in the application of hyper-filtration technology in the protein separation process.
Description
Technical field
The present invention relates to a kind of separation method of life field of engineering technology, particularly a kind ofly adopt ultrasound-enhanced charged ultrafiltration to improve the method for protein separation behavior.
Background technology
Ultrafiltration is to be the membrane filtration technique of motivating force with pressure, a kind of sepn process of high throughput be can under lower process cost, realize, the separation of protein, enzyme, Interferon, rabbit, toxoid, toxinicide, Blood Preparations etc., concentrated, the refining filter wash etc. that reaches have been widely used in.But the low selectivity of film and pollution problem have seriously restricted the application of ultra-filtration technique in the protein separation process, are still to substitute the isolating main reason of other high-value protein at present.Traditional ultrafiltration is based on physics screening principle and realizes the solute separating process.The highly selective of ultra-filtration membrane must be that the flux with expendable film is a cost, if will obtain higher product yield, then must select the film of smaller aperture due, but less membrane pore size must make film forming transmission coefficient and reduce, thereby has influenced the velocity of separation of product.Therefore must weigh the selectivity and the relation of the choice between the permeation flux of film in the substantial sepn process, this has finally limited the velocity of separation and the yield of product.
The minimizing of being polluted the transmission coefficient that causes by film is another key issue of restriction ultrafiltration broader applications in protein separation.The pre-treatment feed liquid, to select modified membrane, operation conditions optimization and cleaning film for use be present control, reduce the major measure that film pollutes.The technology such as turbulence promoter, eddy current mixing, unstable state stream, gas blowout stream that much studies show that also can effectively reduce the film pollution and obtain certain development.In recent years, the research that utilizes external force field to come enhancing ultrafiltration, minimizing film to pollute has caused increasing concern.The correlative study report of extra electric field enhancing ultrafiltration is more, but because the object of handling (need be with charged) is limited, the easy electrolysis of electrode surface, the complicacy of feed liquid intermediate ion and high process energy consumption etc. makes it the industrial applications popularization and is restricted.Utilize the ultrasonic cavatition that in the fluid transmittance process, produces, acoustic streaming, characteristics such as the dispersion of radiation pressure and solute, aggegation and variation, ultrasonic technique have obtained widespread use at suspend separation, cytoclasis extraction, extraction, crystallization, emulsification and each commercial run such as breakdown of emulsion, ultrasonic cleaning.The existing formation speed of utilizing the enhancing ultrafiltration that adds the ultrasonic field effect can slow down pollution layer that studies show that is carried out automatically, in time, clean online established pollution layer simultaneously, improves ultrasiltrated rate effectively.At the low selectivity of film in the protein ultra-filtration and separation with pollute two large problems, present measure only lays particular emphasis on basically to utilize and ultrasound-enhancedly is used for improving single proteinic flux, and selectivity and flux is not worked in coordination with consideration.
Find by prior art documents, people such as S.Muthukumaran (are published in Journal of Membrane Science " membrane science magazine " in Mechanisms forthe ultrasonic enhancement of dairy whey ultrafiltration (mechanism of a ultrasound-enhanced whey ultrafiltration) literary composition, 2005,258:106-114) adopt polysulphones hyperfiltration membrane, to be about the concentration of 24000g/mol be 6% whey to the ultrafiltration molecular weight under room temperature (22-25 ℃) condition, load ultrasonic wave (ultrasonic frequency 50kHz, ultrasound intensity 0.02W/cm
2) find after 4 hours that membrane flux has improved about 50% than the ultra-filtration process that does not load under hyperacoustic similarity condition.But the process and the separation selectivity problem that do not relate to two kinds of protein separation in the article only improve single protein ultrafiltration flux and investigate ultrasonic.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, providing a kind of adopts ultrasound-enhanced charged ultrafiltration to improve the method for protein separation behavior, make it can realize the method that proteinic velocity of separation and yield improve simultaneously, can improve proteinic separation selectivity effectively, reduce film and pollute, remove film automatically, in time, online and pollute also effective membrane flux that improves.Thereby realize that molecular weight is close, the separation of high-value protein, the minimizing of the prolongation in the work-ing life of film and whole sepn process expense.
The present invention is achieved by the following technical solutions, the present invention includes following steps:
The first step, realize on the isolating ultra-filtration membrane in traditional utilization screening principle, adopt charged modification technology to make film become charged ultra-filtration membrane, determine the envrionment conditions of protein soln to be separated by the electric weight of proteinic molecular weight size to be separated, iso-electric point and charged ultra-filtration membrane, can make full use of the electrostatic interaction between charged ultra-filtration membrane and the protein like this, improve the proteinic yield of proteinic separation selectivity and product effectively, also may realize the effective separation between the close protein of molecular weight.
The charged modification technology of described employing makes film become charged ultra-filtration membrane, can adopt existing techniques in realizing, as surface chemical reaction, surface-coated, surface adsorption, chemical graft, surface light graft polymerization modification method, plasma technique and electron beam irradiation grafting method etc., make film become charged ultra-filtration membrane.In modifying process, also can obtain to have the film of different carrying capacities according to different modified conditions.
Described charged ultra-filtration membrane can be that positively charged membrane also can be the negative electricity film, and its shape can be flat, rolling, tubular fibre formula or tubular type.
The envrionment conditions of described protein soln to be separated is specially: pH value 2-11, ionic strength 1mM-1000mM.
Second step placed the charged ultra-filtration membrane for preparing on the ultra-filtration equipment, and ultra-filtration equipment is loaded ultrasonic wave.In the ultra-filtration process under the ultrasound environments, ultrasonic cavitation phenomenon and the ultrasonic wave that produces at charged ultra-filtration membrane near surface makes liquid produce the more coefficient result of high flow rate formation acoustic streaming in communication process, can reduce concentration polarization and film and pollute, reach the effect of enhancing ultrafiltration; Utilize characteristics such as ultrasonic cavatition that in the fluid transmittance process, produces and acoustic streaming, can reduce pollutent effectively, effectively improve the life-span of membrane flux and prolongation film in the deposition on film surface and the absorption in the fenestra; In addition, even pollutent is at film surface deposition and fenestra internal adsorption, utilize characteristics such as ultrasonic cavatition that in the fluid transmittance process, produces and acoustic streaming, can realize removing automatically, in time, online the striping surface contaminant, even enter removal pollutent in the fenestra, thereby effectively improve the life-span of membrane flux and prolongation film.
In the above-mentioned steps, can select suitable ultrasonic loading regime, ultrasound parameter and ultra-filtration process operating parameters as required, guarantee ultrasonicly, improve protein separation speed and while optionally, do not cause protein and the irreversible destruction of mould material at enhancing ultrafiltration.Here ultrasonic loading regime comprises whole ultra-filtration equipment is placed the ultrasonic generator water-bath that ultrasonic generator directly is affixed on the membrane module surface, or the ultra-sonic generator rod is as stirring rod.Ultrasound parameter is specially: ultrasound intensity is 0.01W/cm
2-30W/cm
2, frequency is 10kHz-2000kHz, and ultrasonic sounding position is upside, downside, left side or the right side on membrane sepn surface, and the ultrasonic loading time is that 10min is to 24hr.Ultrasonic loading can be continuously, intermittently or pulsed.The ultrafiltration transmembrane pressure is 0.05MPa-0.5MPa, and the ultrafiltration temperature is 20 ℃ to 85 ℃.The ultrafiltration mode can dead end or cross-flow operation mode.
The selectivity that the present invention improves charged ultra-filtration membrane film effectively combines with the flux advantage separately that adds ultrasonic field raising film, has formed ultrasound-enhanced charged ultra-filtration membrane ultrafiltration new technology, has realized the selectivity of film and the collaborative raising of flux.The invention discloses a kind of convenience, feasible, economic, solved the two big bottleneck problems of ultrafiltration in the protein separation process is used to a certain extent simultaneously: the low selectivity of film and the separation method of pollution problem.For obtaining of charged ultra-filtration membrane, the present invention adopts the method for chemical reaction, promptly uses on the regenerated cellulose ultra-filtration membrane in commerce, by carrying out the reaction of different time with the chemical reagent that is fit to, makes the positive electricity or the negative electricity of different electric weight on the film band.Simple, the easy row of the method for the charged modified ultrafiltration membrane of this acquisition.For the realization of ultrasonic technique, only need whole ultra-filtration equipment is placed the ultrasonic generator water-bath, or ultrasonic generator directly is affixed on the membrane module surface, or the ultra-sonic generator rod need not additionally to add complex apparatus as stirring rod, it is quite convenient to operate.In a word, the invention provides a kind of effective method of improving the protein separation behavior, can improve the selectivity and the flux of film simultaneously, improve proteinic yield of product and velocity of separation simultaneously, under lower process cost, realize high throughput.The present invention can slow down the formation speed of film surface or fenestra internal contamination layer very effectively, can carry out automatically established pollution layer simultaneously, in time, clean online, improves ultrasiltrated rate effectively, prolongs the work-ing life of film, reduces process cost.At protein system to be separated,, also can realize effective separation of the close molecular weight protein matter that traditional ultrafiltration can't realize by the operational condition of the charged ultra-filtration membrane of choose reasonable, solution environmental, ultrasound parameter and ultra-filtration process.
Embodiment
Below embodiments of the invention are elaborated: present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiments of the invention are separate object with bovine serum albumin and D-tryptophane.
Embodiment 1
(1) choose the flat regenerated cellulose film that molecular weight cut-off is 100kDa, its surface zeta potential current potential is-0.8V.Adopt the method for modifying of surface chemical reaction, make the reaction of this film and sulfonic acid substance obtain the zeta current potential after 12 hours to be-the modification bear electro ultrafiltration film of 10.4V.According to the foundation of choosing pH value and ionic strength in the step (1), choose finally that protein to be separated is dissolved in pH=7.0 and ionic strength is in the solution of 10mM, can make full use of the electrostatic interaction between charged ultra-filtration membrane and the protein like this, improve the separation selectivity of bovine serum albumin and D-tryptophane effectively, improve the yield of bovine serum albumin.
(2) the charged ultra-filtration membrane that step (1) is obtained places on the dead end ultra-filtration equipment, and whole ultrafiltration system is placed 20 ℃ of ultrasound baths.Transmembrane pressure in the ultra-filtration process is 0.07MPa, and ultrasound intensity is 2W/cm
2, frequency is 28kHz, and ultrasonic sounding position is at the downside 10cm on membrane sepn surface, and ultrasonic phonation time is 60min and loads continuously.The ultrasound-enhanced charged ultrafiltration of carrying out under this ultrafiltration system and operating parameters, membrane flux are 1.2 * 10
-4M/s, the flux that uses unmodified regenerated cellulose ultra-filtration membrane and do not load ultrasonic ultra-filtration process has improved 44%; The separation factor of D-tryptophane and bovine serum albumin (being the ratio of the transmitance of D-tryptophane and bovine serum albumin) is 135, and the separation factor of using unmodified regenerated cellulose ultra-filtration membrane and not loading ultrasonic ultra-filtration process has improved 4.1 times.
Embodiment 2
(1) choose the flat regenerated cellulose film that molecular weight cut-off is 100kDa, its surface zeta potential current potential is-0.8V.Adopt the method for modifying of surface chemical reaction, make the reaction of this film and sulfonic acid substance obtain the zeta current potential after 48 hours to be-the modification bear electro ultrafiltration film of 16.8V.According to the foundation of choosing pH value and ionic strength in the step (1), choose finally that protein to be separated is dissolved in pH=7.0 and ionic strength is in the solution of 10mM, can make full use of the electrostatic interaction between charged ultra-filtration membrane and the protein like this, improve the separation selectivity of bovine serum albumin and D-tryptophane effectively, improve the yield of bovine serum albumin.
(2) the charged ultra-filtration membrane that step (1) is obtained places on the dead end ultra-filtration equipment, and whole ultrafiltration system is placed 20 ℃ of ultrasound baths.Transmembrane pressure in the ultra-filtration process is 0.07MPa, and ultrasound intensity is 2W/cm
2, frequency is 28kHz, and ultrasonic sounding position is at the downside 10cm on membrane sepn surface, and ultrasonic phonation time is 60min and loads continuously.The ultrasound-enhanced charged ultrafiltration of carrying out under this ultrafiltration system and operating parameters, membrane flux are 1.16 * 10
-4M/s, the flux that uses unmodified regenerated cellulose ultra-filtration membrane and do not load ultrasonic ultra-filtration process has improved 40%; The separation factor of D-tryptophane and bovine serum albumin is 146, and the separation factor of using unmodified regenerated cellulose ultra-filtration membrane and not loading ultrasonic ultra-filtration process has improved 4.6 times.
Embodiment 3
(1) choose the flat regenerated cellulose film that molecular weight cut-off is 100kDa, its surface zeta potential current potential is-0.8V.Adopt the method for modifying of surface chemical reaction, make the reaction of this film and sulfonic acid substance obtain the zeta current potential after 12 hours to be-the modification bear electro ultrafiltration film of 10.4V.According to the foundation of choosing pH value and ionic strength in the step (1), choose finally that protein to be separated is dissolved in pH=7.0 and ionic strength is in the solution of 10mM, can make full use of the electrostatic interaction between charged ultra-filtration membrane and the protein like this, improve the separation selectivity of bovine serum albumin and D-tryptophane effectively, improve the yield of bovine serum albumin.
(2) the charged ultra-filtration membrane that step (1) is obtained places on the dead end ultra-filtration equipment, and whole ultrafiltration system is placed 20 ℃ of ultrasound baths.Transmembrane pressure in the ultra-filtration process is 0.07 MPa, and ultrasound intensity is 2W/cm
2, frequency is 45kHz, and ultrasonic sounding position is at the downside 10cm on membrane sepn surface, and ultrasonic phonation time is 60min and loads continuously.The ultrasound-enhanced charged ultrafiltration of carrying out under this ultrafiltration system and operating parameters, membrane flux are 1.1 * 10
-4M/s, the flux that uses unmodified regenerated cellulose ultra-filtration membrane and do not load ultrasonic ultra-filtration process has improved 33%; The separation factor of D-tryptophane and bovine serum albumin is 130, and the separation factor of using unmodified regenerated cellulose ultra-filtration membrane and not loading ultrasonic ultra-filtration process has improved 4.0 times.
Embodiment 4
(1) choose the flat regenerated cellulose film that molecular weight cut-off is 100kDa, its surface zeta potential current potential is-0.8V.Adopt the method for modifying of surface chemical reaction, make the reaction of this film and sulfonic acid substance obtain the zeta current potential after 12 hours to be-the modification bear electro ultrafiltration film of 10.4V.According to the foundation of choosing pH value and ionic strength in the step (1), choose finally that protein to be separated is dissolved in pH=7.0 and ionic strength is in the solution of 10mM, can make full use of the electrostatic interaction between charged ultra-filtration membrane and the protein like this, improve the separation selectivity of bovine serum albumin and D-tryptophane effectively, improve the yield of bovine serum albumin.
(2) the charged ultra-filtration membrane that step (1) is obtained places on the dead end ultra-filtration equipment, and whole ultrafiltration system is placed 20 ℃ of ultrasound baths.Transmembrane pressure in the ultra-filtration process is 0.07MPa, and ultrasound intensity is 2W/cm
2, frequency is 28kHz, and ultrasonic sounding position is at the downside 10cm on membrane sepn surface, and the ultrasonic loading time is intermittent type ground (loaded once every 5 minutes and a loading time is 5 minutes).The ultrasound-enhanced charged ultrafiltration of carrying out under this ultrafiltration system and operating parameters, membrane flux are 1.15 * 10
-4M/s, the flux that uses unmodified regenerated cellulose ultra-filtration membrane and do not load ultrasonic ultra-filtration process has improved 35%; The separation factor of D-tryptophane and bovine serum albumin (being the ratio of the transmitance of D-tryptophane and bovine serum albumin) is 130, and the separation factor of using unmodified regenerated cellulose ultra-filtration membrane and not loading ultrasonic ultra-filtration process has improved 3.9 times.
Claims (1)
1. a method that adopts ultrasound-enhanced charged ultrafiltration to improve the protein separation behavior is characterized in that: comprise the steps:
The first step, choosing molecular weight cut-off is the flat regenerated cellulose film of 100kDa, adopt the method for modifying of surface chemical reaction, make the reaction of this film and sulfonic acid substance obtain the charged ultra-filtration membrane of zeta current potential after 12 hours for-10.4V, determine the envrionment conditions of protein soln to be separated by the electric weight of proteinic molecular weight size to be separated, iso-electric point and charged ultra-filtration membrane, utilize the electrostatic interaction between charged ultra-filtration membrane and the protein to improve proteinic separation selectivity and the proteinic yield of product;
Second step, the charged ultra-filtration membrane of preparation is placed on the ultra-filtration equipment, and to ultra-filtration equipment loading ultrasonic wave, in the ultra-filtration process under the ultrasound environments, ultrasonic charged ultra-filtration membrane near surface produce cavitation phenomenon and ultrasonic wave in communication process, make liquid produce more high flow rate forms acoustic streaming, utilize their acting in conjunction to remove the striping surface contaminant, thereby further improve proteinic disintegrate-quality and velocity of separation;
Described protein is bovine serum albumin and D-tryptophane;
The shape of described charged ultra-filtration membrane is flat, rolling, tubular fibre formula or tubular type;
The envrionment conditions of described protein soln to be separated is specially: pH value 7, ionic strength 10mM; Described loading ultrasonic wave, its time loads once for the continuous 60min of loading or every 5 minutes and a loading time is 5 minutes; Described ultrasonic, its parameter is: ultrasound intensity is 2W/cm
2, frequency is 28kHz-45kHz; Described ultrafiltration, its transmembrane pressure are 0.07MPa, and temperature is 20 ℃;
Described loading ultrasonic wave places the ultrasonic generator water-bath with whole ultra-filtration equipment, or ultrasonic generator directly is affixed on the membrane module surface, or the ultra-sonic generator rod is as stirring rod;
Described ultrasonic, upside, downside, left side or right side that its sounding position is the membrane sepn surface;
Described ultrafiltration, its mode are dead end or cross-flow operation mode.
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| CN101555048B (en) * | 2009-05-21 | 2011-04-06 | 上海交通大学 | Method for removing natural organism and heavy metal simultaneously from water |
| CN103007759A (en) * | 2012-12-03 | 2013-04-03 | 北京清大国华环保科技有限公司 | Ultrasound vibration type rolling type ultrafiltration membrane group device and ultrafiltration device thereof |
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Non-Patent Citations (5)
| Title |
|---|
| S. Muthukumaran et al..Mechanisms for the ultrasonic enhancement of dairy wheyultrafiltration.Journal of Membrane Science258.2005,258第106页右栏第2段、第107页左栏第1段和Fig.1，第108页左栏倒数第2行、第9-12行、第6-9行和第12-14行，第112页右栏第6段第3-4行. * |
| S. NAKAO et al..SEPARATION OF PROTEINSBYCHARGEDULTRARILTRATION MEMBRANES.Desalination 70.1988,(70),摘要和第191页第3段第3-5行. |
| S. NAKAO et al..SEPARATION OF PROTEINSBYCHARGEDULTRARILTRATION MEMBRANES.Desalination 70.1988,(70),摘要和第191页第3段第3-5行. * |
| 汪勇等.荷正电膜的研究.天津工业大学学报20 1.2001,20(1),第79-83页. |
| 汪勇等.荷正电膜的研究.天津工业大学学报20 1.2001,20(1),第79-83页. * |
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