CN101781645B - Preparation method of fixed enzyme membrane - Google Patents
Preparation method of fixed enzyme membrane Download PDFInfo
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- CN101781645B CN101781645B CN201010300675XA CN201010300675A CN101781645B CN 101781645 B CN101781645 B CN 101781645B CN 201010300675X A CN201010300675X A CN 201010300675XA CN 201010300675 A CN201010300675 A CN 201010300675A CN 101781645 B CN101781645 B CN 101781645B
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- methyl methacrylate
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Abstract
The invention relates to a preparation method of a fixed enzyme membrane. The invention solves the problems of low enzyme protein carrying capacity and low catalytic activity of a fixed enzyme membrane prepared by the traditional method. The method comprises the following steps of: (1) preparing a methyl methacrylate grafted membrane; (2) preparing a methyl methacrylate grafted membrane with absorbed hexanediamine; and (3) soaking and processing the methyl methacrylate grafted membrane with the absorbed hexanediamine into a bacillus licheniformis alkaline protease solution or a transglutaminase solution so as to obtain a fixed enzyme membrane. The fixed enzyme membrane prepared by the invention has good enzyme protein carrying capacity, bacillus licheniformis alkaline protease carrying capacity larger than 35mg/g membrane and transglutaminase carrying capacity larger than 20mg/g membrane. The fixed enzyme membrane prepared by the method has good absorption capability, catalytic activity and thermal stability.
Description
Technical field
The present invention relates to a kind of preparation method of immobilized enzyme.
Background technology
Immobilized enzyme is with certain material organized enzyme to be fettered or is limited in certain zone, but still can carry out the peculiar catalyzed reaction of enzyme, and recyclable and reusable a kind of new technology.Compare with water-soluble enzyme, immobilized enzyme is beyond enzymic catalytic reaction characteristic such as keep that it is efficient, single-minded, the activity of gentleness and enzyme can be regulated control, also have Separation and Recovery easily, can repeat repeatedly to use, operate continuously and controlled, serial advantage such as technology is easy, therefore developed rapidly in association areas such as biology and biotechnology, medicine, food, medical science and life sciences.
Since membrane separation technique entered industrialization from the fifties in last century, through the development of decades, it extensively was penetrated into chemical industry, metallurgy, environmental protection, bioengineering field.Separatory membrane not only can effectively separate gas, organism, all kinds of biotechnological formulations etc., concentrate and purifying, but also becomes the carrier of immobilized enzyme.Fixed enzyme membrane can organically combine the catalysis characteristics of enzyme and the good separation performance of film, thereby constitutes the enzyme membrane bio-reactor.Microporous membrane is the film with countless intercommunication micropores (aperture is generally 0.01～10 μ m), and its base material can be polyolefine, polyethers alum, polyurethane etc.In above-mentioned base material, polypropylene has excellent properties such as high-melting-point, high heat resistance, and polypropylene material is cheap, thereby the microporous membrane that polypropylene material is made has obtained widespread use.The existing polypropylene material making fixed enzyme membrane making method of utilizing has physics method and chemical method two big classes, the advantage of physics method is that enzyme do not participate in chemical reaction, one-piece construction remains unchanged, the catalytic activity of enzyme obtains fine reservation, but the bearing capacity that this method is made the zymoprotein on the fixed enzyme membrane that obtains is lower, only is about the 15mg/g film, the bearing capacity of zymoprotein is lower, has influenced the use of fixed enzyme membrane; Chemical method be receive natural by chemical bond-linking enzyme or the synthetic polymer carrier on, use coupling agent enzyme is crosslinked by the group on enzyme surface, relative molecular weight is bigger, the method for insoluble immobilized enzyme and form, but, this method is destroyed serious in making processes to the enzymic activity group, greatly reduce the catalytic activity of enzyme, the fixed enzyme membrane catalytic activity that this made obtains is low, has limited fixed enzyme membrane application in practice greatly.
Summary of the invention
The present invention makes the problem that the zymoprotein bearing capacity is low and catalytic activity is low of the fixed enzyme membrane that obtains in order to solve existing method, and the preparation method of fixed enzyme membrane is provided.
The preparation method of fixed enzyme membrane of the present invention carries out according to following steps: one, polypropylene screen soaks 22～26h in acetone; putting into the acetone soln that concentration is the 0.2mol/L benzophenone behind dry 22～26h under 25～35 ℃ of conditions then; with UV-irradiation 14～16min, UV-irradiation intensity is 90～110 μ W/cm under nitrogen protection
2Irradiation distance 11～13cm, film being dried naturally the back immerses in the ethanolic soln that mass concentration is 10%～30% methyl methacrylate again, water-bath vibration 22～26h under 28～32 ℃ of conditions, vacuum-drying 22～26h promptly obtains the methyl methacrylate-grafted film under 28～32 ℃ condition then, and wherein the solvent of the ethanolic soln of methyl methacrylate is that mass concentration is 90%～99% ethanol; Two, to be immersed in mass concentration be in 13%～17% the hexanediamine solution to the methyl methacrylate-grafted film, obtained being adsorbed with the methyl methacrylate-grafted film of hexanediamine behind the 35～45min that vibrates under 45～55 ℃ of conditions with washed with de-ionized water; Three, 4.5 the bacillus licheniformis alkali protease of～5.5g or the trans-glutaminases of 4.5～5.5g are dissolved in 480～520ml, concentration is 0.05mol/L, pH obtains bacillus licheniformis alkali protease solution or transglutamin-ase 9 enzyme solution in 6.0 the phosphate buffer soln, the methyl methacrylate-grafted film that will be adsorbed with hexanediamine then is immersed in bacillus licheniformis alkali protease solution or the transglutamin-ase 9 enzyme solution, reacting 23～25h under 3.8～4.2 ℃ of conditions, is 0.05mol/L with concentration, pH has promptly obtained fixed enzyme membrane after 6.0 phosphate buffer soln cleans.
The present invention is carrier with the microporous polypropylene membrane, adopts the process for fixation of UV-irradiation initiation grafting hydroxyethyl methylacrylate that bacillus licheniformis alkali protease and microbial transglutaminase are carried out immobilization.The structure of employed hydroxyethyl methylacrylate is in the present embodiment:
Owing to contain a plurality of hydroxyls in the structure of hydroxyethyl methylacrylate; more binding site is provided after UV-irradiation, can for the immobilization of follow-up enzyme; and in the enzyme immobilization process, added polyol as protective material; weakened the destruction of glutaraldehyde greatly to enzymic activity group such as carboxyl and Serine hydroxyl etc.; improve the zymoprotein bearing capacity of fixed enzyme membrane of the present invention greatly, guaranteed that the catalytic activity of enzyme is constant.The bearing capacity of the bacillus licheniformis alkali protease of the fixed enzyme membrane that the present invention's making obtains is greater than the 35mg/g film, the bearing capacity of L-Glutamine deaminase is greater than the 20mg/g film, the proteolytic enzyme of comparing with existing method carrying has improved more than 10%, the present invention makes the zymoprotein bearing capacity height of being fixed enzyme membrane, and the fixed enzyme membrane catalytic activity that the inventive method making obtains is good, the catalytic activity that the present invention that compares with the fixed enzyme membrane that existing method making obtains makes the fixed enzyme membrane that obtains has improved about 20%, and the inventive method is made the adsorptive power and the Heat stability is good of the fixed enzyme membrane that obtains.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the preparation method of present embodiment fixed enzyme membrane carries out according to following steps: one, polypropylene screen soaks 22～26h in acetone; putting into the acetone soln that concentration is the 0.2mol/L benzophenone behind dry 22～26h under 25～35 ℃ of conditions then; with UV-irradiation 14～16min, UV-irradiation intensity is 90～110 μ W/cm under nitrogen protection
2Irradiation distance 11～13cm, film being dried naturally the back immerses in the ethanolic soln that mass concentration is 10%～30% methyl methacrylate again, water-bath vibration 22～26h under 28～32 ℃ of conditions, vacuum-drying 22～26h promptly obtains the methyl methacrylate-grafted film under 28～32 ℃ condition then, and wherein the solvent of the ethanolic soln of methyl methacrylate is that mass concentration is 90%～99% ethanol; Two, to be immersed in mass concentration be in 13%～17% the hexanediamine solution to the methyl methacrylate-grafted film, obtained being adsorbed with the methyl methacrylate-grafted film of hexanediamine behind the 35～45min that vibrates under 45～55 ℃ of conditions with washed with de-ionized water; Three, 4.5 the bacillus licheniformis alkali protease of～5.5g or the trans-glutaminases of 4.5～5.5g are dissolved in 480～520ml, concentration is 0.05mol/L, pH obtains bacillus licheniformis alkali protease solution or transglutamin-ase 9 enzyme solution in 6.0 the phosphate buffer soln, the methyl methacrylate-grafted film that will be adsorbed with hexanediamine then is immersed in bacillus licheniformis alkali protease solution or the transglutamin-ase 9 enzyme solution, reacting 23～25h under 3.8～4.2 ℃ of conditions, is 0.05mol/L with concentration, pH has promptly obtained fixed enzyme membrane after 6.0 phosphate buffer soln cleans.
Acetone is solvent in the acetone soln of the benzophenone of present embodiment step 1, and benzophenone is a solute.
The bearing capacity of the bacillus licheniformis alkali protease of the fixed enzyme membrane that the present embodiment making obtains is greater than the 35mg/g film, the bearing capacity of L-Glutamine deaminase is greater than the 20mg/g film, the proteolytic enzyme carrying of comparing with the fixed enzyme membrane that existing method making obtains has improved more than 10%, present embodiment is made the zymoprotein bearing capacity height of being fixed enzyme membrane, and the fixed enzyme membrane catalytic activity that the inventive method making obtains is good, and the catalytic activity that the present embodiment of comparing with the fixed enzyme membrane that existing method making obtains is made the fixed enzyme membrane that obtains has improved about 20%.The inventive method is made the adsorptive power and the Heat stability is good of the fixed enzyme membrane that obtains.
Present embodiment is made in the separation preparation that the fixed enzyme membrane obtain can be applied in soybean protein, separate soybean protein purity and the good dispersity that obtains, the molecular structure integrity of soybean protein is good.
Embodiment two: what present embodiment and embodiment one were different is: polypropylene screen soaks 24h in the step 1 in acetone.Other step and parameter are identical with embodiment one.
Embodiment three: what present embodiment and embodiment one to two were different is: in the step 1 under 30 ℃ of conditions dry 24h.Other step and parameter are identical with embodiment one to two.
Embodiment four: what present embodiment and embodiment one to three were different is: use UV-irradiation 15min in the step 1, UV-irradiation intensity is 100 μ W/cm
2, irradiation distance 12cm.Other step and parameter are identical with embodiment one to three.
Embodiment five: what present embodiment and embodiment one to four were different is: 40min vibrates under 50 ℃ of conditions in the step 2.Other step and parameter are identical with embodiment one to four.
Embodiment six: what present embodiment and embodiment one to five were different is: react 24h in the step 3 under 4 ℃ of conditions.Other step and parameter are identical with embodiment one to five.
Embodiment seven: the preparation method of present embodiment fixed enzyme membrane carries out according to following steps: one, polypropylene screen soaks 24h in acetone; behind dry 24h under 30 ℃ of conditions, put into the acetone soln that concentration is the 0.2mol/L benzophenone then; use UV-irradiation 15min under nitrogen protection, UV-irradiation intensity is 100 μ W/cm
2Irradiation distance 11～13cm, film being dried naturally the back immerses in the ethanolic soln that mass concentration is 20% methyl methacrylate again, water-bath vibration 24h under 30 ℃ of conditions, vacuum-drying 24h promptly obtains the methyl methacrylate-grafted film under 30 ℃ condition then, and wherein the solvent of the ethanolic soln of methyl methacrylate is that mass concentration is 95% ethanol; Two, to be immersed in mass concentration be in 15% the hexanediamine solution to the methyl methacrylate-grafted film, obtained being adsorbed with the methyl methacrylate-grafted film of hexanediamine behind vibration 40min under 50 ℃ of conditions with washed with de-ionized water; Three, to be dissolved in 500ml, concentration be that 0.05mol/L, pH obtain bacillus licheniformis alkali protease solution or transglutamin-ase 9 enzyme solution in 6.0 the phosphate buffer soln to the bacillus licheniformis alkali protease of 5g, the methyl methacrylate-grafted film that will be adsorbed with hexanediamine then is immersed in bacillus licheniformis alkali protease solution or the transglutamin-ase 9 enzyme solution, reacting 24h under 4 ℃ of conditions, is that 0.05mol/L, pH have promptly obtained fixed enzyme membrane after 6.0 phosphate buffer soln cleans with concentration.
Acetone is solvent in the acetone soln of the benzophenone of present embodiment step 1, and benzophenone is a solute.
The bearing capacity that present embodiment is made the bacillus licheniformis alkali protease of the fixed enzyme membrane that obtains is the 42mg/g film, the proteolytic enzyme carrying of comparing with the fixed enzyme membrane that existing method making obtains has improved 12%, present embodiment is made the zymoprotein bearing capacity height of being fixed enzyme membrane, and the fixed enzyme membrane catalytic activity that the inventive method making obtains is good, and the catalytic activity that the present embodiment of comparing with the fixed enzyme membrane that existing method making obtains is made the fixed enzyme membrane that obtains has improved 25%.Present embodiment is made the adsorptive power and the Heat stability is good of the fixed enzyme membrane that obtains.
Embodiment eight: what present embodiment and embodiment seven were different is: to be dissolved in the phosphate buffer soln other step and parameter identical with embodiment seven for the trans-glutaminases of 5g in the step 3.
The bearing capacity that present embodiment is made the trans-glutaminases of the fixed enzyme membrane that obtains is the 29mg/g film, the proteolytic enzyme carrying of comparing with the fixed enzyme membrane that existing method making obtains has improved 11.5%, present embodiment is made the zymoprotein bearing capacity height of being fixed enzyme membrane, and the fixed enzyme membrane catalytic activity that the inventive method making obtains is good, and the catalytic activity that the present embodiment of comparing with the fixed enzyme membrane that existing method making obtains is made the fixed enzyme membrane that obtains has improved 35%.Present embodiment is made the adsorptive power and the Heat stability is good of the fixed enzyme membrane that obtains.
Claims (6)
1. the preparation method of fixed enzyme membrane; the preparation method who it is characterized in that fixed enzyme membrane carries out according to following steps: one, polypropylene screen soaks 22～26h in acetone; putting into the acetone soln that concentration is the 0.2mol/L benzophenone behind dry 22～26h under 25～35 ℃ of conditions then; with UV-irradiation 14～16min, UV-irradiation intensity is 90～110 μ W/cm under nitrogen protection
2Irradiation distance 11～13cm, film being dried naturally the back immerses in the ethanolic soln that mass concentration is 10%～30% methyl methacrylate again, water-bath vibration 22～26h under 28～32 ℃ of conditions, vacuum-drying 22～26h promptly obtains the methyl methacrylate-grafted film under 28～32 ℃ condition then, and wherein the solvent of the ethanolic soln of methyl methacrylate is that mass concentration is 90%～99% ethanol; Two, to be immersed in mass concentration be in 13%～17% the hexanediamine solution to the methyl methacrylate-grafted film that is prepared by step 1, obtained being adsorbed with the methyl methacrylate-grafted film of hexanediamine behind the 35～45min that vibrates under 45～55 ℃ of conditions with washed with de-ionized water; Three, 4.5 the bacillus licheniformis alkali protease of～5.5g or the trans-glutaminases of 4.5～5.5g are dissolved in 480～520ml, concentration is 0.05mol/L, pH obtains bacillus licheniformis alkali protease solution or transglutamin-ase 9 enzyme solution in 6.0 the phosphate buffer soln, to be immersed in by the methyl methacrylate-grafted film that is adsorbed with hexanediamine that step 2 prepares in bacillus licheniformis alkali protease solution or the transglutamin-ase 9 enzyme solution then, reacting 23～25h under 3.8～4.2 ℃ of conditions, is 0.05mol/L with concentration, pH has promptly obtained fixed enzyme membrane after 6.0 phosphate buffer soln cleans.
2. the preparation method of fixed enzyme membrane according to claim 1 is characterized in that polypropylene screen soaks 24h in the step 1 in acetone.
3. the preparation method of fixed enzyme membrane according to claim 1 and 2 is characterized in that in the step 1 dry 24h under 30 ℃ of conditions.
4. the preparation method of fixed enzyme membrane according to claim 3 is characterized in that using in the step 1 UV-irradiation 15min, and UV-irradiation intensity is 100 μ W/cm
2, irradiation distance 12cm.
5. according to the preparation method of claim 1,2 or 4 described fixed enzyme membranes, it is characterized in that the 40min that under 50 ℃ of conditions, vibrates in the step 2.
6. the preparation method of fixed enzyme membrane according to claim 5 is characterized in that reacting 24h in the step 3 under 4 ℃ of conditions.
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| CN201010300675XA CN101781645B (en) | 2010-01-25 | 2010-01-25 | Preparation method of fixed enzyme membrane |
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| CN201010300675XA CN101781645B (en) | 2010-01-25 | 2010-01-25 | Preparation method of fixed enzyme membrane |
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| CN101781645B true CN101781645B (en) | 2011-11-16 |
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| WO2017019874A1 (en) * | 2015-07-30 | 2017-02-02 | North Carolina State University | Grafted islands-in-the-sea nonwoven for high capacity ion exchange bioseparation |
| CN108545835B (en) * | 2018-04-16 | 2021-04-30 | 成都大学 | Method for removing protein in yellow serofluid by ultrafiltration by using enzyme membrane reactor |
| CN111100894A (en) * | 2019-12-31 | 2020-05-05 | 江苏苏南药业实业有限公司 | Method for carrying out fixed-point polyethylene glycol long-acting modification on interferon IFN α -2b |
| CN116426021B (en) * | 2023-03-30 | 2024-06-21 | 淮阴工学院 | Preparation method and application of targeting gallium-containing protein modified SEBS (styrene-ethylene-butylene-styrene) film |
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