CN101260395A - Method for preparing double-pore enzyme immobilization reactor - Google Patents
Method for preparing double-pore enzyme immobilization reactor Download PDFInfo
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- CN101260395A CN101260395A CNA2007100568664A CN200710056866A CN101260395A CN 101260395 A CN101260395 A CN 101260395A CN A2007100568664 A CNA2007100568664 A CN A2007100568664A CN 200710056866 A CN200710056866 A CN 200710056866A CN 101260395 A CN101260395 A CN 101260395A
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Abstract
The invention provides a preparation method for a diplopore enzyme immobilization reactor, wherein, alkoxy silanes are used as silicon sources; polyethylene glycols are used as macroporous template agents; cetyltrimethylammonium bromides, ammonia or citric acids are used as mesopore template agents; a diplopore silica gel overall carrier is prepared through demoulding, cleaning, drying and baking by adoption of the improved sol-gel method; the silica gel overall carrier is arranged inside a heat contraction tube and closely coated after heating; the silica gel overall carrier after coating is cleaned by utilization of reagents which contain 3-amino propyl triethoxysilane; amido is bonded on the surface of silica gel; the aminated silica gel is cleaned by glutaraldehyde solution and then cleaned by water solution which contains horse-radish catalases, and then the diplopore enzyme immobilization reactor is prepared. The product of the invention can be used for processing waste water which contains persistent organic pollutants and has the advantages of low cost, high efficiency, safe reaction and capability of being reused.
Description
Technical field
The present invention relates to a kind of preparation method of double-pore enzyme immobilization reactor.
Background technology
Be accompanied by economy, fast development of society, the harm of people's environmental pollution is growing interest also.The essence of environmental pollution is in the mankind's activity a large amount of pollutents to be entered environment, influences its self-purification capacity, has upset the function of the ecosystem.In the various chemical of introducing environment, persistence organic pollutant is particularly noticeable.Because of it has extended residual, biological accumulation, half volatile and high toxicity, environment and human health had serious harm.
Degradation technique to persistence organic pollutant has carried out research widely both at home and abroad, and method commonly used has: biological process, chemical method.But existing treatment technology all comes with some shortcomings.As adopt single biological process to be difficult to the pollutent of high density is degraded fast, and adopt chemical method often to need the certain reaction condition, there is contradiction between its degradation efficiency and the processing cost.
People find that under study for action enzyme is as a kind of biological catalyst, and its katalysis has characteristics such as highly selective, high catalytic activity, reaction conditions gentleness, non-environmental-pollution.Along with the technical progress of enzyme industry, the cost of enzyme extraction greatly reduces, and zymotechnic has prospect in the application in pollutant control field.Peroxidase such as horseradish catalase have been used to the chlorophenol pollutant in the catalyzed degradation waste water.
But the enzyme of unbound state is to heat, highly basic, strong acid, high ionic strength, relatively poor, the easy inactivation of organic solvent equistability, and easily sneaks into catalysate after the reaction, and purification difficult can not be reused.In order to overcome these problems, enzyme immobilization technology arises at the historic moment.Traditional enzyme immobilization method is broadly divided into four classes: absorption method, crosslinking, entrapping method, covalent attachment method.In use respectively there are some shortcomings in these methods.As absorption method, a little less than the bonding force, enzyme is easy to come off between enzyme and the carrier; Crosslinking need adopt linking agent, can only be as the supplementary means of other immobilization methods; Often there are problems such as diffusional limitation in the using embedding immobilization enzyme, unsuitable catalysis macromolecule substrate; The covalent attachment method needs violent reaction, and the activity of the immobilized enzyme loss is serious.
Therefore, it is particularly necessary that the method for development of new immobilized enzyme seems, its guiding theory is: be implemented in the immobilization of carrying out enzyme under the comparatively gentle condition, reduce as far as possible or avoid the loss of enzyme activity.
Because carrier has considerable influence to immobilized enzyme, make the ideal immobilized enzyme, should select the rational and effective process for fixation for use, good carrier is arranged again simultaneously.Chinese scholars has been carried out a large amount of work round synthetic, the modification of novel carriers material.There are some researches show that having big specific surface area is expected to become the ideal enzyme immobilization carrier with the mesoporous molecular sieve that the surface has high density slightly acidic silanol groups.
The research of mesopore material in recent years is very active, has obtained considerable progress aspect synthetic.Wherein the surfactant templates method has obtained using widely, has synthesized many mesopore materials with heterogeneous networks composition and pore structure.Yet, also have the problem of many worth researchs and solution about mesopore material.Since they normally particle diameter be the fine powder of 1-15 μ m, though have big surface-area, also have very big defective in actual applications.Such as, in liquid phase reaction, not easily separated, recovery will be loaded into the enzyme post usually and be used behind immobilized enzyme.The particle diameter of powder carrier is thin more, and the amount of immobilized enzyme is high more, but the resistance to mass transfer of water is also high more.
The present invention is exactly at the problems referred to above, and a kind of preparation method of double-pore enzyme immobilization reactor is provided.
Summary of the invention
Technical scheme provided by the invention is: a kind of double-pore enzyme immobilization reactor, with prepared diplopore monolithic silica column is carrier, by this solid support material is coated, the silica gel surface carries out amination modified, crosslinked, fixing horseradish catalase, makes double-pore enzyme immobilization reactor.Prepared enzyme immobilization reactor covers one deck engineering plastics outward, and whole silica-gel carrier has the skeleton that is cross-linked with each other and the macropore of perforation.In the silica gel skeleton mesopore is arranged.Big hole on framework is 0.3-15 μ m, macropore diameter 0.5-10 μ m, mesopore aperture 2-50nm.The horseradish catalase is immobilized onto diplopore monolithic silica gel material internal.
The present invention also provides above-mentioned monolithic silica gel preparation methods: with the organoalkoxysilane is the silicon source, with the polyoxyethylene glycol is the macropore template, with cetyl trimethylammonium bromide or ammoniacal liquor or citric acid is the mesopore template, adopt improved sol-gel process, make the monolithic silica gel carrier by the demoulding, washing, drying, roasting.The monolithic silica gel carrier is placed in the heat-shrinkable tube, after the heating monolithic silica gel material is closely coated.With coating good monolithic silica gel material, amino on the silica gel surface bond with the reagent flushing that contains the 3-aminopropyltriethoxywerene werene.After washing amidized silica-gel carrier with glutaraldehyde, wash, promptly get made double-pore enzyme immobilization reactor to contain the catalatic aqueous solution of horseradish.
Above-mentioned organoalkoxysilane is tetramethoxy-silicane or tetraethoxysilane.
Above-mentioned heat shrinkable pipe material is other fluoro-containing plastics of tetrafluoroethylene or tool red shortness.
Double-pore enzyme immobilization reactor of the present invention has unique double-pore structure, has fluid permeability height, characteristics that the enzyme charge capacity is big.Immobilized enzyme is difficult for inactivation, can repeatedly use.The application of diplopore, integral material can also overcome the shortcoming of conventional powder material filling collimation difference, and is easier to recycle, and the diplopore adjustment and control of pore diameter can make catalyzed reaction have good selectivity simultaneously.
Embodiment
Be better understanding content of the present invention, the present invention is further illustrated below by embodiment, but the cited case does not limit protection scope of the present invention.
Embodiment 1: in beaker, adding pH is 5.0 acetic acid aqueous solution 50mL, adds the 4.9g polyoxyethylene glycol again, stirs, and adds the 24mL tetramethoxy-silicane, stirs 10min in the time of 0 ℃, adds the 0.1g cetyl trimethylammonium bromide, and 30min is stirred in continuation in the time of 0 ℃.The gained mixture is poured in several the tetrafluoroethylene test tubes, in 40 ℃ of water-baths, reach gelation point after, ageing 24h, silicagel column must wet.With the silicagel column after the demoulding, the rinsing, place in the thermostat container, 70 ℃ of dry 12h, strict control colloid dehydrating speed, roasting in chamber type electric resistance furnace afterwards is with 1 ℃ of min
-1Speed be warming up to 550 ℃, constant temperature 4h makes the diplopore monolithic silica column.With the diplopore integral post that makes, place in the tetrafluoroethylene heat-shrink tube, in 180 ℃ of reaction 4h, finish coating to the diplopore integral post.With coating good silicagel column volume percent is that the toluene solution of 25% 3-aminopropyltriethoxywerene werene washes, flow velocity with 0.1mL/min washes 10h down at 80 ℃, then with sealed at both ends, at room temperature place 12h, carry out vacuum-drying afterwards, then respectively with toluene, ether flushing, vacuum-drying 10h subsequently, to constant weight, get amidized silicagel column.In the time of 4 ℃, be 1% glutaraldehyde phosphate buffer soln flushing 12h with amidized silicagel column concentration, flow velocity is 0.1mL/min.The catalatic phosphate buffer soln of horseradish that with concentration is 1mg/mL then with 0.1mL/min flow velocity flushing 12h, promptly gets prepared double-pore enzyme immobilization reactor in the time of 4 ℃.
Embodiment 2: in beaker, adding pH is 5.0 acetic acid aqueous solution 50mL, adds the 4.0g polyoxyethylene glycol again, stirs, and adds the 30mL tetramethoxy-silicane, stirs 10min in the time of 0 ℃.The gained mixture is poured in several the tetrafluoroethylene test tubes, in 40 ℃ of water-baths, reach gelation point after, ageing 24h, silicagel column must wet.With the silicagel column after the demoulding, the rinsing, be dipped in the ammoniacal liquor of 0.01mol/L, 120 ℃ of hydrothermal treatment consists 6h place in the thermostat container then, 70 ℃ of dry 12h, strict control colloid dehydrating speed, roasting in chamber type electric resistance furnace afterwards is with 1 ℃ of min
-1Speed be warming up to 550 ℃, constant temperature 4h makes the diplopore monolithic silica column.With the diplopore integral post that makes, place in the tetrafluoroethylene heat-shrink tube, in 160 ℃ of reaction 6h, finish coating to the diplopore integral post.With coating good silicagel column volume percent is that the toluene solution of 20% 3-aminopropyltriethoxywerene werene washes, flow velocity with 0.2mL/min washes 10h down at 80 ℃, then with sealed at both ends, at room temperature place 12h, carry out vacuum-drying afterwards, then respectively with toluene, ether flushing, vacuum-drying 10h subsequently, to constant weight, get amidized silicagel column.In the time of 4 ℃, be 2.5% glutaraldehyde phosphate buffer soln flushing 12h with amidized silicagel column concentration, flow velocity is 0.1mL/min.The catalatic phosphate buffer soln of horseradish that with concentration is 5mg/mL then with 0.1mL/min flow velocity flushing 12h, promptly gets prepared double-pore enzyme immobilization reactor in the time of 4 ℃.
Embodiment 3: in beaker, adding pH is 5.0 acetic acid aqueous solution 50mL, adds the 5.2g polyoxyethylene glycol again, stir, add the 36mL tetraethoxysilane, in the time of 0 ℃, stir 10min, add the 0.29g citric acid, continue to stir 30min in the time of 0 ℃, transferring the pH of solution with ammoniacal liquor is 5.0.The gained mixture is poured in several the tetrafluoroethylene test tubes, in 40 ℃ of water-baths, reach gelation point after, ageing 24h, silicagel column must wet.With the silicagel column after the demoulding, the rinsing, place in the thermostat container, 70 ℃ of dry 12h, strict control colloid dehydrating speed, roasting in chamber type electric resistance furnace afterwards is with 1 ℃ of min
-1Speed be warming up to 550 ℃, constant temperature 4h makes the diplopore monolithic silica column.With the diplopore integral post that makes, place in the tetrafluoroethylene heat-shrink tube, in 180 ℃ of reaction 4h, finish coating to the diplopore integral post.With coating good silicagel column volume ratio is that the toluene solution of 15% 3-aminopropyltriethoxywerene werene washes, wash 12h with the 0.1mL/min flow velocity down at 70 ℃, then with sealed at both ends, at room temperature place 12h, carry out vacuum-drying afterwards, then respectively with toluene, ether flushing, vacuum-drying 10h subsequently, to constant weight, get amidized silicagel column.In the time of 4 ℃, be 5% glutaraldehyde phosphate buffer soln flushing 12h with amidized silicagel column concentration, flow velocity is 0.1mL/min.Then with the catalatic phosphate buffer soln of the horseradish of 10mg/mL 4 ℃ the time, with 0.1mL/min flow velocity flushing 10h, promptly get prepared double-pore enzyme immobilization reactor.
Embodiment 4: in beaker, adding pH is 5.0 acetic acid aqueous solution 50mL, adds the 6.0g polyoxyethylene glycol again, stirs, and adds the 35mL tetramethoxy-silicane, stirs 10min in the time of 0 ℃.The gained mixture is poured in several the tetrafluoroethylene test tubes, in 40 ℃ of water-baths, reach gelation point after, ageing 24h, silicagel column must wet.With the silicagel column after the demoulding, the rinsing, be dipped in the ammoniacal liquor of 0.1mol/L, 120 ℃ of hydrothermal treatment consists 6h place in the thermostat container then, 80 ℃ of dry 10h, strict control colloid dehydrating speed, roasting in chamber type electric resistance furnace afterwards is with 1 ℃ of min
-1Speed be warming up to 550 ℃, constant temperature 4h makes the diplopore monolithic silica column.With the diplopore integral post that makes, place in the tetrafluoroethylene heat-shrink tube, in 200 ℃ of reaction 2h, finish coating to the diplopore integral post.With coating good silicagel column volume percent is that the toluene solution of 25% 3-aminopropyltriethoxywerene werene washes, wash 12h with the 0.1mL/min flow velocity down at 60 ℃, then with sealed at both ends, at room temperature place 12h, carry out vacuum-drying afterwards, then respectively with toluene, ether flushing, vacuum-drying 10h subsequently, to constant weight, get amidized silicagel column.In the time of 4 ℃, be 5.0% glutaraldehyde phosphate buffer soln flushing 12h with amidized silicagel column concentration, flow velocity is 0.1mL/min.The catalatic phosphate buffer soln of horseradish that with concentration is 5mg/mL then with 0.1mL/min flow velocity flushing 12h, promptly gets prepared double-pore enzyme immobilization reactor in the time of 4 ℃.
Claims (6)
1. the preparation method of a double-pore enzyme immobilization reactor is characterized in that, is carrier with the diplopore monolithic silica column, coats integral carriers with the engineering plastics with red shortness.Fix by amination modified, crosslinking Treatment, horseradish catalase, make double-pore enzyme immobilization reactor the silica gel surface.
2. the preparation method of a kind of double-pore enzyme immobilization reactor as claimed in claim 1 is characterized in that, diplopore monolithic silica gel column support has the skeleton that is cross-linked with each other and the macropore of perforation.The silica gel skeleton has mesopore.Big hole on framework is 0.3-15 μ m, macropore diameter 0.5-10 μ m, mesopore aperture 2-50nm.
3. the preparation method of a kind of double-pore enzyme immobilization reactor as claimed in claim 1 is characterized in that, the material that coats diplopore monolithic silica gel column support is to have the polyfluortetraethylene pipe of thermal contraction performance or other fluoro-containing plastics of tool red shortness.
4. the preparation method of a kind of double-pore enzyme immobilization reactor as claimed in claim 1, it is characterized in that, with the toluene solution of 3-aminopropyltriethoxywerene werene, the flushing silica-gel carrier, the silica-gel carrier surface is carried out amination modified, flow velocity is 0.1-1.0mL/min.The temperature of reaction is 50-100 ℃.
5. the preparation method of a kind of double-pore enzyme immobilization reactor as claimed in claim 1 is characterized in that, handles with the silica-gel carrier of glutaraldehyde as cross linker after to amination.
6. the preparation method of a kind of double-pore enzyme immobilization reactor as claimed in claim 1 is characterized in that, with the catalatic phosphate buffer soln of horseradish silica-gel carrier is washed, and realizes the immobilization of horseradish catalase on the diplopore silica-gel carrier.Flow velocity is 0.1-1.0mL/min.The temperature of reaction is 50-100 ℃.
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| CNA2007100568664A CN101260395A (en) | 2007-03-06 | 2007-03-06 | Method for preparing double-pore enzyme immobilization reactor |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101825615B (en) * | 2009-03-04 | 2012-09-05 | 复旦大学 | Preparation method of integral column for in situ protein quick enzymolysis and application thereof |
| CN103694382A (en) * | 2012-09-28 | 2014-04-02 | 中国石油天然气股份有限公司 | Preparation method of double-mould-pore-size-distribution silica-gel carrier |
| CN108640132A (en) * | 2018-04-10 | 2018-10-12 | 江苏金茂源生物化工有限责任公司 | A kind of macroporous-mesoporous alumina and its preparation method and application |
-
2007
- 2007-03-06 CN CNA2007100568664A patent/CN101260395A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101825615B (en) * | 2009-03-04 | 2012-09-05 | 复旦大学 | Preparation method of integral column for in situ protein quick enzymolysis and application thereof |
| CN103694382A (en) * | 2012-09-28 | 2014-04-02 | 中国石油天然气股份有限公司 | Preparation method of double-mould-pore-size-distribution silica-gel carrier |
| CN103694382B (en) * | 2012-09-28 | 2016-05-11 | 中国石油天然气股份有限公司 | The preparation method of bimodulus pore-size distribution silica-gel carrier |
| CN108640132A (en) * | 2018-04-10 | 2018-10-12 | 江苏金茂源生物化工有限责任公司 | A kind of macroporous-mesoporous alumina and its preparation method and application |
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Open date: 20080910 |