CN101096665A - Application of halloysite in enzyme immobilization carrier - Google Patents
Application of halloysite in enzyme immobilization carrier Download PDFInfo
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- CN101096665A CN101096665A CNA2007100545588A CN200710054558A CN101096665A CN 101096665 A CN101096665 A CN 101096665A CN A2007100545588 A CNA2007100545588 A CN A2007100545588A CN 200710054558 A CN200710054558 A CN 200710054558A CN 101096665 A CN101096665 A CN 101096665A
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Abstract
The invention discloses an application of halloysite as enzyme biological catalyst carrier material in the enzyme fixing carrier, which comprises the following steps: adopting halloysite as enzyme biological catalyst carrier material; fining the halloysite; dispersing to do chemical disposal; adding certain quantity of enzyme liquid; solidifying under certain temperature; separating and settling the composite system; washing; drying; obtaining the product.
Description
Technical field
The present invention relates to chemical field, relate in particular to the application of halloysite as a kind of enzyme immobilization carrier.
Background technology
Enzyme is as a kind of natural polymer catalyst, has high selectivity, the catalytic reaction condition gentleness, pollution-free, but the enzyme of unbound state is relatively poor to heat, strong acid, highly basic, high ionic strength, organic solvent equistability, easy inactivation, and sneak into material such as catalysate after the reaction, purification difficult can not be reused.And enzyme is easy to preserve through overcoming above shortcoming after the immobilization simultaneously, and the stability of heat, pH etc. is also improved a lot, and the susceptibility of inhibitor is reduced, and the enzyme that has has also possessed the characteristic of protease inhibitor decomposition.Through the fixed enzyme, through simple filtering or centrifugal, enzyme just can reclaim after reaction is finished, and can recycle, and the enzyme activity reduction is less, has so just reduced production cost.Therefore, the immobilization technology of resolvase is the focus of studying recently always.Immobilized enzyme is answered industrial demand and is produced, in industrial production, biochemical pharmacy, obtained widespread use, and recently at the most active biosensor of research, wastewater treatment and environmental monitoring aspect have also obtained application, for this reason, the high fixed rate of research preparation, high enzyme immobilized enzyme alive, high stability have very high using value.
Carrier Materials of Immobilized Enzyme commonly used has polymer carrier, inorganic carrier and composite carrier, and inorganic carrier material has good stability, physical strength height, is difficult for by Institute of Micro-biology's decomposition, acid and alkali-resistance, advantage such as cost is low, the life-span is long.Domestic researchist has carried out extensive studies aspect immobilization technology, also developed some unique immobilization technologies, and solid support material still is unrealized large-scale synthetic.Paul Takhistov has prepared the aluminum oxide with nanoporous with anonizing, makes the enzyme catalysis biosensor with its absorption penicillinase, and the existence of nano grade pore structure has strengthened the fixed action of aluminum oxide to enzyme; Humphrey H.P. etc. has utilized three kinds of molecular sieve MCM-41, MCM-48 and SBA-15 that trypsinase has been carried out immobilization research; Usefulness 3-glycerine propyl trimethoxy silicanes such as AntonellaPetri make epoxidation silica gel, are used for fixing muriate, catalase; People such as Li Zeng pass through in magnetic Fe
3O
4The copolymerization on surface has been synthesized prepared in reaction in dendroid composite magnetic particle (MS type carrier), with the method immobilized porcine pancreatic lipase of simple absorption and glutaraldehyde covalent cross-linking.The immobilized enzyme stability of above method gained all is higher than resolvase, but required carrier all needs through the synthetic preparation, the technology relative complex, and cost is very high, therefore, it is significant that research is suitable for the novel carriers and the corresponding enzyme immobilization technology of enzyme immobilization.
Summary of the invention
The object of the invention be to provide a kind of cheap and easy to get, be easy to industrialization and can prepare high fixed rate, high enzyme is lived, the novel enzyme immobilization carrier of high stability immobilized enzyme is applied to enzyme and fixes, and all can immobilization to the enzyme of different molecular weight.
Technical solution of the present invention is as follows: select natural mineral for use---and the nanometer halloysite is a Carrier Materials of Immobilized Enzyme, is applied to enzyme and fixes.
Because the hollow tubular structure that halloysite has complete form, even size distribution is stable, pipe range 0.5～3 μ m, about 30～the 80nm of external diameter of pipe, about 6～the 40nm of bore, the hydroxy functional group that has high negative charged surface and can react with enzyme has high specific surface area and vesicular structure, can improve the binding ability between carrier and the enzyme greatly, thereby make carrier easily and mutually crosslinked operability and the stability that improves curing degree and immobilized enzyme of enzyme.For this reason, the present invention selects for use halloysite as Carrier Materials of Immobilized Enzyme.
Concrete applying step: at first halloysite is carried out thinning processing, use to sieve to obtain the halloysite powder greater than 200 purposes; The enzyme liquid that halloysite powder and buffer preparation by pH=5～8 are formed mixes, and stirs, and under 0～35 ℃ of temperature, carries out immobilized reactant, then with mixed system centrifugal settling, washing, drying, and being fixed enzyme; Perhaps for improving the enzyme fixed effect better, can carry out halloysite cross-linking modified earlier, be about to soak in the cross-linking agent solution that the halloysite powder joins 0.5%～7% percentage concentration, fully washing then, add the enzyme liquid that the buffer preparation by pH value=5～8 forms again and carry out immobilized reactant, mixed system centrifugal settling subsequently, washing, drying, being fixed enzyme.
The buffered soln of pH value=5～8 is: phosphate buffer solution or acetate or acetate/acetic buffered soln.Linking agent is: glutaraldehyde etc.
The present invention adopts the carrier of natural mineral halloysite as enzyme immobilization first, and key problem in technology is:
1, uses by the above tubulose halloysite powder that sieves of 200 orders as enzyme immobilization material.
2, select to be suitable for the buffered soln of free enzyme.
3, the pH value of regulation system, the pH value plays crucial effects to the enzyme immobilization process, and the pH value can change the ionization state of enzyme molecule and fixation support, when the pH value was low, solidification effect was undesirable, if pH is too high, enzymatic structure there is destruction, can causes the active decline of immobilized enzyme catalysis.
4, the temperature of Controlling System, temperature is also very important for the enzyme immobilization process, and temperature is low excessively, and fixed rate and enzyme are lived all undesirable, and temperature is too high, and the protein structure of meeting destructive enzyme causes protein denaturation, even inactivation.
The invention has the advantages that: use natural mineral to be support material, cheap and easy to get, good stability, the physical strength height is easy to separate with product, and is recyclable and use repeatedly, reduced the running cost of immobilized enzyme; And carrier has big specific surface area and vesicular structure, hole internal diameter 6～40nm, the immobilization of the suitable various enzymes of the existence of nano grade pore structure, gained immobilized enzyme fixed rate height, excellent in stability.
The present invention utilizes the coomassie brilliant blue staining method to calculate the fixed rate of enzyme, it is alive that different enzymes takes corresponding method to measure its enzyme, through experiment, the enzyme of immobilized enzyme specific ionization attitude has the wideer temperature applicable range and the pH scope of application, and immobilized enzyme has good reusability and shelf stability.
Embodiment
For the present invention is better illustrated, as follows for embodiment:
Embodiment 1
Halloysite is carried out fragmentation, milled processed, sieve, obtain the halloysite powder with 250 mesh sieves.0.25g halloysite powder is mixed with 14mg/mL L-Aminoacylase liquid (by the phosphate buffer solution configuration of pH=7.0) 5mL; stir; under 4 ℃ of conditions; carried out immobilized reactant 12 hours, and perhaps halloysite was joined in the glutaraldehyde solution of 1% percentage concentration and soak, then fully washing; add above-mentioned L-Aminoacylase liquid again; carry out immobilized reactant, mixed system centrifugal settling subsequently, washing, drying, being fixed enzyme.The protein bound rate reaches 0.112～0.252mg (g halloysite)
-1, enzyme work reaches 0.105～0.253IU (mghalloysite)
-1, the pH value scope of application 4～14,40～100 ℃ of temperature applicable ranges, after immobilized enzyme used 15 times, remaining activity still reached 80～85%, does not lose activity.Sample is after depositing 3 months under 4 ℃, and enzymic activity still can keep 92～95% of original catalytic activity.
Embodiment 2
Halloysite is carried out suitable fragmentation, milled processed, and utilize 200 mesh sieves to sieve, obtain the halloysite powder.The halloysite powder of 0.25g is mixed with 12mg/mL urase liquid (by the phosphate buffer solution configuration of pH=7.0) 5mL, stir, under 4 ℃ of conditions, carried out immobilized reactant 12 hours, and perhaps halloysite was joined in the glutaraldehyde solution of 1% percentage concentration and soak, then fully washing, add above-mentioned urase liquid again, carry out immobilized reactant, mixed system centrifugal settling subsequently, washing, drying, being fixed urase.Enzymatic activity recovery can reach 52～85%, the pH value scope of application 5～9,32～90 ℃ of temperature applicable ranges are after immobilized enzyme uses 10 times, remaining activity still reaches the enzymic activity of depositing 2 months immobilized enzyme under 87～95%, 4 ℃ still can keep 80～95% of original catalytic activity.。
Embodiment 3
Halloysite is carried out suitable fragmentation, milled processed, and utilize 300 mesh sieves to sieve, obtain the halloysite powder.The halloysite powder of 0.25g is mixed with 16mg/mL horseradish peroxidase liquid (by the acetate/acetic buffered soln configuration of pH=6.0) 5mL, stir, under 14 ℃ of conditions, carried out immobilized reactant 12 hours, and perhaps halloysite was joined in the glutaraldehyde solution of 3% percentage concentration and soak, then fully washing, add above-mentioned horseradish peroxidase liquid again, carry out immobilized reactant, mixed system centrifugal settling subsequently, washing, drying are fixed horseradish peroxidase.Immobilization efficiency can be up to 95%, and immobilized enzyme work reaches 1.0U/g, the pH value scope of application 5.5～9.5, and 40～85 ℃ of temperature applicable ranges, after immobilized enzyme used 3 times, remaining activity still reached 80～85%, does not lose activity.Sample is after depositing 4 months under 4 ℃, and enzymic activity still can keep 75% of original catalytic activity.
Embodiment 4
Halloysite is carried out suitable fragmentation, milled processed, and utilize 400 mesh sieves to sieve, obtain the halloysite powder.The halloysite powder of 0.25g is mixed with 14mg/mL laccase liquid (by the phosphate buffer solution configuration of pH=5.0) 5mL, stir, under 24 ℃ of conditions, carried out immobilized reactant 12 hours, and perhaps halloysite was joined in the glutaraldehyde solution of 5% percentage concentration and soak, then fully washing, add above-mentioned laccase liquid again, carry out immobilized reactant, mixed system centrifugal settling subsequently, washing, drying are fixed laccase.Immobilization efficiency 60～86%, the pH value scope of application 2.5～9,25 ~ 65 ℃ of temperature applicable ranges are after immobilized enzyme uses 15 times, remaining activity still reaches the enzymic activity of depositing 2 months immobilized enzyme under 60～70%, 4 ℃ still can keep 80～95% of original catalytic activity.
Embodiment 5
Halloysite is carried out suitable fragmentation, milled processed, and utilize 250 mesh sieves to sieve, obtain the halloysite powder.The halloysite powder of 0.25g is mixed with 12mg/mL cellulase solution (by the phosphate buffer solution configuration of pH=8.0) 5mL, stir, under 2 ℃ of conditions, carried out immobilized reactant 12 hours, and perhaps halloysite was joined in the glutaraldehyde solution of 7% percentage concentration and soak, then fully washing, add above-mentioned cellulase solution again, carry out immobilized reactant, mixed system centrifugal settling subsequently, washing, drying are fixed cellulase.Immobilized enzyme activity 600～650U/g, the pH value scope of application 3.9～5.8,40～80 ℃ of temperature applicable ranges are after immobilized enzyme uses 15 times, remaining activity still reaches the enzymic activity of depositing 2 months immobilized enzyme under 70～83%, 4 ℃ still can keep 80～90% of original catalytic activity.
Embodiment 6
Halloysite is carried out suitable fragmentation, milled processed, and utilize 250 mesh sieves to sieve, obtain the halloysite powder.The halloysite powder of 0.25g is mixed with 14mg/mL penicillin acylase liquid (by the acetate/acetic buffered soln configuration of pH=7.0) 5mL; stir; under 4 ℃ of conditions; carried out immobilized reactant 12 hours, and perhaps halloysite was joined in the glutaraldehyde solution of 0.5% percentage concentration and soak, then fully washing; add above-mentioned penicillin acylase liquid again; carry out immobilized reactant, mixed system centrifugal settling subsequently, washing, drying obtain penicillin acylase.Immobilization efficiency 60～86%, immobilized enzyme work reaches 1330～1680IU/g, the pH value scope of application 6～10,20～60 ℃ of temperature applicable ranges, after immobilized enzyme used 8 times, remaining activity still reached 72～85%, still kept vigor 80% in 2 hours 60 ℃ of insulations.
Claims (5)
1, the application of halloysite in enzyme immobilization carrier is characterized in that, is applied to enzyme immobilization carrier.
2, halloysite as claimed in claim 1 is in the application of enzyme immobilization carrier, it is characterized in that, at first the enzyme liquid that refinement, dispersive halloysite and buffer preparation by pH value=5-8 are formed mixes, under 0-35 ℃ of condition, stir and carry out physical adsorption, mixed system centrifugal settling then, washing, drying, being fixed enzyme.
3, halloysite as claimed in claim 1 is in the application of enzyme immobilization carrier, it is characterized in that, earlier refinement, dispersive halloysite are joined in the cross-linking agent solution of 0.5%-7% percentage concentration and soak, fully after the washing, the enzyme liquid that adding is formed by the buffer preparation of pH value=5-8, under 0-35 ℃ of condition, carry out immobilized reactant, mixed system centrifugal settling then, washing, drying, being fixed enzyme.
4, as claim 2 or 3 described halloysites in the application of enzyme immobilization carrier, it is characterized in that described buffered soln is: phosphate buffer solution or acetate or acetate/acetic buffered soln.
5, halloysite as claimed in claim 3 is characterized in that in the application of enzyme immobilization carrier, and described linking agent is: glutaraldehyde.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2007100545588A CN101096665A (en) | 2007-06-12 | 2007-06-12 | Application of halloysite in enzyme immobilization carrier |
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| CNA2007100545588A CN101096665A (en) | 2007-06-12 | 2007-06-12 | Application of halloysite in enzyme immobilization carrier |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146675A (en) * | 2013-03-06 | 2013-06-12 | 昆明理工大学 | Preparation method of immobilized lipase regarding red halloysite as carrier |
| CN103172402A (en) * | 2011-12-20 | 2013-06-26 | 上海风享环保科技有限公司 | Multifunctional porous purifying ceramic granular material and preparation method |
| CN104911224A (en) * | 2015-06-26 | 2015-09-16 | 南京工业大学 | Method for catalytic synthesis of atazanavir intermediate |
| CN109097355A (en) * | 2018-08-30 | 2018-12-28 | 浙江农林大学 | A kind of method of laccase support material and its fixing laccase |
| CN113429142A (en) * | 2021-05-14 | 2021-09-24 | 深圳大学 | Enzyme modified halloysite nanotube and preparation method and application thereof |
| CN117778116A (en) * | 2024-02-28 | 2024-03-29 | 山东海化集团有限公司 | Preparation method and application of Pickering microemulsion sterilization and decontamination agent |
-
2007
- 2007-06-12 CN CNA2007100545588A patent/CN101096665A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103172402A (en) * | 2011-12-20 | 2013-06-26 | 上海风享环保科技有限公司 | Multifunctional porous purifying ceramic granular material and preparation method |
| CN103172402B (en) * | 2011-12-20 | 2015-02-11 | 上海风享环保科技有限公司 | Multifunctional porous purifying ceramic granular material and preparation method |
| CN103146675A (en) * | 2013-03-06 | 2013-06-12 | 昆明理工大学 | Preparation method of immobilized lipase regarding red halloysite as carrier |
| CN103146675B (en) * | 2013-03-06 | 2014-07-30 | 昆明理工大学 | Preparation method of immobilized lipase regarding red halloysite as carrier |
| CN104911224A (en) * | 2015-06-26 | 2015-09-16 | 南京工业大学 | Method for catalytic synthesis of atazanavir intermediate |
| CN104911224B (en) * | 2015-06-26 | 2018-12-25 | 南京工业大学 | Method for catalytic synthesis of atazanavir intermediate |
| CN109097355A (en) * | 2018-08-30 | 2018-12-28 | 浙江农林大学 | A kind of method of laccase support material and its fixing laccase |
| CN109097355B (en) * | 2018-08-30 | 2022-07-12 | 浙江农林大学 | Laccase immobilized material and method for immobilizing laccase by using same |
| CN113429142A (en) * | 2021-05-14 | 2021-09-24 | 深圳大学 | Enzyme modified halloysite nanotube and preparation method and application thereof |
| CN117778116A (en) * | 2024-02-28 | 2024-03-29 | 山东海化集团有限公司 | Preparation method and application of Pickering microemulsion sterilization and decontamination agent |
| CN117778116B (en) * | 2024-02-28 | 2024-06-07 | 山东海化集团有限公司 | Preparation method and application of Pickering microemulsion sterilization and decontamination agent |
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