CN105907744A - Method for preparing acylating chlorination modified magnetic silicon dioxide pellet immobilized laccase - Google Patents
Method for preparing acylating chlorination modified magnetic silicon dioxide pellet immobilized laccase Download PDFInfo
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- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
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Abstract
The invention discloses a method for preparing acylating chlorination modified magnetic silicon dioxide pellet immobilized laccase, and belongs to the technical field of laccase preparation. A magnetic silicon dioxide pellet carrier is prepared from a silicon dioxide macroporous material, due to the fact that the surfaces of magnetic silicon dioxide particles are provided with rich carboxyl groups, the carboxyl groups can react with phosphorus trichloride, the carboxyl groups are converted into acyl chloride groups, magnets are separated, washed and dried after reacting, and acylating chlorination modified magnetic silicon dioxide pellets are obtained; the acylating chlorination modified magnetic silicon dioxide pellets are added into a laccase solution prepared from a buffer solution, oscillation filtering is carried out, and after filter residues are washed with the buffer solution, the acylating chlorination modified magnetic silicon dioxide pellet immobilized laccase can be obtained. Living examples prove that the prepared immobilization carrier material keeps the catalytic activity and stability of the laccase, meanwhile, separation is easy, the price is low, preparation is convenient, the activity of the finally prepared magnetic silicon dioxide pellet immobilized laccase reaches up to 280 U/mL, the total output is increased to 89% or above, and wide market prospects are achieved.
Description
Technical field
The invention discloses the preparation method of a kind of chloride modified magnetic silicon dioxide microsphere immobilization laccase, belong to laccase preparing technical field.
Background technology
Laccase is a kind of cupric polyphenol oxidase, it is not only present in higher plant, and some polypor and saprophytic bacteria and insecticide and antibacterial etc. also can produce laccase, and great majority are by fungus secretion, in most of funguses, whiterot fungi is the lignin degradation person the strongest, topmost generally acknowledged at present, and can make the final mineralising of lignin is CO2And H2O.Laccase can be catalyzed oxidation gas chromatography, including phenol, methoxyl group phenol, amino phenols etc. are difficult to the aromatic compound degraded, molecular oxygen is reduced into water simultaneously, recent research indicate that, laccase has important effect in paper industry, can degrade the lignin in wood pulp, and a large amount of pollutant in paper industry sewage can be removed, owing to laccase is at Catalytic lignin, the degraded of many environmental contaminants such as lignin precursor substance molecule and structure homologue thereof has special effect, in recent years, about the research of laccase by extensive concern both domestic and external, demonstrate bigger researching value and application potential.
But owing to laccase is soluble in water, laccase free in water is the most recyclable can not be preserved for a long time, and it is difficult to keep in the stability of high temperature or other extreme environment laccases, thus limits the actual application of laccase.Immobilization technology is while keeping the catalysis activity single-minded, efficient of free laccase, improve laccase reusing, the handling and effective means of stability especially, conventional fixation support includes the multiple materials such as activated carbon, chitosan, Bio-sil and micro-filtration membrane, but existing immobilization material exists, and immobilization efficiency is low, processing technique is complicated, separation is difficult and the most high defect of operability.
Summary of the invention
nullThe technical problem that present invention mainly solves: can not preserve for a long time for the most free laccase，High temperatures is poor，Therefore its actual application is limited，And though conventional immobilization technology can keep the catalysis activity of laccase，Improve usability and stability，But the material immobilized efficiency of fixation support used is low，Separate difficulty and the present situation of complex process，Provide a kind of magnetic silica microballoon carrier utilizing silicon dioxide large pore material to prepare，Owing to magnetic silica microparticle surfaces is rich in carboxylic group，After can reacting with Phosphorous chloride.，Carboxylic group is changed into acid chloride groups，Separate with Magnet after reaction，Clean separator lyophilization，Obtain chloride modified magnetic silicon dioxide microsphere，And add with the laccase solution of buffer，Vibration is filtered，And use wash buffer filtering residue，I.e. can get chloride modified magnetic silicon dioxide microsphere immobilization laccase.The fixation support material that the present invention prepares is easily isolated while keeping Laccase Catalyzed activity, stability, and cheap, preparation convenience, the final magnetic silica microballoon immobilization laccase vigor prepared is up to 280U/mL, total output is also promoted to more than 89%, has wide market prospect.
In order to solve above-mentioned technical problem, the technical solution adopted in the present invention is:
(1) 30～50g bulk silicon dioxide large pore materials are weighed, put it into and mass fraction 5% hydrochloric acid soaks 1～2h, filter after immersion and take out, bulk silicon dioxide large pore material after acid treatment is put in polytetrafluoroethyllining lining reactor, adding mixed organic solvents by solid-to-liquid ratio 1:5, arranging temperature is 80～120 DEG C, insulated and stirred reaction 10～12h, reaction is cooled to room temperature after terminating, filter silicon dioxide large pore material cleans 3～5 times with acetone and deionized water respectively；Described mixed organic solvents is to be mixed by γ-(2,3-glycidoxy) propyl trimethoxy silicane and normal hexane 1:2 by volume；
(2) the silicon dioxide large pore material after above-mentioned cleaning is mixed with deionized water by solid-to-liquid ratio 1:3, and add 0.5～0.8g nanometer Fe 3 O 4 magnetic granule, 15～20mL mass fraction 8% dodecylbenzene sodium sulfonate solution, ultrasonic disperse 20～30min after mixing, ultrasonic backward dispersion liquid adds 50～70mL Semen Maydis oil and Semen Maydis oil quality 2～5% sorbitan fatty acid ester, at 12000～15000r/min rotating speeds emulsified 5～10min after mixing；
(3) 30～40mL glutaraldehydes are added during above-mentioned emulsifying terminates backward emulsion mixture, the fixing reaction 5～6h of stirring under 200～400r/min rotating speeds, after fixing reaction, under 8000～8500r/min rotating speeds, centrifugation obtains precipitate, and clean 2～3 times with mass fraction 3% sorbitan fatty acid ester solution, cleaning postprecipitation thing is put into baking oven is dried at 50～60 DEG C 6～8h, obtain magnetic silica microballoon carrier；
(4) above-mentioned magnetic silica microballoon carrier is added in mass fraction 5% Phosphorous chloride. solution by solid-to-liquid ratio 1:2, it is stirred at room temperature reaction 15～20min, reaction separates with Magnet after terminating, separator deionized water is cleaned 4～6 times, separate and lyophilization with Magnet again after cleaning, i.e. can get magnetic silica microballoon after chloride is modified；
(5) after above-mentioned chloride being modified in the magnetic silica microballoon laccase solution by the configuration of solid-to-liquid ratio 1:3 addition buffer, oscillating reactions 6～8h at 15～35 DEG C, reaction is filtered after terminating, filtering residue pH4.5 disodium hydrogen phosphate-citrate buffer solution is rinsed 3～5 times, i.e. can get chloride modified magnetic silicon dioxide microsphere immobilization laccase；The laccase solution of described buffer configuration is to have the laccase liquid 1:2 by volume mixing that pH is 4.5 disodium hydrogen phosphates-citrate buffer solution and concentration is 1mg/mL.
The application process of the present invention is: by solid-to-liquid ratio 1: 1000～3000, the magnetic silica microballoon immobilization laccase present invention prepared is poured in waste water, it is sufficiently mixed uniformly, Air Exposure 2～3h, detect water source afterwards, the COD that can record in waste water is reduced to 400～550mg/L from original 3000～5000mg/L, and clearance is up to more than 90%, and effect is the most notable.
The invention has the beneficial effects as follows:
(1) present invention is easy and simple to handle, and method is unique, under conditions of keeping Laccase Catalyzed activity, stability, simple process, fixation support material is easily isolated；
(2) the invention enables prepared magnetic silica microballoon immobilization laccase vigor to be up to 280U/mL, total output is also promoted to more than 89%；
(3) the magnetic silica microballoon immobilization laccase that the present invention prepares is widely used, and can be used for the aspects such as textile printing and dyeing, papermaking, energy regeneration, feedstuff, waste water process.
Detailed description of the invention
First 30～50g bulk silicon dioxide large pore materials are weighed, put it into and mass fraction 5% hydrochloric acid soaks 1～2h, filter after immersion and take out, bulk silicon dioxide large pore material after acid treatment is put in polytetrafluoroethyllining lining reactor, adding mixed organic solvents by solid-to-liquid ratio 1:5, arranging temperature is 80～120 DEG C, insulated and stirred reaction 10～12h, reaction is cooled to room temperature after terminating, filter silicon dioxide large pore material cleans 3～5 times with acetone and deionized water respectively；Described mixed organic solvents is to be mixed by γ-(2,3-glycidoxy) propyl trimethoxy silicane and normal hexane 1:2 by volume；Then the silicon dioxide large pore material after above-mentioned cleaning is mixed with deionized water by solid-to-liquid ratio 1:3, and add 0.5～0.8g nanometer Fe 3 O 4 magnetic granule, 15～20mL mass fraction 8% dodecylbenzene sodium sulfonate solution, ultrasonic disperse 20～30min after mixing, ultrasonic backward dispersion liquid adds 50～70mL Semen Maydis oil and Semen Maydis oil quality 2～5% sorbitan fatty acid ester, at 12000～15000r/min rotating speeds emulsified 5～10min after mixing；The most above-mentioned emulsifying terminates to add in backward emulsion mixture 30～40mL glutaraldehydes, the fixing reaction 5～6h of stirring under 200～400r/min rotating speeds, after fixing reaction, under 8000～8500r/min rotating speeds, centrifugation obtains precipitate, and clean 2～3 times with mass fraction 3% sorbitan fatty acid ester solution, cleaning postprecipitation thing is put into baking oven is dried at 50～60 DEG C 6～8h, obtain magnetic silica microballoon carrier；Next above-mentioned magnetic silica microballoon carrier is added in mass fraction 5% Phosphorous chloride. solution by solid-to-liquid ratio 1:2, it is stirred at room temperature reaction 15～20min, reaction separates with Magnet after terminating, separator deionized water is cleaned 4～6 times, separate and lyophilization with Magnet again after cleaning, i.e. can get magnetic silica microballoon after chloride is modified；After finally above-mentioned chloride being modified in the magnetic silica microballoon laccase solution by the configuration of solid-to-liquid ratio 1:3 addition buffer, oscillating reactions 6～8h at 15～35 DEG C, reaction is filtered after terminating, filtering residue pH4.5 disodium hydrogen phosphate-citrate buffer solution is rinsed 3～5 times, i.e. can get chloride modified magnetic silicon dioxide microsphere immobilization laccase；The laccase solution of described buffer configuration is to have the laccase liquid 1:2 by volume mixing that pH is 4.5 disodium hydrogen phosphates-citrate buffer solution and concentration is 1mg/mL.
Example 1
First 30g bulk silicon dioxide large pore material is weighed, put it into immersion 1h in mass fraction 5% hydrochloric acid, filter after immersion and take out, bulk silicon dioxide large pore material after acid treatment is put in polytetrafluoroethyllining lining reactor, adding mixed organic solvents by solid-to-liquid ratio 1:5, arranging temperature is 80 DEG C, insulated and stirred reaction 10h, reaction is cooled to room temperature after terminating, filter silicon dioxide large pore material cleans 3 times with acetone and deionized water respectively；Described mixed organic solvents is to be mixed by γ-(2,3-glycidoxy) propyl trimethoxy silicane and normal hexane 1:2 by volume；Then the silicon dioxide large pore material after above-mentioned cleaning is mixed with deionized water by solid-to-liquid ratio 1:3, and add 0.5g nanometer Fe 3 O 4 magnetic granule, 15mL mass fraction 8% dodecylbenzene sodium sulfonate solution, ultrasonic disperse 20min after mixing, ultrasonic backward dispersion liquid adds 50mL Semen Maydis oil and Semen Maydis oil quality 2% sorbitan fatty acid ester, at the emulsified 5min of 12000r/min rotating speed after mixing；The most above-mentioned emulsifying terminates to add in backward emulsion mixture 30mL glutaraldehyde, stirring fixing reaction 5h under 200r/min rotating speed, after fixing reaction, under 8000r/min rotating speed, centrifugation obtains precipitate, and clean 2 times with mass fraction 3% sorbitan fatty acid ester solution, cleaning postprecipitation thing is put into baking oven is dried at 50 DEG C 6h, obtain magnetic silica microballoon carrier；Next above-mentioned magnetic silica microballoon carrier is added in mass fraction 5% Phosphorous chloride. solution by solid-to-liquid ratio 1:2, it is stirred at room temperature reaction 15min, reaction separates with Magnet after terminating, separator deionized water is cleaned 4 times, separate and lyophilization with Magnet again after cleaning, i.e. can get magnetic silica microballoon after chloride is modified；After finally above-mentioned chloride being modified in the magnetic silica microballoon laccase solution by the configuration of solid-to-liquid ratio 1:3 addition buffer, oscillating reactions 6h at 15 DEG C, reaction is filtered after terminating, filtering residue pH4.5 disodium hydrogen phosphate-citrate buffer solution is rinsed 3 times, i.e. can get chloride modified magnetic silicon dioxide microsphere immobilization laccase；The laccase solution of described buffer configuration is to have the laccase liquid 1:2 by volume mixing that pH is 4.5 disodium hydrogen phosphates-citrate buffer solution and concentration is 1mg/mL.
This example operation is simple, during use, by solid-to-liquid ratio 1: 1000, the magnetic silica microballoon immobilization laccase present invention prepared is poured in waste water, is sufficiently mixed uniformly, Air Exposure 2h, detect water source afterwards, the COD that can record in waste water is reduced to 300mg/L from original 3000mg/L, and clearance is up to 90%, and effect is the most notable.
Example 2
First 40g bulk silicon dioxide large pore material is weighed, put it into immersion 1.5h in mass fraction 5% hydrochloric acid, filter after immersion and take out, bulk silicon dioxide large pore material after acid treatment is put in polytetrafluoroethyllining lining reactor, adding mixed organic solvents by solid-to-liquid ratio 1:5, arranging temperature is 100 DEG C, insulated and stirred reaction 11h, reaction is cooled to room temperature after terminating, filter silicon dioxide large pore material cleans 4 times with acetone and deionized water respectively；Described mixed organic solvents is to be mixed by γ-(2,3-glycidoxy) propyl trimethoxy silicane and normal hexane 1:2 by volume；Then the silicon dioxide large pore material after above-mentioned cleaning is mixed with deionized water by solid-to-liquid ratio 1:3, and add 0.65g nanometer Fe 3 O 4 magnetic granule, 18mL mass fraction 8% dodecylbenzene sodium sulfonate solution, ultrasonic disperse 25min after mixing, ultrasonic backward dispersion liquid adds 60mL Semen Maydis oil and Semen Maydis oil quality 4% sorbitan fatty acid ester, at the emulsified 8min of 13500r/min rotating speed after mixing；The most above-mentioned emulsifying terminates to add in backward emulsion mixture 35mL glutaraldehyde, stirring fixing reaction 5.5h under 300r/min rotating speed, after fixing reaction, under 8300r/min rotating speed, centrifugation obtains precipitate, and clean 2 times with mass fraction 3% sorbitan fatty acid ester solution, cleaning postprecipitation thing is put into baking oven is dried at 55 DEG C 7h, obtain magnetic silica microballoon carrier；Next above-mentioned magnetic silica microballoon carrier is added in mass fraction 5% Phosphorous chloride. solution by solid-to-liquid ratio 1:2, it is stirred at room temperature reaction 18min, reaction separates with Magnet after terminating, separator deionized water is cleaned 5 times, separate and lyophilization with Magnet again after cleaning, i.e. can get magnetic silica microballoon after chloride is modified；After finally above-mentioned chloride being modified in the magnetic silica microballoon laccase solution by the configuration of solid-to-liquid ratio 1:3 addition buffer, oscillating reactions 7h at 25 DEG C, reaction is filtered after terminating, filtering residue pH4.5 disodium hydrogen phosphate-citrate buffer solution is rinsed 4 times, i.e. can get chloride modified magnetic silicon dioxide microsphere immobilization laccase；The laccase solution of described buffer configuration is to have the laccase liquid 1:2 by volume mixing that pH is 4.5 disodium hydrogen phosphates-citrate buffer solution and concentration is 1mg/mL.
During use, by solid-to-liquid ratio 1: 2000, the magnetic silica microballoon immobilization laccase present invention prepared is poured in waste water, it is sufficiently mixed uniformly, Air Exposure 2.5h, detects water source afterwards, and the COD that can record in waste water is reduced to 350mg/L from original 4000mg/L, clearance is up to 91.2%, and effect is the most notable.
Example 3
First 50g bulk silicon dioxide large pore material is weighed, put it into immersion 2h in mass fraction 5% hydrochloric acid, filter after immersion and take out, bulk silicon dioxide large pore material after acid treatment is put in polytetrafluoroethyllining lining reactor, adding mixed organic solvents by solid-to-liquid ratio 1:5, arranging temperature is 120 DEG C, insulated and stirred reaction 12h, reaction is cooled to room temperature after terminating, filter silicon dioxide large pore material cleans 5 times with acetone and deionized water respectively；Described mixed organic solvents is to be mixed by γ-(2,3-glycidoxy) propyl trimethoxy silicane and normal hexane 1:2 by volume；Then the silicon dioxide large pore material after above-mentioned cleaning is mixed with deionized water by solid-to-liquid ratio 1:3, and add 0.8g nanometer Fe 3 O 4 magnetic granule, 20mL mass fraction 8% dodecylbenzene sodium sulfonate solution, ultrasonic disperse 30min after mixing, ultrasonic backward dispersion liquid adds 70mL Semen Maydis oil and Semen Maydis oil quality 5% sorbitan fatty acid ester, at the emulsified 10min of 15000r/min rotating speed after mixing；The most above-mentioned emulsifying terminates to add in backward emulsion mixture 40mL glutaraldehyde, stirring fixing reaction 6h under 400r/min rotating speed, after fixing reaction, under 8500r/min rotating speed, centrifugation obtains precipitate, and clean 3 times with mass fraction 3% sorbitan fatty acid ester solution, cleaning postprecipitation thing is put into baking oven is dried at 60 DEG C 8h, obtain magnetic silica microballoon carrier；Next above-mentioned magnetic silica microballoon carrier is added in mass fraction 5% Phosphorous chloride. solution by solid-to-liquid ratio 1:2, it is stirred at room temperature reaction 20min, reaction separates with Magnet after terminating, separator deionized water is cleaned 6 times, separate and lyophilization with Magnet again after cleaning, i.e. can get magnetic silica microballoon after chloride is modified；After finally above-mentioned chloride being modified in the magnetic silica microballoon laccase solution by the configuration of solid-to-liquid ratio 1:3 addition buffer, oscillating reactions 8h at 35 DEG C, reaction is filtered after terminating, filtering residue pH4.5 disodium hydrogen phosphate-citrate buffer solution is rinsed 5 times, i.e. can get chloride modified magnetic silicon dioxide microsphere immobilization laccase；The laccase solution of described buffer configuration is to have the laccase liquid 1:2 by volume mixing that pH is 4.5 disodium hydrogen phosphates-citrate buffer solution and concentration is 1mg/mL.
During use, by solid-to-liquid ratio 1: 3000, the magnetic silica microballoon immobilization laccase present invention prepared is poured in waste water, it is sufficiently mixed uniformly, Air Exposure 3h, detects water source afterwards, and the COD that can record in waste water is reduced to 550mg/L from original 5000mg/L, clearance is up to 89%, and effect is the most notable.
Claims (1)
1. the preparation method of a chloride modified magnetic silicon dioxide microsphere immobilization laccase, it is characterised in that concrete preparation process is:
(1) 30～50g bulk silicon dioxide large pore materials are weighed, put it into and mass fraction 5% hydrochloric acid soaks 1～2h, after immersion, 0 filters taking-up, bulk silicon dioxide large pore material after acid treatment is put in polytetrafluoroethyllining lining reactor, adding mixed organic solvents by solid-to-liquid ratio 1:5, arranging temperature is 80～120 DEG C, insulated and stirred reaction 10～12h, reaction is cooled to room temperature after terminating, filter silicon dioxide large pore material cleans 3～5 times with acetone and deionized water respectively；Described mixed organic solvents is to be mixed by γ-(2,3-glycidoxy) propyl trimethoxy silicane and normal hexane 1:2 by volume；
(2) the silicon dioxide large pore material after above-mentioned cleaning is mixed with deionized water by solid-to-liquid ratio 1:3, and add 0.5～0.8g nanometer Fe 3 O 4 magnetic granule, 15～20mL mass fraction 8% dodecylbenzene sodium sulfonate solution, ultrasonic disperse 20～30min after mixing, ultrasonic backward dispersion liquid adds 50～70mL Semen Maydis oil and Semen Maydis oil quality 2～5% sorbitan fatty acid ester, at 12000～15000r/min rotating speeds emulsified 5～10min after mixing；
(3) 30～40mL glutaraldehydes are added during above-mentioned emulsifying terminates backward emulsion mixture, the fixing reaction 5～6h of stirring under 200～400r/min rotating speeds, after fixing reaction, under 8000～8500r/min rotating speeds, centrifugation obtains precipitate, and clean 2～3 times with mass fraction 3% sorbitan fatty acid ester solution, cleaning postprecipitation thing is put into baking oven is dried at 50～60 DEG C 6～8h, obtain magnetic silica microballoon carrier；
(4) above-mentioned magnetic silica microballoon carrier is added in mass fraction 5% Phosphorous chloride. solution by solid-to-liquid ratio 1:2, it is stirred at room temperature reaction 15～20min, reaction separates with Magnet after terminating, separator deionized water is cleaned 4～6 times, separate and lyophilization with Magnet again after cleaning, i.e. can get magnetic silica microballoon after chloride is modified；
(5) after above-mentioned chloride being modified in the magnetic silica microballoon laccase solution by the configuration of solid-to-liquid ratio 1:3 addition buffer, oscillating reactions 6～8h at 15～35 DEG C, reaction is filtered after terminating, filtering residue pH4.5 disodium hydrogen phosphate-citrate buffer solution is rinsed 3～5 times, i.e. can get chloride modified magnetic silicon dioxide microsphere immobilization laccase；The laccase solution of buffer configuration is to have the laccase liquid 1:2 by volume mixing that pH is 4.5 disodium hydrogen phosphates-citrate buffer solution and concentration is 1mg/mL.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107034207A (en) * | 2017-04-06 | 2017-08-11 | 江苏大学 | Biologic inorganic hybridized film preparation method and application based on foil carriers fixing laccase |
| CN114457063A (en) * | 2022-03-11 | 2022-05-10 | 安徽黑娃食品科技有限公司 | Aspergillus flavus toxin B in degradation peanut1Preparation method of immobilized enzyme |
-
2016
- 2016-06-26 CN CN201610469755.5A patent/CN105907744A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107034207A (en) * | 2017-04-06 | 2017-08-11 | 江苏大学 | Biologic inorganic hybridized film preparation method and application based on foil carriers fixing laccase |
| CN107034207B (en) * | 2017-04-06 | 2020-02-21 | 江苏大学 | Preparation method and application of bio-inorganic hybrid membrane based on copper foil carrier immobilized laccase |
| CN114457063A (en) * | 2022-03-11 | 2022-05-10 | 安徽黑娃食品科技有限公司 | Aspergillus flavus toxin B in degradation peanut1Preparation method of immobilized enzyme |
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Application publication date: 20160831 |