CN102206624A - Magnetic composite microsphere immobilized laccase and preparation method thereof - Google Patents
Magnetic composite microsphere immobilized laccase and preparation method thereof Download PDFInfo
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- CN102206624A CN102206624A CN2011100947493A CN201110094749A CN102206624A CN 102206624 A CN102206624 A CN 102206624A CN 2011100947493 A CN2011100947493 A CN 2011100947493A CN 201110094749 A CN201110094749 A CN 201110094749A CN 102206624 A CN102206624 A CN 102206624A
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Abstract
The invention relates to a magnetic composite microsphere immobilized laccase and a preparation method thereof, and belongs to the field of environmental microbiology. In the immobilized laccase, metal-chelated magnetic composite microspheres are taken as a carrier, and laccase is immobilized on the carrier through coordination bonding action. The preparation method comprises two steps of: preparing the carrier, namely synthesizing magnetic ferroferric oxide nanoparticles by using a chemical precipitation process, preparing core-shell magnetic silica by taking the nanoparticles as cores through a sol-gel method, grafting polymer containing carbonyl or amino on the surface of the magnetic silica by taking gamma-chloropropyltrimethoxysilane as a medium, and preparing a functional carrier capable of immobilizing the laccase by chelating transition metal ions (such as Cu2+. Zn2+, Ni2+ or Co2+); and immobilizing the laccase, namely performing coordination adsorption of the laccase on the surface of the carrier, washing, and drying to obtain the finished product of immobilized laccase. The immobilized laccase prepared by the method has high stability and activity and quick magnetic response, is easily recovered from a reaction system and can be repeatedly used; and the preparation process is simple and the cost is low.
Description
Technical field
The present invention relates to the preparation method of a kind of magnetic composite microsphere immobilization laccase and this immobilization laccase.But a kind of rapid magnetic of preparation of saying so more specifically separates the magnetic composite microsphere immobilization laccase that operational stability is good.
Background technology
Laccase is a kind of copper bearing polyphenoloxidase, extensively exists in the multiple organism.The oxygen to directly oxidize of laccase in can catalytic air decomposes various phenols dyestuffs, substituted phenol, chlorophenol, thiophenol, dihydroxyphenyl propane, aromatic amine etc., in the presence of laccase amboceptor (ABTS, HBT etc.), the ditan that laccase is can also catalyzed degradation relevant with xylogen, (agricultural chemicals) is Ji dioxin etc. to phenyl diamines, organo phosphorous compounds in the N-replacement.Owing to have Substratspezifitaet and stability quite widely preferably, laccase has important use value at aspects such as paper industry, wastewater treatment, bio-bleaching, aromatic compound conversion, biosensor structure and foodstuffs industry.But because laccase is made up of protein, its higher structure is very responsive to environment of living in, in use is subject to changes in environmental conditions influence and causes sex change inactivation, particularly resolvase and reaction product to mix, can not reclaim after the reaction, be difficult to realize recycling.And the immobilized enzyme of making after enzyme fixed can overcome above-mentioned shortcoming.Compare with resolvase, also there are series of advantages such as stability is high, Separation and Recovery is easy, it is continuous and controlled to be repeatedly used, to operate, technology is easy in immobilized enzyme in its efficient, single-minded and gentle enzymic catalytic reaction characteristic of maintenance.
The key of preparation immobilized enzyme is that the selection of carrier, the fixation support that can be used for biological enzyme that reported in literature is more mainly contain the surface and be rich in the organic carrier of functional group and mechanical stability inorganic carrier etc. preferably.But these solid support material ubiquities synthetic costing an arm and a leg, complete processing complexity, shortcomings such as inferior separating effect.In recent years, growing along with magnetic separation technique, the immobilization that magnetic particle is used for biological enzyme gets more and more people's extensive concerning gradually.Yet magnetic nano-particle has high-specific surface area, high-ratio surface energy, and thereby the anisotropic moment of dipole effect of particle is easy to reunite, and exposed magneticsubstance acid-proof alkaline is poor, easy deterioration by oxidation in air, thereby cause to form stable dispersion system, it is restricted in the application of magnetic resolution.This just requires to carry out modification for these magneticsubstances, need superscribe protective layer outside usually, to improve the resistance of oxidation of magneticsubstance, obtains dispersiveness composite particles preferably.Silicon-dioxide has good biocompatibility, good acid and alkali-resistance, organic solvent-resistant and high-temperature stability, finishing to magnetic nano-particle can reduce its surface energy, improve the resistance of oxidation of particle, obtain the nano-complex particle of good dispersity, available various silane coupling agents are introduced various functional groups to be used for the immobilization of biomacromolecules such as enzyme on its surface simultaneously.Therefore, the coated with silica magnetic particle has incomparable advantage.
In recent years, the research of enzyme immobilization technology has obtained vigorous growth, and the method for being used for fixing enzyme mainly contains absorption method, crosslinking, covalent bonds method, entrapping method etc. at present.Wherein, crosslinking and covalent bonds method are comparatively violent because of reaction conditions, and the enzyme activity loss is bigger; Loss is bigger because substrate binding site and active centre are sealed by carrier thereby enzyme is lived for entrapping method; Absorption method has mild condition, enzyme activity position and space conformation thereof and is difficult for ruined characteristics, but in the absorption method by ionic linkage, hydrogen bond, dipolar bond and hydrophobic bond fixed enzyme be subject to reaction medium pH, ionic strength etc. influence and come off from carrier.Adopt the metal-chelating carrier then can overcome the some shortcomings of using always at present in the process for fixation with the coordination immobilized enzyme.It utilizes the imidazolyl of the sub-amino acid-Histidine of power supply of metal-chelating part and protein surface, the mercapto alcohol radical of halfcystine etc. with the coordination immobilized enzyme, enzyme combines firmly difficult drop-off with carrier, the chemical damage of having avoided the use of linking agent that the enzyme molecule is caused simultaneously, the native conformation that has kept the enzyme molecule to a greater extent can keep enzyme to a greater extent and live.
Therefore, in the present invention, by at a kind of transition metal ion of magnetic silica chelated surface (as Cu
2+, Zn
2+, Ni
2+Or Co
2+But) make the functional magnetic carrier of fixing laccase, by the coordination immobilization laccase, this immobilization laccase not only can be realized sharp separation under extraneous the action of a magnetic field with this carrier, recyclablely uses repeatedly, has also strengthened the stability of laccase simultaneously.
Summary of the invention
But the object of the present invention is to provide a kind of rapid magnetic Separation and Recovery, the magnetic composite microsphere immobilization laccase that operational stability is good.Another purpose of the present invention is to provide the preparation method of said fixing laccase.
Purpose of the present invention is achieved through the following technical solutions:
A kind of magnetic composite microsphere immobilization laccase, with a kind of transition metal ion of chelated surface (as Cu
2+, Zn
2+, Ni
2+Or Co
2+) magnetic composite microsphere be carrier, the interaction that utilizes metal-chelating part and protein surface to power between sub-amino acid is fixed on laccase on this carrier.
A kind of preparation method of magnetic composite microsphere immobilization laccase, this method comprise two steps: preparing carriers and laccase are fixed.
The preparing carriers step comprises:
(1) will be dissolved with FeCl
24H
2O and FeCl
36H
2The sodium dodecyl sulfate aqueous solution uniform mixing of O mixture, dripping 2-5mol/L NaOH solution to pH of mixed under agitation condition is 10-12, products therefrom behind the slaking 30-60min, 50-70 ℃ of oven dry down, obtains ferroferric oxide powder by grinding in 80-100 ℃ of water-bath;
(2) with the ferroferric oxide powder ultra-sonic dispersion in secondary water, stir the mixing solutions that adds tetraethoxy and methyl alcohol down, drip strong aqua after continuing to stir 15min, continued stirring reaction 4 hours, 50-70 ℃ of oven dry down obtains the core-shell structure magnetic SiO 2 powder;
(3) magnetic silica powder and γ-r-chloropropyl trimethoxyl silane are dissolved in methyl alcohol and the glycerol mixing solutions, 80 ℃ of following stirring reactions were dried down, are obtained the magnetic silica of silanization for 50-70 ℃ after 3 hours;
(4) magnetic silica of 1.4g silanization and 175mL 0.5-10mg/L a kind of contained carbonyl or amino polymers soln in 50 ℃ of following stirring reactions 3 hours, 50-70 ℃ of oven dry down; 0.5g should be dried powder and 75mL 0.02-0.3mol/L contains a kind of transition metal ion (as Cu
2+, Zn
2+, Ni
2+Or Co
2+) sulfate liquor mix concussion, temperature is 25-30 ℃, shake time 8-12 hour, dries down at 50-70 ℃ then and promptly getting the carrier finished product.
The laccase fixing step comprises:
Will be through preparing carriers step carrier finished product that makes and the free laccase 5mL thorough mixing that is dissolved in Sodium phosphate dibasic-citric acid solution, at pH is that 2.5-6.0, temperature are to stir 1-16 hour under the 5-55 ℃ of condition, after leaving standstill 8-12 hour under 4 ℃ of conditions, with pH is that Sodium phosphate dibasic-citric acid solution of 3.0 repeatedly washs, in washings, do not have albumen to detect, make the immobilization laccase finished product through low-temperature vacuum drying again.
In said process, be dissolved with FeCl described in the preparing carriers step (1)
24H
2O and FeCl
36H
2The FeCl that consists of 0.25mol/L of the sodium dodecyl sulfate aqueous solution of O mixture
24H
2The FeCl of O, 0.45-0.5mol/L
36H
2The sodium laurylsulfonate of O, 0.2-0.5% massfraction; The strong aqua of the methyl alcohol that consists of the 80-120 parts by volume of the methyl alcohol described in the preparing carriers step (2), secondary water and strong aqua, the secondary water of 40-60 parts by volume, 4-6 parts by volume, the concentration of ferroferric oxide powder is 8.06g/L, and the concentration of tetraethoxy is 16.12-32.24g/L; The quality of the magnetic silica powder described in the preparing carriers step (3) is 1.5g, and the volume of γ-r-chloropropyl trimethoxyl silane is 5-10mL, and the volume of methyl alcohol and glycerol is respectively 87.5mL and 37.5mL; A kind of polymkeric substance that contains carbonyl or amino described in the preparing carriers step (4) is polyacrylamide or polymine; In the laccase fixing step, the free laccase concentration that is dissolved in Sodium phosphate dibasic-citric acid solution is 0.2-1.4g/L, and the blending ratio of free laccase and carrier finished product is 10-70mg/1g.
Characteristics of the present invention are:
(1) do not use linking agent in the laccase immobilization process, the chemical damage of having avoided linking agent that the enzyme molecule is caused, enzyme is fixed on the native conformation that can keep the enzyme molecule on the carrier to a greater extent with coordination, keeps enzyme to a greater extent and lives.
(2) prepared immobilization laccase can separate rapidly from reaction system under the action of a magnetic field, helps reclaiming and recycling, and has significantly improved the operational stability of immobilization laccase.
Embodiment
Below with specific embodiment embodiment is described.
Embodiment 1
A kind of magnetic composite microsphere immobilization laccase, the preparation method mainly comprises two steps:
Preparing carriers
(1) getting mol ratio is 1: 2 FeCl
24H
2O and FeCl
36H
2It is in 0.5% the sodium dodecyl sulfate solution that the mixed powder of O is dissolved in the 50mL massfraction, stirs the NaOH solution that drips 2mol/l down pH is transferred to about 12.Products therefrom utilizes the distilled water repetitive scrubbing to neutral in 80 ℃ of water-baths behind the slaking 30min, dry, grind under 50 ℃ ferroferric oxide powder;
(2) get Fe after the grinding
3O
41.5g ultra-sonic dispersion in 60ml secondary water, simultaneously with 6.4ml tetraethoxysilane ultra-sonic dispersion in 120ml methyl alcohol, the methanol solution with tetraethoxysilane under mechanical stirring joins Fe
3O
4Suspension in, add the 6ml strong aqua behind the vigorous stirring 15min, continued at room temperature stirring reaction 4 hours, wash solid sediment to neutral with methyl alcohol, secondary water washing respectively, dry down, obtain the core-shell structure magnetic SiO 2 powder for 50 ℃;
(3) get 1.5g magnetic silica powder and be scattered in the 37.5ml methyl alcohol, add 37.5ml glycerine behind the ultra-sonic dispersion 20min, put into the 250ml there-necked flask behind the ultra-sonic dispersion 10min again; Simultaneously 5mL γ-chloropropyl trimethyl silane is scattered in the 50ml methyl alcohol, also be added in this there-necked flask after mixing, mix with magnetic silica, 80 ℃ of following stirring reactions 3 hours, use methyl alcohol respectively, secondary water washing is washed solid precipitation 6 times, dries down, obtains the magnetic silica of silanization for 50 ℃;
(4) with the polyacrylamide solution of the magnetic silica of 1.4g silanization and 175mL2mg/L in 50 ℃ of following stirring reactions 3 hours, 50 ℃ of oven dry down; Get 0.5g and should dry powder and 75mL 0.2mol/L copper-bath 30 ℃ of concussions 10 hours down, 50 ℃ down oven dry promptly get the carrier finished product.
Laccase is fixed
Get 0.1g carrier finished product and the free laccase 5mL thorough mixing that is dissolved in Sodium phosphate dibasic-citric acid solution, be 4.0 at pH, temperature is to stir 1 hour under 25 ℃ of conditions, after leaving standstill 8 hours under 4 ℃ of conditions, with pH is that Sodium phosphate dibasic-citric acid solution of 3.0 repeatedly washs, in washings, do not have albumen to detect, make the immobilization laccase finished product through low-temperature vacuum drying again.The blending ratio of wherein free laccase and carrier finished product is 35mg/1g.
Embodiment 2
The preparation method of carrier such as example 1, different is that concentration of polyacrylamide is 0.5mg/L, and the concentration of copper-bath is 0.1mol/L, and duration of oscillation is 12 hours.Laccase fixing means such as example 1, different is 0.1g carrier finished product be dissolved in the free laccase 5mL thorough mixing of Sodium phosphate dibasic-citric acid solution after, be 3.0 at pH, temperature is to stir 3 hours under 5 ℃ of conditions.The blending ratio of wherein free laccase and carrier finished product is 20mg/1g.
Embodiment 3
Preparing carriers method such as example 1, different is that concentration of polyacrylamide is 5mg/L, and the concentration of copper-bath is 0.05mol/L, and duration of oscillation is 8 hours; Laccase fixing means such as example 1, different is 0.1g carrier finished product be dissolved in the laccase 5mL thorough mixing of Sodium phosphate dibasic-citric acid solution after, be 5.0 at pH, temperature is to stir 5 hours under 15 ℃ of conditions.The blending ratio of wherein free laccase and carrier finished product is 50mg/1g.
Claims (7)
1. a magnetic composite microsphere immobilization laccase is characterized in that, with a kind of transition metal ion of chelated surface (as Cu
2+, Zn
2+, Ni
2+Or Co
2+) magnetic composite microsphere be carrier, the interaction that utilizes metal-chelating part and protein surface to power between sub-amino acid is fixed on laccase on this carrier.
2. the preparation method of the described magnetic composite microsphere immobilization laccase of claim 1, this method comprises two steps: preparing carriers and laccase are fixed.Wherein the preparing carriers step comprises:
(1) will be dissolved with FeCl
24H
2O and FeCl
36H
2The sodium dodecyl sulfate aqueous solution uniform mixing of O mixture, dripping 2-5mol/L NaOH solution to pH of mixed under agitation condition is 10-12, products therefrom behind the slaking 30-60min, 50-70 ℃ of oven dry down, obtains ferroferric oxide powder by grinding in 80-100 ℃ of water-bath;
(2) with the ferroferric oxide powder ultra-sonic dispersion in secondary water, stir the mixing solutions that adds tetraethoxy and methyl alcohol down, drip strong aqua after continuing to stir 15min, continued stirring reaction 4 hours, 50-70 ℃ of oven dry down obtains the core-shell structure magnetic SiO 2 powder;
(3) magnetic silica powder and γ-r-chloropropyl trimethoxyl silane are dissolved in methyl alcohol and the glycerol mixing solutions, 80 ℃ of following stirring reactions were dried down, are obtained the magnetic silica of silanization for 50-70 ℃ after 3 hours;
(4) magnetic silica of 1.4g silanization and 175mL 0.5-10mg/L a kind of contained carbonyl or amino polymers soln in 50 ℃ of following stirring reactions 3 hours, 50-70 ℃ of oven dry down; Get that 0.5g should be dried powder and 75mL 0.02-0.3mol/L contains a kind of transition metal ion (as Cu
2+, Zn
2+, Ni
2+Or Co
2+) sulfate liquor mix concussion, temperature is 25-30 ℃, shake time 8-12 hour, dries down at 50-70 ℃ then and promptly getting the carrier finished product.
The laccase fixing step comprises:
Will be through preparing carriers step carrier finished product that makes and the free laccase 5mL thorough mixing that is dissolved in Sodium phosphate dibasic-citric acid solution, at pH is that 2.5-6.0, temperature are to stir 1-16 hour under the 5-55 ℃ of condition, after leaving standstill 8-12 hour under 4 ℃ of conditions, with pH is that Sodium phosphate dibasic-citric acid solution of 3.0 repeatedly washs, in washings, do not have albumen to detect, make the immobilization laccase finished product through low-temperature vacuum drying again.
3. according to the method described in the claim 2, it is characterized in that the FeCl that is dissolved with described in the preparing carriers step (1)
24H
2O and FeCl
36H
2The FeCl that consists of 0.25mol/L of the sodium dodecyl sulfate aqueous solution of O mixture
24H
2The FeCl of O, 0.45-0.5mol/L
36H
2The sodium laurylsulfonate of O, 0.2-0.5% massfraction.
4. according to the method described in the claim 2, it is characterized in that the methyl alcohol that consists of the 80-120 parts by volume of the methyl alcohol described in the preparing carriers step (2), secondary water and strong aqua, the secondary water of 40-60 parts by volume, the strong aqua of 4-6 parts by volume, the concentration of ferroferric oxide powder is 8.06g/L, and the concentration of tetraethoxy is 16.12-32.24g/L.
5. according to the method described in the claim 2, the quality that it is characterized in that the magnetic silica powder described in the preparing carriers step (3) is 1.5g, the volume of γ-r-chloropropyl trimethoxyl silane is 5-10mL, and the volume of methyl alcohol and glycerol is respectively 87.5mL and 37.5mL.
6. according to the method described in the claim 2, it is characterized in that a kind of polymkeric substance that contains carbonyl or amino described in the preparing carriers step (4) is polyacrylamide or polymine.
7. according to the method described in the claim 2, it is characterized in that in the laccase fixing step that the free laccase concentration that is dissolved in Sodium phosphate dibasic-citric acid solution is 0.2-1.4g/L, the blending ratio of free laccase and carrier finished product is 10-70mg/1g.
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Application publication date: 20111005 |