CN110438116A - A kind of process for fixation of laccase - Google Patents
A kind of process for fixation of laccase Download PDFInfo
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- CN110438116A CN110438116A CN201910821287.7A CN201910821287A CN110438116A CN 110438116 A CN110438116 A CN 110438116A CN 201910821287 A CN201910821287 A CN 201910821287A CN 110438116 A CN110438116 A CN 110438116A
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
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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 present invention relates to enzyme engineering field, specifically a kind of process for fixation of laccase.Has magnetic Fe by coprecipitation preparation3O4Particle；By Fe3O4Particle is added in 3- aminopropyl triethoxysilane aqueous solution, and pure and mild lye is added after reaction, adds ethyl orthosilicate, attracts powder to obtain Fe with being ultrasonically treated after washes of absolute alcohol powder with magnet3O4/SiO2‑NH2Particle distributes it to phosphate buffer, adds veratryl alcohol, and laccase solution is poured into above-mentioned reaction system, attracts magnetic-particle, as immobilization laccase with magnet after oscillating reactions.This method operating procedure is simple, and required chemicals additional amount is few, and required reaction condition is mild, and preparation efficiency is high, can be improved the catalytic activity and recycling rate of waterused of laccase, improves laccase stability.
Description
Technical field
The present invention relates to enzyme engineering field, specifically a kind of process for fixation of laccase.
Background technique
Laccase is the cupric polyphenol oxidase of a kind of degradable lignin, be distributed widely in plant, fungi, a small number of insects and
In bacterium, laccase is had also discovered in some animal kidneys (such as pig kidney) and serum.Since Laccase Catalyzed substrate is wide in range, can be catalyzed more
The oxidation of kind structure is complicated substrate, in forestry products processing, paper pulp bleaching, bio-pulping, industrial textile, dye decolored, waste water
Removing toxic substances, bio-sensing, research and application, biological prosthetic, food industry and bio-fuel etc. show good application potential,
The always very active research hotspot in the fields such as biology, medicine, chemistry, environmental pollution improvement receives significant attention and high
Degree is paid attention to.
Due to the laccase that dissociates stability is poor, poor resistance, and be not easily recycled recycling, make laccase using by
Very big limitation, therefore raising laccase stability, reusable property are suffered from a problem that in laccase research.Document
" Pleurotus ostreatus is in Fe3O4/SiO2The decoloration of immobilization and azo dyes on nanoparticle " (Dai Jianan
.Pleurotus ostreatus is in Fe3O4/SiO2The Harbin decoloration [D] of immobilization and azo dyes on nanoparticle
Normal university, 2016.) and " magnetic Fe3O4/SiO2Compound particle immobilization laccase and catalytic elimination phenolic comp ' ds pollution " (Ma Li,
Zhao Donglei, Zheng Xiaobing, He Ying magnetic Fe3O4/SiO2Compound particle immobilization laccase and the river catalytic elimination phenolic comp ' ds pollution [J]
Northern polytechnical university's journal, 2016,45 (01): 85-89.) disclose method using magnetic compound particles fixing laccase, method compared with
Simply, but the load enzyme amount of immobilization and recycling number are to be improved.Document " Fe3O4/SiO2The Schiff of carrier is modified
The decoloration of phenols azo dyes is studied with immobilization laccase " (Wang Yingying .Fe3O4/SiO2The Schiff of carrier is modified and is consolidated
Surely change laccase and [D] .2017 studied to the decoloration of phenols azo dyes) disclose it is a kind of using magnetic compound particles fixing laccase
Method, crosslinking agent used in immobilization process is more common glutaraldehyde, and crosslinking agent and immobilization step need into one
Step is improved.Document " mesoporous SiO2/ densification SiO2/Fe3O4Tiny balloon preparation and performance study " (the mesoporous SiO of Zhou Yunlu2/ fine and close
SiO2/Fe3O4Tiny balloon preparation and performance study [D] Beijing University of Technology, 2017.) it discloses a kind of using hollow magnetic
The method of microballoon fixing laccase.Document " yolk-shell structure Fe3O4@SiO2The preparation of@PMO magnetic microsphere and fixation to laccase
Change " (Duan Bingyi, Wang Yu, Guo Ningning, Wang Runwei, ancestor's Tao, Qiu Shilun yolk-shell structure Fe3O4@SiO2@PMO magnetic microsphere
Preparation and to paint enzyme immobilizatio [J] Chemical Journal of Chinese Universities, 2019,40 (02): 210-215.) disclose a kind of benefit
With the method for magnetic compound particles fixing laccase, but in its material preparation process step, drug using more, energy consumption is high.
Summary of the invention
In view of the above-mentioned problems, in order to solve the deficiencies in the prior art, the present invention provides a kind of paint enzyme immobilizatio
Method, while improving the catalytic activity and recycling rate of waterused of immobilization laccase.
Technical solution of the present invention is as follows:
A kind of process for fixation of laccase, the specific steps are as follows:
Step A: has magnetic Fe by coprecipitation preparation3O4Particle；
Step B: by Fe in step A3O4Particle is added in 3- aminopropyl triethoxysilane aqueous solution, after standing reaction
Pure and mild lye is added, ethyl orthosilicate is added dropwise under stirring condition, has attracted powder with magnet after reaction, it is clear to outwell upper layer
Liquid twice or more than twice with dehydrated alcohol oscillation cleaning powder is then sonicated, obtains evenly dispersed Fe3O4/SiO2-
NH2, collect spare；
Step C: with Fe in step B3O4/SiO2-NH2Particle is base core, distributes it to phosphate buffer, adds
Laccase solution is poured into above-mentioned reaction system by veratryl alcohol, attracts magnetic-particle, as immobilization with magnet after oscillating reactions
Laccase.
The step A has magnetic Fe by coprecipitation preparation3O4Particle；The specific method is as follows: will be soluble sub-
Molysite and soluble ferric iron salt are dissolved into deionized water, are stirred and evenly mixed, and are heated to 80 DEG C, alkali soluble is added dropwise in whipping process
Liquid；Continue to be stirred to react after adding lye, then recycling the sediment of black by magnet is magnetic Fe3O4Particle, spend from
Sub- water washing falls impurity in sediment, dry.
The aqueous slkali is that concentrated ammonia liquor or concentration are no more than 0.2mol/L NaOH solution.
The concentration of the 3- aminopropyl triethoxysilane aqueous solution is not less than a ten thousandth.
The alcohol is hydroxyl not in the monohydric alcohol or polyalcohol at straight chain end, and volume is that 3- aminopropyl triethoxysilane is water-soluble
The 1/3-2/3 of liquid product.
The lye is that concentrated ammonia liquor or concentration are no more than 0.2mol/L NaOH solution.
The content of the veratryl alcohol in the reaction system is not less than 0.05%.
Present invention has the main advantage that this method operating procedure is simple, required chemicals additional amount is few, required reaction item
Part is mild, and preparation efficiency is high, can be improved the catalytic activity and recycling rate of waterused of laccase, improves laccase stability.
Specific embodiment
The present invention is described in detail below by embodiment.
Embodiment 1:
(1) Fe is prepared3O4Magnetic nano-particle
At room temperature, by 0.005mol FeCl2·4H2O and 0.01mol FeCl3·6H2O is added to 300mL deionization
It in water, stirs and evenly mixs, is then heated to 80 DEG C, concentration is added dropwise as 0.2mol/L NaOH solution after being stirred for 25min
250mL.Using the stirring of 30min, black precipitate, i.e. magnetic Fe are recycled by magnet3O4Particle is washed with deionized
The sediment for several times, stops washing when the pH value of cleaning solution is 7, finally that the magnetic nanoparticle being collected into is (magnetic
Fe3O4Particle) 8h is dried in vacuo at 60 DEG C, it is spare.
(2) magnetic Fe containing amino is prepared3O4/SiO2-NH2Particle
20 μ L 3- aminopropyl triethoxysilanes are taken to be added in the 500mL conical flask equipped with 200mL distilled water, ultrasound
After handling 5min, 400mg Fe is added into conical flask3O4After standing 40min, 100mL 2- butanol is added in magnetic nanoparticle
With concentrated ammonia liquor (25%, m/m), pH to 11 or more is adjusted, 2mL ethyl orthosilicate is then slowly added dropwise under agitation and (is added dropwise
Speed is no more than 0.5mL/40min), with magnet solid phase reaction product is attracted after reacting 12h, has outwelled reaction solution and (use magnet
Having attracted solid phase reaction product can guarantee that solid phase reaction product is attracted in reaction container bottom during toppling over reaction solution,
Toppling process does not lose solid phase reaction product), 30mL dehydrated alcohol is used after cleaning solid phase reaction product twice with ethanol solution again
Ultrasonic treatment 30min is carried out, then solution is removed in the case where having attracted the solid phase reaction product state with magnet, obtains evenly dispersed
Magnetic Fe3O4/SiO2-NH2Particle is collected spare.
(3) immobilization laccase is prepared
Take the above-mentioned magnetic Fe of 50mg3O4/SiO2-NH2Particle is distributed to the phosphate-buffered of 10mL (0.12M, pH=7.0)
In liquid (PBS), be added 5mL concentration be 0.2% black false hellebore alcoholic solution, add 5mL gold ground ganoderma lucidum laccase crude enzyme liquid, 25 DEG C,
3h is vibrated under the conditions of 100r/min, then outwells solution in the case where having attracted solid particulate matter state using magnet, obtained solid
Particulate matter is immobilization laccase.
Embodiment 2:
(1) Fe is prepared3O4Magnetic nano-particle
At room temperature, by 0.01mol FeCl2·4H2O and 0.02mol FeCl3·6H2O is added to the ion that 400mL is gone
It in water, stirs and evenly mixs, is then heated to 80 DEG C, be stirred for that the 0.2mol/L NaOH solution of 500mL is added dropwise after 25min.Again
By the stirring of 30min, black precipitate, i.e. magnetic Fe are recycled by magnet3O4Sediment number is washed with deionized in particle
It is secondary, stop washing, the nano particle (magnetic Fe being finally collected into when the pH value of cleaning solution is 73O4Particle) at 60 DEG C
It is dried in vacuo 8h, it is spare.
(2) magnetic Fe containing amino is prepared3O4/SiO2-NH2Particle
30 μ L 3- aminopropyl triethoxysilanes are taken to be added in the 500mL conical flask equipped with 200mL distilled water, ultrasound
After handling 5min, 500mg magnetic Fe is added into conical flask3O4After standing 40min, 80mL isobutanol and concentrated ammonia liquor is added in particle
(25%, m/m) adjusts pH to 11 or more, and 2mL ethyl orthosilicate is then slowly added dropwise under agitation, and (rate of addition cannot
More than 0.5mL/40min), with magnet solid phase reaction product is attracted after reacting 12h, has outwelled reaction solution and (attracted with magnet solid
Phase reaction product can guarantee that solid phase reaction product is attracted in reaction container bottom, toppling process during toppling over reaction solution
Solid phase reaction product is not lost), ultrasound is carried out with 30mL dehydrated alcohol again after cleaning solid phase reaction product twice with ethanol solution
30min is handled, then solution is removed in the case where having attracted the solid phase reaction product state with magnet, obtains evenly dispersed magnetism
Fe3O4/SiO2-NH2Particle is collected spare.
(3) immobilization laccase is prepared
Take the above-mentioned magnetic Fe of 50mg3O4/SiO2-NH2Particle is distributed to the phosphate-buffered of 10mL (0.12M, pH=7.0)
In liquid (PBS), be added 1mL concentration be 3% black false hellebore alcoholic solution, add 10mL gold ground ganoderma lucidum laccase crude enzyme liquid, 25 DEG C,
Under the conditions of 100r/min, 4h is vibrated, then outwells solution in the case where having attracted solid particulate matter state using magnet, obtained solid
Particulate matter is immobilization laccase.
Embodiment 3:
(1) Fe is prepared3O4Magnetic nano-particle
At room temperature, by 0.005mol FeCl2·4H2O and 0.01mol FeCl3·6H2O be added to that 300mL goes from
It in sub- water, stirs and evenly mixs, is then heated to 80 DEG C, be stirred for that the 0.2mol/L NaOH solution of 250mL is added dropwise after 25min.
Using the stirring of 30min, black precipitate, i.e. magnetic Fe are recycled by magnet3O4Sediment is washed with deionized in particle
For several times, stop washing, the nano particle (magnetic Fe being finally collected into when the pH value of cleaning solution is 73O4Particle) in 60 DEG C
Lower vacuum drying 8h, it is spare.
(2) magnetic Fe containing amino is prepared3O4/SiO2-NH2Particle
30 μ L 3- aminopropyl triethoxysilanes are taken to be added in the 500mL conical flask equipped with 200mL distilled water, ultrasound
After handling 5min, 400mg Fe is added into conical flask3O4Magnetic nanoparticle, stand 40min after, be added 80mL isopropanol and
Concentrated ammonia liquor (25%, m/m) adjusts pH to 11 or more, and 2mL ethyl orthosilicate is then slowly added dropwise under agitation and (speed is added dropwise
Degree is no more than 0.5m L/40min), it reacts and has attracted solid phase reaction product with magnet after 12h, outwell reaction solution and (inhaled with magnet
Drawing solid phase reaction product can guarantee that solid phase reaction product is attracted in reaction container bottom during toppling over reaction solution, is inclined
Process does not lose solid phase reaction product), after cleaning solid phase reaction product twice with ethanol solution again with 30mL dehydrated alcohol into
Row ultrasonic treatment 30min, then removes solution in the case where having attracted the solid phase reaction product state with magnet, obtains evenly dispersed
Magnetic Fe3O4/SiO2-NH2Particle is collected spare.
(3) immobilization laccase is prepared
Take the above-mentioned magnetic Fe of 50mg3O4/SiO2-NH2Particle is distributed to the phosphate-buffered of 10mL (0.12M, pH=7.0)
In liquid (PBS), be added 1mL concentration be 5% black false hellebore alcoholic solution, add 10mL gold ground ganoderma lucidum laccase crude enzyme liquid, 25 DEG C,
Under the conditions of 100r/min, 2h is vibrated, then outwells solution in the case where having attracted solid particulate matter state using magnet, obtained solid
Particulate matter is immobilization laccase.
Comparative example
Come from document: Wang Yingying .Fe3O4/SiO2The Schiff of carrier is modified and immobilization laccase is to phenols azo dyes
Decoloration study [D] .2017
Note: NPs: nano particle
(1) has magnetic Fe by coprecipitation preparation3O4Particle；
(2) magnetic Fe3O4/SiO2The preparation of NPs
Firstly, (1) takes Fe3O4500mg adds distilled water to be dissolved in 150mL distilled water to 50mL, (2) 30 μ LAPS, (2) is fallen
Enter quiet in (1) (not put on magnet) to 40min.It is added 100mL isopropanol, 25mL concentrated ammonia liquor (25%) adjusts pH 11 or more,
It is reacted under blender, directly dropwise addition 2mLTEOS (0.5mL/40min), attracts 20min with magnet after reaction 12h, it is clear to outwell upper layer
Liquid is washed twice with ethyl alcohol, and 30mL dehydrated alcohol is added to be respectively charged into 8 test tubes, is centrifuged (10000rpm) 10min, and about 6-7 times, most
Ultrasound 30min after primary centrifugation afterwards, obtains evenly dispersed Fe3O4/SiO2NPs is collected and is surveyed density, spare.
(3) preparation of immobilization laccase
Take the magnetic Fe of 50mg3O4/SiO2NPs, ultrasound are dispersed in 20mL ethyl alcohol, add 2.5mL distilled water,
100 μ L APS, are stirred at room temperature 12h for mixture, obtain Fe3O4/SiO2-NH2NPs；By above-mentioned Fe3O4/SiO2-NH2
NPs is distributed in the phosphate buffer (PBS) of 10mL (0.1M pH=7.0), and the glutaraldehyde (GA) of 1mL (2.5%), room is added
Temperature stirring 3h, obtains the carrier that modification is completed, i.e. Fe3O4/SiO2-NH2-GA NPs；Finally, weighing 2mg laccase is dissolved in 10mL
In PBS, in the carrier after adding it to above-mentioned modification, 12h is stirred at room temperature, and obtained immobilization laccase (is expressed as IM-
La), distribute it in a small amount of PBS, be stored in 4 DEG C of refrigerator, it is spare.
In order to improve contrast effect, according to Wang Yingying (Fe3O4/SiO2Schiff modification and the immobilization laccase pair of carrier
The decoloration of phenols azo dyes is studied) way, experiment condition control (maximum difference: in fact identical with the preparation condition of embodiment 3
It applies example 3 and uses veratryl alcohol, be glutaraldehyde used in comparative example).
It is specific as follows:
The utility model has the advantages that finding that the immobilization laccase of this method preparation is catalyzed 0.5mmol/L ABTS (2'- by contrast test
Bis- -3- ethyl benzo thiazole phenanthroline -6- the sulfonic acid of hydrazine -) efficiency mentions compared with the immobilization laccase catalytic efficiency prepared in Wang Yingying method
It is 18 percentage points high；The methylene blue dye wastewater of 50mg/L is handled, the immobilization laccase of this method preparation reuses 46
Catalytic efficiency still has original 50% after secondary, and equally operates, and reuses 41 with the immobilization laccase of Wang Yingying method preparation
Catalytic efficiency is original 50% after secondary.
The above is only a specific embodiment of the invention, but scope of protection of the present invention is not limited thereto, any to be familiar with
Those skilled in the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, and should all cover
Within protection scope of the present invention.Therefore, protection scope of the present invention should be subject to the protection scope in claims.
Claims (7)
1. a kind of process for fixation of laccase, which is characterized in that specific step is as follows:
Step A: has magnetic Fe by coprecipitation preparation3O4Particle；
Step B: by Fe in step A3O4Particle is added in 3- aminopropyl triethoxysilane aqueous solution, is added after standing reaction
Pure and mild lye is added dropwise ethyl orthosilicate under stirring condition, has attracted powder with magnet after reaction, outwelled supernatant liquor, uses
Dehydrated alcohol oscillation cleaning powder twice or more than twice, is then sonicated, obtains evenly dispersed Fe3O4/SiO2-NH2, receive
Collect spare；
Step C: with Fe in step B3O4/SiO2-NH2Particle is base core, distributes it to phosphate buffer, adds black false hellebore
Laccase solution is poured into above-mentioned reaction system by alcohol, attracts magnetic-particle, as immobilization paint with magnet after oscillating reactions
Enzyme.
2. by the process for fixation of laccase described in claim 1, which is characterized in that the step A is prepared by coprecipitation and had
Magnetic Fe3O4Particle；The specific method is as follows: soluble ferrite and soluble ferric iron salt being dissolved into deionized water, stirred
It mixes, is heated to 80 DEG C, aqueous slkali is added dropwise in whipping process；Continue to be stirred to react after adding lye, be returned by magnet
The sediment for receiving black is magnetic Fe3O4Impurity in sediment is washed with deionized in particle, dry.
3. by the process for fixation of laccase described in claim 2, which is characterized in that the aqueous slkali be concentrated ammonia liquor or concentration not
More than 0.2mol/L NaOH solution.
4. by the process for fixation of laccase described in claim 1, which is characterized in that the 3- aminopropyl triethoxysilane is water-soluble
The concentration of liquid is not less than a ten thousandth.
5. by the process for fixation of laccase described in claim 1, which is characterized in that the alcohol is hydroxyl not in the unitary at straight chain end
Alcohol or polyalcohol, volume are the 1/3-2/3 of 3- aminopropyl triethoxysilane aqueous solution volume.
6. by the process for fixation of laccase described in claim 1, which is characterized in that the lye is that concentrated ammonia liquor or concentration are no more than
0.2mol/L NaOH solution.
7. by the process for fixation of laccase described in claim 1, which is characterized in that the content of the veratryl alcohol in the reaction system
Not less than 0.05%.
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Cited By (4)
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| CN111896529A (en) * | 2020-06-17 | 2020-11-06 | 安徽师范大学 | Preparation method and application of one-dimensional ferroferric oxide @ silicon dioxide magnetic nanochain and immobilized glucose oxidase thereof |
| RU2741010C1 (en) * | 2020-06-04 | 2021-01-22 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Тверской государственный технический университет" | Heterogeneous catalyst for liquid-phase oxidation of organic compounds and method for production thereof |
| CN112680436A (en) * | 2020-12-23 | 2021-04-20 | 陕西科技大学 | Immobilized enzyme for extracting anthocyanin from black rice and preparation and use methods thereof |
| CN113501644A (en) * | 2021-06-29 | 2021-10-15 | 华南理工大学 | Preparation for improving organic solid waste anaerobic fermentation efficiency and preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2741010C1 (en) * | 2020-06-04 | 2021-01-22 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Тверской государственный технический университет" | Heterogeneous catalyst for liquid-phase oxidation of organic compounds and method for production thereof |
| CN111896529A (en) * | 2020-06-17 | 2020-11-06 | 安徽师范大学 | Preparation method and application of one-dimensional ferroferric oxide @ silicon dioxide magnetic nanochain and immobilized glucose oxidase thereof |
| CN112680436A (en) * | 2020-12-23 | 2021-04-20 | 陕西科技大学 | Immobilized enzyme for extracting anthocyanin from black rice and preparation and use methods thereof |
| CN113501644A (en) * | 2021-06-29 | 2021-10-15 | 华南理工大学 | Preparation for improving organic solid waste anaerobic fermentation efficiency and preparation method and application thereof |
| CN113501644B (en) * | 2021-06-29 | 2023-02-14 | 华南理工大学 | Preparation for improving organic solid waste anaerobic fermentation efficiency and preparation method and application thereof |
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