CN110468120A - A kind of copper nano flower and preparation method thereof containing laccase - Google Patents
A kind of copper nano flower and preparation method thereof containing laccase Download PDFInfo
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- CN110468120A CN110468120A CN201910793318.2A CN201910793318A CN110468120A CN 110468120 A CN110468120 A CN 110468120A CN 201910793318 A CN201910793318 A CN 201910793318A CN 110468120 A CN110468120 A CN 110468120A
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- laccase
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- 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 a kind of copper nano flower and preparation method thereof containing laccase, belongs to field of enzyme immobilization.This method is to add copper ion inorganic salt solution after mixing phosphate buffer solution with laccase solution, obtains the copper nano flower containing laccase.The copper nano flower containing laccase that the present invention synthesizes has bigger specific surface area compared with resolvase, and application effect is more preferably good, and preparation method is simple, cheap.
Description
Technical field
The present invention mainly passes through the complexation reaction of protein and copper ion, the aggressiveness formed between protein and copper ion
Nucleation site is provided for phosphoric acid copper nanocrystalline, has gradually formed the nanometer floral material using phosphoric acid copper nanocrystalline as petal.
The present invention relates to a kind of copper nano flower and preparation method thereof containing laccase, belongs to field of enzyme immobilization.
Background technique
Laccase (Laccases) is a kind of covellite oxidizing ferment, and more copper ion is combined as protein, source phase
To relatively extensively, it is primarily present in mushroom, bacterium and plant.It can aoxidize including phenolic substances, polyphenols, aniline, lignin, more
A series of substances such as cycloaromatics even inorganic matter, using molecular oxygen as electron acceptor.Laccase is substantially a kind of environment-friendly type ferment, and
Itself has certain enzyme activity, can rely on air existence in nature, catalysis can occur with other many kinds of substance and react, finally
Obtained product is pollution-free, is under normal circumstances all water, is a kind of pollution-free, green, environmentally friendly enzyme element.Laccase mainly by copper from
3 son, peptide chain and sugared aglucon parts form, and protein molecular weight is between 60-900kDa, and peptide chain is generally by 500-550 ammonia
Base acid composition, sugared aglucon is mainly made of 5 kinds of carbohydrates, accounts for the 10%-80% of total molecular weight, laccase under the conditions of weakly acidic,
There is certain specificity to substrate, most suitable reaction temperature (30-40 DEG C) is relatively low, and the efficiency of catalysis oxidation is higher.One
As be added laccase when control pH in the range of 6.0-4.8, enzyme activity within this range is higher, and is not easy deactivation in itself.
There are many kinds of the fixing means of enzyme, including absorption method, investment, combined techniques, cross-linking method etc..Every kind of immobilization side
Method all respectively has its advantage and disadvantage, how to make enzyme fixed while to increase its enzyme activity be research hotspot in recent years.Wherein, with receiving
Popped rice carrys out immobilised enzymes and achieves breakthrough, and fixed enzyme not only has stronger stability but also enzyme activity in this way
It is very high.Because nano flower has bigger surface area, this has sizable advantage in terms of enzyme-to-substrate contact.
Summary of the invention
The object of the present invention is to provide a kind of copper nano flower containing laccase, by the complexation reaction of protein and copper ion,
The aggressiveness formed between protein and copper ion provides nucleation site for phosphoric acid copper nanocrystalline, has gradually formed with phosphoric acid
Copper nanocrystalline is the nanometer floral material of petal.
The preparation method of the present invention also provides a kind of copper nano flower containing laccase, this method comprises:
(1) it is prepared to obtain the laccase solution that concentration is 0.1~0.5mg/mL with aqueous solution；
(2) it prepares and obtains the copper-bath of 120mM, and be filtered to remove impurity；
(3) copper-bath obtained in step (2) is added drop-wise in the laccase solution that step (1) obtains dropwise, is stirred
0.5~6.0 hour is stood in the environment of 20~30 DEG C after uniformly, synthesizes mixed liquor containing laccase by way of self assembly
Copper nano flower collects precipitating, by washing, being freeze-dried to obtain the copper nano flower containing laccase.
Above-mentioned technical proposal, in step (1), aqueous solution is the phosphate buffer solution that pH is 7.4 under 25 DEG C of environment
(PBS).The phosphate buffer solution is to be dissolved in disodium hydrogen phosphate (Shi Ershui) 7.16g, sodium dihydrogen phosphate 3.12g respectively
Disodium phosphate soln and sodium dihydrogen phosphate are formed in 100mL deionized water, then take disodium phosphate soln respectively
81.00ml, sodium dihydrogen phosphate 19.00mL simultaneously mix obtained solution.
Above-mentioned technical proposal, in step (2), the filter device is injector drive filter, and filter membrane is hydrophilic
Property PVDF, pore size be 0.22 μm.
The copper nano flower containing laccase that the present invention synthesizes has bigger specific surface area compared with resolvase, and application effect is more
It is good good, and preparation method is simple, it is cheap.
Specific embodiment
The present invention is described in further detail combined with specific embodiments below, following embodiment be it is descriptive,
It is not restrictive, these specific embodiments cannot be limited the invention to.In following embodiments, unless otherwise specified, institute
The experimental method used is conventional method, and reagent used etc. can chemically or biological reagent company is bought.
The preparation method of the present invention provides a kind of copper nano flower containing laccase, which is characterized in that its method includes:
(1) it is prepared to obtain the laccase solution that concentration is 0.1~0.5mg/mL with aqueous solution；
(2) it prepares and obtains the copper-bath of 120mM, and be filtered to remove impurity；
(3) copper-bath obtained in step (2) is added drop-wise in the laccase solution that step (1) obtains dropwise, is stirred
0.5~6.0 hour is stood in the environment of 20~30 DEG C after uniformly, synthesizes mixed liquor containing laccase by way of self assembly
Copper nano flower collects precipitating, by washing, being freeze-dried to obtain the copper nano flower containing laccase.
Above-mentioned technical proposal, in step (1), aqueous solution is the phosphate buffer solution that pH is 7.4 under 25 DEG C of environment
(PBS).The phosphate buffer solution is to be dissolved in disodium hydrogen phosphate (Shi Ershui) 7.16g, sodium dihydrogen phosphate 3.12g respectively
Disodium phosphate soln and sodium dihydrogen phosphate are formed in 100mL deionized water, then take disodium phosphate soln respectively
81.00mL, sodium dihydrogen phosphate 19.00mL simultaneously mix obtained solution.
Above-mentioned technical proposal, in step (2), used filter device is injector drive filter, and filter membrane is parent
Aqueous PVDF, pore size are 0.22 μm.
Embodiment 1
(1) disodium hydrogen phosphate (Shi Ershui) 7.16g, sodium dihydrogen phosphate 3.12g are dissolved in shape in 100mL deionized water respectively
At disodium phosphate soln and sodium dihydrogen phosphate, then disodium phosphate soln 81.00mL, sodium dihydrogen phosphate are taken respectively
19.00mL is simultaneously mixed to get phosphate buffer solution (PBS)；
(2) laccase for weighing 6.0mg, is dissolved in the PBS of 60ml, is ultrasonically treated 1min；
(3) 1.5g Salzburg vitriol is weighed, is dissolved in 50mL deionized water, shakes up, with injector drive filter mistake
Filter removes the impurity in solution；
(4) 150 μ L copper-baths are added dropwise dropwise in 3mL enzyme solutions, shake up, stand 1.0h, centrifugation is precipitated, and is used
Deionization is washed 3 times, and the powder being finally freeze-dried is the copper nano flower containing laccase.
Embodiment 2
(1) disodium hydrogen phosphate (Shi Ershui) 7.16g, sodium dihydrogen phosphate 3.12g are dissolved in shape in 100mL deionized water respectively
At disodium phosphate soln and sodium dihydrogen phosphate, then disodium phosphate soln 81.00mL, sodium dihydrogen phosphate are taken respectively
19mL is simultaneously mixed to get phosphate buffer solution (PBS)；
(2) laccase for weighing 12.0mg, is dissolved in the PBS of 60mL, is ultrasonically treated 1min；
(3) 1.5g Salzburg vitriol is weighed, is dissolved in 50mL deionized water, shakes up, with injector drive filter mistake
Filter removes the impurity in solution；
(4) 150 μ L copper-baths are added dropwise dropwise in 3mL enzyme solutions, shake up, stand 2.0h, centrifugation is precipitated, and is used
Deionization is washed 3 times, and the powder being finally freeze-dried is the copper nano flower containing laccase.
Claims (3)
1. a kind of copper nano flower and preparation method thereof containing laccase, it is characterized in that including the following steps: in order
(1) it is prepared to obtain the laccase solution that concentration is 0.1~0.5mg/mL with aqueous solution；
(2) it prepares and obtains the copper-bath of 120mM, and be filtered to remove impurity；
(3) copper-bath obtained in step (2) is added drop-wise in the laccase solution that step (1) obtains dropwise, is stirred evenly
0.5~6.0 hour is stood in the environment of 20~30 DEG C afterwards, so that mixed liquor is synthesized the copper containing laccase by way of self assembly and receives
Popped rice collects precipitating, by washing, being freeze-dried to obtain the copper nano flower containing laccase.
2. according to the method described in claim 1, it is characterized in that the aqueous solution is the phosphate that pH is 7.4 under 25 DEG C of environment
Buffer solution (PBS).The phosphate buffer solution is by disodium hydrogen phosphate (Shi Ershui) 7.16g, sodium dihydrogen phosphate 3.12g points
It is not dissolved in 100ml deionized water and forms disodium phosphate soln and sodium dihydrogen phosphate, then take disodium hydrogen phosphate molten respectively
Liquid 81.00ml, sodium dihydrogen phosphate 19.00ml simultaneously mix obtained solution.
3. according to the method described in claim 1, it is characterized in that the used filter device is injector drive filter,
Filter membrane is hydrophilic PVDF, and pore size is 0.22 μm.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110742065A (en) * | 2019-10-25 | 2020-02-04 | 山东农业大学 | Nano flower-loaded pesticide preparation and preparation method thereof |
| CN111676213A (en) * | 2020-06-15 | 2020-09-18 | 吉林农业大学 | laccase-nanoflower-gamma-Fe2O3Copper alginate gel bead and preparation method and application thereof |
| CN114015068A (en) * | 2021-11-23 | 2022-02-08 | 中国医学科学院生物医学工程研究所 | Flower-shaped MOF material based on coordination of baicalein and copper ions and preparation method and application thereof |
| CN114410404A (en) * | 2022-02-16 | 2022-04-29 | 胡春霞 | Storable red wine and preparation method thereof |
| CN114480365A (en) * | 2022-01-18 | 2022-05-13 | 南昌大学 | Macromolecule-enzyme-inorganic hybrid nano flower, preparation method thereof and application thereof in degrading mycotoxin in edible oil |
| CN114606223A (en) * | 2022-04-19 | 2022-06-10 | 浙江工业大学 | D-amino acid oxidase-inorganic hybrid nanoflower and preparation method and application thereof |
| CN114874831A (en) * | 2022-05-25 | 2022-08-09 | 武汉材料保护研究所有限公司 | Method for improving lubricating performance of lubricating oil |
| CN114956028A (en) * | 2022-05-25 | 2022-08-30 | 武汉材料保护研究所有限公司 | Copper phosphate with porous structure and preparation method thereof |
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2019
- 2019-08-27 CN CN201910793318.2A patent/CN110468120A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110742065A (en) * | 2019-10-25 | 2020-02-04 | 山东农业大学 | Nano flower-loaded pesticide preparation and preparation method thereof |
| CN111676213A (en) * | 2020-06-15 | 2020-09-18 | 吉林农业大学 | laccase-nanoflower-gamma-Fe2O3Copper alginate gel bead and preparation method and application thereof |
| CN114015068A (en) * | 2021-11-23 | 2022-02-08 | 中国医学科学院生物医学工程研究所 | Flower-shaped MOF material based on coordination of baicalein and copper ions and preparation method and application thereof |
| CN114015068B (en) * | 2021-11-23 | 2023-02-10 | 中国医学科学院生物医学工程研究所 | Flower-shaped MOF material based on coordination of baicalein and copper ions and preparation method and application thereof |
| CN114480365A (en) * | 2022-01-18 | 2022-05-13 | 南昌大学 | Macromolecule-enzyme-inorganic hybrid nano flower, preparation method thereof and application thereof in degrading mycotoxin in edible oil |
| CN114480365B (en) * | 2022-01-18 | 2024-05-28 | 南昌大学 | Polymer-enzyme-inorganic hybrid nanoflower, preparation method thereof and application thereof in degrading mycotoxin in edible oil |
| CN114410404A (en) * | 2022-02-16 | 2022-04-29 | 胡春霞 | Storable red wine and preparation method thereof |
| CN114606223A (en) * | 2022-04-19 | 2022-06-10 | 浙江工业大学 | D-amino acid oxidase-inorganic hybrid nanoflower and preparation method and application thereof |
| CN114874831A (en) * | 2022-05-25 | 2022-08-09 | 武汉材料保护研究所有限公司 | Method for improving lubricating performance of lubricating oil |
| CN114956028A (en) * | 2022-05-25 | 2022-08-30 | 武汉材料保护研究所有限公司 | Copper phosphate with porous structure and preparation method thereof |
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Application publication date: 20191119 |