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Method for preparing MGO-laccase

A technology of laccase and graphene, applied in biochemical equipment and methods, chemical instruments and methods, enzymes, etc., can solve problems affecting the accessibility of enzymes and mediators, and achieve industrial production, separable, high The effect of the bleaching effect

Pending Publication Date: 2019-09-03
NANJING FORESTRY UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the above method, it is necessary to carry out complex aminosilanization modification on the carrier magnetic graphene oxide, and then use glutaraldehyde as a cross-linking agent to co-immobilize the laccase and mediator system. Glutaraldehyde itself has low environmental toxicity, and the laccase and mediator system Simultaneous immobilization of the body on the material facilitates the recovery of enzyme and mediator, but at the same time affects the accessibility of enzyme and mediator contact

Method used

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  • Method for preparing MGO-laccase
  • Method for preparing MGO-laccase
  • Method for preparing MGO-laccase

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Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1 Preparation of Magnetic Graphene Oxide Immobilized Laccase

[0035] 1 Extraction of CotA crude enzyme solution

[0036] Expression of CotA laccase protein. A strain of Bacillus subtilis cjp3 with laccase activity was isolated from the black liquor of a paper mill, and the CotA laccase gene was cloned from the cjp3 strain and induced to express in E. mM CuSO4, 200rpm, induced for 10h at 25°C, and ultrasonically crushed to obtain a crude enzyme solution. The medium is LB+Kan liquid medium.

[0037] LB+Kan liquid medium: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, pH=7±0.2, sterilized at 121°C under high temperature and high pressure for 20 minutes. After cooling, filter-sterilized Kanamycin Sulfate (Kan) was added to a final concentration of 100 μΜ.

[0038] 2 Preparation of Graphene Oxide

[0039] GO is usually prepared according to a modified Hummers method. The preparation of specific graphene oxide is divided into three stages, namely...

Embodiment 2

[0063] The characterization of embodiment 2 MGO-laccase

[0064] The samples of ferric oxide, graphene oxide, MGO and MGO-laccase were ultrasonically obtained, and the low-concentration and uniform dispersion liquid was added dropwise to the copper grid, and the samples were photographed under the electron microscope after drying. The morphology and structural characteristics of the sample on the porous carbon-coated copper grid were further observed by using a microscope system operated at 120 kV with a transmission electron microscope JEM-1400 produced by Japan JEOL Company.

[0065] Such as Figure 4 As shown in (a), the TEM image clearly shows that graphene oxide is stacked together in a single layer or several layers, and the surface of the graphene oxide sheet is very smooth, as thin as a gauze. Depend on Figure 4 (b) It can be seen that Fe 3 o 4 The particles are spherical, and its average particle diameter is about 20nm. while GO and Fe 3 o 4 The composite imag...

Embodiment 3

[0066] Embodiment 3 MGO-laccase degradation dye

[0067] 1 Conditions for the decolorization of malachite green by MGO-laccase

[0068] The ability of MGO-laccase to decolorize MG dye was measured using a TU1810 UV-Vis spectrophotometer. The decrease in absorbance due to MG loss was measured at a wavelength of maximum absorption of 617 nm. Suspend 0.9 mg / mL free laccase, immobilized laccase (about 10 mg) or heat-inactivated immobilized enzyme in 10 mL of 50 mg / L dye solution in the presence or absence of ABTS. The reaction mixture was incubated with constant shaking (150 rpm). The absorbance of the dye is measured at regular time intervals.

[0069] When the temperature was 30°C, about 10 mg of immobilized laccase was incubated in malachite green dye (50 mg / L) in a series of different pH buffers for 1 h. Such as Figure 5 As shown in (a), it shows that the decolorization rate of the immobilized laccase is the best at pH 6, as high as 83.3%. After determining the most sui...

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Abstract

The invention discloses a method for preparing MGO-laccase. The graphene oxide is prepared by a modified Hummers method, and the ferroferric oxide is deposited on the graphene oxide by a coprecipitation technology to obtain magnetic graphene oxide. The CotA laccase from Bacillus subtilis cjp3 is immobilized on magnetic graphene oxide by covalent bonding with EDC / NHS. EDC / NHS is convenient and quick as a cross-linking agent, amino-functionalization is not required on a carrier material, the cost and time of immobilization are reduced, and industrial production is easy to realize. At the same time, MGO-laccase has a large loading range, strong fixing ability and wide pH and temperature suitability. The MGO laccase is applied to the decolorization of a malachite green dye, a decolorization rate can reach as high as 98.5%, the reusability is high, the toxicity is markedly reduced, and the MGO-laccase is a promising composite biocatalyst.

Description

technical field [0001] The invention relates to the field of enzyme immobilization, in particular to a preparation method of MGO-laccase. Background technique [0002] Laccase is a member of the multicopper oxidase protein family, distributed in fungi, bacteria and plants. Due to its wide range of substrates and excellent catalytic efficiency, laccase has been widely used in the decolorization of dyes and pulp in textile, papermaking, printing and dyeing industries. However, it is difficult to separate water-soluble laccase from the reaction system, hindering its reuse. In addition, laccase activity is difficult to maintain under harsh operating environments, including high temperature, wide pH range, and the presence of organic solvents, and these considerations inhibit the industrial application of laccases. [0003] To overcome the aforementioned drawbacks of laccase usage, immobilization of the enzyme is considered as a promising new strategy to improve its activity, s...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12N9/02C02F3/34C01G49/08C01B32/198C02F101/38
CPCC12N11/14C12N9/0061C12Y110/03002C02F3/342C01G49/08C01B32/198C02F2101/308C02F2101/38
Inventor 乔维川刘海付博
Owner NANJING FORESTRY UNIV
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