Immobilized laccase and preparation method thereof, and application of immobilized laccase in antibiotic degradation

A technology for immobilizing laccase and laccase, applied in the direction of immobilization on or in inorganic carriers, on/in organic carriers, biochemical equipment and methods, etc., can solve the problems of low immobilization efficiency and enzyme activity Loss, instability and other problems, to achieve effective removal, improve enzyme activity, no secondary pollution effect

Pending Publication Date: 2020-04-17
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the conventional immobilization technology has defects such as low immobilization efficiency, large loss of enzyme activity, and instability.

Method used

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  • Immobilized laccase and preparation method thereof, and application of immobilized laccase in antibiotic degradation
  • Immobilized laccase and preparation method thereof, and application of immobilized laccase in antibiotic degradation
  • Immobilized laccase and preparation method thereof, and application of immobilized laccase in antibiotic degradation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] A preparation method for immobilized laccase, comprising the steps of:

[0040] Add 2U laccase to 1 mL of 10 mM copper acetate solution (the solvent of the solution is citric acid buffer solution with pH=5.5) and mix well, then add 0.1 mL of 50 mM trimesic acid dimethyl sulfoxide solution , after mixing for 5 minutes, centrifuge at 1000 rpm for 10 minutes to remove the supernatant, resuspend and wash with water for 3 times, and dry in vacuo to obtain immobilized laccase (Laccase@Cu-BTC).

[0041] Weigh the dried solid, the solid mass is 20 mg, the enzyme loading of the immobilized carrier is 20 mg / g, and the enzyme activity is 13 U. In the case of the same amount of enzyme, the enzyme activity of the immobilized laccase is higher than that of the free enzyme Enzyme activity increased by 6.5 times.

[0042] On the determination of enzyme activity of free laccase and immobilized laccase and the calculation of laccase immobilization rate:

[0043] Take two parts of 2μg l...

Embodiment 2

[0051] A preparation method for immobilized laccase, comprising the steps of:

[0052] Add 2U laccase to 200mL of 0.5mM cupric chloride solution (acetic acid buffer with solvent pH=5.0) and mix evenly, then add 200mL of 0.5mM trimesic acid ethanol solution, mix Homogenize for 5 minutes, centrifuge at 1000 rpm for 10 minutes to remove the supernatant, resuspend and wash with pH=5.5 citrate buffer for 3 times, and dry in vacuo to obtain immobilized laccase (Laccase@Cu-BTC).

[0053] Weigh the dried solid, the solid mass is 30.5mg, the enzyme loading of the immobilized carrier is 13.1mg / g, and the total enzyme activity is 32U. Enzyme activity is 16 times higher than that of free enzyme.

Embodiment 3

[0055] An immobilization method for improving laccase activity, comprising the following steps:

[0056] Add 2U laccase to 150mL concentration of 5mM copper sulfate solution (solvent of this solution is glycine buffer solution with pH=5.5) and mix evenly, then add 10mL concentration of 15mM trimesic acid solution (solvent of this solution is Tris-HCl buffer solution with pH=7.8), after mixing for 5 minutes, centrifuge at 1000 rpm for 10 minutes to remove the supernatant, resuspend and wash the solid with acetic acid buffer solution with pH=5.5 for 3 times, and fix it after vacuum drying Laccase (Laccase@Cu-BTC).

[0057] The dried solid was weighed, the solid mass was 60.5mg, the enzyme load of the immobilized carrier was 6.7mg / g, and the total enzyme activity was 60U. The activity of the free enzyme is 30 times higher than that of the free enzyme.

[0058] Experiments have shown that water, pH=4.5 citric acid buffer, pH=7.0 citric acid buffer, pH=4.5 acetate buffer, pH=7.0 ...

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Abstract

The invention discloses immobilized laccase and a preparation method thereof, and application of the immobilized laccase in antibiotic degradation. The preparation method comprises the following steps: adding laccase into a solution containing copper ions, carrying out uniform mixing, then adding a trimesic acid solution, carrying out uniform mixing, performing centrifuging to remove a supernatant, conducting cleaning with a cleaning solution, and carrying out drying in vacuum to obtain the immobilized laccase. The method is simple to operate; the laccase can be coated in a carrier; and an organic framework structure provides shell protection for the laccase, and damage to the structure of the laccase due to external factors such as friction and shearing force in the use process is avoided. According to the invention, reduction in enzyme activity avoided, and the enzyme activity is greatly improved (the enzyme activity of the immobilized laccase obtained by the immobilization method is1.5-30 times of the activity of equivalent free laccase). The immobilized laccase disclosed by the invention has efficient catalytic degradation capability on antibiotics such as tetracycline, ampicillin, tetracycline derivatives and derivatives of ampicillin, can achieve an effect close to complete degradation in an extremely short time, and is good in reusability and free of secondary pollution.

Description

technical field [0001] The invention belongs to the technical field of sewage treatment, and in particular relates to an immobilized laccase, a preparation method and an application in antibiotic degradation. Background technique [0002] Antibiotics are a class of chemical substances that inhibit or kill pathogenic microorganisms produced by microorganisms (including bacteria, fungi, and actinomycetes) or higher animals and plants during their life. In recent years, antibiotics have been widely used in medicine, animal husbandry, planting and aquaculture and other fields. Due to the excessive use of antibiotics and the inability to be completely metabolized, a large amount of antibiotics enter the environment, causing pollution to water bodies and soil. Antibiotics entering the soil and water will not only be enriched in animals and plants through the circulation of the ecosystem, but also promote the reproduction of drug-resistant pathogens and even induce the production ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12N11/02C02F3/34
CPCC12N11/14C12N11/02C12N9/0061C12Y110/03002C02F3/342
Inventor 齐崴尤生萍张成玉苏荣欣
Owner TIANJIN UNIV
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