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Immobilized enzyme method for improving stability of horseradish peroxidase and application

A technology of horseradish peroxidase and immobilized enzyme, applied in the directions of enzyme stabilization, oxidoreductase, biochemical equipment and methods, etc., can solve problems such as insufficient stability of natural enzymes, and achieve improved storage stability and protection performance. The effect of enhancing, enhancing stability

Active Publication Date: 2019-09-10
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The invention solves the defects of insufficient stability of natural enzymes, and realizes the immobilization of natural horseradish peroxidase (HRP@ZIF-8) through the zeolite imidazolate framework-8 / graphene composite material (ZIF-8 / GO). / GO), and applied it to the colorimetric detection of kanamycin (KAN)

Method used

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  • Immobilized enzyme method for improving stability of horseradish peroxidase and application
  • Immobilized enzyme method for improving stability of horseradish peroxidase and application
  • Immobilized enzyme method for improving stability of horseradish peroxidase and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Configure the determination of kanamycin content in water samples:

[0028] (1) Preparation of graphene oxide:

[0029] Graphene oxide was prepared using a modified Hummers chemistry. The specific steps are as follows: Weigh 1.0g of graphite powder and slowly add it to 23mL of concentrated sulfuric acid. After fully stirring, slowly add 3g of KMnO 4 , slowly added while fully stirring, and then continuously ultrasonicated for 7 hours to obtain a dark brown solution, then slowly added 46mL of deionized water, heated and boiled for 15min, then added 140mL of high-purity water and 10mL of hydrogen peroxide to terminate the reaction, and obtained bright yellow graphene oxide aqueous solution. After centrifugation, use 5% dilute hydrochloric acid 8000r / min centrifugal washing 2 times to remove impurities, and then use high-purity water 8000r / min centrifugal washing 3 times to remove impurities. After washing, the purified graphite oxide was taken out, put into a dialysis ...

Embodiment 2

[0040] Configure the determination of kanamycin content in water samples:

[0041] (1) Preparation of graphene oxide:

[0042] Graphene oxide was prepared using a modified Hummers chemistry. The specific steps are as follows: Weigh 1.0g of graphite powder and slowly add it to 23mL of concentrated sulfuric acid. After fully stirring, slowly add 3g of KMnO 4 , slowly added while fully stirring, and then continuously ultrasonicated for 6 hours to obtain a dark brown solution, then slowly added 46mL of deionized water, heated and boiled for 15min, then added 140mL of high-purity water and 10mL of hydrogen peroxide to terminate the reaction, and obtained bright yellow graphene oxide aqueous solution. After centrifugation, use 5% dilute hydrochloric acid 10000r / min centrifugal washing 2 times to remove impurities, and then use high-purity water 10000r / min centrifugal washing 5 times to remove impurities. After washing, the purified graphite oxide was taken out, put into a dialysi...

Embodiment 3

[0053] Determination of Kanamycin Content in Tap Water Samples:

[0054] (1) Preparation of graphene oxide:

[0055] Graphene oxide was prepared using a modified Hummers chemistry. The specific steps are as follows: Weigh 1.0g of graphite powder and slowly add it to 23mL of concentrated sulfuric acid. After fully stirring, slowly add 3g of KMnO 4 , slowly added while fully stirring, and then continuously ultrasonicated for 5 hours to obtain a dark brown solution, then slowly added 46mL of deionized water, heated and boiled for 15min, then added 140mL of high-purity water and 10mL of hydrogen peroxide to terminate the reaction, and obtained bright yellow graphene oxide aqueous solution. After centrifugation, use 5% dilute hydrochloric acid 10000r / min centrifugal washing 2 times to remove impurities, and then use high-purity water 10000r / min centrifugal washing 5 times to remove impurities. After washing, the purified graphite oxide was taken out, put into a dialysis bag (MW=...

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Abstract

The invention belongs to the technical field of environmental monitoring, and provides an immobilized enzyme method for improving the stability of horseradish peroxidase and application. At normal temperature and normal pressure, by using a co-precipitation method, a biological composite material of zeolite imidazolate skeleton-8 / graphene immobilized natural horseradish peroxidase is synthesized in an aqueous phase. Embedding of the ZIF-8 / GO composite material retains the excellent peroxidase catalytic activity of HRP, and meanwhile, the stability of the HRP is enhanced. Compared with a singleZIF-8 mineralized HRP material, the introduction of GO increases the tolerance of the HRP against complex environments such as high temperature, organic solvents and denaturants, and meanwhile, the storage stability and recycling capability of the HRP are enhanced. The peroxidase catalytic activity of the HRP@ZIF-8 / GO biological composite material is regulated by utilizing DNA molecules; a non-marked colorimetric sensing method is established, and specific detection of kanamycin is realized.

Description

technical field [0001] The invention belongs to the technical field of environmental monitoring, and relates to an immobilized enzyme method and application for improving the stability of horseradish peroxidase. Background technique [0002] Natural enzymes are proteins or RNAs produced by living cells that are highly specific for their substrates and highly catalytically efficient. Natural enzymes have the advantages of wide sources, various types, good substrate affinity, and high catalytic efficiency, and are widely used in various aspects of production and life such as catalytic analysis, disease diagnosis, and clinical treatment. However, as a typical class of biomolecules, natural enzymes have relatively poor stability, are easily affected by the external environment, and have strict requirements on storage conditions. For example, natural enzymes are easily damaged by extreme external conditions such as high temperature, strong acid, and strong alkali, and lose some ...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12N11/02C12N9/04C12N9/96G01N21/31G01N21/78
CPCC12N11/14C12N11/02C12N9/96C12N9/0065C12Y111/01007G01N21/78G01N21/3103
Inventor 赵慧敏谭冰
Owner DALIAN UNIV OF TECH
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