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Preparation of malathion hydrolase by immobilization method of cross-linked enzyme aggregates

A malathion and hydrolase technology, applied in the direction of immobilized enzymes, hydrolase, chemical instruments and methods, can solve the problems of reducing the mass transfer efficiency between enzymes and substrates, changing the conformation of enzymes, and reducing enzyme activity, etc., to achieve Good degradation effect, good activity and stability, and high immobilization rate

Active Publication Date: 2022-05-13
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the immobilized enzyme also has certain defects: (1) the mass transfer efficiency between the enzyme and the substrate is reduced; (2) the conformation of the enzyme is changed, which reduces the activity of the enzyme; (3) the process of immobilization and use will cause enzyme Loss

Method used

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  • Preparation of malathion hydrolase by immobilization method of cross-linked enzyme aggregates
  • Preparation of malathion hydrolase by immobilization method of cross-linked enzyme aggregates
  • Preparation of malathion hydrolase by immobilization method of cross-linked enzyme aggregates

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Organophosphate hydrolase (that is, malathion hydrolase Pseudomonas oleovorans DSM50188) was dissolved in 1 mL of 50 mM phosphate buffer solution (pH 7.0) buffer, the concentration of malathion hydrolase was 20 mg / ml, and ammonium sulfate was first added for aggregation Then add polyether Pluronic F127, the mass ratio of polyether Pluronic F127 to malathion hydrolase 1:4, stirred at 4°C for 30 min; finally added glutaraldehyde, the concentration of glutaraldehyde was 90 mM, and cross-linked at 4°C for 3 h. After the reaction, the reaction solution was collected and centrifuged at 12000 rpm for 3 minutes to separate the immobilized enzyme.

[0034] Enzyme activity assay:

[0035] The immobilized enzyme was placed in a constant temperature shaking reactor at 50°C for 7 hours. Add a certain amount of immobilized enzyme to 1ml of 50mM Tris-HCL (pH 9.0, containing 2mM Ellman reagent) buffer solution containing 0.5mM malathion at a final concentration of 0.5mM malathion for...

Embodiment 2

[0043] A method for immobilizing malathion hydrolase by cross-linking enzyme aggregates comprises the following steps:

[0044] (1) Add a certain amount of ammonium sulfate to the malathion hydrolase solution and stir evenly, and obtain aggregates of malathion hydrolase by salting out;

[0045] (2) adding a certain concentration of polyether to the aggregate to obtain a combination of aggregate and polyether;

[0046] (3) Finally, a certain concentration of glutaraldehyde is added to carry out the cross-linking reaction, and the insoluble particles obtained by centrifugation or filtration after the end are the immobilized enzymes.

[0047] figure 1 It is a comparison chart of the effect of the traditional immobilization method and the new cross-linked aggregate immobilization method, which shows that the specific activity of all cross-linking methods after adding polyether Pluronic F127 is higher than that of conventional CLEA, and the best addition method is: PoOPHM9 Precip...

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Abstract

The invention relates to a method for preparing malathion hydrolase by immobilizing cross-linked enzyme aggregates. The method comprises the following steps: (1) adding a certain amount of ammonium sulfate to the malathion hydrolase solution and stirring evenly, Obtain aggregates of malathion hydrolase by salting out; (2) add a certain concentration of polyether to the above aggregates to obtain a combination of aggregates and polyether; (3) finally add a certain concentration of glutaraldehyde After the cross-linking reaction, the insoluble particles obtained by centrifugation or filtration are the immobilized enzymes. Compared with the prior art, the activity of the malathion hydrolase of the present invention is increased by 4 times, and the stability and detergent tolerance are obviously improved, and the activity of the enzyme is basically unchanged after being recycled and used for 10 times, showing a very Good vitality and stability.

Description

technical field [0001] The invention relates to the development and preparation of an organophosphatase immobilization method, in particular to a preparation method and application of a novel cross-linked aggregate immobilized enzyme. Background technique [0002] Organophosphates are a class of highly toxic compounds that are artificially synthesized, including phosphoric acid esters, phosphothiol esters, etc., and are widely used in pesticides, insecticides, herbicides, etc. Among them, malathion is the most important in the world. It is widely used in many countries to prevent and control pests and diseases on crops such as grain, vegetables, tobacco, and tea. Due to the limited ability of the naturally evolved biocatalytic system to degrade this synthetic compound, organophosphorus compounds pose a great threat to environmental safety and human health. [0003] Organophosphorus hydrolase reduces the toxicity by hydrolyzing the phosphoester bonds of organophosphorus comp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/089B09C1/10C02F3/34C11D3/386C02F101/34
CPCC11D3/38672C12N9/18C12N11/08C02F3/342B09C1/10C02F2101/40C02F2101/306
Inventor 白云鹏赵玉莲张晓彦徐殿胜
Owner EAST CHINA UNIV OF SCI & TECH
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