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Modified glutathione peroxidase and preparation method thereof

A technology of glutathione peroxidase and glutathione peroxide, applied in the biological field, can solve the problems of easy oxidase, limited practicability, short half-life, etc., achieve high modification enzyme activity, eliminate enzyme protein The effect of antigen-antibody reaction

Inactive Publication Date: 2010-09-15
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sulfhydryl group of the enzyme is easily oxidized at room temperature to inactivate the enzyme, the half-life is very short, and the stability is poor, which limits the practicability of the enzyme in a normal form.

Method used

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  • Modified glutathione peroxidase and preparation method thereof
  • Modified glutathione peroxidase and preparation method thereof
  • Modified glutathione peroxidase and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] 1. Activation of mPEG 1 The acquisition of:

[0024] Take 5.5 g of cyanuric chloride recrystallized twice (recrystallized twice with anhydrous benzene first) and dissolve it in 400 mL of anhydrous benzene containing 10 g of anhydrous sodium carbonate, add 50 g of mPEG-5000, stir overnight at room temperature, filter , take about 400mL of filtrate and stir and slowly add 600mL of diethyl ether, continue to stir for 20min after the white product precipitates, filter with suction, and dissolve the precipitate with 400mL of anhydrous benzene. up to the absorption peak. Place the activated mPEG in a vacuum desiccator to dry it up to obtain a white powder, that is, to obtain activated mPEG 1 .

[0025] 2. Modification of glutathione peroxidase:

[0026] The reaction temperature is 15°C, the pH is 9.0, the reactant ratio is 1 mg of enzyme, 0.08 g of modifier is added for reaction, after 45 min, GSH is added as the terminator of GPx modification reaction to end the reaction...

Embodiment 2

[0028] 1. Activation of mPEG 1 Acquisition: as in Example 1

[0029] 2. Modification of glutathione peroxidase:

[0030] The reaction temperature is 36°C, pH 7.0, and the reactant ratio is 1 mg of enzyme and 0.04 g of modifier for reaction. After 30 minutes, GSH is added as the terminator of GPx modification reaction to end the reaction, and the modified glutathione is obtained after ultrafiltration to remove the terminator. Glycerin peroxidase.

Embodiment 3

[0032] 1. Activation of mPEG 1 Acquisition: as in Example 1

[0033] 2. Modification of glutathione peroxidase:

[0034] The reaction temperature is 5°C, the pH is 10.0, and the reactant ratio is 1 mg of enzyme and 0.02 g of modifier for reaction. After 60 minutes, GSH is added as the terminator of GPx modification reaction to end the reaction, and the modified glutathione is obtained after ultrafiltration to remove the terminator. Glycerin peroxidase.

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Abstract

The invention belongs to the technical field of biology, relating to a modified glutathione peroxidase and a preparation method thereof. The preparation method comprises the following steps of: reacting single-chain monomethoxypolyethylene glycol (mPEG1) with amino-groups of glutathione peroxidase through free chlorine atoms so as to connect the mPEG1 to the glutathione peroxidase, wherein the activated single-chain mPEG1 is used as a modifier to react with the glutathione peroxidase at 4-37 DEG C with a pH value of 7.0-10.0, and the mass ratio of the single-chain mPEG1 to the glutathione peroxidase is (20-100):1; adding glutathione (GSH) by a proportion of 1:1 of the modifier to the GSH used as a terminator to terminate the reaction after reacting for 15-60min; and then removing the terminator by using 30 thousands of ultrafiltration membranes for ultrafiltering to obtain the modified glutathione peroxidase. The amino-group modification rate of the modified glutathione peroxidase is controlled to be 40-45 percent, and the enzymatic activity retention rate exceeds 100 percent.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a modified glutathione peroxidase and a preparation method thereof. Background technique [0002] Glutathione peroxidase (glutathione peroxidase, EC1.11.1.9, referred to as GPx) is one of the three important antioxidant enzymes in biological tissues, and it is also an important selenium-containing enzyme. Selenium is the Components of the active center of an enzyme. It can eliminate hydrogen peroxide and lipid peroxide in the body, block the further damage of active oxygen free radicals to the body, and is an important active oxygen free radical scavenger in the body. It uses selenocysteine ​​(Sec ) to act in the form of glutathione (GSH) as a reducing agent to decompose lipid peroxides in the body, thereby preventing cell membranes and other biological tissues from being damaged by peroxidation; it disproportionates with superoxide in the body Enzymes (SOD) and catalase (CAT) togeth...

Claims

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Application Information

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IPC IPC(8): C12N9/08C12N9/96
Inventor 陈芳艳冯定远王林川
Owner SOUTH CHINA AGRI UNIV
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