Modified glutathione peroxidase and preparation method thereof

A technology of glutathione peroxidase and glutathione peroxidase, which is applied in the direction of fixing on/in the organic carrier, can solve the problems of easily oxidized enzymes, limited practicability, and short half-life, etc. Achieve the effect of modifying the enzyme with high activity and eliminating the antibody reaction of the enzyme protein antigen

Inactive Publication Date: 2011-10-19
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

[0024] 1. Activation of mPEG 1 The acquisition of:

[0025] Take 5.5g of secondary recrystallized cyanuric chloride (recrystallized twice with anhydrous benzene first) and dissolve it in 400mL of anhydrous benzene containing 10g of anhydrous sodium carbonate, add 50g 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 .

[0026] 2. Acquisition of pig lung-derived glutathione peroxidase: take 500g of healthy pig lung, add 1000mL pre-cooled 20mmol / L pH7.2 phosphate buffer (containing 2mmol / L mercaptoethanol, 1mmol / L EDTA), Homogenate, centrifuge at 12 000r / min at 4°C for 15min, the enzyme liquid is d...

Embodiment 2

[0030] 1. Activation of mPEG 1 Acquisition: as in Example 1

[0031] 2. Acquisition of pig lung-derived glutathione peroxidase: take 500g of healthy pig lung, add 1000mL pre-cooled 20mmol / L pH7.2 phosphate buffer (containing 2mmol / L mercaptoethanol, 1mmol / L EDTA), Homogenate, centrifuge at 12 000r / min at 4°C for 15min, the enzyme liquid is divided into three layers, the upper layer is the fat layer, and the lower layer is the precipitate, remove the precipitate and fat layer to obtain the crude enzyme liquid; then precipitate with 30-50% ethanol, DEAE- Cellulose (DE 52 ), DEAE-Sephadex A50, and the second DE 52 treatment, and finally treated with Sephadex G-150, the pure enzyme with reasonable yield and high specific activity can be obtained.

[0032] 3. Modification of pig lung-derived glutathione peroxidase:

[0033] The reaction temperature is 36°C, pH 7.0, and the reactant ratio is 1 mg of enzyme and 0.04 g of modifying agent for reaction. After 30 minutes, GSH is adde...

Embodiment 3

[0035] 1. Activation of mPEG 1 Acquisition: as in Example 1

[0036] 2. Acquisition of pig lung-derived glutathione peroxidase: take 500g of healthy pig lung, add 1000mL pre-cooled 20mmol / L pH7.2 phosphate buffer (containing 2mmol / L mercaptoethanol, 1mmol / L EDTA), Homogenate, centrifuge at 12 000r / min at 4°C for 15min, the enzyme liquid is divided into three layers, the upper layer is the fat layer, and the lower layer is the precipitate, remove the precipitate and fat layer to obtain the crude enzyme liquid; then precipitate with 30-50% ethanol, DEAE- Cellulose (DE 52 ), DEAE-Sephadex A50, and the second DE 52 treatment, and finally treated with Sephadex G-150, the pure enzyme with reasonable yield and high specific activity can be obtained.

[0037] 3. Modification of porcine lung-derived glutathione peroxidase:

[0038] 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 the reaction. After 60 minutes, GSH...

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Abstract

The invention belongs to the technical field of biology, and relates to a modified glutathione peroxidase and a preparation method thereof. The modified glutathione peroxidase is prepared by the following steps: reacting single-chain mPEG1 (Methoxy Polyethylene Glycol) with amino-group of glutathione peroxidase through free chlorine atoms, and then connecting the ingle-chain mPEG1 to the glutathione peroxidase; taking the activated mPEG1 as a modifier, and reacting the activated mPEG1 with glutathione peroxidase under the temperature of 4-37 DEG C and the pH of 7.0-10.0, wherein the mass ratio of mPEG1 to glutathione peroxidase is 20-100:1; after the reaction is carried out for 15-60min, adding GSH (glutathione) as a terminator into the mixture according to the proportion of 1:1 of the modifier and the terminator of GSH to terminate the reaction; then filtering the terminator through a thirty-thousand ultrafiltration membrane; and finally, obtaining the modified glutathione peroxidase. The amino-group modification rate of the modified glutathione peroxidase is controlled between 40-50%, and the enzymatic activity rate is over 100%.

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, thus preventing cell membranes and other biological tissues from being damaged by peroxidation; it disproportionates with superoxide in the body Enzymes (SOD) and catalase (CAT) together ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/08
Inventor 陈芳艳冯定远王林川
Owner SOUTH CHINA AGRI UNIV
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