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Cryoprotectant for xanthine oxidase and application thereof

A technology of xanthine oxidase and freeze-drying protective agent, applied in the field of freeze-dried preparations of xanthine oxidase, can solve problems such as limited reports, and achieve the effect of accelerating stability

Active Publication Date: 2015-01-28
SHANGHAI DIAZYME
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The xanthine oxidase substrate isolated and purified from Arthrobacter is more specific, but there are limited reports on its freeze-dried preparation, only mentioned in Japanese Patent No. 62-55081

Method used

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  • Cryoprotectant for xanthine oxidase and application thereof
  • Cryoprotectant for xanthine oxidase and application thereof
  • Cryoprotectant for xanthine oxidase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Xanthine oxidase sample preparation:

[0043] The purified xanthine oxidase was dialyzed into 20mM Tris-HCl pH7.2 buffer system, and the enzyme concentration was about 10mg / ml. Preparation of xanthine oxidase lyophilized preparation:

[0044] Preparation 1: take more than 100ml of the prepared sample, add 2g of sucrose and 2g of glycine, fully dissolve each component and store at 2-8°C.

[0045] Preparation 2: Take more than 100ml of the prepared sample, add 4g of trehalose and 1g of PEG2000, fully dissolve each component and store at 2-8°C.

[0046] Preparation 3: Take more than 100ml of the prepared sample, add 2g of trehalose and 0.1ml of Tween80, fully dissolve each component and store at 2-8°C.

[0047] Preparation 4: Take more than 100ml of the prepared sample, add 2gBSA and 1g sodium glucate, fully dissolve each component and store at 2-8°C.

[0048] Preparation 5: Take more than 100ml of the prepared sample, add 2g of trehalose and 1g of histidine, fully diss...

Embodiment 2

[0060] The purified xanthine oxidase was dialyzed into 20mM Tris-HCl pH7.2 buffer system, the enzyme concentration was about 5mg / ml.

[0061] Preparation of xanthine oxidase lyophilized preparation:

[0062] Preparation 7: Take more than 100ml of the prepared sample, add 1g trehalose, 1.5g histidine, 50mg iron porphyrin to fully dissolve each component and store at 2-8°C.

[0063] The above preparations were freeze-dried, the specific process: pre-freezing stage: -40 ℃ pre-freezing for 4 hours; sublimation stage: rise the product from -40 ℃ to -10 ℃ for 1 hour and maintain it for 12 hours; secondary sublimation stage: 0.5 hours The product is raised from -10°C to 10°C and maintained for 10 hours; the last 0.5 hour is raised to 25°C and maintained for 2 hours out of the box. The entire freeze-drying process takes 30 hours.

[0064] The freeze-dried xanthine oxidase powder was stored at -20°C and 37°C respectively, and its shape and activity were regularly observed.

[0065] ...

Embodiment 3

[0070] The purified xanthine oxidase was dialyzed into 20mM Tris-HCl pH7.2 buffer system, and the enzyme concentration was about 15mg / ml.

[0071] Preparation of xanthine oxidase lyophilized preparation:

[0072] Preparation 7: Take the sample prepared above 100ml, add 3g trehalose, 0.5g histidine, 70mg iron porphyrin to fully dissolve each component, and store at 2-8°C.

[0073] The above preparations were freeze-dried, the specific process: pre-freezing stage: -40 ℃ pre-freezing for 4 hours; sublimation stage: rise the product from -40 ℃ to -10 ℃ for 1 hour and maintain it for 12 hours; secondary sublimation stage: 0.5 hours The product is raised from -10°C to 10°C and maintained for 10 hours; the last 0.5 hour is raised to 25°C and maintained for 2 hours out of the box. The entire freeze-drying process takes 30 hours.

[0074] The freeze-dried xanthine oxidase powder was stored at -20°C and 37°C respectively, and its shape and activity were regularly observed.

[0075] T...

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Abstract

The invention belongs to the technical field of preservation of enzymes and specifically relates to a cryoprotectant for xanthine oxidase and an application thereof. The invention provides the cryoprotectant for the xanthine oxidase. Each 100ml of buffer solution contains the following components: 0.5-10g of trehalose, 0.1-5g of L-histidine and 0.01-0.1g of iron porphyrin. The using effects of the cryoprotectant disclosed by the invention show that by using the cryoprotectant, more than 90% of the activity of the xanthine oxidase can be retained at 37 DEG C after 7 days, and 90% of the activity of the xanthine oxidase can be still retained at -20 DEG C after 2 years. In addition, compared with the formulas in the existing patents, the preparation formula disclosed by the invention has better stability in acceleration.

Description

technical field [0001] The invention belongs to the technical field of enzyme preservation, and in particular relates to a xanthine oxidase freeze-drying protectant and application thereof, and a xanthine oxidase freeze-dried preparation prepared by the freeze-drying protectant. Background technique [0002] Xanthine oxidase (English name is xanthine oxidase, referred to as XOD, EC1.2.3.11) is a kind of low specificity, which can not only catalyze hypoxanthine to generate xanthine, and then generate uric acid, but also directly catalyze the generation of xanthine An enzyme that produces uric acid. Xanthine oxidase, as a flavin enzyme containing molybdenum, non-heme iron, inorganic sulfide, and FAD, mainly exists in milk, animals, especially the liver and kidney of birds, insects, and bacteria. It is mainly used for For biochemical research, it is a commonly used enzyme for diagnosis, and can be specifically applied to the determination of xanthine, inosine and guanosine. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/96C12N9/02
Inventor 汪海霞杨武剑陆婷郑颖
Owner SHANGHAI DIAZYME
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