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Preparation method and application of uricase immobilized resin

A uricase and resin technology, applied in the field of enzyme immobilization, can solve problems such as health risks, influence on adsorption performance, chemical structure damage, etc., and achieve the effect of stable enzyme spatial structure, good biocompatibility, and increased stability

Pending Publication Date: 2022-05-27
顾凌巍
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This will lead to the loss of beneficial body fluid components, causing some potential health risks
[0005] At present, some users have modified and reprocessed solid adsorbents to varying degrees. However, since hemoperfusion devices belong to the third category of medical devices, they are disposable after sterilization. It is difficult to maintain its adsorption performance stably. At the same time, after the modified adsorbent is sterilized and stored for a long time, the modified chemical structure will be damaged to varying degrees, which will affect the adsorption performance and fail to achieve the original adsorption effect.
Moreover, the basic structure of the resin itself has not undergone fundamental changes, and the problem of non-specific adsorption has not been well resolved.

Method used

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  • Preparation method and application of uricase immobilized resin

Examples

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Effect test

Embodiment 1

[0030] A preparation method of uricase immobilized resin, comprising the following steps:

[0031] (1) The purified recombinant uricase was prepared into 25 mL of enzyme solution with a concentration of 2 g / L at room temperature, followed by adding a phosphate buffer (75 mL) with a pH of 7.0 and a concentration of 8 mmol / LEDTA solution (50 mL), and the mixture was shaken and mixed. uniform for 30min, and then EDTA-Cu was separated by molecular sieve chromatography. 2+ The chelate is removed from the reaction solution to obtain a Cu-free 2+ uricase solution, continue to add zinc sulfate solution (150 mL) with a concentration of 8 mmol / L, shake at a constant temperature of 20 ° C for 1 h, and separate and purify to obtain Zn 2+ Replaced uricase;

[0032](2) Disperse 70g of polystyrene resin particles in water, then add 30mL of concentrated nitric acid and 10mL of concentrated sulfuric acid, react at 45°C for 12h, centrifuge after the reaction is completed, wash with ultrapure ...

Embodiment 2

[0037] A preparation method of uricase immobilized resin, comprising the following steps:

[0038] (1) At room temperature, the purified recombinant uricase was prepared into 25 mL of an enzyme solution with a concentration of 4 g / L, followed by adding a phosphate buffer solution (75 mL) with a pH of 7.0 and a concentration of 12 mmol / L EDTA solution (60 mL), shaking Mixed for 45min, then EDTA-Cu was separated by molecular sieve chromatography 2+ The chelate is removed from the reaction solution to obtain a Cu-free 2+ uricase solution, continue to add zinc sulfate solution (175 mL) with a concentration of 12 mmol / L, shake at constant temperature for 1.5 h at 25 °C, and separate and purify to obtain Zn 2+ Replaced uricase;

[0039] (2) Disperse 70 g of polystyrene resin particles in water, then add 50 mL of concentrated nitric acid and 30 mL of concentrated sulfuric acid, react at 50°C for 14 hours, centrifuge after the reaction is completed, wash with ultrapure water to neut...

Embodiment 3

[0044] A preparation method of uricase immobilized resin, comprising the following steps:

[0045] (1) At room temperature, the purified recombinant uricase was configured into 25 mL of an enzyme solution with a concentration of 3 g / L, followed by adding a phosphate buffer solution (75 mL) with a pH of 7.0 and a concentration of 10 mmol / L EDTA solution (75 mL), shaking Mixed for 30-60min, then EDTA-Cu was separated by molecular sieve chromatography 2+ The chelate is removed from the reaction solution to obtain a Cu-free 2+ uricase solution, continue to add zinc sulfate solution (200 mL) with a concentration of 10 mmol / L, shake at a constant temperature of 20 ° C for 2 h, and separate and purify to obtain Zn 2+ Replaced uricase;

[0046] (2) Disperse 70g of polystyrene resin particles in water, then add 40mL of concentrated nitric acid and 20mL of concentrated sulfuric acid, react at 45°C for 10h, centrifuge after the reaction is complete, wash with ultrapure water to neutral...

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Abstract

The invention discloses a preparation method of uricase immobilized resin. The preparation method mainly comprises the following steps: (1) replacing uricase copper ions Cu < 2 + > into zinc ions Zn < 2 + >; (2) aminating the resin particles; (3) modifying uricase with dextran; (4) connecting aminated resin particles with dextran dialdehyde on the surface of uricase; and (5) wrapping the finished product resin with chitosan. According to the uricase immobilized resin provided by the invention, the uricase is modified and optimized, the stability of the uricase is improved, the uricase can tolerate a relatively violent reaction environment, and the optimized uricase surface group and aminated polystyrene resin particles are subjected to cross-linking combination through dextran dialdehyde, so that the uricase immobilized resin is obtained. According to the method, uricase is taken as a substrate to achieve immobilization of uricase, chitosan is used for wrapping resin particles of immobilized enzyme, so that the space structure of the enzyme is further stable, the enzyme is resistant to irradiation sterilization, and after irradiation sterilization of a conventional dosage, a chitosan molecular chain is broken and degraded to fall off.

Description

technical field [0001] The invention belongs to the technical field of enzyme immobilization, and in particular relates to a preparation method and application of a uricase immobilized resin. Background technique [0002] Enzymes are biological macromolecules with catalytic activity and high selectivity. Compared with traditional chemical catalysts, enzymatic catalysis has the advantages of high efficiency, substrate specificity, and mild reaction conditions, and is widely used in the synthesis of biomedical products, environmental monitoring, food processing, and biosensors. However, the higher-order structural parts of proteins in most enzymes are extremely unstable in the environment of strong acid, strong base, high temperature, and organic solvents, and free enzymes are easily deactivated under these reaction conditions, which greatly limits the practical application of enzymes. . In addition, although the activity of the enzyme in aqueous solution is high, the lytic ...

Claims

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Application Information

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IPC IPC(8): C12N11/082C12N11/10C12N9/06A61M1/36
CPCC12N11/082C12N11/10C12N9/0048A61M1/3679A61M1/3687C12Y107/03003A61M2207/00
Inventor 顾凌巍陆雪峰陈情忠王蕾丁小燕
Owner 顾凌巍
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