Preparation method of polyphenol oxidase immobilizing carrier

A technology of polyphenol oxidase and immobilized carrier, which is applied in the direction of fixing on or in the inorganic carrier, fixing on/in the organic carrier, etc., which can solve the separation difficulties and unfavorable immobilization of chitosan-gold nanoparticles Eliminate the problems of repeated use of enzymes, and achieve the effects of high immobilization efficiency, high enzyme activity, and simple preparation methods

Active Publication Date: 2015-04-22
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the separation of chitosan-gold nanoparticles is difficult, which is not conducive to the reuse of immobilized enzymes

Method used

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  • Preparation method of polyphenol oxidase immobilizing carrier
  • Preparation method of polyphenol oxidase immobilizing carrier
  • Preparation method of polyphenol oxidase immobilizing carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Add 4.0 g of Na-based montmorillonite into 100 mL of distilled water and stir at room temperature for 2 h to make it fully swell. Dissolve 3.3 g of chitosan in 250 mL of 1% (v / v) acetic acid solution, and adjust the pH value to 4.9 with 20% (w / v) NaOH solution after fully dissolving. The chitosan solution was slowly added to the montmorillonite suspension, heated to 60°C, and stirred for 6 h. After the reaction, centrifuge, wash and dry to obtain the chitosan / montmorillonite intercalation complex.

[0025] Dissolve chloroauric acid in distilled water to prepare a chloroauric acid solution with a concentration of 0.01 mM, and adjust the pH to 9.0 with 1 M NaOH solution. The chitosan / montmorillonite intercalation complex was added to the above chloroauric acid solution, so that the mass ratio of chloroauric acid to chitosan / montmorillonite intercalation complex was 0.15% (w / w). Stir fully at 80°C for 1 h under constant temperature and magnetic force. After the reaction,...

Embodiment 2

[0032] Dissolve chloroauric acid in distilled water to prepare a chloroauric acid solution with a concentration of 0.05 mM, and adjust the pH to 3.0 with 1 M HCl solution. The chitosan / montmorillonite intercalation complex was added to the above chloroauric acid solution, so that the mass ratio of chloroauric acid to chitosan / montmorillonite intercalation complex was 0.50% (w / w). Stir at 60°C for 2 h under constant temperature and magnetic force. After the reaction, centrifuge and wash the product with distilled water until the pH of the supernatant is neutral to obtain the chitosan-nanogold / montmorillonite intercalation complex.

[0033] Add chitosan-nanogold / montmorillonite intercalation complex to the polyphenol oxidase buffer solution of pH 7.0, so that the mass ratio of polyphenol oxidase to chitosan-nanogold / montmorillonite intercalation complex carrier is 80 mg / g. Shake and adsorb at 25°C for 5 h, remove the enzyme solution by filtration, wash the immobilized enzyme wi...

Embodiment 3

[0035] Dissolve chloroauric acid in distilled water to prepare a chloroauric acid solution with a concentration of 0.10 mM, and adjust the pH to 6.0 with 1 M NaOH solution. The chitosan / montmorillonite intercalation complex was added to the above chloroauric acid solution, so that the mass ratio of chloroauric acid to chitosan / montmorillonite intercalation complex was 1.00% (w / w). Stir at 60°C for 1.5 h under constant temperature and magnetic force. After the reaction, centrifuge and wash the product with distilled water until the pH of the supernatant is neutral to obtain the chitosan-nanogold / montmorillonite intercalation complex.

[0036] Add chitosan-nanogold / montmorillonite intercalation complex to the polyphenol oxidase buffer solution of pH 6.0, so that the mass ratio of polyphenol oxidase to chitosan-nanogold / montmorillonite intercalation complex carrier is 60 mg / g. Shake and adsorb at 30°C for 4 h, remove the enzyme solution by filtration, wash the immobilized enzyme...

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Abstract

The invention discloses a preparation method of a polyphenol oxidase immobilizing carrier and a method for immobilizing polyphenol oxidase by using the same. The method comprises the following steps: inserting a chitosan molecular chain into a montmorillonite lamella by adopting a solution intercalation method so as to prepare a chitosan/montmorillonite intercalation compound; performing in-situ reduction on chlorauric acid by using chemical structures with multiple hydroxyl groups and multiple amino groups of chitosan as a stabilizing agent and a reducing agent so as to realize one-step green synthesis of nano-gold particles; and preparing a chitosan-nano-gold/montmorillonite intercalation compound, and taking the intercalation compound as a carrier to immobilize polyphenol oxidase. The intercalation compound is an immobilizing carrier for polyphenol oxidase, and a prepared immobilized enzyme has the advantages of high immobilizing efficiency, high enzymatic activity, good stabilizer and the like.

Description

technical field [0001] The invention relates to a preparation method of an immobilized enzyme carrier, in particular to a preparation method of an immobilized polyphenol oxidase carrier, belonging to the field of biotechnology. Background technique [0002] Polyphenol oxidase is a kind of oxidoreductase, which has dual properties of hydroxylase and oxidase. Under aerobic conditions, it can catalyze the hydroxylation of monophenolic compounds to generate catechol, and then oxidize catechol to generate This characteristic makes polyphenol oxidase widely used in the fields of industry, food and environmental protection. Free polyphenol oxidase has defects such as low stability, inability to be reused, and susceptibility to environmental conditions to inhibit enzyme activity during use, which hinders its popularization and application. [0003] The enzyme immobilization technology can enable the enzyme to maintain its high-efficiency, specific and mild enzyme-catalyzed reaction...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12N11/08
Inventor 李瑾王涵梁红波马启敏
Owner OCEAN UNIV OF CHINA
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