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Method for fixing polyphenol oxidase with ion exchange resin support

A technology of ion exchange resin and polyphenol oxidase, which is applied in the direction of fixing on/in the organic carrier, can solve the problems of increasing steric hindrance and reducing catalytic efficiency, and achieves reduced steric hindrance, good water insolubility, Highly stable effect

Inactive Publication Date: 2009-07-29
TEA RES INST CHINESE ACAD OF AGRI SCI
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AI Technical Summary

Problems solved by technology

These immobilization methods embed enzyme protein molecules in water-insoluble calcium alginate and chitosan carriers, which can make enzyme molecules insoluble in water and achieve t

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Weigh 10 g of unactivated ion exchange resin D113, add 50 mg of crude polyphenol oxidase after activation (polyphenol oxidase is dissolved in 50 ml of phosphate buffer solution with pH 5.6 at 4°C), and shake at a constant temperature at 30°C to Immobilized by adsorption at 120rpm for 2h, then filtered, washed 5 times with phosphate buffer solution at pH 5.6 at 4°C, and then added 50ml of 0.04% glutaraldehyde solution and cross-linked at 120rpm for 2h under a constant temperature shaker at 30°C. Afterwards, it was filtered and washed 5 times with buffer solution of pH 5.6 at 4°C to obtain immobilized polyphenol oxidase. The unit enzyme activity of the obtained immobilized polyphenol oxidase is 0.222U / g*min, and the immobilization efficiency is 98.75%.

Embodiment 2

[0016] Weigh 10 g of unactivated ion exchange resin carrier D152 (fine), add 10 mg of pure polyphenol oxidase after activation (polyphenol oxidase is dissolved in 50 ml of phosphate buffer solution at 4 °C pH 5.6), and keep at a constant temperature of 30 °C Under the shaking table, adsorb and immobilize at 80rpm for 2h, then filter, wash with phosphate buffer solution of pH 5.6 at 4°C for 5 times, after that, add 50ml of 0.04% glutaraldehyde solution at 30°C on a constant temperature shaker, at 120rpm After cross-linking for 2 hours, it was filtered and washed five times with pH 5.6 buffer at 4°C to obtain immobilized polyphenol oxidase. The unit enzyme activity of the obtained immobilized polyphenol oxidase was 0.348 U / g*min, and the immobilization efficiency was 96.08%.

Embodiment 3

[0018] Weigh 10g of the activated ion-exchange resin carrier JK110, add 50mg of relatively pure polyphenol oxidase (polyphenol oxidase is dissolved in 50ml of 4°C pH 5.6 buffer), and adsorb at 180rpm under a constant temperature shaker at 30°C After immobilization for 2 hours, add 50ml of 0.04% glutaraldehyde solution and cross-link at 120rpm for 2 hours under a constant temperature shaker at 30°C, then filter and wash 5 times with phosphate buffer solution of pH 5.6 at 4°C to obtain immobilization Polyphenol oxidase. The unit enzyme activity of the obtained immobilized polyphenol oxidase was 0.383 U / g*min, and the immobilization efficiency was 95.37%.

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Abstract

The invention discloses a method for immobilizing polyphenol oxidase by taking duolite as a carrier. The current carries for immobilizing polyphenol oxidase are mainly sodium alginate and chitosan, can realize that enzyme molecules are insoluble in water and achieve the immobilized effect of the enzyme molecules, but increase a steric clash for binding action of the enzyme molecules and a substrate and reduce the catalytic efficiency of enzyme. The invention is characterized in that activation is carried out on the duolite by employing a sequence of acid washing- alkali wash-acid washing; then a zymoprotein solution is added to carry out absorption immobilization in a constant-temperature shaker; then a cross linker is added to carry out crosslinking; then the mixture is filtered and washed by a buffer liquid to obtain micro-spherical grains, which namely are the immobilized polyphenol oxidase with high enzyme activity. The invention reduces the steric clash for the action of the enzyme molecules and the substrate, and has high catalytic efficiency of enzyme.

Description

technical field [0001] The invention relates to a method for immobilizing polyphenol oxidase with an ion exchange resin as a carrier. Background technique [0002] Polyphenol oxidase is a kind of oxidoreductase, which is composed of copper ion prosthetic group and main enzyme. The copper ion prosthetic group cannot be dialyzed. Because polyphenol oxidase is closely related to food processing and vegetable preservation, storage and transportation, polyphenol oxidase has always been a research hotspot, especially in its biochemical and physiological properties. Polyphenol oxidase can selectively catalyze the hydroxylation of phenols and oxidize them into quinones under aerobic conditions. This feature makes polyphenol oxidase widely used in industries such as industry, food and environmental protection. Phenol oxidase is used to catalyze the oxidation of catechins to form theaflavins and unique flavor substances in tea beverages, which are used in tobacco to form unique quali...

Claims

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Application Information

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IPC IPC(8): C12N11/06
Inventor 江和源袁新跃张建勇刘晓辉
Owner TEA RES INST CHINESE ACAD OF AGRI SCI
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