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Method for quickly extracting and separating polyphenol oxidase from plant leaves

A technology of polyphenol oxidase and plant leaves, which is applied in the direction of biochemical equipment and methods, enzymes, hydrolytic enzymes, etc., can solve the problems of high equipment requirements, large loss of enzyme activity, long purification time, etc., and achieve simple process and high enzyme activity. High, high-volume effects

Active Publication Date: 2014-03-05
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Polyphenol oxidase can be purified by ammonium sulfate fractional precipitation, sephadex G-75 and other purification methods. The purification process of polyphenol oxidase is complicated, the equipment requirements are high, the purification time is long, the impurities are not easy to remove, and the enzyme activity is lost larger

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Wash the fresh mulberry leaves for later use, weigh 100g of mulberry leaves, add 400mL of 0.1mol / L phosphate buffer solution with a pH of 6.0 (each 100ml of phosphate buffer solution contains 1.368g of sodium dihydrogen phosphate and 0.44g of disodium hydrogen phosphate), and use Grind it into a paste with a tissue masher, place it in a 4°C refrigerator for low-temperature extraction for 6 hours, and then filter it with 4 layers of gauze, discard the filter residue and collect the filtrate. Refrigerated and centrifuged at 12000 r / min for 30 min at 4°C, and the centrifuged liquid was collected as the crude polyphenol oxidase enzyme liquid.

[0021] Use an ultrafiltration centrifuge tube with a membrane diameter of 30ku to centrifuge the crude polyphenol oxidase enzyme solution at 6000r / min for 20min to concentrate the enzyme solution, and use the CTAB-isooctane reverse micelles system to extract the polyphenol oxidase enzyme solution to obtain 50mmol / LCTAB-isooctane reve...

Embodiment 2

[0024] Wash the fresh tea tree leaves for later use, weigh 100g of tea tree leaves, add 400mL of 0.1mol / L phosphate buffer solution with a pH of 6.0 (each 100ml of phosphate buffer solution contains 1.368g of sodium dihydrogen phosphate and 0.44g of disodium hydrogen phosphate), and use Grind it into a paste with a tissue masher, place it in a refrigerator at 4°C for 6 hours, and then filter it with 4 layers of gauze, and discard the filter residue to collect the filtrate. Refrigerated and centrifuged at 12000 r / min for 30 minutes at 4°C, and the centrifuged liquid was collected as the crude polyphenol oxidase enzyme liquid.

[0025] Use the organic solvent acetone to carry out protein precipitation on the crude enzyme solution, add -20°C pre-cooled acetone to the crude enzyme solution, the volume ratio of the crude enzyme solution to acetone is 1:3, put it in a refrigerator at 4°C for 3 hours, and centrifuge at 6000r / min Finally, dissolve the precipitate with 50ml of pH6.0 ph...

Embodiment 3

[0028] Wash the fresh tea tree leaves for later use, weigh 100g of tea tree leaves, add 400mL of 0.1mol / L phosphate buffer solution with a pH of 6.0 (each 100ml of phosphate buffer solution contains 1.368g of sodium dihydrogen phosphate and 0.44g of disodium hydrogen phosphate), and use Grind it into a paste with a tissue masher, place it in a refrigerator at 4°C for 6 hours, and then filter it with 4 layers of gauze, and discard the filter residue to collect the filtrate. Refrigerated and centrifuged at 12000 r / min for 30 minutes at 4°C, and the centrifuged liquid was collected as the crude polyphenol oxidase enzyme liquid.

[0029] Use the organic solvent acetone to carry out protein precipitation on the crude enzyme solution, add -20°C pre-cooled acetone to the crude enzyme solution, the volume ratio of the crude enzyme solution to acetone is 1:3, put it in a refrigerator at 4°C for 3 hours, and centrifuge at 6000r / min Finally, dissolve the precipitate with 50ml of pH6.0 ph...

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PUM

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Abstract

The invention relates to a method for quickly extracting and separating polyphenol oxidase from plant leaves, and belongs to the field of bioseparation. The method for quickly extracting and separating the polyphenol oxidase from the plant leaves comprises the following steps: cleaning plant leaves; adding a buffer solution, mashing, leaching and carrying out suction filtering; collecting a filtrate, centrifugally collecting crude enzyme; extracting by using reverse micelles after ultra-filtration of the crude enzyme; and concentrating, freezing and drying after extraction is ended, so as to obtain polyphenol oxidase powder. The polyphenol oxidase crude enzyme is extracted by using the reverse micelles, and the obtained polyphenol oxidase is high in purity, fewer in impurities, and high in enzyme activity, and the processing method is simple in technology, large in handling capacity and applicable to industrial production.

Description

technical field [0001] The invention relates to a method for quickly extracting and separating plant leaf polyphenol oxidase, which belongs to the field of biological separation. Background technique [0002] Polyphenol oxidase (polyphenol oxidase, PPO) is a kind of metalloprotease widely distributed in nature. It is ubiquitous in the plastids of plants, fungi, and insects, and polyphenols can even be detected on rotting plant residues in soil. Oxidase activity. In plant tissues (such as apples, lychees, spinach, potatoes, beans, tea leaves, mulberry leaves, tobacco, etc.), polyphenol oxidase is combined with endothelial membranes, and is inactive in its natural state. After pulp or injury, polyphenol oxidase is activated, thus showing activity. In fruit and vegetable cell tissues, the location of polyphenol oxidase varies with the type, variety and maturity of raw materials. Most of the polyphenol oxidase activity in green leaves exists in the chloroplast; almost all subc...

Claims

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Application Information

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IPC IPC(8): C12N9/50
CPCC12N9/0059C12N9/0061C12N9/0071
Inventor 杨洋严东赵芬芬叶琴马万良胡振兴
Owner GUANGXI UNIV
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