Method for separating and extracting peroxidase in sweet potato peels

A technology of peroxidase and sweet potato peel, applied in biochemical equipment and methods, enzymes, enzymes, etc., can solve the problems of increased cost investment and restriction of raw material sources, and achieves reduction of activity decline, cheap raw material sources, and easy industrial scale-up. Effect

Inactive Publication Date: 2011-08-10
HANGZHOU NORMAL UNIVERSITY
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  • Abstract
  • Description
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Problems solved by technology

[0003] Horseradish peroxidase (Horseradish peroxidase, HR) is by far the most widely used enzyme-linked immunoassay for labeling enzymes, but horseradish needs to be planted specially as a raw material, and horseradish is a non-planting plant, so the source of raw materials is limited. Increased relative cost investment

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  • Method for separating and extracting peroxidase in sweet potato peels
  • Method for separating and extracting peroxidase in sweet potato peels

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Effect test

Embodiment 1

[0034] (1) Preparation and purification of sweet potato peroxidase

[0035] Take sweet potatoes, wash them, collect 500g of sweet potato skins, first add 0.5L of 10mM pH 8.0 phosphate buffer solution and mince, keep the temperature at 4°C during the mincer process, then add 2L of 10mM pH 8.0 phosphate buffer solution, stir well Then, put it in a refrigerator at 4°C and extract overnight. The filtrate was collected by filtration, the residue was extracted once more, and the filtrates were combined. Centrifuge the filtrate at 4°C and 5000rpm for 30min to remove starch and small tissue particles, collect the supernatant, add solid ammonium sulfate and PEG6000 while stirring, so that the mass percentage concentration of ammonium sulfate is 30%, and the mass percentage concentration of PEG6000 is 8%. , the addition was completed within about 1 hour, and transferred to a separatory funnel to stand still, keeping the temperature at 4°C. After standing and stratifying, the upper phas...

Embodiment 2

[0045] (1) Preparation and purification of sweet potato peroxidase

[0046] Take sweet potatoes, wash them, collect 500g of sweet potato skins, first add 0.5L of 10mM pH 8.0 phosphate buffer saline to grind, keep the temperature at 4°C during the grinding process, then add 2L of 10mM pH 8.0 phosphate buffer, stir well , placed in a refrigerator at 4°C, and leached overnight. The filtrate was collected by filtration, the residue was extracted twice, and the filtrate was combined. Centrifuge the filtrate at 4°C and 5000rpm for 30min to remove starch and small tissue particles, collect the supernatant, add solid ammonium sulfate and PEG6000 while stirring, so that the mass percentage concentration of ammonium sulfate is 30%, and the mass percentage concentration of PEG6000 is 8%. , the addition was completed within about 1 hour, and transferred to a separatory funnel to stand still, keeping the temperature at 4°C. After standing and stratifying, the upper phase solution was main...

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Abstract

The invention discloses a method for separating and extracting peroxidase in sweet potato peels, comprising the following steps of: (1) taking peels after sweet potatoes are cleaned, adding a phosphate buffer with pH value of 7.0-8.0 to homogenate and extract at 0-15 DEG C, and filtering after extracting to obtain a filter liquor; (2) adding two aqueous phase extractants including solid ammonium sulfate and PEG6000 (Polyethylene Glycol 6000) into the filter liquor at the same time stirring so that the mass percentage concentration of the ammonium sulfate is 20-30% and the mass percentage concentration of the PEG6000 is 6-8%, standing for delaminating at 0-15 DEG C after dissolution under sufficient stirring, and taking a lower layer of solution which is a crude enzyme solution; (3) directly carrying out hydrophobic chromatography on the crude enzyme solution obtained in the step (2) by a Phenyl Sepharose6 fast flow, carrying out gradient elution by using an ammonium sulfate solution of 0-1 mol / L after sample application, and lyophilizing after collecting an elution solution with enzyme activity; (4) carrying out gel chromatography and ion chromatography on lyophilized powder to obtain the peroxidase. The invention preferably solves the problem on material sources and can increase the efficiency and the enzyme activity.

Description

(1) Technical field [0001] The invention relates to a peroxidase preparation technology, in particular to a method for extracting peroxidase from sweet potatoes. (2) Background technology [0002] Peroxidase (Peroxidase, POD) is widely distributed in nature and abundant in resources, and it is a commonly used marker enzyme for clinical detection and diagnosis. Peroxidase and various antibody markers, combined with enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA) as a routine method, can be used for localization and quantitative detection of various diseases, and the operation is simple. [0003] Horseradish peroxidase (Horseradish peroxidase, HR) is by far the most widely used enzyme-linked immunoassay for labeling enzymes, but horseradish needs to be planted specially as a raw material, and horseradish is a non-planting plant, so the source of raw materials is limited. The relative cost of investment increases. (3) Contents of the invention ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/08
Inventor 李霞韩凤何睿陈灿玉焦艳华梁媛媛黄志坚谢恬
Owner HANGZHOU NORMAL UNIVERSITY
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