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Method for rapidly extracting tyrosinase from potato

A tyrosinase and potato technology, applied in the direction of oxidoreductase and the like, can solve the problems of large experimental error, high cost, cumbersome process, etc., and achieve the effects of maintaining tyrosinase activity, fast operation and convenient source.

Inactive Publication Date: 2009-05-20
DONGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is costly, cumbersome, time-consuming, and not reproducible, which can easily lead to the inactivation of TYR and large experimental errors.

Method used

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  • Method for rapidly extracting tyrosinase from potato
  • Method for rapidly extracting tyrosinase from potato
  • Method for rapidly extracting tyrosinase from potato

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] 1. Extraction of potato TYR crude enzyme:

[0027] (1) Peel and chop fresh potatoes, put them into a pulverizer and perform high-speed homogenization with 0.2 mol / L sodium acetate buffer solution (pH5.6) for 2 minutes, and filter.

[0028] (2) The above-mentioned filtrate was centrifuged at 8000 rpm for 20 min at 4°C, and the supernatant was taken, and 65% saturated ammonium sulfate solution was added to the supernatant for precipitation, and left overnight at 4°C.

[0029] (3) Centrifuge (10000r / min) the above precipitated solution for 20min, retain the precipitate, and dissolve the precipitate with a small amount of 0.2mol / L sodium acetate buffer.

[0030] (4) Use 0.02mol / L Tris-HCl to fully dialyze the above solution until there is no NH 4 + and SO 4 2- , centrifuged to remove a small amount of precipitate, available potato TYR crude enzyme solution.

[0031] 2. FPLC purification of potato TYR:

[0032] (1) Molecular sieve gel chromatography. The above crude e...

Embodiment 2

[0038] 1. Extraction of potato TYR crude enzyme:

[0039] (1) Peel and chop fresh potatoes, put them into a pulverizer and perform high-speed homogenization with 0.2 mol / L sodium acetate buffer solution (pH5.6) for 2 minutes, and filter.

[0040] (2) The above-mentioned filtrate was centrifuged at 8000 rpm for 20 min at 4°C, and the supernatant was taken, and 65% saturated ammonium sulfate solution was added to the supernatant for precipitation, and left overnight at 4°C.

[0041] (3) Centrifuge (10000r / min) the above precipitated solution for 20min, retain the precipitate, and dissolve the precipitate with a small amount of 0.2mol / L sodium acetate buffer.

[0042] (4) Use 0.02mol / L Tris-HCl to fully dialyze the above solution until there is no NH 4 + and SO 4 2- , centrifuged to remove a small amount of precipitate, available potato TYR crude enzyme solution.

[0043] 2. FPLC purification of potato TYR:

[0044]Molecular sieve gel chromatography. The above crude enzyme...

Embodiment 3

[0047] 1. Extraction of potato TYR crude enzyme:

[0048] (1) Peel and chop fresh potatoes, put them into a pulverizer and perform high-speed homogenization with 0.2 mol / L sodium acetate buffer solution (pH5.6) for 2 minutes, and filter.

[0049] (2) The above-mentioned filtrate was centrifuged at 8000 rpm for 20 min at 4°C, and the supernatant was taken, and 65% saturated ammonium sulfate solution was added to the supernatant for precipitation, and left overnight at 4°C.

[0050] (3) Centrifuge (10000r / min) the above precipitated solution for 20min, retain the precipitate, and dissolve the precipitate with a small amount of 0.2mol / L sodium acetate buffer.

[0051] (4) Use 0.02mol / L Tris-HCl to fully dialyze the above solution until there is no NH 4 + and SO 4 2- , centrifuged to remove a small amount of precipitate, available potato TYR crude enzyme solution.

[0052] 2. FPLC purification of potato TYR:

[0053] Ion-exchange chromatography: the above crude enzyme soluti...

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Abstract

The invention relates to a method for quickly purifying tyrosinase (TYR) from potatoes and comprises the following steps: (1) the potatoes are smashed into homogenate and salted out, precipitate is dissolved and dialyzed so as to extract a raw TYR enzyme; (2) a FPLC gel molecular sieve or anion exchanging chromatography are used for purifying the TYR; and (3) polyacrylamide gel electrophoresis and enzymatic activity measurement with an ultraviolet spectrophotometer are used for representing the TYR. The method is quick, simple and convenient, and has low cost and higher purified TYR activity.

Description

technical field [0001] The invention belongs to a method for purifying tyrosinase in plants, in particular to a method for quickly purifying tyrosinase from potatoes. Background technique [0002] Tyrosinase (TYR) is a copper-containing polyphenol oxidase widely present in animal, plant and microbial cells. In mammals, it can be used as a dual-function biocatalyst with oxygenase and deoxidase Hydrogenase has two activities, which can not only catalyze the selective hydroxylation of monophenolic compounds to generate catechol, but also oxidize the dehydrogenation of catechol to generate o-benzoquinone, and in microorganisms, it can microdegrade phenolic substances , so it has a wide application in the fields of medicine, environment, food, fine chemical industry and so on. [0003] However, the content of tyrosinase in the organism is low, the price is high, and the enzyme activity is also limited by conditions such as temperature and pH. Therefore, the source of enzymes ha...

Claims

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Application Information

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IPC IPC(8): C12N9/04
Inventor 朱利民柴艳倩董唯静李树白张玉高周立明
Owner DONGHUA UNIV
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