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
0 Cites 3 Cited by

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Problems solved by technology

However, this method is costly, cumbersome, time-consuming, and not reproducible...
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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.

Application Domain

Oxidoreductases

Technology Topic

ChemistryMolecular sieve +7

Image

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

Examples

  • Experimental program(3)

Example Embodiment

[0025] Example 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 (pH 5.6) for 2 min, and filter.
[0028] (2) Centrifuge the above filtrate at 8000 rpm for 20 min at 4°C, take the supernatant, add 65% saturated ammonium sulfate solution to the supernatant for precipitation, and store at 4°C overnight.
[0029] (3) Centrifuge the above-mentioned precipitation solution (10000 r/min) for 20 min, take out the precipitate, and dissolve the precipitate with a small amount of 0.2 mol/L sodium acetate buffer.
[0030] (4) Fully dialyze the above solution with 0.02mol/L Tris-HCl until there is no NH 4 + and SO 4 2- , and centrifuged to remove a small amount of precipitate to obtain potato TYR crude enzyme solution.
[0031] 2. FPLC purification of potato TYR:
[0032] (1) Molecular sieve gel chromatography. The above crude enzyme solution was chromatographed in the FPLC system under the following conditions: Molecular sieve gel column: SephodexG75; Eluent: 0.02M Tris-HCl buffer solution (pH7.0); Equilibrium: 2 column beds; Elution: 1 column bed; flow rate: 0.5 ml/min. peak spectrum figure 1 , the second peak is the vitality peak.
[0033] (2) Ion exchange chromatography. The above crude enzyme solution was separated by ion exchange column in FPLC system, the conditions were: ion exchange column: HiTrip Q FF 1ml; eluent A: 0.02M Tris-HCl buffer solution (pH7.0); eluent B: 0.02M Tris-HCl buffer solution (pH7.0) + 1M NaCl; Equilibration: 20 column beds; Washing: 5 column beds; Elution gradient: eluent B in 5 column beds for 3 %, then 10% in 5 beds, then 20% in 5 beds, then 40% in 5 beds; flow rate: 1 ml/min. Anion exchange chromatography yielded three peaks, figure 2 , the second peak is the vitality peak.
[0034] Polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC enzyme activity characterization:
[0035] (1) SDS-PAGE gel electrophoresis: The collected peaks obtained by anion chromatography and molecular sieve gel chromatography were characterized by electrophoresis. Among them, molecular sieve gel chromatography obtained three bands after electrophoresis; anion exchange chromatography obtained one band. The conditions are: separating gel: 15%; stacking gel: 5%; current: 10 mA (stacking gel), 20 mA (separating gel); electrophoresis time: 60 min; electrophoresis tank: Ready Gel Precast Gel System (Bio-Rad). Electrophoresis results showed that the peak obtained by anion exchange chromatography was a pure enzyme, image 3.
[0036] (2) Determination of enzyme activity by ultraviolet spectrophotometry: the amount of TYR catalyzed by unit protein to catalyze the oxidation of L-DOPA is regarded as one unit of enzyme activity. The oxide of L-DOPA is red and can be determined by UV spectrophotometry.

Example Embodiment

[0037] Example 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 (pH 5.6) for 2 min, and filter.
[0040] (2) Centrifuge the above filtrate at 8000 rpm for 20 min at 4°C, take the supernatant, add 65% saturated ammonium sulfate solution to the supernatant for precipitation, and store at 4°C overnight.
[0041] (3) Centrifuge the above-mentioned precipitation solution (10000 r/min) for 20 min, take out the precipitate, and dissolve the precipitate with a small amount of 0.2 mol/L sodium acetate buffer.
[0042] (4) Fully dialyze the above solution with 0.02mol/L Tris-HCl until there is no NH 4 + and SO 4 2- , and centrifuged to remove a small amount of precipitate to obtain potato TYR crude enzyme solution.
[0043] 2. FPLC purification of potato TYR:
[0044]Molecular sieve gel chromatography. The above crude enzyme solution was chromatographed in the FPLC system under the following conditions: Molecular sieve gel column: SephodexG75; Eluent: 0.02M Tris-HCl buffer solution (pH7.0); Equilibrium: 2 column beds; Elution: 1 column bed; flow rate: 0.5 ml/min. Obtain the peak spectrum.
[0045] The enzymatic activity of TYR was characterized by polyacrylamide gel electrophoresis (SDS-PAGE) and UV spectrophotometry.

Example Embodiment

[0046] Example 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 (pH 5.6) for 2 min, and filter.
[0049] (2) Centrifuge the above filtrate at 8000 rpm for 20 min at 4°C, take the supernatant, add 65% saturated ammonium sulfate solution to the supernatant for precipitation, and store at 4°C overnight.
[0050] (3) Centrifuge the above-mentioned precipitation solution (10000 r/min) for 20 min, take out the precipitate, and dissolve the precipitate with a small amount of 0.2 mol/L sodium acetate buffer.
[0051] (4) Fully dialyze the above solution with 0.02mol/L Tris-HCl until there is no NH 4 + and SO 4 2- , and centrifuged to remove a small amount of precipitate to obtain potato TYR crude enzyme solution.
[0052] 2. FPLC purification of potato TYR:
[0053] Ion-exchange chromatography: The above crude enzyme solution was separated by an ion-exchange column in the FPLC system under the following conditions: ion-exchange column: HiTrip Q FF1ml; eluent A: 0.02M Tris-HCl buffer solution (pH7.0) ; Eluent B: 0.02M Tris-HCl buffer solution (pH 7.0) + 1M NaCl; Equilibration: 20 column beds; Washing: 5 column beds; Elution gradient: Eluent B in 5 columns 3% in bed, then 10% in 5 beds, then 20% in 5 beds, then 40% in 5 beds; flow rate: 1 ml/min. Anion exchange chromatography yielded three peaks.
[0054] The enzymatic activity of TYR was characterized by polyacrylamide gel electrophoresis (SDS-PAGE) and UV spectrophotometry.

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