Method for rapidly extracting tyrosinase from potato

[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.

[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.

[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.