Isolation, Purification And Properties Of Tyrosinase From Potato

Tyrosinase(EC.1 .14.18.1) was isolated and purified from potato and its biochemical properties was studied. Potato tyrosinase was purified to electrophoretic homogeneity by homogenate,salting-out, chromatography with DEAE-Sepharose F.F and Sephadex G-150. The purification multiple was 50 and the specific activity was 170.03U/mg. The molecular weight is 42KD with SDS-PAGE. The enzyme kinetic properties against L-DOPA were studied. The Michaelis constant (Km) was 3.70 mmol/L, the optimum pH value of the enzyme was 7.5 and the optimum temperature was 30℃.The enzyme was stable within pH 5.60-8.00 and below 40℃ . The relative enzyme activity only decreased 4% when temperature increased from 20℃ to 40℃, while rapidly decreased from 84% to 5% when temperature increased from 50℃ to 70℃ .Tyrosinase from potato exhibited monophenols and diphenols with different substrates(tyrosine, catechol, L-DOPA and chlorogenic acid). The activity of monophenols is lower than diphenols and unstable during purification. The enzyme showed high substrate specificity towards catechol and was very sensitive to metal ions, Its activity is strongly enhanced byZn2+, Fe2+and inhibited by Hg2+, K+, Ca2+, Mg2+, while Na+ have no distinct inhibition. Ca2+, K+ were selected for determining types of inhibition and the results showed that KCl was a competitive inhibitor with inhibition constant of 10mmol/L, CaCl2 was a noncompetitive inhibitor, with inhibition constant of 11.68 mmol/L.Sulfiting compounds, ascorbic acid and kojic acid exhibited strong inhibition on potato tyrosinase.Theorder of inhibition ability is DTT>β -Mercaptoethanol >L-glutathione>cysteine>ascorbic acid> kojic acid>thiourea. The inhibition mechanism by kojic acid was investigated. The preincubation time and temperature did not significantly affect tyrosinase inhbition by kojic acid Kojic acid can inhibited monooxygenase by increasing the lag period of catalysing for L-Tyrosine. Kojic acid exhibited a competitive inhibition against L-DOPA with inhibition constant of 0.17 mmol/L.Sectrophotometric methods demonstrated that kojic acid was capable of reducing quinones to diphenols to prevent the final melanin forming.