TRITON X-114-AIDED PURIFICATION OF LATENT TYROSINASE

Mushroom tyrosinase was partially purified using an aqueous two-phase system with Triton X-114. The purification achieved was 5.5-fold from a crude extract of mushroom pileus, with a high recovery of 84%. The p henols were reduced to 8% of the original content, avoiding pre- and p ost-purification tanning of the enzyme. The enzyme obtained was latent and was activated 3-fold by trypsin, 2.7-fold by changes in the pH an d to different extents by cationic and anionic detergents, the latter being the more effective. There was also a synergistic effect between trypsin and detergent, at low detergent concentrations. When kinetical ly characterized, latent enzyme showed both monophenolase and diphenol ase activities, the latter activity displaying an unexpected lag perio d before reaching the steady-state rate. This behaviour is characteris tic of a hysteretic enzyme, acid has not been previously described for this enzyme. In addition, inhibition studies with substrate analogues were carried out, tropolone being found to be the most effective inhi bitor.