Polyphenoloxidases from lobster (Homarus americanus) : extraction, purification and characterization

Polyphenoloxidases (EC 1.10.3.1) (PPO) were extracted from the skin layer between the muscle and exoskeleton, of lobster (Homarus americanus) by the successive steps of (NH$ sb4) sb2$SO$ sb4$ fractionation, ion-exchange chromatography and isoelectric focusing. The lobster 'PPO-fraction' recovered by the 40-80% (NH$ sb4) sb2$SO$ sb4$ fractionation step was found to exist in the inactive precursor form which required trypsin for activation and was inhibited by polyvinyl pyrrolidone (PVP). A comparative study of the lobster 'PPO-fraction' and the commercial tyrosinase from mushroom (EC 1.14.18.1) indicated that the two enzymes were similar with respect to substrate specificity, response to pH and selected inhibitors (cysteine, EDTA and PABA). However, both enzymes differed from one another in terms of their thermal and pH stabilities. Their catalytic efficiencies indicated that tyrosinase was better suited to catalyze oxidation of DOPA than lobster 'PPO-fraction'. Also, the activity of both enzymes were enhanced by copper.Further purification of the 'PPO-fraction' by ion-exchange chromatography and preparative isoelectric focusing with a Rotofor unit yielded three isozymes designated PPO 1, PPO 11 and PPO 111. These isozymes had pI values of 3.89, 4.26 and 4.54 and molecular weights of 32,180, 35,480 and 39,300 respectively. The isozymes were similar with respect to their pH and temperature activity profiles but differed from one another (especially PPO 1 versus PPOs 11 and 111) in terms of their pH and thermal stabilities. They also exhibited varying degrees of differences in their catalytic efficiencies. All three isozymes were inhibited by PABA, EDTA and cysteine, with PABA being a competitive inhibitor, and EDTA or cysteine a noncompetitive inhibitor. Lower concentrations of ascorbate and mercaptoethanol had no effect on the isozymes. The free amino acid analysis suggested that melanosis might originate from the skin of the lobster