Biochemical And Enzymatic Characterization Of The Phenoloxidase From The Sea Urchin Strongylocentrotus Nudus

In this study, phenoloxidases(POs) were purified from the coelomic fluid supernatant of sea urchin Strongylocentrotus nudus with the method of linear-gradient native-PAGE combined with catechol staining. The results showed that: three POs were detected in the coelomic fluid supernatant of S. nudus and exhibited brown, yellow and purple respectively after reaction with catechol in polyacrylamide gel and named as SnPO1, SnPO2 and SnPO3 according to their molecular mass from high to low.The SnPOs were characterized for substrate specificity and the effects of temperature, pH, divalent metal ions and inhibitors on PO activities. They showed oxidative activities to L-3,4-dihydroxyphenylalanine.(L-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine, which illustrated the three proteins had laccase-like PO activities. The optimum temperature for the activities of SnPO1, SnPO2 and SnPO3 was 75 ℃, 70 ℃, 40 ℃, and the optimum pH was 7.0, 9.0, 8.0, respectively. The SnPOs were notably activated after being incubated in boiled water for 60 min, suggesting that the three proteins are thermophilic. The activity of SnPO1 was greatly enhanced by Cu2+, Mn2+ and Fe2+ and inhibited by Pb2+, Cd2+, EDTA, DETC, sodium sulfite and ascorbic acid, but SnPO2 and SnPO3 were not obviously affected by Pb2+ and Cd2+, suggesting the three proteins are copper-containing, and the catalytic properties of SnPO1 might be different from those of SnPO2 and SnPO3. Taken together, SnPO1, SnPO2 and SnPO3 might play different roles in the immune and physiological processes of S. nudus.The antibacterial characteristics of S. nudus PO oxidation products were studied using the method of bacterial growth curve determination. The results showed that: using dopamine as the substrate, the oxidation products of SnPO1 showed antibacterial activities to Vibrio splendidus, Vibrio harveyi and Staphylococcus aureus, the oxidation products of SnPO2 showed antibacterial activities to V. harveyi, S. aureus and Nocardiopsis sp., while the oxidation products of SnPO3 only showed antibacterial activities to V. harveyi and Nocardiopsis sp.; using L-3,4-dihydroxyphenylalanine as the substrate, only the oxidation products of SnPO2 showed antibacterial activity to Nocardiopsis sp.; the oxidation products of S. nudus POs had no obvious effects on the growth of Pseudoalteromonas nigrifaciens, Shewanella baltica and Micrococcus lysodeikticus. The results described above suggested that in S. nudus, PO oxidation products had limited antibacterial spectrum, the antibacterial activities in dopamine-derived oxidation products were stronger than those in L-3,4-dihydroxyphenylalanine-derived oxidation products, and different PO isoforms had different antibacterial spectrum and characteristics.