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| Biochemical and Enzymatic Characteristics of Phenoloxidase in Sea Cucumber Apostichopus japonicus |
| XIAO Yao, JIANG Jingwei, LI Shilei, DONG Ying, ZHOU Zunchun |
| Liaoning Ocean and Fisheries Science Research Institute, Dalian 116023, China |
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Abstract In order to understand the biochemical and enzymatic characteristics of phenoloxidase (PO) in the coelomocytes of sea cucumber Apostichopus japonicus, the coelomocytes of females and males were used as samples, and then using the method of linear-gradient native-PAGE combined with catechol staining, the four kinds of POs were purified and characterized, and the biochemical and enzymatic properties of different POs were determined by spectrophotometry. The optimum pH for the activity was 9.0 in AjPO4, 8.0 in AjPO5, 9.0 in AjPO6 and 8.0 in AjPO7. The activities of AjPO4, AjPO5 and AjPO6 in boiling water were stable, indicating that the heat resistance of AjPO4, AjPO5 and AjPO6 was better than that of AjPO7. The four POs catalyzed catechol, L-dopa, dopamine and hydroquinone, among which the optimal substrate was catechol, but failed to catalyze tyrosine, indicating that the four POs belonged to laccase-type phenoloxidase, and had the highest affinity to catechol.The four AjPOs were activated by Mn2+ and Fe2+, indicating that the function of PO system in sea cucumber was regulated by the divalent metal ions in vivo. In addition, the activity of the four POs was inhibited not only by citric acid, sodium sulfite and ascorbic acid, but also by sodium diethyl dithiocarbamate (DETC), a copper chelating agent, suggesting that all the four POs be copper-containing enzymes. The enzymology characteristics of AjPOs provide references for further understanding of the characteristics and functional mechanism of AjPO system.
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Received: 13 February 2020
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