Purification of Hemocyanin‐Derived Phenoloxidase and Comparison of Its Properties With Polyphenoloxidase in the Shrimp Litopenaeus vannamei

Postmortem blackening of shrimp caused by phenoloxidase activity has resulted in enormous economic losses for aquaculture. Apart from polyphenoloxidase, the hemocyanin‐derived phenoloxidase also proved its ability in possessing phenoloxidase activity. In this study, the nature of hemocyanin‐derived phenoloxidase in Litopenaeus vannamei was explored and analyzed. The results highlighted the purification of a hemocyanin‐derived phenoloxidase with a molecular weight of 80 kDa, similar to the molecular weight of expressed polyphenoloxidase. The optimum temperature and pH of this enzyme were 35°C and pH 5.0, in general agreement with natural polyphenoloxidase and expressed polyphenoloxidase. The protein sequence identity and similarity between hemocyanin‐derived phenoloxidase and phenoloxidase were 33.8% and 48.3%, respectively. However, hemocyanin‐derived phenoloxidase cannot bind to the polyclonal antibody specific to polyphenoloxidase and expressed polyphenoloxidase. The specific enzyme activity of hemocyanin‐derived phenoloxidase was 1/4.83 of polyphenoloxidase. This study provided a theoretical basis to further analyses of hemocyanin‐derived phenoloxidase and phenoloxidase contributions to shrimp melanosis, respectively.