Molecular Characterization, Activation Mechanism, and Antimelanosis Implications of Polyphenol Oxidase in Litopenaeus vannamei.

Polyphenol oxidase (PPO) plays a central role in the quality deterioration of shrimp, particularly in melanosis development. However, the mechanisms triggering melanosis in Litopenaeus vannamei (L. vannamei) remain poorly understood. This study purified natural PPO from L. vannamei and investigated its molecular characteristics and activation mechanism. PPO exhibited phenoloxidase activity in the form of a 210 kDa heterotetramer. Under heat-denaturing conditions, it dissociated into two 62 kDa PPOα chains and two 45 kDa PPOβ chains. During cold storage, the onset of melanosis was accompanied by hepatopancreatic autolysis, the release of serine proteinases (SPs) from hepatopancreas elevated the activation of prophenoloxidase (proPPO) to the active form PPO. A 28 kDa SP derived from hepatopancreas was identified as one of the prophenoloxidase activating proteinases (PAPs), enhancing the activity of recombinant L. vannamei proPPO (rLv-PPO) through partial peptide cleavage. Serine proteinase inhibitor phenylmethanesulfonyl fluoride (PMSF) effectively delayed melanosis in shrimp, demonstrating the feasibility of retarding shrimp melanosis by blocking the SP-mediated activation pathway of proPPO. Our present study exhibited the structural characteristics of shrimp PPO and its activation mechanism, providing a theoretical foundation for developing antimelanosis agents in the future.