Biochemical properties of polyphenoloxidase from the cephalothorax of Pacific white shrimp (Litopenaeus vannamei)

Melanosis is a major problem associated with the quality loss of Pacific white shrimp. This is mediated by polyphenoloxidase (PPO), in which biochemical and molecular properties can be varied with species, molting period, etc. The better understanding of PPO characteristics should pave a way for lowering melanosis, thereby extending the shelf life of Pacific white shrimp during handling and storage. PPO was extracted from the cephalothorax of Pacific white shrimp, partially purified by ammonium sulphate precipitation (0% to 40% saturation) and diethylaminoethyl-Sephacel anion exchange chromatography, and characterised. Partially purified PPO (83.8-fold purity) showed the maximal activity using L-β-(3,4-dihydroxylphenyl)alanine (L-DOPA) as a substrate at pH 6 and at 55°C. PPO was stable over a pH range of 5 to 10 but unstable at a temperature greater than 60°C. Based on the activity staining with L-DOPA, the apparent molecular weight of PPO was 210 kDa. The Michaelis constant (Km) of PPO for the oxidation of L-DOPA was 2.43 mM. Trypsin, copper acetate and sodium dodecyl sulphate were unable to activate PPO, suggesting that the enzyme was in the active form. Cysteine, ethylenediaminetetraacetic acid and p-aminobenzoic acid showed PPO inhibitory activity in a dose-dependent manner. At the same concentration used (1 and 10 mM), cysteine exhibited a higher inhibitory effect towards PPO. Active PPO with higher stability could be responsible for the rapid melanosis formation in Pacific white shrimp, especially at the cephalothorax portion during postmortem storage. The use of an effective PPO inhibitor, especially cysteine, in combination with chilled storage could prevent melanosis in shrimp.