Investigations on tyrosinase activity in melanin-free ink from Sepia officinalis: potential for food proteins cross-linking

Abstract

Protein modification via enzymatic cross-linking is an attractive way for altering food structure so as to create products with increased quality and nutritional value. In this study, enzymatic cross-linking of β-casein was performed by tyrosinase activity, from melanin-free ink from Sepia officinalis, which was monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) techniques. The melanin-free ink contains a strong tyrosinase activity with pH 7 and 58 °C as optima of pH and temperature, respectively. Such activity is stimulated by ferrous ions and strongly inhibited by Mn2+, EDTA, H2O2, arbutin, and p-coumaric acid. We also show that 2 Mercapto-ethanol (14 mM) quickly and completely inactivated sepia tyrosinase. The melanin-free ink exhibits a major protein on SDS–PAGE with an N-terminal sequence matching perfectly with an internal sequence of the sepia peroxidase. The zymogram confirmed the inactive state of this truncated protein and the presence of an active tyrosinase enzyme. Interestingly, this activity was able to cross-link the β-casein protein. The tyrosinase implication in reticulation was demonstrated by the addition of its inhibitors, with 2-mercaptoethanol being the most effective, followed by arbutin, p-coumaric acid, and hydrogen peroxide.