Fish have a complex self-defense mechanism against microbial invasion. Recently, L-lysine α-oxidases have been identified from a number of fish species as a novel type of antibacterial protein in the integument. These enzymes exhibit strict substrate specificity for L-lysine, but the underlying mechanisms and details of their catalytic properties remain unknown. In this study, a synthetic gene coding for Scomber japonicus L-lysine α-oxidase, originally termed AIP (for apoptosis-inducing protein), was expressed in Pichia pastoris, and the recombinant enzyme (rAIP) was purified and characterized. rAIP exhibited essentially the same substrate specificity as the native enzyme, catalyzing the oxidative deamination of L-lysine as an exclusive substrate. rAIP was N-glycosylated and remained active over a wide range of pH, with an optimal pH of 7.5. The enzyme was stable in the pH range from 4.5 to 10.0 and was thermally stable up to 60°C. A molecular modelling of rAIP and a comparative structure/sequence analysis with homologous enzymes indicate that Asp220 and Asp320 are the substrate-binding residues that are likely to confer exclusive substrate specificity for L-lysine on the fish enzymes.