Discovery of serum L-amino acid oxidase in the rockfish Sebastes schlegeli: Isolation and biochemical characterization

Abstract

Fish have a complex innate defense mechanism against microbial invasion. In particular, epidermal mucus and serum in fish play important roles in innate immunity and contain a variety of bioactive substances such as complements, lectins and lysozymes, involved in host defense. Recently, L-amino acid oxidases (LAOs) with antibacterial activity were isolated from the skin and/or gill mucous secretions of rockfish, great sculpin and flounder, and were identified to be a novel type of antibacterial protein in the integument of fish. In the present study, we found LAO activity in the serum of rockfish Sebastes schlegeli. The LAO was isolated from the serum by sequential column chromatography of Con-A lectin affinity chromatography, anion exchange HPLC, hydroxyapatite HPLC and gel filtration HPLC, and characterized. The LAO (a molecular mass of 160 kDa) comprised subunits with a molecular mass of 53 kDa and showed strict substrate specificity, catalyzing only L-lysine with Km 0.37 mM and kcat 57.1s(-1). The serum LAO exhibited a broad antibacterial activity against Gram positive and negative bacteria, most potently against Aeromonas hydrophila and Aeromonas salmonicida with a minimum inhibitory concentration of 0.078 μg/mL. This is the first report of LAO in the serum of fish and its involvement in innate immunity in the rockfish body.