A putative carotenoid oxygenase from Novosphingobium aromaticivorans was purified with a specific activity of 0.8 U/mg by His-Trap affinity chromatography. The native enzyme was estimated to be a 52 kDa monomer. Enzyme activity for beta-apo-8'-carotenal was maximal at pH 8.0 and 45 A degrees C, with a half life of 15.3 h, K (m) of 21 mu M, and k (cat) of 25 l/min. The enzyme exhibited cleavage activity only for carotenoids containing one beta-ionone ring and its catalytic efficiency (k (cat)/K (m)) followed the order beta-apo-8'-carotenal > beta-apo-4'-carotenal > gamma-carotene. The enzyme converted these carotenoids to beta-apo-13-carotenones by cleaving their C-13-C-14 double bonds. The oxygen atom of beta-apo-13-carotenone originated not from water but from molecular oxygen. Thus, the enzyme was an apo-carotenoid 13,14-dioxygenase.