L-amino acid oxidase (L-AAO) from Rhodococcus opacus is a highly enantioselective enzyme with a broad substrate specificity that catalyses the oxidation of L-amino acids to keto acids. The lao-gene (AY053450) from R. opacus was cloned into different Escherichia coli and Streptomyces lividans expression vectors. Expression in E. coli resulted in the accumulation of insoluble protein, but S. lividans was a suitable host for the heterologous production Of L-AAO. When using the thiostrepton-inducible vector pIlaao, a specific activity of 0.18 U mg(-1) was obtained in the crude extract of S. lividans 1326. For the vector pUlaao, which contains the constitutive ermEp* promoter, a specific activity of 0.05 U mg(-1) was reached. Both the wild type and the recombinant L-AAO were purified to homogeneity. The expression systems described here now allow the structural and biochemical analysis of the L-AAO using genetic engineering methods. (C) 2002 Elsevier Science (USA). All rights reserved.