The expression, purification and characterization of zebrafish monoamine oxidase (zMAO) using the methylotropic yeast Pichia pastoris expression system is described. A 1 L fermentation culture of Pichia postoris containing the gene encoding zMAO under control of the methanol oxidase promotor expresses similar to 200 mg of zMAO exhibiting 300 U of total activity. The enzyme is found in the mitochondrial fraction of the expression host and is purified in a 30% yield as a homogenous species with a M-r of similar to 60,000 on SDS-PAGE and a mass of 58,525 +/- 40 Da from MALDI-TOF measurements. The zMAO preparation contains one mole of covalent flavin cofactor per mole of enzyme and exhibits >80% functionality. The covalent flavin exhibits fluorescence and EPR spectral properties consistent with known properties of 8 alpha-S-cysteinylFAD. Chemical degradation of the flavin peptide results in the liberation of FAD. zMAO exhibits no immuno-chemical cross-reactivity with polyclonal anti-sera raised against human MAO A. The enzyme preparation exhibits reasonable thermostability up to a temperature of 30 degrees C. Benzylamine is oxidized with a k(cat) value of 4.7 +/- 0.1 min(-1) (K-m = 82 +/- 9 mu M) and the enzyme oxidizes phenylethylamine with a k(cat) value of 204 min(-1) (K-m = 86 +/- 13 mu M). The K-m (O-2) values determined for zMAO using either benzylamine or phenylethylamine as substrates ranges from 108(+/-5) to 140(+/-21) mu M. The functional behavior of this teleost MAO relative to human MAO A and MAO B is discussed. (C) 2010 Elsevier Inc. All rights reserved.