The membrane-bound [NiFe]-hydrogenase from Hydrogenooibrio marinus (HmMBH) was purified homogeneously under anaerobic conditions. Its molecular weight was estimated as 110 kDa, consisting of a heterodimeric structure of 66 kDa and 37 kDa subunits. The purified enzyme exhibited high activity in a wide temperature range: 185 U/mg at 30 degrees C and 615 U/mg at 85 degrees C (the optimum temperature). The Km and k(cat)/K(m) values for H(2) were, respectively, 12 mu M and 8.58 x 10(7) M(-1) s(-1). The optimum reaction pH was 7.8, but its stability was particularly high at pH 4.0-7.0. Results show that HmMBH was remarkably thermostable and oxygen-resistant: its half-life was 75 h at 80 degrees C under H(2), and more than 72 h at 4 degrees C under air. The air-oxidized HmMBH for 72 h showed only weak EPR signals of Ni-B, suggesting a structural feature in which the active center is not easily oxidized. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.