An oxidatively stable liquefying a-amylase (Amy204) was found in the culture of a Bacillus isolate, named strain JAMB-204, from the deep-sea at a depth of about 6000 m. The enzyme activity was maintained almost completely even after 1-h incubation with 1.0 M of H2O2. The molecular mass deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 55 kDa. The enzyme had a high isoelectric point of 8.6. The specific activity of purified Amy204 was very high at approximately 4200 units/mg of protein at 60 degrees C and pH 6.5 when soluble potato starch was used as the substrate. This enzyme efficiently hydrolyzed various carbohydrates to yield maltopentaose, maltotriose, and maltose as the major end products after completion of the reaction. The gene for Amy204 encodes 513 amino acids including a signal peptide of 29 amino acids. Amy204 exhibited amino acid homology to other known alpha-amylases: 80.4% identity with the enzyme from a Cytophaga sp., 77.8% identity with the enzyme from Bacillus licheniformis, and 77.7% identity with the enzyme from Bacillus amyloliquefaciens. Mutagenesis studies showed that the oxidative stability of Amy204 resulted from the amino acid at position 198 being a non-oxidizable amino acid of leucine, unlike the other liquefying alpha-amylases that have methionines at the equivalent positions. (c) 2006 Elsevier Inc. All rights reserved.