Catalase from the facultatively psychrophilic bacterium Vibrio rumoiensis S-1 T , which was isolated from an environment exposed to H 2 O 2 and exhibited high catalase activity, was purified and characterized, and its localization in the cell was determined. Its molecular mass was 230 kDa, and the molecule consisted of four identical subunits. The enzyme, which was not apparently reduced by dithionite, showed a Soret peak at 406 nm in a resting state. The catalytic activity was 527,500 U ⅐ mg of protein ؊1 under standard reaction conditions at 40°C, 1.5 and 4.3 times faster, respectively, than those of the Micrococcus luteus and bovine catalases examined under the same reaction conditions, and showed a broad optimum pH range (pH 6 to 10). The catalase from strain S-1 T is located not only in the cytoplasmic space but also in the periplasmic space. There is little difference in the activation energy for the activity between strain S-1 T catalase and M. luteus and bovine liver catalases. The thermoinstability of the activity of the former catalase were significantly higher than those of the latter catalases. The thermoinstability suggests that the catalase from strain S-1 T should be categorized as a psychrophilic enzyme. Although the catalase from strain S-1 T is classified as a mammal type catalase, it exhibits the unique enzymatic properties of high intensity of enzymatic activity and thermoinstability. The results obtained suggest that these unique properties of the enzyme are in accordance with the environmental conditions under which the microorganism lives.

A novel facultatively psychrophilic bacterium, strain S-1, which exhibits extraordinarily high catalase activity was isolated from the drain pool of a fish product processing plant that uses H2O2 as a bleaching and microbicidal agent. The catalase activity of the isolate was 1 or 2 orders of magnitude higher than those of Corynebacterium glutamicum,Staphylococcus aureus, Pseudomonas fluorescens, and five other species tested in this study. The strain seemed to possess only one kind of catalase, according to the results of polyacrylamide gel electrophoresis of the cell extract. The optimum temperature for catalase activity was about 30°C, which was about 20°C lower than that for bovine catalase activity. Electron microscopic observation revealed that the surface of the microorganism was covered by blebs. Although the isolate was nonflagellated, its taxonomic position on the basis of physiological and biochemical characteristics and analysis of 16S rRNA sequence and DNA-DNA relatedness data indicated that strain S-1 is a new species belonging to the genus Vibrio. Accordingly, we propose the nameVibrio rumoiensis. The type strain is S-1 (FERM P-14531).

A catalase that exhibits a high level of activity and a rapid reaction with organic peroxides has been purified from Exiguobacterium oxidotolerans T-2-2T (EKTA catalase). The amino acid sequence of EKTA catalase revealed that it is a novel clade 1 catalase. Amino acid residues in the active site around the protoheme are conserved in the primary structure of EKTA catalase. Although the general interactions of molecules larger than hydrogen peroxide with catalases are strongly inhibited because of the selection role of long and narrow channels in the substrate reaching the active site, the formation rate of reactive intermediates (compound I) in the reaction of EKTA catalase with peracetic acid is 77 times higher than that of bovine liver catalase (BLC) and 1200 times higher than that of Micrococcus luteus catalase (MLC). The crystal structure of EKTA catalase has been determined and refined to 2.4 A resolution. The main channel structure of EKTA catalase is different from those of BLC and MLC. The rate constant of compound I formation in catalases decreased with an increase in the molecular size of the substrate. For EKTA catalase with a larger bottleneck 15 A from the iron (entrance of narrow channel) in the main channel, a lower rate of reduction in compound I formation rate with an increase in the molecular size of substrates was found. The increase in the rate constant of compound I formation in these catalases was directly proportional to the increase in the size of the bottleneck in the main channel when molecules of substrates larger than H2O2, such as organic peroxides, are used in the reaction. The results indicate that the size of the bottleneck in the main channel in catalase is an important factor in defining the rate of compound I formation corresponding to the molecular size of the substrates, and this was demonstrated. The Leu149-Ile180 and Asp109-Met167 combinations at the entrance of the narrow channel in EKTA catalase determine the size of the bottleneck, and each atom-to-atom distance for the combination of residues was larger than those of corresponding combinations of amino acid residues in BLC and MLC. The combination of these four amino acids is quite specific in EKTA catalase as compared with the combinations in other catalases in the gene database (compared with more than 432 catalase genes in the database).