Alkali- and halo-tolerant catalase from Halomonas sp SK1:: Overexpression in Escherichia coli, purification, characterization, and genetic modification

yA catalase gene, ohktA, from an alkali- and halo-tolerant bacterium, Halomonas sp. SKI, on the pKK223-3, was expressed in the catalase-lacking Escherichia coli strain UM2. Highly purified catalase showing a single band on SDS-PAGE was obtained by two liquid chromatography steps on DEAE-Toyopear1 and Chelating-Sepharose Fast Flow. The enzyme, oHk-tA, shows high catalase activity with a pH optimum at 10, and the activity was stable in 4 m KC1. This enzyme is thermo-sensitive, showing a significant loss of activity within 5 minutes at 37degreesC. To modify the stability of the catalase, the addition of domain II of the heat stable Mn catalase from Thermus thermophilus to the C-terminus was made. When coexpressed with a chaperone (PhFKBP29) gene product, peptidyl-prolyl cis-trans isomerase, from a thermophilic bacterium, a chimeric catalase was produced in the soluble fraction. The stability of this catalase in the range of 37degrees-45degreesC was improved and it was stable for more than 1 h at 37degreesC.