Alkali- and Halo-tolerant Catalase from Halomonas sp. SK1: Overexpression in Escherichia coli, Purification, Characterization, and Genetic Modification

抄録

A catalase gene, ohktA, from an alkali- and halo-tolerant bacterium, Halomonas sp. SK1, 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, oHktA, 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 37 °C. 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 37°–45 °C was improved and it was stable for more than 1 h at 37 °C.