Alkali- and halo-tolerant catalase from Halomonas sp. SK1: overexpression in Escherichia coli, purification, characterization, and genetic modification.
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 degrees 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 degrees -45 degrees C was improved and it was stable for more than 1 h at 37 degrees C.
Thuy
Le Huyen Ai
le HA
Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu, Japan. shinozawa@bce.gunma-u.ac.jp
Phucharoen
Krisana
K
Ideno
Akira
A
Maruyama
Tadashi
T
Shinozawa
Takao
T
D000468:Alkalies / Q000737:chemistry / Q000494:pharmacology; D000595:Amino Acid Sequence; D002374:Catalase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D020632:Halomonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011489:Protein Denaturation / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D016415:Sequence Alignment; D013696:Temperature
D000468:Alkalies; D002374:Catalase