|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1997 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1996 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Bioremediation is emerging as one of the most promising technologies for the removal of hazardous organochemicals from the environment. For this, the screening of potential organochemicals degrading bacteria is one of the key step. Cytochrome P450 are widely distributed in the biosphere and catalyze a remarkably broad range of chemical reactions on an equally broad range of substrates. Therefore, the bacteria producing cytochrome P450 are good candidates for biodegradation of hazadous organochemicals.
In this study, we isolated 7 strains of bacteria producing cytochrome P450 from environmental soil and water by using classical enrichment techniques. Six strains of isolated bacteria grew on 2-ethoxyphenol as sole carbon and energy sources. Another one grew on camphor as sole carbon and energy sources. A possibility exists that the bacteria producing cytochrome P450 are widespread in nature and bacterial cytochrome P450 is involved in the biodegradation of hazardous chemicals in nature.
rain EJ that isolated from soil using 2-ethoxyphenol containing medium was investigated. The growth of EJ was observed on 2-ethoxyphenol, 4-ethoxybenzoic acid, 2-methoxyphenol, 4-methoxybenzoic acid and n-hexadecane. The organism could also be cultivated on lactose, glucose, succinic acid, ethanol and glycerol. Of the growth substrates tested, the presence of P450 was detected only in extracts of cell grown on 2-ethoxyphenol or 4-ethoxybenzoic acid.
The kinetics of P450 induction by 2-ethoxyphenol was investigated. The enzyme was induced during the exponential growth phase and reached a maximum level of 0.5 nmol/mg-protein at the end of exponential growth phase. The P450 level then rapidly decreased at stationery phase. The cell free extract of the 2-ethoxyphenol-grown EJ metabolized not only 2-ethoxyphenol but also benzene, xylene, toluene, 1,3-dichloropropene, carbon tetrachloride, 1,1,1-trichloroethane. The P450 can display a remarkable chemical flexibility toward substrate. Thus, the P450 from strain EJ may be useful for the removal of hazardous organochemicals from environment.
The cell free extract from EJ was subjected to gel filtration and anion exchange chromatography. The P450 fraction from the anion exchange column had a specific content of P450 of 17.1 nmol/mg/protein. Molecular weight calibration of gel filtration indicated an approximate molecular weight of 45,000 for the P450 containing peak. Less