Abstract
Bacillus megaterium (ATCC 13368) exhibits cytochrome P-450 monooxygenase activity (referred to herein as Cyt P-450 meg) catalyzing 15 beta-steroid hydroxylation. This activity belongs to the widespread ferredoxin reductase-ferredoxin-Cyt P-450 type of monooxygenases, providing a representative model system for this type of activity. The level of Cyt P-450 meg activity reaches its maximum in the cells during the stationary phase of the growth curve and is not affected by Cyt P-450 inducers. Here we present the development of an approach for stabilizing the Cyt P-450 meg system so that it performs continuous steroid hydroxylation and will be a model system for Cyt P-450-based detoxification. It is based on cell immobilization and simulation of stationary-phase conditions in a continuously operated fluidized-bed bioreactor. The combination of an appropriate immobilization technique, operational conditions, and medium composition provided a stabilized cell environment resulting in "freezing" of a physiological steady-state analog under stationary phase conditions, allowing stable performance of continuous hydroxylation for several weeks. It is suggested that this approach may be extended for use with other environmentally induced enzymatic activities.
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- Berg A., Gustafsson J. A., Ingelman-Sundberg M. Characterization of a cytochrome P-450-dependent steroid hydroxylase system present in Bacillus megaterium. J Biol Chem. 1976 May 10;251(9):2831–2838. [PubMed] [Google Scholar]
- Berg A., Ingelman-Sundberg M., Gustafsson J. A. Purification and characterization of cytochrome P-450meg. J Biol Chem. 1979 Jun 25;254(12):5264–5271. [PubMed] [Google Scholar]
- Berg A., Rafter J. J. Studies on the substrate specificity and inducibility of cytochrome P-450meg. Biochem J. 1981 Jun 15;196(3):781–786. doi: 10.1042/bj1960781. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bouwer E. J., Zehnder A. J. Bioremediation of organic compounds--putting microbial metabolism to work. Trends Biotechnol. 1993 Aug;11(8):360–367. doi: 10.1016/0167-7799(93)90159-7. [DOI] [PubMed] [Google Scholar]
- Freeman A. Gel entrapment of whole cells in cross-linked prepolymerized polyacrylamide-hydrazide gels. Methods Enzymol. 1987;135:216–222. doi: 10.1016/0076-6879(87)35079-7. [DOI] [PubMed] [Google Scholar]
- Guengerich F. P. Enzymatic oxidation of xenobiotic chemicals. Crit Rev Biochem Mol Biol. 1990;25(2):97–153. doi: 10.3109/10409239009090607. [DOI] [PubMed] [Google Scholar]
- Kuchar D. L., Rottman J., Berger E., Freeman C. S., Garan H., Ruskin J. N. Prediction of successful suppression of sustained ventricular tachyarrhythmias by serial drug testing from data derived at the initial electrophysiologic study. J Am Coll Cardiol. 1988 Oct;12(4):982–988. doi: 10.1016/0735-1097(88)90465-2. [DOI] [PubMed] [Google Scholar]
- Käppeli O. Cytochromes P-450 of yeasts. Microbiol Rev. 1986 Sep;50(3):244–258. doi: 10.1128/mr.50.3.244-258.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sariaslani F. S. Microbial cytochromes P-450 and xenobiotic metabolism. Adv Appl Microbiol. 1991;36:133–178. doi: 10.1016/s0065-2164(08)70453-2. [DOI] [PubMed] [Google Scholar]
- Wiseman A. Genetically-engineered mammalian cytochromes P-450 from yeasts--potential applications. Trends Biotechnol. 1993 Apr;11(4):131–136. doi: 10.1016/0167-7799(93)90087-P. [DOI] [PubMed] [Google Scholar]