期刊
BIOCATALYSIS AND BIOTRANSFORMATION
卷 27, 期 5-6, 页码 348-359出版社
TAYLOR & FRANCIS LTD
DOI: 10.3109/10242420903286141
关键词
CYP metabolism; recombinant expression; Escherichia coli; immobilization; alginate
资金
- Flemish Province-IWT
Milligram amounts of metabolites of drug candidates arc required to identify toxic products. Human drug metabolites are currently produced selectively in a time- and cost-efficient manner in bioreactor systems containing recombinant Escherichia coli co-expressing a human cytochrome P450 isoenzyme/NADPH cytochrome P450 reductase (hCYP/HR) complex. For further optimization, immobilization of the catalytic system in Ca-alginate microbeads was considered. This new concept was designed for CYP3A4 with testosterone as substrate. Immobilized E. coli cells had a high maximal and homogeneously distributed biomass. Viability was stable over at least I week of culture and even longer during storage. Gene expression was ideally initiated 6 h after immobilization. Although immobilized E. coli cells expressed a highly functional enzyme system after 2 days, they did not metabolize testosterone, probably due to cell permeability problems resulting from immobilization. Therefore, immobilized cell membranes displaying testosterone bioconversion activity, even after long-term storage, will be used in bioreactors with high organic solvent content.
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