Abstract
Baeyer–Villiger biooxidation of 4-methylcyclohexanone–5-methyloxepane-2-one catalysed by recombinant Escherichia coli overexpressing cyclopentanone monooxygenase encapsulated in polyelectrolyte complex capsules was used to investigate effect of substrate conversion on the viability of cells. Confocal laser scanning microscopy (CLSM) was used to assess cell viability using propidium iodide fluorescence marker for necrosis, and flavin autofluorescence to identify living bacteria. Viability of encapsulated cells decreased with increasing substrate concentration from 99 ± 1 to 83 ± 4%, while substrate conversions from decreased 100 to 6 ± 1%. Storage stabilization of encapsulated cells was observed by increased substrate conversion form 68 ± 2 to 96 ± 3%. Measurements by CLSM with standard deviations up to 5% may be regarded as powerful tool for recombinant cell viability determination during Baeyer–Villiger biooxidations.
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Acknowledgments
This work was supported by the Slovak Grant Agency for Science VEGA 1/0335/10, the Slovak Research and Development Agency under the contract APVV-51-033205, COST Action 865 and COST Action CM0701. This contribution/publication is the result of the project implementation: Centre for materials, layers and systems for applications and chemical processes under extreme conditions–Stage II supported by the Research & Development Operational Program funded by the ERDF. We thank to prof. Marko D. Mihovilovic (VUT, Vienna, Austria) for delivery of the expression strain E. coli overexpressing CPMO.
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Schenkmayerová, A., Bučko, M., Gemeiner, P. et al. Viability of free and encapsulated Escherichia coli overexpressing cyclopentanone monooxygenase monitored during model Baeyer–Villiger biooxidation by confocal laser scanning microscopy. Biotechnol Lett 34, 309–314 (2012). https://doi.org/10.1007/s10529-011-0765-7
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DOI: https://doi.org/10.1007/s10529-011-0765-7