Department of Glycobiotechnology, Institute of Chemistry-Center for Glycomics, Slovak Academy of Sciences, Dúbravská Cesta 9, 845 38, Bratislava, Slovakia.
Biotechnol Lett. 2012 Feb;34(2):309-14. doi: 10.1007/s10529-011-0765-7. Epub 2011 Oct 8.
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.
包埋在聚电解质复合物胶囊中的过表达环戊酮单加氧酶的重组大肠杆菌对 4-甲基环己酮-5-甲基环氧戊烷-2-酮的 Baeyer-Villiger 生物氧化作用，用于研究底物转化率对细胞活力的影响。利用碘化丙啶荧光标记坏死的方法和黄素自发荧光来识别活菌，通过共聚焦激光扫描显微镜（CLSM）评估细胞活力。随着底物浓度从 99±1%增加到 83±4%，包埋细胞的活力逐渐降低，而底物转化率从 100%降低到 6±1%。通过增加从 68±2%到 96±3%的底物转化率，观察到包埋细胞的储存稳定性增加。标准偏差高达 5%的 CLSM 测量可被视为 Baeyer-Villiger 生物氧化过程中重组细胞活力测定的有力工具。
