Woo H j, Sanseverino J, Cox C D, Robinson K G, Sayler G S
Center for Environmental Biotechnology, The University of Tennessee, Knoxville TN 37996, USA.
J Microbiol Methods. 2000 Apr;40(2):181-91. doi: 10.1016/s0167-7012(00)00123-8.
Toluene dioxygenase (Tod) enzyme activity can be measured by the conversion of indole to indigo. Indigo is measured spectrophotometrically at 600 nm. However, this method is inadequate to measure the whole-cell enzyme activity when interference by suspended biomass is present. Indoxyl is a highly fluorescent intermediate in the conversion of indole to indigo by Tod. A fluorescence-based assay was developed and applied to monitor Tod activity in whole cells of Pseudomonas putida F1 biofilm from a continuously operated biofilter. Suspended growth studies with pure cultures indicated that indoxyl, as measured by fluorescence, correlated with indigo production (r(2)=0.89) as measured by spectrophotometry. Whole-cell enzyme activity was followed during growth on a minimal medium containing toluene. The maximum normalized whole cell enzyme activity of 19+/-1.5x10(-4) mg indigo (mg protein)(-1) min(-1) was reached during early stationary phase. P. putida F1 cells from a biofilm grown on vapor phase toluene had a normalized whole-cell enzyme activity of 5.0+/-0.2x10(-4) mg indigo (mg protein)(-1) min(-1). The half-life of whole-cell enzyme activity was estimated to be between 5.5 and 8 h in both suspended and biofilm growth conditions.
甲苯双加氧酶（Tod）的酶活性可以通过吲哚向靛蓝的转化来测定。靛蓝通过分光光度法在600nm处进行测定。然而，当存在悬浮生物质的干扰时，这种方法不足以测定全细胞的酶活性。吲哚酚是Tod将吲哚转化为靛蓝过程中的一种高荧光中间体。开发了一种基于荧光的测定方法，并将其应用于监测来自连续运行生物滤池的恶臭假单胞菌F1生物膜全细胞中的Tod活性。纯培养物的悬浮生长研究表明，通过荧光测定的吲哚酚与通过分光光度法测定的靛蓝生成量相关（r² = 0.89）。在含有甲苯的基本培养基上生长期间跟踪全细胞酶活性。在稳定期早期达到了最大归一化全细胞酶活性，为19±1.5×10⁻⁴ mg靛蓝（mg蛋白质）⁻¹ min⁻¹。在气相甲苯上生长的生物膜中的恶臭假单胞菌F1细胞的归一化全细胞酶活性为5.0±0.2×10⁻⁴ mg靛蓝（mg蛋白质）⁻¹ min⁻¹。在悬浮和生物膜生长条件下，全细胞酶活性的半衰期估计在5.5至8小时之间。
Zotero 插件