2.7 Quantification of active rrBChE
Fresh biomass was washed and extracted with cold (4˚C) 100 mM sodium
phosphate + 100 mM NaCl buffer pH 7.4 at a ratio of 1 mL extraction
buffer per 1 gram FW biomass as previously described (Corbin et al.,
2016). The extracted mixture was then centrifuged, and the supernatant
was transferred to a new tube and stored at 4˚C until analysis. A
modified Ellman activity assay (Ellman et al., 1961) was used to
quantify active rrBChE in the cell extract (cell-associated rrBChE) and
the medium (culture medium rrBChE). The assay was performed by adding a
150 µL of Ellman’s substrate to a 50 µL of sample, diluted with the
extraction buffer if necessary, in a 96-well plate and measuring the
absorbance at 405 nm for 3 min at 25˚C with a spectrophotometer
(SpectraMax 340PC, Molecular Devices, Sunnyvale, CA) where the initial
rate was in the linear range of 200-500 mOD/min. Three replicates of
each sample were measured. The specific activity of 260 U/mg crude
rrBChE was assumed throughout this study (Alkanaimsh et al., 2016).