2.4 Analytical methods
The enzyme activity under the different acetate concentrations was
determined in a 200 μL mixture composed of 170 μL DCPIP, 20 μL 100 g/L
substrate sorbitol, and 10 μL G. oxydans with
OD600=2. The 96-well plate was placed in a Microplate
Reader (INIFINITE 200 PRO, Tecan Austria GmbH) set at 220 rpm and 30℃,
and the absorbance measured at 600 nm after every 30 seconds. The
absorbance was then linearly fitted to obtain a slope. The enzyme
activity was finally calculated using formula (1)(Peters et al., 2013):
\begin{equation}
U=\frac{V_{t}\frac{A}{t}}{V_{s}\text{lε}}\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ (1)\backslash n\nonumber \\
\end{equation}The titer of EG, 1,3-PG, 1,4-BG, 1,5-PG, 1,6-HG, hydroxyl acid and
diacid catalyzed by G. oxydans were detected by high performance
liquid chromatography (HPLC) (Agilent 1260 series) equipped with
differential Detector. The separation column used was Aminex Bio-Rad
HPX-87H column and 5 mM H2SO4 was used
as mobile phase at a flow rate of 0.6 mL/min.
Due to the difference of experiment data, three parallel assays were
performed for each experiment to ensure reliability of results.