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.