Analytical methods for fermentation process

Biomass growth was controlled by optical density (OD₆₀₀) measurement at 600 nm (BioSpectrophotometer, Eppendorf, Hamburg, Germany). The concentration of glycerol and methanol in the cell-free supernatant during the fermentation was determined by HPLC (Agilent Technologies 1220 Infinity LC System with Agilent Technologies 1260 Infinity RI detector, Agilent Technologies, Santa Clara, CA, USA) using a WATREX Polymer IEX H form column and guard column. A flow rate of 0.8 mL/min of 9 mM sulfuric acid at 45 °C was used [38 (link)]. Sinigrin concentration was determined by HPLC (Agilent 1260 Infinity LC System with Quaternary Pump and UV detector, Agilent Technologies, Santa Clara, CA, USA) using a Phenomenex Gemini^® NX 5μm C18 110Å (150 × 4.6 mm) column according to Tsao et al. [39 (link)]. The nano-liquid chromatography and mass spectrometry analysis (LC-MS-MS) of the tryptic digest of myrosinase (myr-Δ19) was modified from [23 (link)]. The detailed method description can be found in Supplementary Materials. Protein concentration in cell-free supernatants was determined using the Bradford method [40 (link)]. Bradford reagent was purchased from Sigma-Aldrich (St. Louis, MO, USA).