Db waxetr column

A UV–vis spectrophotometer was used to measure the cell growth of Y. lipolytica at OD₆₀₀ (Mecasys, Seoul, Korea). After the cultured samples were centrifuged at 13,000 rpm for 3 min, the supernatant was taken for machine analysis. Isopropanol was analyzed using a high-performance gas chromatograph (GC) (model GC7890; Agilent, Santa Clara, CA) equipped with a flame ionization detector (FID) and a DB-WAXetr column (Agilent, 30 m, 0.32 mm, 0.25 µm). The GC oven temperature was set at 40 °C for 1 min, then gradually increased to 100 °C using a 4 °C/min gradient and held for 1 min. The detector temperature was kept constant at 250 °C. Nitrogen was used as a carrier gas. Glycerol was analyzed using a high-performance liquid chromatography (HPLC) system (binary HPLC pump Model 1528, autosampler Model 2707, Refractive Index Detector, Waters, MA, U.S.A.) using an HPLC column (HPLC column Hi-Plex H, 8 µm, 7,7 × 300 mm). 0.0085 M sulfuric acid was used as the mobile phase with a flow rate of 0.6 ml/min. The oven temperature was set to 75 °C, and the RID detector temperature was set to 50 °C.

Before starting the reaction, a mixture of glycine buffer (100 mM, pH 8.6) and the appropriate substrate (1-bromocyclohexane, final concentration: 8.1 mM or 1-chlorohexane, final concentration: 7.2 mM) was incubated in a Screw Top Mini Flask with Hole Cap and Septum (Sigma-Aldrich, USA) on a shaker for 30 min (37°C, 275 rpm). Thereafter, the protein was introduced to the mixture, and the reaction was carried out for 20 min. The samples were withdrawn from the enzymatic reaction mixture every 4 min using a syringe and were mixed with the same volume of a mixture of methanol and IS (1,2-dichloroethane) in a 1000:1 ratio (v:v) to quench the catalyst. The increase in product was then analysed by gas chromatography (Hawlett Packard 5890 Series II GC, USA) on the 30 m x 0.32 mm LD (film thickness 0.5 μm) DB-WAXETR column (Agilent, USA). The concentration of the compounds was calculated based on prepared calibration curves. Conditions of the analysis were as follows: cyclohexane: oven 150°C, injector 140°C, detector 260°C; hexane: oven 120°C, injector 110°C, detector 260°C; air to hydrogen ratio: 10:1, gas flow rate: 1.3 mL/min in 30°C, split: 1:100. The enzymatic reactions for each enzyme and each substrate were prepared in at least 3 replicates, and the final enzymatic activity was presented as an average value.

VOCs analysis was performed with a 7890B gas chromatography system (Agilent, Palo Alto, CA, USA) equipped with a CombiPal auto-sampler (CTC Analytics, Zwingen, Switzerland) coupled to an Agilent 5975C triple quadrapole mass detector. The SPME fibre was desorbed into an ultra-inert straight SPME liner (0.75 mm, Agilent Technologies Inc., USA) at 250 °C in splitless mode for 2 min, and separation of compounds achieved through a DB-Waxetr column (60 m × 250 µm inner diameter × 0.25 µm film thickness; Agilent Technologies Ltd., USA) with helium at a flow rate of 1 mL min⁻¹. The oven temperature was set to 40 °C for 3 min and then ramped from 40 to 90 °C at 10 °C min⁻¹, 90 to 180 °C at 5 °C min⁻¹, 180 to 250 °C at 20 °C min⁻¹ and held for 2 min, resulting in a total run time of 31.5 min. The mass spectrometer was operated in an electron impact (EI) ionization at 70 eV with an ion source temperature of 250 °C, to scan a mass range from 35 m/z to 350 m/z.