5890 series 2 gc

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 The samples were withdrawn from the enzymatic reaction mixture every 4 min using a syringe and were mixed with the same volume of a mixture of methanol and IS (1,2-dichloroethane) in a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.

For the separation, a Hewlett Packard 5890 series II GC (Agilent Technologies, Santa Clara, CA, USA) was used, which was equipped with an HP-5 MS capillary column (300.25 mm, film thickness, 0.25 m) and an HP 5972 mass selective detector. With a mass scan range of m/z 30 to 550 at 70 eV, the mass selective detector was operated in electron-impact ionization (EI) mode. At a flow rate of 1 mL/min, helium was used as the carrier gas. The temperature was initially set at 70 °C, held for 1 min, then ramped at 3 °C/min to 180 °C, kept for 3 min, and finally increased at 5 °C/min to 230 °C, held for 5 min. Temperatures for the injector and MS transfer line were set at 230 and 250 degrees Celsius, respectively. A 1:10 split ratio was used to manually inject a sample of 1 mL of 1% essential oil.