Agilent 8860

The individual composition of the fatty acids present in the oil was determined by gas chromatography (GC) [55 ]. The oil (100 mg) was subjected to transesterification with 5 mL of methanol in the presence of sulfuric acid to obtain the fatty acid methyl esters (FAME) [56 ]. They were extracted from the solution with 20 mL of n-hexane. Then, the solvent was evaporated on a rotary vacuum evaporator and the dry residue was dissolved with petroleum ether (1% solution of FAME in petroleum ether). After that, 1 μL was injected in the GC unit (Agilent 8860, Santa Clara, CA, USA). The column used was a capillary column DB-FastFAME with the following characteristics: 30 m × 0.25 mm × 0.25 μm (film thickness). The detector was a flame ionization detector (FID) and the carrier gas was nitrogen. The conditions of the analysis were as follows: 70 °C (holding at this temperature for 1 min), then increasing the temperature up to 180 °C at a rate of 6 °C/min, and increasing the temperature up to 250 °C at a rate of 5 °C/min. Identification of the individual FAME was performed by comparing their retention times with that of a standard solution of FAME (Supelco, FAME mix 37 components, Bellefonte, PA, USA). The limit of detection of the GC was 0.5%.

Cecal contents (100 mg) were diluted in 2 mL of distilled water to obtain a slurry, and 0.5 mL of H₃PO₄ and 1 mL of diethyl ether were added to it. The slurry was centrifuged at 4 °C and 7000 rpm for 10 min after vortexing for 4 min. A 0.22 µm microporous membrane was used to filter the organic phase. Gas chromatography (GC–MS; Agilent-8860, Santa Clara, CA, USA) was used to synthesize and determine concentrations of various SCFAs in organic fractions.

The photocatalytic CO₂ reduction was performed in a 125 mL homemade photoreactor, which was irradiated with a 300 W Xe lamp (HSXF300, Beijing NBET Technology Co., Ltd.) equipped with a 420 nm cutoff filter at 25 °C. Typically, 5 mg catalyst samples were spread on a quartz filter membrane which was placed on the sample holder built in the center of the reacting vessel, then 5 mL pure water was added below the holder, avoiding direct contact with the samples. Then the reactor was filled with 0.1 MPa of high‐purity CO₂. The gas products after the reaction were quantitatively analyzed by Agilent 8860 gas chromatography equipped with thermal conductivity detectors TCD and FID detector. Helium was used as the carrier gas to detect H₂, and nitrogen was the carrier gas used to detect other gas products. The liquid phase product was detected by 1H‐NMR (Bruker, 400 MHz).

The concentration of hydrogen, methane and carbon dioxide was measured by injecting 50 µL of gaseous samples with a gastight syringe (Hamilton, Reno, NV, USA) into a gas-chromatograph equipped with a thermal conductivity detector (TCD, Agilent 8860, GC system, Santa Clara, CA, USA) as reported in the Supporting Information. Gas quantification was performed using external standards of hydrogen, methane and carbon dioxide. Standards were prepared to start from pure gases and suitably diluted to different concentrations with nitrogen.

Analysis of fatty acid methyl esters was performed with a gas chromatograph (Agilent 8860; Agilent Technologies. Inc., Santa Clara, CA, USA) equipped with a flame ionization detector (FID) and ALS. The temperature of the injector was 200 °C and the temperature of the detector was 240 °C. Chromatography was performed on a capillary column DB-23 (Agilent Technologies, Santa Clara, CA, USA), length 60 m, inner diameter of the column 0.25 mm, active layer thickness 0.25 μm). The initial column temperature was 150 °C for 2 min; it was then increased to 230 °C by heating at 5 °C/min and held at that temperature for 20 min. Hydrogen at a flow rate of 1 mL/min was used as the carrier gas. Collection and processing of the results were conducted with the computer program OpenLAB CDS ChemStation Workstation VL. Fatty acids were identified by comparing the retention times with methyl standards (Sigma Aldrich Chemie, GmbH and Supelco, St. Louis, MI, USA). Quantification was performed using nonadecanoic acid methyl ester (C19:0). The fatty acid composition was calculated as the percentage of each individual fatty acid relative to the total fatty acids.

The reduction reaction was initiated in a 400 μL solution containing 50 mM KH₂PO₄, 10% sorbitol, 1 mM DTT, 10 mM NADPH tetrasodium salt hydrate, 6 mM glucose-6-phosphate, 20 U glucose-6-phosphate dehydrogenase (Solarbio), together with the wild-type and mutated MpPR, ArPR, and NtPR (Supplementary Table 1). Other conditions are the same as the in vitro enzyme catalysis assay. Quantitative analysis was performed by the comparison of the peak areas of products to the standards of known concentrations obtained from chiral GC analysis (Agilent 8860 equipped with a HP-5 capillary column; the column condition is the same as described above). All biotransformation reactions were performed in at least duplicates, and the results are averages of the data. The yields of menthone and iso-menthone at each concentration were calculated by the comparison of the peak areas of products to the standards of known concentrations. The kinetic parameters K_m and k_cat were calculated using Equation 1.
where V₀ is the initial velocity, [E] is the enzyme concentration, [S] is the substrate concentration, V_max is the maximum velocity; K_m is the Michaelis constant and k_cat is calculated from V_max/[E].

The bigels’ (150 mg of sample or 500 µL of digested sample) and correspondent oils’ (15 mg) fatty acid profile was evaluated via gas chromatography (GC) after transesterification according to the method described by Machado et al. 2022 [28 (link)]. GC analyses were performed in a gas chromatograph Agilent 8860 (Agilent, Santa Clara, CA, USA) with a flame ionization detector (FID), using a BPX70 capillary column (60 m × 0.25 mm × 0.25 μm; SGE Europe Ltd., Paris, France). The following operating conditions were employed. Injector (split 25:1) and FID temperatures were 250 °C and 275 °C, respectively. Hydrogen (carrier gas) was used at a flow rate of 1 mL/min (20.5 psi). The oven temperature program started at 60 °C (held for 5 min), raised 15 °C/min to 165 °C (held for 1 min), and finally 2 °C/min to 225 °C (held for 2 min). Supelco 37 and individual standards from CLNA were used for the identification of fatty acids. The GC analysis was performed in triplicate.