Micromass gct premier

G. oxydans cells were grown to an OD₆₀₀ of 1.3, centrifuged (4,000 × g, 5 min) and washed twice with 50 mM phosphate buffer (pH 6). After the second washing step, cells were resuspended in biotransformation buffer (6.6 g L⁻¹ Na₂HPO₄, 3 g L⁻¹ KH₂PO₄, 1 g L⁻¹ NH₄Cl, 0.5 g L⁻¹ NaCl, 0.49 g L⁻¹ MgSO₄, 0.02 g L⁻¹ CaCl₂) supplemented with 2% (w/v) l-rhamnose and incubated for 24 h at 30°C and 200 rpm. Then, the cells were removed from the buffer (4,000 × g, 5 min) and the supernatant was used for analysis by gas chromatography (Agilent 6,890 N, Agilent Technologies) coupled to a Waters Micromass GCT Premier high-resolution time-of-flight mass spectrometer (Waters). Sample handling for derivatization, GC-TOF-MS operation, and peak identification were carried out as described (Paczia et al., 2012 (link)). As a control, samples from biotransformation buffer with l-rhamnose and without cells as well as biotransformation buffer without l-rhamnose yet with cells were prepared.

Samples were thawed, diluted with extraction mix to 1.7 mL, and 5 µL IS were added. Aliquots of 0.7 mL per sample were processed as described above. Derivatization was achieved in a reduced volume of 25 µL methoxymation solution and 25 µL silylation solution. Samples with low IS signal were re-derivatized with MSTFA as described above. GC–MS measurements were conducted using a 6890 N GC equipped with a 7683B auto sampler (Agilent) coupled to a Micromass GCT Premier (Waters) mass spectrometer. The GC was operated with glass liners (Agilent, 4 × 6.3 × 78.5 mm inner × outer diameter × length), split 1, and 250 °C injector temperature. The MS was used with 300 °C source temperature and dynamic range extension mode. Resolution was > 6.000 at m/z 501.97.