Model 996 photodiode array detector

Purification of viscosin was performed as described previously by Bak et al. (2015) (link). Briefly, P. fluorescens SBW25 was cultivated on King's B agar plates (King et al., 1954 (link)) in darkness at 28 °C for 1 day before being transferred to 20 °C and incubated for another 3 days. Colony material was suspended in demineralized water (MilliQ; Millipore) and homogenized by shaking. Cells and supernatant were separated twice by centrifugation at 4700 r.p.m. for 20 min at 4 °C in a Sigma 3-18K centrifuge (Sciquip). The supernatant was acidified to pH 2.0 with 1 M HCl and left overnight on ice for a precipitate to form. The solution including the precipitate was centrifuged for 27 min at 7000 r.p.m. and 4 °C in a Sigma 3-18K centrifuge. The supernatant was discarded and the precipitate was washed four times with MilliQ water at pH 2.0. The precipitate was dissolved in MilliQ water and pH was adjusted to 8.0 with 1 M NaOH to fully dissolve the precipitate. The solution was lyophilized and the purity of the lipopeptide preparations was verified by HPLC. HPLC analysis was carried out using a Waters Alliance series 2695 system and a Waters model 996 photodiode array detector. The procedure was carried out as described previously by Bak et al. (2015) (link). The same HPLC protocol was used for quantification of viscosin produced in liquid cultures of P. fluorescens SBW25.

The liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) experiments were performed on a system consisting of a Waters Alliance model 2690 separations module and model 996 photodiode array detector (Waters, Eschborn, Germany) controlled with a Compaq AP200 workstation coupled to a Micromass model ZMD mass detector (Micromass, Altrincham, UK). The samples were automatically injected on a Waters narrow bore Nova-Pak column C₁₈ (2.1 × 150 mm, 60 Å pore size, 3.5 μm particle size). The elution was carried out with solvents A (0.1% TFA/H₂O) and B (60% acetonitrile/0.1% TFA/H₂O) at a flow rate of 0.4 mL/min using a linear gradient from 5% to 95% B in 30 min. The condition used for mass spectrometry measurements was a positive ESI. Specifically for the case of monitoring enzymatic reactions, a different linear gradient of 35 to 50% (v/v) of solution B in 15 min, with flow rate of 1.5mL/min was used.

Liquid chromatography-mass spectrometry (LC/MS) was developed on a Waters Alliance model 2690 system and model 996 photodiode array detector (Waters; Eschborn, Germany) controlled by a Micromass model ZMD mass detector (Micromass; Altrincham, UK). Peptide samples were applied to a 150 × 2.1 mm C18 column, 3.5 µm particle size, 60 Å pore size (Nova-Pak, Waters, Milford, MA, USA). Elution was performed with 0.1% TFA:H₂O as solvent A and 60% acetonitrile:0.1% TFA:H₂O as solvent B, at a flow rate of 0.4 mL·min⁻¹, using a linear gradient from 5% to 95% for solvent B for 30 min at 220 nm and a mass range of 500–3930 Da.