G1312b binary pump sl

For HPLC/MS measurements, an Agilent Technologies 1200 series (Santa Clara, CA) equipped with a G1379B degasser, G1312B binary pump SL, G1367C HiP-ALS SL autosampler, a G1314C VWD SL UV detector, G1316B TCC SL column oven, and a G1956B MSD mass selective detector was used. The mass spectrometer was operated in positive electrospray ionization mode. The analytes were separated on an Atlantis® dC18 column (5 μm, 4.6 × 250 mm, Waters) at 25 °C by using aqueous eluent (0.1% formic acid) and acetonitrile at a flow rate of 1.0 ml min⁻¹. The column was equilibrated with 7% acetonitrile in water (0.1% formic acid), and the following gradient was used for analysis: 0–2 min, 7% acetonitrile; 2–10 min, 7–100% acetonitrile; 10–12 min, 100% acetonitrile; 12–14 min, 7% acetonitrile. 10 μl of 300 μm HPLC purified 8f-FAD or 300 μm FAD solution for control, dissolved in water, were injected for each run.

Tris was purchased from Carl Roth (Karlsruhe, Germany), isopropyl β-d-1-thiogalactopyranoside (IPTG) from Serva (Heidelberg, Germany), methacrylonitrile from Fluka (Buchs, Switzerland) and CoCl₂ and FeSO₄*7H₂O from Merck. HPLCMS grade acetonitrile was purchased from J.T.Baker/Avantor Performance Materials (Deventer, The Netherlands). All other chemicals were obtained from Sigma–Aldrich (St. Luis, MO, USA) and used without further purification.
E. coli cells were cultivated in an RS 306 shaker (Infors, Bottmingen, Switzerland), a Multitron shaker (Infors AG Bottmingen, Switzerland) and a Certomat BS-1, and the cells were harvested with an Avanti J-20 XP centrifuge (Beckman Coulter, Brea, CA, USA). Cell pellets were disrupted by a 102C converter with a Sonifier 250 (Branson, Danbury, CT, USA), and the cell-free extract was obtained by centrifugation in an Avanti J-20 XP centrifuge (Beckman Coulter). Reactions were performed on a Thermomixer comfort (Eppendorf, Hamburg, Germany). HPLC/MS analysis was carried out on an Agilent Technologies (Santa Clara, CA, USA) 1200 Series equipped with G1379B degasser, G1312B binary pump SL, G1367C HiP-ALS SL autosampler, a G1314C VWD SL UV detector, G1316B TCC SL column oven and a G1956B MSD. A positive electrospray ionization mode was used as ionization method.