1260 infinity hplc system

Bacterial cultures were grown in 250 ml flasks and at the relevant time points, 200 μl sample aliquots were clarified at 10,000 rpm for 5 min. 100 μl of the resulting supernatant was subsequently added to 900 μl of 10 mM H₂SO₄. Carbohydrate content was analysed using an Aglient (California, USA) 1260 Infinity HPLC system with a Rezex monosaccharide H⁺ column (Phenomenex, California, USA), equilibrated with 0.005% H₂SO₄ mobile phase at 50 °C and 0.6 ml min^-1. 40 μl of sample was injected and carbohydrates were eluted from the column using an isocratic elution over 30 min. Carbohydrates were detected by a refractive index (RI) detector with an initial temperature of 40 °C, RI detector range of 500 μRIU/V and recorder range of 512.00 μRIU.

¹H and ¹³C NMR spectra were recorded at 20 °C on a 300 MHz Bruker NMR. The conversions and regiomeric ratio of all biotransformations were measured by reversed‐phase HPLC with an Agilent 1260 Infinity HPLC system at 25 °C, using a Phenomenex Luna® 5 mm, C18, 100 A (250×4.6 mm) column. Detection was performed with a diode array detector (G4212B). TLC was carried out with pre‐coated aluminum sheets (TLC Silica gel 60 F254, Merck) with detection by UV (254 nm) and/or by staining with cerium molybdate solution. All chemicals and solvents were obtained from commercial suppliers (TCI, Sigma Aldrich/Fluka, VWR International/Merck, Roth) and used as received unless stated otherwise. Commercial available compounds were utilized as obtained as references for the quantitative and qualitative determination of the isomers produced during the biotransformation. In the case that the references were not commercially available, they were synthesized. 3‐Hydroxytyrosol (1 f) was synthesized following a procedure from literature.^[25] Units are defined as converted substrate in μmol per min.

A 36 h old Brevibacillus laterosporus DSM 25 culture was centrifuged at 15,000 g for 15 min, and the supernatant was collected and adjusted the pH to 7. After that, the culture was applied to a CM Sephadex^TM C-25 column (GE Healthcare) equilibrated with distilled water. The flow-through was discarded, and the column was subsequently washed with 12 column volumes (CV) of distilled water. The peptide was eluted with 6 CV 2 M NaCl. The eluted peptide was then applied to a SIGMA-ALDRICH C18 Silica gel spherically equilibrated with 10 CV of 5% aq. MeCN containing 0.1% trifluoroacetic acid. After washing with a 10 CV of 5% aq. MeCN containing 0.1% trifluoroacetic acid, peptides were eluted from the column using up to 10 CV of 50% aq. MeCN containing 0.1% trifluoroacetic acid. Fractions containing the eluted peptides were freeze-dried and dissolved in MQ water. After filtration through a 0.2 μm filter, the cyclic lipopeptides were purified on an Agilent 1260 Infinity HPLC system with a Phenomenex Aeris C18 column (250 × 4.6 mm, 3.6 μm particle size, 100 Å pore size). Acetonitrile was used as the mobile phase, and a gradient of 25-35% aq. MeCN over 30 min at 1 mL per min was used for separation. The purified lipo-tridecapeptides were eluted with a gradient of 28 to 32% aq. MeCN. The expression levels for Bre and BreB were 6 mg/L and 0.5 mg/L, respectively.