Strain fermentation and purification of dynobactin A.

Photorhabdus australis was grown as 5 ml overnight culture in starter culture tubes incubated at 28 °C with 200 r.p.m. shaking, inoculated 1:100 into a 2 l Erlenmeyer flask with 500 ml tryptic soy broth and incubated for 8–10 d. Culture supernatant (10 l) was collected by centrifugation at 10,000 g for 10 min, and incubated overnight with agitation with 2 kg polymeric resin XAD16N (20–60 mesh, Sigma-Aldrich). Resin was washed using 20 l of doubly deionized water (ddH₂O), and dynobactin was eluted from XAD16N resin utilizing 4 l of acidified methanol (0.1% (v/v) formic acid). The eluate was concentrated using a rotary evaporator, exchanged into water containing 0.1% formic acid and applied to a cation exchange chromatography column. The eluate was loaded onto the cation exchange resin (200 ml of SP Sepharose Fast Flow) and flushed with 0.1% formic acid (v/v) in ddH₂O at pH 3. The bound dynobactin A was then eluted using a step gradient of 50 mM ammonium acetate: pH 5 and then pH 7. The resin was then washed with 0.5 M NaOH and 0.5 M NaCl. Each gradient step consisted of 10 column volumes of the respective buffers. The pH 7 eluate, showing biological activity, was concentrated by rotary evaporator or lyophilised, and further purified by reverse-phase high-performance liquid chromatography (RP-HPLC) using a C18 column (Agilent 1200, XBridge Prep C18, 5 μm OBD, 19 × 250 mm column). RP-HPLC conditions were as follows: solvent A, ddH₂O with 0.1% (v/v) formic acid; solvent B, acetonitrile with 0.1% (v/v) formic acid. A flow rate of 15 ml min⁻¹ was used over a gradient of 2–20% solvent B, incrementing 1% per minute. A photodiode array enables UV monitoring (210 to 400 nm) for peak picking. Dynobactin A could be collected with purity in excess of 98% by UV analysis.