Nerfilin I, a novel microbial metabolite inducing neurite outgrowth of PC12 cells.

A preculture of S. halstedii 2723-SV2 was inoculated into three 50-liters jar fermentors containing 27 liters of the culture mediumconsisting of glycerol 2%, molasses 1%, casein 0.5%, polypepton 0.1% and CaCO3 0.4% (pH 7.0 before sterilization), and fermentation was carried out at 27°C for 24 hours with agitation at 150 rpm and an air flow at 30 liters/minute. Nerfilin I was purified from the supernatant by monitoring the induction activity of neurite outgrowth of PC12 cells. The supernatant collected by centrifugation was subjected to a Diaion HP-20 column, which was washed with 30% aqueous methanol and then eluted with 70% aqueous methanol. After evaporation, the active fraction was applied to an MPLCLiChroprep RP-18 (MERCK)column with 50% aqueous methanol. The active fraction was further purified by an HPLC TSKgel ODS-80TM (TOSOH) column with 50% aqueous methanol. Active compound nerfilin I and its derivative nerfilin II were concentrated to dryness separately to yield white powders of nerfilin I 3mg and nerfilin II lOOmg from the total of 80 liters broth. The physico-chemical properties of nerfilin I and nerfilin II are summarized in Table 1. Their molecular formulae were determined as C28H37N3O5 and