RESUMO
In this work, a robust brefeldin A-synthesizing fungus, Eupenicillium brefeldianum ZJB082702, was bred from a Murraya paniculata endophytic fungus E. brefeldianum A1163. Using one-factor-at-a-time experimental design, optimization of media composition for E. brefeldianum ZJB082702 fermenting brefeldin A was conducted. Outcomes indicated that mixed carbon source and mixed nitrogen source were of c ritical importance to brefeldin A fermentation. After 6d culture in the optimized fermentation media, composed of (gl(-1)) 13.33 starch, 26.67 glucose, 1.0 yeast extract powder, 1.0 corn steep liquor, 0.5 soybean meal, 0.75 NaNO(3), 2.5 malt extract, 6.0 CaCO(3), 3.0 MgSO(4), 4.0 KH(2)PO(4), 1.0 × 10(-2) CuSO(4), brefeldin A yield peaked at 1304.7 mgl(-1), 648.2 mgl(-1) in 500 ml baffled flask and 15 l stirred fermentor respectively, formed as a growth associated type of secondary metabolite based on fermentation profile analysis.
Assuntos
Reatores Biológicos , Brefeldina A/metabolismo , Eupenicillium/metabolismo , Antibacterianos/biossíntese , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Brefeldina A/isolamento & purificação , Carbono/metabolismo , Carbono/farmacologia , Meios de Cultura/química , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Eupenicillium/efeitos dos fármacos , Eupenicillium/crescimento & desenvolvimento , Fermentação/efeitos dos fármacos , Nitrogênio/metabolismo , Nitrogênio/farmacologiaRESUMO
Brefeldin A (BFA) is a macrolide lactone antibiotic, possessing antitumor, antiviral, antifungal activities. In this work, a separation strategy involving one-step macroporous resin adsorption chromatography combined with crystallization was established for BFA purification from Eupenicillium brefeldianum CCTCC M 208113 fermentation broth. Among six macroporous resin adsorbents tested, the non-polar resin HZ830 had the best adsorption and desorption performance. The static equilibrium adsorption data fitted well with the Freundlich equation, and the adsorption kinetic followed the pseudo-second order model. Through experimental optimization of column adsorption and desorption, BFA in purity of 90.4% (w/w), 92.1% (w/w) yield was obtained by a one-step macroporous resin adsorption chromatography, using a stepwise elution protocol. Furthermore, high purity (>99%, w/w) of BFA crystals were prepared from E. brefeldianum CCTCC M 208113 fermentation broth in an overall recovery of 67.0% (w/w), using a combination of adsorption chromatography packed with non-polar macroporous adsorbent HZ830 and crystallization in acetone.