Removal of endotoxin during purification of poll(3-hydroxybutyrate) from gram-negative bacteria

Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several gram-nega tive bacteria, including Ralstonia eutropha, Alcaligenes latus, and recombi nant Escherichia coli. PHB was recovered from these bacteria by two differe nt methods, and the endotoxin levels were determined. When PHB was recovere d by the chloroform extraction method, the endotoxin level was less than 10 endotoxin units: (EU) per g of PHB irrespective of the bacterial strains e mployed and the PHB content in the cell. The NaOH digestion method, which w as particularly effective for the recovery of PHB from recombinant E. coli, was also examined for endotoxin removal. The endotoxin level present in PR E recovered by 0.2 N NaOH digestion for 1 h at 30 degrees C was higher than 10(4) EU/g of PHB. Increasing the digestion time or NaOH concentration red uced the endotoxin level to less than 1 EU/g of PHB. It was concluded that PHB with a low endotoxin level, which can be used for various biomedical ap plications, could be produced by chloroform extraction. Furthermore, PHB wi th a much lower endotoxin level could be produced from recombinant E. coli by simple NaOH digestion.