AQUEOUS RELEASE AND PURIFICATION OF POLY(BETA-HYDROXYBUTYRATE FROM ESCHERICHIA-COLI

The poly(P-hydroxybutyrate) (PHB) biosynthetic genes of Ralstonia eutr opha that are organized in a single operon (phaCAB) have been cloned i n Escherichia coli, where the expression of the genes in the wild-type pha operon from plasmid pTZ18U-PHB leads to the formation of 50-80% P HB/celldry mass when the cells are grown in Luria-Bertani medium suppl emented with 1% glucose (w/v). In combination with the phaCAB genes, e xpression of cloned lysis gene E of bacteriophage PhiX174 from plasmid pSH2 has been used to release PHB granules produced in E. coli. It wa s shown that small PHB granules in a semiliquid stage are squeezed out of the cells through the E-lysis tunnel structure which is characteri zed by a small opening in the envelope with borders of fused inner and outer membranes. All envelope components remain intact after E-lysis and can be removed from the mixture of released PHB granules by densit y gradient centrifugation. In addition, a modified E-lysis procedure i s described which enables the release of PHB from cell pellets in pure water or low ionic strength buffer. PHB granules in aqueous solution can be aggregated by divalent cations. Addition of glassmilk speeds up the agglomeration of PHB granules and binding to glass beads can eith er be used for collection or further purification of PHB in aqueous so lutions. (C) 1998 Elsevier Science B.V. All rights reserved.