Improving selectivity in protein extraction by subtle interactions and environmental changes

Abstract: The challenges for efficient bioprocesses lie in developing and adapting further current technologies or in creating innovative separation technologies. The thesis is based on the studies on protein recovery from crude feedstocks by extraction in aqueous two-phase systems and detergent foam, respectively. The separation technologies have been adapted to suit individual proteins by utilizing different forms of molecular interactions and environmental changes in a very simple way. Interaction of the lipolytic enzyme, cutinase with it's substrate, butyrate resulted in sufficient conformational changes to change it's extraction behavior in a polyethylene glycol (PEG)/salt system. The ability of the polymer, polyvinylpyrrolidone (PVP) to complex reactive dyes and metal ions was used to set up a selective extraction process for the proteins (porcine Lactate dehydrogenase (LDH) and His6-LDH) binding these entities in a two-phase system employing PVP as one of the phase forming components. Extraction in aqueous two-phase systems was found to be an efficient separation technique for cold-active, highly labile enzyme molecules. Cold-active ß-galactosidase was purified with high yields using extraction as a primary separation sep. In case of cold-active ß-lactamase cloned in a mesophilic host, partial denaturation of the labile enzyme under conditions when the host proteins were still intact, allowed selective extraction in a PEG/salt system. Foam, generated by stirring of a non-ionic detergent Triton X-114 was used as a separation tool for isolation of cutinase mutants. The mutant with the hydrophobic fusion tag, (Trp-Pro)4 was recovered with high yields due to the stronger interaction with the detergent. Protein extraction into the foam was made more selective by integration of affinity interactions. The foam prepared from detergent modified with Cibacron blue could selectively extract hydrophilic LDH from muscle extract. The thermoseparating property of Triton X-114 further helped easy separation of the target enzyme from the bulk detergent.