Surfactant precipitation applied as a surfactant mediated protein purification
technique has considerable potential in protein extraction, and therefore the
understanding of the interactions involved and the folding behaviour in the
precipitated protein was the first aim of this thesis. The key system parameters such as
buffer salt concentration, molar ratio of surfactant to protein and pH which determines
the protein stability in protein-surfactant complex formation were evaluated. The
surfactant:protein ratio determines saturation of protein binding sites while pH
determines the strength of affinity for ionic binding which influences hydrophobic
binding with surfactant monomers causing the protein to lose its conformation. The
protein-surfactant binding varied for lysozyme, cytochrome c and ribonuclease A with
trypsin and α -chymotrypsin, and hence the denaturation profile.
In the second aim, protein recovery from surfactant precipitation was enhanced by
improving the solvent recovery method and, implementing a new and novel
counterionic surfactant recovery method. The effect of a variety of recovery phases
and solution conditions on lysozyme recovery was analysed in terms of their ability in
maintaining protein stability, recovery yield, and activity. It was found that solvent
recovery was limited by solvent polarity and protein solubility, and that the cationic
surfactant, trioctylmethylammonium chloride (TOMAC), used to form nonpolar ion
pairs with sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) was the most efficient
method for recovering protein.
The third aim was to assess the influence of protein properties, such as charge and
hydrophobicity, on protein separation. The selective extraction of a target protein
from mixtures of proteins in both buffer and fermentation broth was investigated. It
appears that the optimum surfactant:protein molar ratio for the extraction of the
proteins from fermentation broth (lysozyme, cytochrome c and ribonuclease A; 16, 17
and 22 respectively) were similar to those in a buffer system. Lysozyme and
ribonuclease A were selectively separated from a binary mixture. The extraction
behaviour was well represented by surface charge distribution which is indifferent to
system conditions. However, certain broth constituents induced the formation of some
unfolded irreversible non-dissolvable precipitate in the recovery process.
Finally, the use of non-ionic surfactants, ionic/non-ionic mixed surfactants, and
cationic surfactants were investigated in surfactant precipitation system. Non-ionic
surfactant does not support direct precipitation of proteins using surfactant or
recovery of protein from a protein-surfactant complex, and has no effect in a mixed
ionic/non-ionic system. The application of cationic surfactant precipitation to separate
trypsin inhibitor was attempted, and good recovery was obtained.