S. Jarudilokkul et al., Selective reverse micellar extraction of three proteins from filtered fermentation broth using response surface methodology, SEP SCI TEC, 35(4), 2000, pp. 503-517
The influence of three system parameters [bis(2-ethylhexyl)sulfosuccinate (
AOT) concentration, pH, and temperature] on the selective separation of cyt
ochrome c, lysozyme, and ribonuclease A from buffer solution and a filtered
fermentation broth was examined. It was found that a minimal AOT concentra
tion exists for greater than or equal to 90% extraction from a buffer solut
ion, and this concentration depends on the pH of solution and hydrophobicit
y of the protein. Extraction from filtered broth resulted in a reduction of
the minimal concentration for both cytochrome c and lysozyme, while for ri
bonuclease A it was unchanged. It appears that certain broth constituents a
ct as cosurfactants and reduce the charge repulsion between the surfactant
headgroups leading to smaller micelles. This is turn led to a reduction in
the pH range, although extraction yields were unaltered. The water content
(W-0) was measured in a reverse micellar phase in contact with buffer, filt
ered fermentation broth, and molecular weight fractionated or extracted bro
th. This data led to the conclusion that the broth constituents responsible
for changes in the minimal AOT concentration and W-0 were low molecular we
ight positively charged amino acids and phospholipids. Finally, response su
rface methodology was used to optimize key system parameters in order to ma
ximize protein extraction, and this technique minimizes the experiments req
uired. By using these optimized conditions, a mixture of the three proteins
could be recovered from a filtered broth with high yields (70-97%) and hig
h purity. Furthermore, the initial protein concentration was found not to i
nfluence protein recovery.