Selective reverse micellar extraction of three proteins from filtered fermentation broth using response surface methodology

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.