Purification of recombinant cutinase by extraction in an aqueous two-phasesystem facilitated by a fatty acid substrate

Purification of recombinant wild-type cutinase from the culture supernatant of Saccharomyces cerevisiae by extraction in aqueous two-phase system was investigated. The partition of the enzyme in a polyethylene glycol (PEG)-po tassium phosphate system to the top phase was increased with lower molecula r weight PEG. Enzyme partition in a 20% PEG/15% phosphate two-phase system was studied in the presence of detergents, fatty acids, and alcohols, respe ctively. Addition of 0.5% (w/w) butyrate increased the partition coefficien t from 17 to 135 and the purification factor from 10 to 23. The effect of b utyrate was also confirmed by using the countercurrent mode of extraction. Recovery of cutinase from the top phase was achieved by a secondary extract ion into a new salt phase at a tower pH or a lower temperature. A specific interaction of butyrate to the active site of the enzyme was demonstrated b y fluorescence spectroscopy. Size exclusion chromatography showed the cutin ase-butyrate complex to be over two times the size of the free enzyme. (C) 2001 John Wiley & Sons, Inc.