PURIFICATION OF THE PENICILLIUM-CITRINUM LIPASE USING AOT REVERSED MICELLES

This work describes the extraction and back-extraction of a lipase fro m crude extract of Penicillium citrinum using AOT reversed micelles in isooctane. The effect of pH, ionic strength, AOT concentration on the protein forward and backward transfer at 20 degrees C was studied. Th e maximum protein forward extraction (32.0%) was achieved at pH 4.0 wi th a 50 mmol dm(-3) acetate buffer containing 100 mmol dm(-3) KCl and 100 mmol dm(-3) AOT in isooctane. Proteins were back-extracted (82.7%) to a new aqueous phase containing 100 mmol dm(-3) pH 8.0 phosphate bu ffer and 1000 mmol dm(-3) KCl. No enzyme activity could be detected ei ther in the micellar phase or in the aqueous phase after protein back- extraction. However, the lipolytic activity was recovered after hydrop hobic interaction chromatography on a Phenyl Superose column. The yiel d obtained for the overall process was 68% for activity, 26.4% for pro tein recovery and the purification factor was 810-fold. A single prote in band at 33 000 Da was obtained for SDS-PAGE analysis for the recove red and purified enzyme.