LIPOLYTIC ENZYMES SEPARATION AND PURIFICATION THROUGH FUNCTIONALIZED SYNTHETIC POLYMERS

Abstract

A new class of functionalized synthetic polymers was prepared for the purification of lipases by a single step affinity chromatography. Polyvinyl alcohol polymers were crosslinked with epichlorohydrin and esterified with fatty acids of different length. The resulting resins were characterized by infrared spectroscopy, electron microscopy and titration of the hydrolysed product to evaluate the degree of esterification. The attention was focused on lauryl ester of polyvinyl alcohol as commercial preparation of Candida cylindracea lipase showed the highest enzyme affinity for esters of fatty acids with a linear chain ranging from 8 to 12 carbon atoms. Chromatographic lipase purification trials were performed on a 7 cm x 1.6 cm i.d. column. Good separation conditions were found by utilizing stepwise increase in CHAPS (3-(3-(cholamido-propyl)-dimetil-ammonio)-1-propan sulfonate) concentration in HEPS/EDTA buffer. The lipase obtained by elution with CHAPS 6.0 mmol/L showed a high purity, as established by SDS-PAGE, where an unique band with a molecular weight of 60.000 was identified.