PARTIAL-PURIFICATION AND SOME PROPERTIES OF BRASSICA-NAPUS LIPASE

Lipase (triacylglycerol acylhydrolase EC 3.1.1.3) from rape (Brassica napus cv. Ceres) was isolated from cotyledons of dark-grown seedlings. The enzyme was partially purified by polyethylene glycol precipitation. Delipidation of the lipase with n-hexane was required prior to further purification by anion exchange chromatography and size exclusion chromatography. A purification factor of 337 was ultimately achieved and the purification process was monitored by SDS-PAGE. Here, at least two protein bands with molecular masses of 62 and 64 kDa respectively were found in the active fraction obtained by size exclusion chromatography. Sodium deoxycholate was found to stimulate the lipase activity, but appeared to cause aggregation of the enzyme. It was not possible to estimate the isoelectric point of the dialyzed rape lipase due to the high molecular mass of the aggregates. Two simple methods to detect lipase activity directly on polyacrylamide gel were applied. No esterase activity was found by using p-nitrophenyl acetate as substrate.