Purification and characterization of the acid lipase from the endosperm of castor oil seeds

The acid lipase from seeds of the castor oil plant (Ricinus communis cv. zanzibarensis) was solubilised from delipidated oil bodies using CHAPS as the detergent. In order to remove residual triacylglycerol from the solubilised protein, the oil body membranes were incubated in buffer at pH 4.2, causing lipase catalysed hydrolysis of the triacylglycerols. The release fatty acids were extracted with diethyl ether thereby minimizing aggregation of the proteins. The lipase was further purified to apparent homogeneity by anion exchange, gel filtration and adsorption chromatography. Peak acid lipase fractions were subjected to SDS-PAGE and the subunit molecular weight of the acid lipase was determined to be about 58 kDa. An approximate native molecular mass of 110 kDa was estimated by gel filtration chromatography, suggesting the lipase exists as a dimer under certain conditions. The isoelectric point of the native protein was determined to be about pH 4.5 using a polyacrylamide-agarose gel containing CHAPS and an acid esterase stain to identify the position of the lipase. The molecular mass of the lipase purified by isoelectric focusing was also 58 kDa as judged by SDS-PAGE.