Purification and characterization of Lip2 and Lip3 isoenzymes from a Candida rugosa pilot-plant scale fed-batch fermentation

Previous purification of a crude extracellular enzyme preparation from Cand ida rugosa ATCC 14830 pilot-plant fed-batch fermentations showed the presen ce of two lipase isoenzymes, Lip2 and Lip3, differing in their molecular ma sses (58 and 62 kDa, respectively). These enzymes were purified but the lip ases were forming active aggregates with a molecular mass higher than 200 k Da. In this work we developed a purification method following three steps: ammonium sulfate precipitation, sodium cholate treatment and ethanol/ether precipitation, and anion exchange chromatography which allowed the sequenti al disaggregation of the isoenzymes. Pure and monomeric Lip2 and Lip3 were characterized according to pi, glycosylation and activity for p-nitrophenol esters and triacylglycerols of varying acyl chain. Lip3 was the best catal yst for the hydrolysis of the simple esters and triacylglycerols with short and medium acyl chains. (C) 2000 Elsevier Science B.V. All rights reserved .