INTRINSIC ACTIVITY AND CATALYTIC RESIDUES OF THE LIPASE FROM GEOTRICHUM CANDIDUM

Abstract

The triacylglycerol hydrolase secreted by Geotrichum candidum reportedly prefers substrates having fatty acids with a cis-double bondin A9-position. A simple and rapid two step methodfor the purification of high amountsofthis lipase was developedstarting from the commercially available "GC-4" lipase powder (Amano, Japan). The molecular massof the purified lipase was 61 kDa, the carbohydrate content 8.8 %. The heterogeneity observedin isoelectric focusing was dueto variable glycosylation of the core protein. With the aim to bioengineer new product oils the lipase was immobilized to an anion exchanger and usedascatalystin the acidolytic modification of subcutaneouscalf fat with isostearic acid. Reaction rates and yields were lower in comparisonto catalysis by other immobilized microbiallipases. With the immobilizate of Geotrichum candidumlipaseoleic acid waspreferentially replaced in the triglycerides by isostearic acid, however. This unique substrate specificity of the enzyme thus prevails in transesterification processes as well. Modification studies with reagents specific for amino acid residues presumedto be active in the catalytic center showedthat the Geotrichum candidumlipase wasvery resistentto inhibition, as only a carbodiimide and photooxydation rendered the lipase inactive. With the aim to enhance the accessibility of the active center, reagents were then applied in the presence of 20 % isopropanol and inhibition with the serine specific reagents phenylmethylsulfony!fluoride and benzene boronic acid was achieved. Thedata indicate that a catalytic triad aspartic acid : histidine : serine may beinvolvedin catalysis.