We have purified and examined the substrate specificity of four lipases from two strains of the mould Geotrichum candidum, ATCC 34614 and CMICC 335426. We have designated the lipases I and I1 (ATCC 34614), and A and B (CMICC 335426). The enzymes are monomeric and have similar molecular masses and pl. Thus, lipases I and I1 have native molecular masses of 50.1 kDa and 55.5 kDa, and p l o f 4.61 and 4.47, respectively.Lipases A and B are very similar to lipases I and I1 with native molecular masses of 53.7 kDa and 48.9 kDa, and p l of 4.71 and 4.50, respectively. Treatment with endo-P-N-acetylglucosaminidase caused a reduction in molecular mass of approximately 4.5 kDa for all four lipases, indicating that these enzymes are glycosylated. Western blotting shows that the lipases are related. However, lipase B from CMICC 335426 shows a remarkable specificity for unsaturated substrates with a double bond at position 9 (cis configuration), and this specificity is not exhibited by the other three lipases. No lipase of this unique specificity has previously been purified to homogeneity. Structural studies using these four lipases should allow insight into the molecular basis of this remarkable specificity.Lipases (glycerol ester hydrolases) catalyse the hydrolysis of ester linkages in lipids with the release of the constituent alcohol and acid moieties. They act at the interface between a substrate phase, which has a very low solubility in water, and an aqueous phase in which the enzyme is dissolved [l]. These enzymes can differ considerably in their positional specificity for the hydrolysis of fatty acid from a triacylglycerol backbone [2, 31. Advantage is taken of this, industrially, in the production of cocoa butter equivalents from cheap starting materials [4].In addition to positional specificity, lipases have been reported to have specificity for the fatty acids which are hydrolyzed. The most extreme example of this is an extracellular lipase isolated by Alford and Pierce from a strain of the mould Geotrichum candidum which has been reported to have a specificity for unsaturated substrates [5]. This enzyme has not been purified and the original strain was thought to be no longer in existence [6]. However, in 1969 our laboratory received a strain of G. candidum from Dr Alford, and this has now been deposited at the Commonwealth Mycological Institute Collection as CMICC 335426.Tsujisaka et al. have purified a lipase from G. candidum ATCC 34614 to homogeneity [7], and this does not appear to show the same substrate specificity as the enzyme isolated by Alford and Pierce [8,9] [19], and these lipases could possess different substrate specificities. Specificity studies of crude extracts can therefore be complicated by the presence of more than one lipase. Sugihara et al. [16] have, however, reported the purification of two lipases from G. candidurn ATCC 34614, but the two enzymes appear to display the same substrate specificity towards a variety of triglycerides.To appreciate the relationship between substrate specific...

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