Determination of lipase specificity.
Specificity of lipases is controlled by the molecular properties of the enzyme, structure of the substrate and factors affecting binding of the enzyme to the substrate. Types of specificity are as follows. I. Substrate: (a) different rates of lipolysis of TG, DG, and MG by the same enzyme; (b) separate enzymes from the same source for TG, DG and MG. II. Positional: (a) primary esters; (b) secondary esters; and (c) all three esters or nonspecific hydrolysis. III. Fatty acid, preference for similar fatty acids. IV. Stereospecificity: faster hydrolysis of one primary sn ester as compared to the other. V. Combinations of I-IV. Lipases with these specificities are: Ia, pancreatic; Ib, postheparin plasma. IIa, pancreatic; IIb, Geotrichum candidum, for fatty acids with cis-9-unsaturation, and IIc, Candida cylindracea. III, G. candidum for unsaturates. IV. sn-1, postheparin plasma and sn-3 human and rat lingual lipases. V. Rat lingual lipase. Methods for determination involve digestion of natural fats of known structure and synthetic acylglycerols followed by analysis of the lipolysis products. All of the types of specificity have been detected with use of synthetic acylglycerols. Detection of stereospecificity requires enantiomeric acylglycerols which are difficult to synthesize, so other methods have been developed. These involve the generation of 1,2-(2,3)DG and resolution of the enantiomers. Trioleoylglycerol or racemic TG can be used as substrates. If the lipase is stereospecific, then either the sn-1,2- or 2,3-enantiomer will predominate. The relative amounts of the enantiomers can be determined by measurement of specific rotation, and nuclear magnetic resonance spectra. The DG can also be separated by conversion to phospholipids and hydrolysis with phospholipases A-2 or C. Applications of these procedures are discussed and data on the specificity of various lipases presented.
D000818:Animals; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D005227:Fatty Acids / Q000378:metabolism; D006801:Humans; D008049:Lipase / Q000378:metabolism*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D005227:Fatty Acids; D008049:Lipase