Determining the regio- and typo-selectivity of calf pregastric lipase

A selection of natural lipids, milk fat, cocoa butter and soybean oil, and three synthetic triglycerides 1,3-dipalmitoyl-2-oleoyl-rac-glycerol (POP), 1(3),2-palmitoyl-3(1)-oleoyl-rac-glycerol (PPO), and 1,3-dipalmitoyl-2-butyryl-rac-glycerol (PBP) were used as substrates for calf pregastric lipase (CPGL) catalysed hydrolysis at pH 6.5, 37.5 °C. CPGL preferentially releases short-chain fatty acids at the sn-3 position and unsaturated fatty acids during the hydrolysis of lipids containing long-chain fatty acids, and its activity increases with increasing unsaturation in the carbon chain of the long-chain fatty acids. The relative rate constants of hydrolysis were 1.56, 0.99, 0.73 h−1 for C4:0, C6:0, C8:0 esters, respectively, at sn-3; 0.0097, 0.0047 h−1 for the C16:0, C18:0 esters at sn-1,3, respectively; 0.0094 h−1 for the C18:1 ester at sn-2; and 0.0073, 0.0085, 0.012 h−1 for C18:1, C18:2, C18:3 esters, respectively, at sn-1,2,3. These last results confirmed that CPGL enzyme does not exhibit positional selectivity towards lipid substrates. The rate constant of hydrolysis was 1.14 h−1 for the C4:0 ester at sn-2. s synthesised in four steps from commercially available glycerol as starting material. Glycerol was first selectively protected as a bis-silylether at the primary alcohol (sn-1,3) positions. The free alcohol at the sn-2 position was then esterified with butyric anhydride. The protecting groups at C1 and C3 were then removed, and the resultant hydroxyl groups esterified with palmitic acid.