The purification and specificity of a lipase fromVernonia anthelmintica seed

Abstract

Acetone powders prepared fromVernonia anthelmintica seed catalyzed the release of 6.4 to 9.6 μ-moles of free fatty acids per milligram of protein when blended with olive oil and phosphate buffer and shaken for 20 min at 43 C. A 20 fold purification was achieved by differential centrifugation of an ammonium hydroxide extract of the acetone powder. Results from Sephadex G-200 chromatography and polyacrylamide gel electrophoresis suggested that the lipase activity was associated with a molecule of molecular weight greater than 200,000. Free fatty acids, 1,2- and 1,3-diglycerides, monoglycerides and glycerol were found in the digestion products. With most substrates the 1,2-to 1,3-diglyceride ratio was approximately 2∶1 and monoglycerides tended to accumulate. Analysis of the digestion products from synthetic triglycerides of known structure indicated that both primary and secondary ester positions of the triglyceride molecule were hydrolyzed and that considerable isomerization of 1,2-diglyceride to 1,3-diglyceride occurred. The monoglyceride was consistently lower than the 1,2-diglyceride and in the majority of cases also lower than the 1,3-diglyceride in the fatty acid originally present in the 2 position of the triglyceride. No fatty acid preference was observed.