J. Sampugna

Studies on the specificity of a lipase system fromGeotrichum candidum

The lipase system fromGeotrichum candidum preferentially hydrolyzed oleic acid, regardless of position, from the four possible racemic triglycerides containing oleate and palmitate. The rate of hydrolysis of these glycerides was most rapid when the substrate contained two moles of oleate. This acid was also preferentially released from a series of triglycerides containing oleate and two moles of a saturated fatty acid. The chain length of the latter did not alter the specificity for oleate.Equimolar quantities of oleic and linoleic acids were released when triolein and trilinolein (equimolar mixture) were hydrolyzed by this lipase. No differentiation between oleate and palmitoleate was observed when racemic glyceryl 1-palmitoleate-2,3-dioleate was the substrate. However, only 7.2 M%cis-vaccenic acid was released from glyceryl 1-cis-vaccenate-2,3-dioleate and 5.4 M% petroselinic acid from glyceryl 1-palmitoleate-2,3-dipetroselinate. It therefore appears that the enzyme may be specific forcis-9-unsaturation as well as forcis-9,cis-12-unsaturation. When specificity was assumed, the fatty acid compositions of the diglycerides obtained from digestions withG. candidum were close to theoretical.

Synthesis of 100-gram quantities of highly purified mixed acid triglycerides

Monopalmitin, mono-olein, and monostearin have been synthesized in quantities of up to 100 grams with recoveries ranging from. 80%-85%. The monoglycerides were reacted with a 0.5 molar excess of pyridine and fatty acid chloride to produce 1, 3,-diglycerides. The recoveries ranged from 60%-70% and, in addition, considerable amounts of triglyceride were obtained from this reaction. Partial glycerides were converted to 100-gram batches of triglyceride in a similar manner with recoveries ranging from 80%-90%. Purification of the partial glycerides was achieved by crystallization. In addition to this technique, certain triglycerides were purified by elution through a column of alumina. Purity of the glycerides, as determined by TLC, G-LC, and pancreatic lipolysis, was estimated to be at least 99%.

Digestion of butyrate glycerides by pancreatic lipase.

The racemic triglycerides, glyceryl-1-palmitate-2,3-dibutyrate (PBB), glyceryl-1-butyrate-2,3-dipalmitate (PPB), glyceryl-2-butyrate-1,3-dipalmitate (PBP), and the diglyceride, racemic glyceryl-1-palmitate-3-butyrate (P-B) were synthesized and digested with pancreatic lipase. Each triglyceride was mixed with equimolar amounts of triolein (OOO) prior to incubation.The following order of digestion rates was observed: PBB>PPB>PBP>P-B. There was no evidence for short-chain fatty acid specificity; however the triglycerides containing butyric acid were hydrolyzed more rapidly than OOO. Based upon the fatty acid composition of partial glycerides, digestion of butyrate glycerides was not a simple phenomenon. For example, in the digestion of PBB, butyric acid accumulated faster than palmitic acid in the diglycerides, and monobutyrin was found to accumulate when the diglyceride, P-B, was digested. As evidenced by the fatty acid composition of the monoglycerides, positional specificity of pancreatic lipase was always maintained.

Specificity of a lipase fromGeotrichum candidum forcis-octadecenoic acid

A lipase fromGeotrichum candidum released mostly oleic acid from glyceryl 1-elaidate-2,3-dioleate and very littletrans fatty acid from margarine. When cod liver, Macadamia nut, peanut and safflower oils were substrates, the oleic acid content of the free acids was always in excess of the amount of the acid in the intact triglycerides. Congo palm oil was digested by bothG. candidum and pancreatic lipases and the fatty acid compositions of the products of hydrolysis compared. The results obtained with the aid ofG. candidum lipase tend to substantiate existence of some of the triglyceride isomers predicted from pancreatic lipase data.

Suitability ofGeotrichum candidum lipase for the stereospecific analysis of some triglycerides

A procedure is described for determining the stereospecific structure of triacid triglycerides containing oleic acid. The method utilizes the unique specificity of the lipase system fromGeotricum candidum for hydrolyzing fatty acids which containcis-9-unsaturation.The procedure involves a pancreatic lipase hydrolysis of 10–20 mg of substrate to determine the fatty acids in the β-position and the incubation of another 50 mg of triglyceride withG. candidum lipase to obtain diglyceride for further treatment. The α,α- and α,β-diglycerides are collected separately, converted to phenyl phosphatides and treated with phospholipase A. The analysis of the monoglycerides, produced with pancreatic lipase and the analysis of the β-lysophosphatides allows the determination of the ratios of the original 2 position oleic acidsn-triglycerides while the analysis of the α-lysophosphatides aids in the determination of the originalsn-triglycerides which contained oleic acid in the 3 position. The 1 position oleic acidsn-triglycerides are calculated by difference.Racemic and enantiomeric triglycerides containing palmitic, stearic and oleic acids were synthesized and used to establish the limits of the new method. The good agreement between the actual and observed values for a mixture of isomers indicates that the procedure will be useful in the analysis of triacid triglycerides which contain oleic acid. The application of the procedure to triacid triglycerides which contain other unsaturated fatty acids is discussed.

The purification and specificity of a lipase fromVernonia anthelmintica seed.

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.

Lipolysis of trivernolin by pancreatic lipase

Vernolic acid (cis-12,13-epoxy-cis-9-oetadece-noic acid) occurs as the triglyceride in the seed ofVernonia anthelmintica. Incubation of the seed produces a 1,3-divernolin. To determine whether the structure of trivernolin is responsible for the apparent secondary ester position specificity of the natural enzyme, trivernolin and tri-olein, were incubated with pancreatic lipase and the free fatty acids and monoglycerides were determined after 5 and 15 min digestion periods. The preponderance of 2-monoglyceride produced by the action of pancreatic lipase was interpreted to indicate that the structure of trivernolin was not solely responsible for the secondary position specificity of theV. anthelmintica lipase toward trivernolin.