Acylglycerol lipase

Second, esterases have broad (or even very broad) and overlapping substrate specificities. For example, carboxylesterase (EC 3.1.1.1) also catalyzes reactions characteristic of a number of other hydrolases. The discovery that individual isoenzymes of carboxylesterases may be identical to or closely related to acylglycerol lipase, acylcamitine hydrolase, and palmitoyl-CoA hydrolase (see Sect. 2.4.3) has increased the confusion surrounding esterase classification [59], Many esterases are able to hydrolyze amides, thiolesters,... 

Conceptually, assays for lipase activity using the colorimetric method (copper-soap procedure Basic Protocol 2) are similar to titrimetry in that liberated fatty acids are being measured however, the colorimetric method is more specific for fatty acids (Lowry and Tinsley, 1976). Quenched subsamples of emulsified acylglycerol/lipase reaction mixtures are combined with the biphasic mixture of cupric acetate/pyridine and benzene. Cupric salts of the fatty acids are formed (molar stoichiometry of fatty acid to Cu2+ of 4 2) and these soaps, which are blue in color, are partitioned into benzene to allow for quantification by measuring absorbance of the clear benzene phase at 715 nm. 

Lipases a group of carboxylesterases, which preferentially hydrolyse emulsifi neutral fats to fatty acids and glycerol or monoacylglycerols. Calcium ions are required for activity. Pancreatic L. also requires taurocholate. Pancreas and certain plant seeds (e.g. Ricinus) contain particularly high activities of L.In addition, high activities are found in adipose tissue, in the stomach (especially in unweaned infants) and in the liver. Pancreatic L. has M, 35,000. Rapid removal of the third and last fatty acid residue from mixed triacylglycerols is catalysed by a specific mono-acylglycerol lipase (EC 3.1.1.23) produced by the intestinal mucosa. 

Acylglycerols can be hydrolyzed by heating with acid or base or by treatment with lipases. Hydrolysis with alkali is called saponification and yields salts of free fatty acids and glycerol. This is how soap (a metal salt of an acid derived from fat) was made by our ancestors. One method used potassium hydroxide potash) leached from wood ashes to hydrolyze animal fat (mostly triacylglycerols). (The tendency of such soaps to be precipitated by Mg and Ca ions in hard water makes them less useful than modern detergents.) When the fatty acids esterified at the first and third carbons of glycerol are different, the sec-... 

Fats (triacylglycerols) are mainly attacked by pancreatic lipase at positions 1 and 3 of the glycerol moiety. Cleavage of two fatty acid residues gives rise to fatty acids and 2-mono-acylglycerols, which are quantitatively the most important products. However, a certain amount of glycerol is also formed by complete hydrolysis. These cleavage products are resorbed by a non-ATP-dependent process that has not yet been explained in detail. 

Cystic fibrosis can obstruct pancreatic ducts due to mucous plugging and impaired secretion of pancreatic enzymes such as lipase and phospholipases, which decreases hydrolysis and uptake oftri-acylglycerols. 

Figure 8-1. Hormonal regulation of fat metabolism. A Control of fatty acid synthesis by reversible phosphorylation of acetyl CoA carboxylase. B Regulation of tri-acylglycerol degradation by reversible phosphorylation of hormone-sensitive lipase. cAMP, cyclic adenosine monophosphate HS, hormone-sensitive.

Other Acylglycerols. If some of the DGs in freshly drawn milk are involved in biosynthesis, it is possible that they are enantiomeric and are probably the sn-1,2 isomer. If so, the constituent fatty acids are long chain. Their configuration can be determined by stereospecific or other analyses, but it is difficult to accumulate enough material for analysis. Nevertheless, Lok (1979) isolated the DGs from freshly extracted cream as the trityl derivatives. Trityl chloride reacts selectively with primary hydroxyls. The stereochemical configuration of the DGs was identified as sn-1,2 therefore, these residual DGs were most likely intermediates of biosynthesis. If the DGs were products of lipol-ysis, they would be a mixture of 1,2/2,3 isomers in a ratio of about 1 2, since milk lipoprotein lipase preferentially attacks the sn-1 position of TGs (Jensen et al. 1983). 

Jensen, R.G. 1983. Detection and determination of lipase (acylglycerol hydrolase) activity from various sources. Lipids 18 650-657. 

Thus, description of a simple change of fatty acid at the primary position required frequent changes of configuration prefixes. Also, the R/S system, like the older D/L one, did not account for the stereospecificity of the acylglycerol derivatives toward lipases (phospholipase A2 in particular). Finally, nonrandom distribution of fatty acids in natural or synthetic enantiomeric acylglycerols could not be systematically correlated by reference to either the R/S or D/L configuration. 

Figure 2.1. Irreversible inactivation of immobilized C. antarctica lipase by contact with MeOH micelles. One reaction mixture ( ) was composed of vegetable oil and MeOH, and the other mixture (O) was composed of acylglycerols/33wt% FAMEs mixture and MeOH. The reaction mixture (10 g) was shaken at 30 °C for 24 h with 4wt% immobilized lipase. The amount of MeOH was expressed as the molar ratio to the amount of total FAs in the system. The conversion was expressed as the amount of MeOH consumed for the ester conversion of acylglycerols (when the amount of MeOH is less than that of FAs in acylglycerols), and as the molar ratio of FAMEs to total FAs in the system (when the amount of MeOH is more than that of FAs in acylglycerols). Arrows indicate the region in which a part of MeOH exists as micelles.

Second-Step Reaction Methanolysis of TAGs After the first-step reaction, immobilized lipase was removed and the oil fraction recovered. MeOH and water in the oil fraction were then removed by evapolation. The resulting mixture was named dehydrated first-step product, which was composed of 80wt% FAMEs, 2wt% FFAs, 10wt% acylglycerols, lwt% sterols, 2wt% FAStEs, 5wt% unknown lipophilic compounds. The second-step reaction is methanolysis of acylglycerols in the dehydrated first-step product. 

Figure 2.5. Stabilization of immobilized C. antarctica lipase in the second-step reaction by addition of vegetable oil and glycerol. The reaction was repeated at 30 °C with 6 wt% immobilized lipase by transferring the lipase to a fresh substrate mixture every 24 h. A, A reaction mixture was composed of dehydrated first-step product, rapeseed oil, and MeOH. Rapeseed oil was added to give the acylglycerol content of 50%, and the amount of MeOH was an equimolar amount to unreacted FAs. B, A reaction mixture was prepared by adding 10wt% glycerol to the mixture used in Figure A. O, The content of FAMEs at 2h , at 4h , at 24h. Dotted lines indicate the content of FAMEs before the reaction (44.1 wt%).

To conduct three reactions (hydrolysis of acylglycerols, esterification of sterols with FFAs, and methyl esterification of FFAs), two lipases from C. 

Figure 2.9. Time course of two-step in situ reaction for purification of tocopherols and sterols in VODDTSC. A mixture of VODDTSC, 5wt% water, and 250 U/g C. rugosa lipase was agitated with dehydration at 20mm Hg. At 24h indicated with dotted line, 20 wt% water, 7 mol MeOH for FFAs, and 25 U/g Alcaligenes lipase were added to the reaction mixture, and the reaction was continued at ordinary pressure. .The content of sterols , FAStEs O, FAMEs , FFAs A, acylglycerols , tocopherols.

Uzawa, H., Noguchi, T., Nishida, Y., Ohrai, H., and Meguro, H. 1993. Determination of the lipase stereospecificities using circular dichroism (CD) lipases produce chiral di-O-acylglycerols from achiral tri-O-acylglycerols Biochim. Biophys. Acta(-Lipids and Lipid Metabolism), 1168, 253-260. 

Figure 2-7 Plane of Symmetry of a Glycerol Molecule (Top) and Mirror Image of Two Enantiomers of a Mono-Acylglycerol (bottom). Source Reprinted with permission from P. Ville-neuve and T.A. Foglia, Lipase Specificities Potential Application in Bioconversions, Inform, 8, pp. 640-650, 1997, AOCS Press.

Because vitamin E is transported in lipoproteins secreted hy the liver, the plasma concentration depends to a great extent on total plasma lipids. Erythrocytes may also he important in transport, because there is a relatively large amount of the vitamin in erythrocyte membranes, and this is in rapid equilibrium with plasma vitamin E. There are two mechanisms for tissue uptake of the vitamin. Lipoprotein lipase releases the vitamin by hydrolyzing the tri-acylglycerol in chylomicrons and VLDL, whereas separately there is receptor-mediated uptake of LDL-bound vitamin E. Studies in knockout mice suggest that the main mechanism for tissue uptake of vitamin E from plasma lipoproteins is byway of the class B scavenger receptor (Mardones et al., 2002). 

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