Ester-exchange reaction between triglycerides with polyethylene glycol-modified lipase

Summary Polyethylene glycol-modified lipase efficiently catalyzed esterexchange reaction between trilaurin and triolein in the straight hydrophobic substrates. Dilauroyl-monooleoyl-glycerol and monolauroyl-dioleoyl-glycerol were formed from two triglyceride-substrates, trilaurin and triolein, in the presence of the modified lipase at 58°C. Consequently, the melting temperature of the mixture of two substrates was decreased from 33–36°C to 11–13°C. Similar ester-exchange reaction took place between fat and oil composed of triglycerides, accompanied by the decrease in the melting temperature of the reaction mixture.     

Rapid thermogravimetric measurements of boiling points and vapor pressure of saturated medium- and long-chain triglycerides

In developing compositional models for biomass-based diesel fuel extenders, volatility properties of medium- and long-chain saturated triglycerides are essential to predict the impact of low levels of these compounds in mixtures with short-chain triglycerides. A thermogravimetric analysis (TGA) method for rapid measurement of boiling points and vapor pressure was used to obtain data for four pure medium- and long-chain triglycerides. Normal boiling points at 1 atm and the temperature dependence of vapor pressure from 760 mm down to 25 mm Hg were obtained for trilaurin (C12:0), trimyristin (C14:0), tripalmitin (C16:0), and tristearin (C18:0). The data showed good agreement with the Clausius-Clapeyron model for temperature dependence of vapor pressure up to 1 atm pressure. The results of this study were consistent with those obtained using differential scanning calorimetry (DSC) and with data previously reported for reduced pressure.     

Randomly interesterified triacylglycerol containing medium- and long-chain fatty acids stimulates fatty acid metabolism in white adipose tissue of rats

We have reported previously that randomly interesterified triacylglycerol containing medium- and long-chain fatty acids in the same glycerol molecule (MLCT) resulted in significantly lower body fat accumulation and higher hepatic fatty acid oxidation than from long-chain triacylglycerol (LCT) in rats. To understand the metabolic changes occurring in white adipose tissue, the fatty acid oxidation and synthesis, and the adipocytokine level were measured in rats fed with MLCT or LCT for 2 weeks. In comparison with LCT, MLCT lowered not only the fatty acid synthase and glycerol-3-phosphate dehydrogenase activities in perirenal adipose tissue, but also the serum insulin and leptin levels, in addition to significantly reducing the body fat accumulation. In contrast, fatty acid oxidation measured as the carnitine palmitoyltransferase activity in the tissue was significantly higher in the MLCT-fed rats than in the LCT-fed rats. It seems that the altered fatty acid metabolism in adipose tissue per se was also responsible for the lower adiposity by dietary MLCT.     

Effect of randomly interesterified triacylglycerols containing medium- and long-chain fatty acids on energy expenditure and hepatic fatty acid metabolism in rats

In our previous studies, medium- and long-chain triacylglycerols (MLCT), randomly interesterified triacylglycerols containing medium-chain and long-chain fatty acids in the same glycerol molecule, significantly reduced body fat accumulation in humans and rats. To clarify mechanism(s) for this effect of MLCT, we measured energy expenditure and hepatic fatty acid metabolism in rats by comparison with long-chain triacylglycerols (LCT) or medium-chain triacylglycerols (MCT). MLCT, compared with LCT, showed significantly lower body fat accumulation, higher 24-h energy expenditure and acyl-CoA dehydrogenase activity measured using octanoyl-CoA as a substrate, and similar lipogenic activity. MCT, compared with LCT, showed significantly higher energy expenditure, but fat accumulation was comparable. Additionally, MCT exhibited significantly higher lipogenic activity than the other oils. These data suggest that enhancement of energy expenditure and medium-chain fatty acids (MCFA) oxidation without activating de novo lipogenesis are responsible at least for the lower body fat accumulation in rats fed MLCT. The activation of hepatic lipogenesis by excessive intake of MCFA might counteract their preventive effects on body fat accumulation.     

Kinetics of the inhibition of hog kidney D-amino acid oxidase by short-, medium- and long-chain fatty acids

Various fatty acids were studied in vitro as inhibitors of pure hog kidney D-amino acid oxidase by means of a spectrophotometric peroxidase-coupling method using D-methionine as a substrate. All the fatty acids tested behaved as substrate-competitive inhibitors of the enzyme. The affinity of the saturated aliphatic acids for D-amino acid oxidase decreased from pentanoate (5:0; Ki = 220 microM) to laurate (12:0; Ki = 675 microM), then rose to a maximum with stearate (18:0; Ki = 36 microM), suggesting the presence of a site in the active center of the enzyme that accepts long-chain fatty acid alkyl groups. Unsaturation did not further increase the affinity of the fatty acid for this binding site.     

Lipase-catalyzed synthesis of medium- and long-chain diesters of 2-oxoglutaric acid

Abstract

The influence of various reaction parameters, such as alcohol-to-substrate ratio, enzyme-to-substrate ratio, solvent and temperature, on the enzymatic preparation of a series of novel medium- and long-chain esters of 2-oxoglutaric acid has been evaluated. Among the tested lipases, those from Candida antarctica and Carica papaya appeared to be the best catalysts. Mild reaction conditions and low environmental impact make the biocatalytic procedure a convenient way to prepare the reported products, which are potential fat substitutes in the food industry.     

Hydrolysis of long-chain, n-3 fatty acid enriched chylomicrons by cardiac lipoprotein lipase

The hydrolysis of chylomicrons enriched in long-chain n-3 fatty acids by cardiac lipoprotein lipase was studied. In 60 min, 24.8% of the triacylglycerol fatty acids were released as free fatty acids. The fatty acids were hydrolyzed at different rates. DHA (docosahexaenoic acid, 22:6n-3) and EPA (eicosapentaenoic acid, 20:5n-3) were released at rates significantly less than average. Stearic acid (18:0), 20:1n-9, and alpha-linolenic acid (18:3n-3) were released significantly faster than average. There was no relationship between the rate of release of a fatty acid and the number of carbons or the number of double bonds. Lipoprotein lipase selectively hydrolyzes the fatty acids of chylomicron triacylglycerols. This selectively will result in remnants that are relatively depleted in 18:0, 20:1, and 18:3 and relatively enriched in 20:5 and 22:6.     

Effect of randomly interesterified triacylglycerol containing medium- and long-chain fatty acids on hepatic fatty acid oxidation after a single administration to rats

MLCTs, which are randomly interesterified triacylglycerol containing medium- and long-chain fatty acids in the same glycerol molecule, showed significantly higher acyl-CoA dehydrogenase activity when measured by using butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA as substrates than long-chain triacylglycerol one hour after a single administration to rats. These results suggest that not only medium-chain fatty acid oxidation, but also long-chain fatty acid oxidation were increased in the liver of rats administered with MLCT.     

Contrasting metabolic effects of medium- versus long-chain fatty acids in skeletal muscle

Dietary intake of long-chain fatty acids (LCFAs) plays a causative role in insulin resistance and risk of diabetes. Whereas LCFAs promote lipid accumulation and insulin resistance, diets rich in medium-chain fatty acids (MCFAs) have been associated with increased oxidative metabolism and reduced adiposity, with few deleterious effects on insulin action. The molecular mechanisms underlying these differences between dietary fat subtypes are poorly understood. To investigate this further, we treated C2C12 myotubes with various LCFAs (16:0, 18:1n9, and 18:2n6) and MCFAs (10:0 and 12:0), as well as fed mice diets rich in LCFAs or MCFAs, and investigated fatty acid-induced changes in mitochondrial metabolism and oxidative stress. MCFA-treated cells displayed less lipid accumulation, increased mitochondrial oxidative capacity, and less oxidative stress than LCFA-treated cells. These changes were associated with improved insulin action in MCFA-treated myotubes. MCFA-fed mice exhibited increased energy expenditure, reduced adiposity, and better glucose tolerance compared with LCFA-fed mice. Dietary MCFAs increased respiration in isolated mitochondria, with a simultaneous reduction in reactive oxygen species generation, and subsequently low oxidative damage. Collectively our findings indicate that in contrast to LCFAs, MCFAs increase the intrinsic respiratory capacity of mitochondria without increasing oxidative stress. These effects potentially contribute to the beneficial metabolic actions of dietary MCFAs.     

Transesterification of phospholipids or triglycerides to fatty acid benzyl esters with simultaneous methylation of free fatty acids for gas-liquid chromatographic analysis

A novel method is presented for transesterification of fatty acid esters in phospholipids and triglycerides to benzyl esters while simultaneously recovering free fatty acids as methyl esters. Transesterification is catalyzed by 0.2 M (m-trifluoromethyl phenyl)trimethyl ammonium hydroxide in methylene chloride, 10% (v/v) benzyl alcohol, and 1% (w/v) potassium tert-butoxide, and is complete in 30 min at room temperature. Methyl esters of all common fatty acids separate from the benzyl esters formed from phospholipids. This method has broad utility and is applicable to the formation of esters optimized for detection by absorbance or fluorescence (high performance liquid chromatography), electron capture (gas-liquid chromatography), or negative ion chemical ionization (gas-liquid chromatography-mass spectrometry).     

Transesterification between triolein and stearic acid catalyzed by lipase in CO2 at various pressures

Transesterification between triolein and stearic acid catalyzed by lipase in pressurized CO2 at 50 °C was classified into three regions according to the pressure. Below 5 MPa which was the non-solvent region, the reaction was limited in the liquid triolein phase and the reaction rate was very slow. In the near critical region, from 5 to 10 MPa, the reaction rate was maximal at 5.9 MPa because of the stabilization of the enzyme-substrate complex. In the supercritical region, above 10 MPa, the reaction rate increased with an increase in pressure reflecting the increase in solubility of substrate in supercritical CO2 © Rapid Science Ltd. 1998     

Characterization of triacsin C inhibition of short-, medium-, and long-chain fatty acid: CoA ligases of human liver

Short-, medium-, and long-chain fatty acid:CoA ligases from human liver were tested for their sensitivity to inhibition by triacsin C. The short-chain fatty acid:CoA ligase was inhibited less than 10% by concentrations of triacsin C as high as 80 microM. The two mitochondrial xenobiotic/medium-chain fatty acid:CoA ligases (XM-ligases), HXM-A and HXM-B, were partially inhibited by triacsin C, and the inhibitions were characterized by low affinity for triacsin C (K(I) values > 100 microM). These inhibitions were found to be the result of triacsin C competing with medium-chain fatty acid for binding at the active site. The microsomal and mitochondrial forms of long-chain fatty acid:CoA ligase (also termed long-chain fatty acyl-CoA synthetase, or long-chain acyl-CoA synthetase LACS) were potently inhibited by triacsin C, and the inhibition had identical characteristics for both LACS forms. Dixon plots of this inhibition were biphasic. There is a high-affinity site with a K(I) of 0.1 microM that accounts for a maximum of 70% of the inhibition. There is also a low affinity site with a K(I) of 6 microM that accounts for a maximum of 30% inhibition. Kinetic analysis revealed that the high-affinity inhibition of the mitochondrial and microsomal LACS forms is the result of triacsin C binding at the palmitate substrate site.The high-affinity triacsin C inhibition of both the mitochondrial and microsomal LACS forms was found to require a high concentration of free Mg(2+), with the EC(50) for inhibition being 3 mM free Mg(2+). The low affinity triacsin C inhibition was also enhanced by Mg(2+). The data suggests that Mg(2+) promotes triacsin C inhibition of LACS by enhancing binding at the palmitate binding site. In contrast, the partial inhibition of the XM-ligases by triacsin C, which showed only a low-affinity component, did not require Mg(2+).     

Transesterification of primary and secondary alcohols using Pseudomonas aeruginosa lipase

Lipases of a newly isolated Pseduomonas aeruginosa MTCC 5113 were assessed for transesterification of benzyl alcohol and vinyl acetate to produce the flavoring agent benzyl acetate. Crude lipase preparations that minimized the cost of the biocatalyst, achieved benzyl alcohol conversion of 89% within 3h at 30 degrees C. In contrast, purified and expensive commercially available lipases of Candida antarctica and porcine pancreas achieved much lower conversions at 80% and 15%, respectively. A well-mixed ( approximately 800 rev.min(-1)) batch reactor having the aqueous phase finely dispersed in heptane was used in these studies. Benzyl alcohol conversion was maximal when the enzyme-containing aqueous phase constituted about 50% of the total reactor volume. Use of solvents such as hexane, benzene, toluene and dimethyl sulfoxide reduced conversion compared with the use of heptane.     

Acidolysis between triolein and short-chain fatty acid by lipase in organic solvents

Ten kinds of lipases were examined as biocatalysts for the incorporation of short-chain fatty acids (acetic, propionic, and butyric acids) into triolein in order to produce one kind of reduced-calorie structured lipids. Trans-esterification (acidolysis) was successfully done in n-hexane by several microbial lipases. Among them, lipase from Aspergillus oryzae was used to investigate the effects of incubation time, substrate molar ratio, and water content on acidolysis. Finally, more than 80% of triolein was incorporated by butyric acid (molar ratio of triolein to butyric acid, 1:10) in the dried n-hexane at 52 degrees C for 72 h. More than 90% of the products was monosubstituent, which was esterified with this short chain fatty acid at the 1-position of the glycerol moiety of triolein. These results suggest that A. oryzae lipase would be a powerful biocatalyst for the synthesis of low caloric oil, such as triacylglycerol containing a mixture of long- and short-chain aliphatic acids.     

Surfactant-Modified lipase for the catalysis of the interesterification of triglycerides and fatty acids

The lipase-catalyzed intresterification of triglycerides and fatty acids in n-hexane was studied. Initially, lipase Saiken was modified with a surfactant of sorbitan esters so that its dispersibility in hydrophobic organic media was improved. The surfactant-modified lipase formed in the modification process carried out in a buffer solution has 1,3-positional specificity and predominantly catalyzed the interesterification reaction in a microaqueous n-hexane system. The modification technique converted inactive lipases to very active biocatalysts for the interesterification of triglycerides and fatty acids. The pH and the weight ratio of surfactant to enzyme used during the lipase modification process have shown significant effects in determining the recoveries of the protein and enzyme activity from the buffer solution, the protein content of the modified lipase complex after being freeze dried, and the interesterification activity of the complex. The water content in the reaction solution has strongly influenced the enzyme activity as well as the distribution of the products. (c) 1995 John Wiley & Sons, Inc.