Lipase-catalyzed esterification of glycerol and polyunsaturated fatty acids from fish and microalgae oils

This paper reports on the synthesis of triglycerides by enzymatic esterification of polyunsaturated fatty acids (PUFA) with glycerol. The lipase Novozym 435 (Novo Nordisk, A/S) from Candida antarctica was used to catalyze this reaction. The main factors influencing the degree of esterification and triglyceride yield were the amount of enzyme, water content, temperature and glycerol/fatty acid ratio. The optimum reaction conditions were established as: 100 mg of lipase; 9 ml hexane; 50°C; glycerol/PUFA concentrate molar ratio 1.2:3; 0% initial water; 1 g molecular sieves added at the start of reaction; and an agitation rate of 200 rpm. Under these conditions, a triglyceride yield of 93.5% was obtained from cod liver oil PUFA concentrate; the product contained 25.7% eicosapentaenoic acid and 44.7% docosahexaenoic acid. These optimized conditions were used to study esterification from a PUFA concentrate of the microalgae Phaeodactylum tricornutum and Porphyridium cruentum. With the first, a triglyceride yield of 96.5%, without monoglycerides and very few diglycerides, was obtained after 72 h of reaction; the resulting triglycerides had 42.5% eicosapentaenoic acid. A triglyceride yield of 89.3% was obtained from a P. cruentum PUFA concentrate at 96 h of reaction, which contained 43.4% arachidonic acid and 45.6% EPA. These high triglyceride yields were also achieved when the esterification reaction was scaled up 5-fold.     

Lipase-catalyzed chemoselective aminolysis of various aminoalcohols with fatty acids

Candida antarctica lipase is well known to convert amines and alcohols into amides and esters. This report describes the development of a solvent-free enzymatic process for the production of fatty alkanolamides. The aminolysis of linoleyl ethyl ester with several aminoalcohols from C2 to C6 (linear or branched compounds), and the very high selectivity of amide compounds have been observed.Our investigation leads us to develop an original biotechnological process for the chemoselective synthesis of new active molecules for cutaneous application.     

Lipase-catalyzed esterification of rutin and naringin with fatty acids of medium carbon chain

Flavonoids rutin and naringin were acylated with fatty acids of medium carbon chain (with 8–12 carbon atoms on their molecule) in a reaction catalyzed by immobilized lipase from Candida antarctica (Novozyme) in various solvent systems. The reaction parameters affecting the acylation rate and the conversion of the enzymatic process, such as the nature of the organic solvent and acyl donor used, the water activity (a_w) of the system, as well as the kinetic of the reaction have been investigated. In all cases studied, only flavonoid monoester is identified as the product, which indicates that this lipase-catalyzed esterification is regioselective. The enzymatic acylation of flavonoids seems to follow Michaelis–Menten kinetics.     

Turnover of the fatty acid and glycerol moieties of acylglycerols and phosphoglycerides during development of Ceratitis capitata

• 1.1. Labelling of free fatty acids, diacylglycerols, triacylglycerols, phosphatidylethanolamine, phosphatidylcholine and their lysoderivatives was followed during the development of Ceratitis capitata after feeding larvae with mixtures of either (³H)glycerol and (¹⁴C)palmitate or (³H)glycerol and (¹⁴C)linoleate. Both, specific activity and dpm/individual were plotted vs the time of development.
• 2.2. Palmitate and linoleate moieties of the diacylglycerol fraction had two exponential components with a similar initial rapid decay component. The turnover times corresponding to the slow decay components were different for both acids in the free form and acylating diacylglycerols.
• 3.3. Triacylglycerols exhibited a monophasic behaviour with different half-life values for either palmitate or linoleate.
• 4.4. Palmitate and linoleate show different turnover in the phosphoglycerides phosphatidylethanolamine and phosphatidylcholine. Palmitate exhibited a monophasic decay curve whereas linoleate exhibited a biphasic decay for both phospholipid classes.

     

A sensitive radioenzymatic assay for glycerol and acylglycerols.

A sensitive radioenzymatic assay for glycerol and acylglycerols is described. The assay depends on the quantitative phosphorylation of glycerol to glycerophosphate by glycerol kinase using [gamma-32P]ATP as a substrate. The 32P content of the formed glycerophosphate is determined and gives a measure of the original glycerol content. Acylglycerols can be determined by prior hydrolysis to glycerol. The assay is sensitive to about 0.1 nmol of glycerol and can be extended to 100 nmol. The assay can be applied to the determination of acylglycerols separated by thin-layer chromatography in amounts as low as 0.5 nmol. The assay is particularly useful in the determination of the specific activity of 14C- or 3H-labeled glycerol moeities.     

Solvent effects on lipase-catalyzed esterification of glycerol and fatty acids

The lipase-catalyzed acylglycerol synthesis with fatty acids of different chain length is studied. Measured ester mole fractions at equilibrium are compared with calculated mole fractions. For these calculations the computer program TREP (Two-phase Reaction Equilibrium Prediction) is used. This program is based on the UNIFAC group contribution method and is developed for nondilute two-phase reaction systems.With one set of equilibrium constants, namely 1.3, 0.8, and 0.6 for monoester, diester, and triester synthesis, respectively, the equilibrium position of the reaction between glycerol and all saturated fatty acids with a chain length from 6 to 18 and oleic acid (cis-9-octadecenoic acid) can be calculated. Deviations, expressed as the ratio between calculated and measured ester mole fractions, usually were between 0.7 and 1.2. In the presence of solvents, the deviations of the monoester mole fractions were higher and rose up to 3. Without addition of a solvent, the ester mole fractions at equilibrium are dependent on the fatty acid chain length. With the short-chain hexanoic acid, the monoester mole fraction is the highest ester mole fraction, while for the long-chain oleic acid, the diester mole fraction is the highest one. The ester mole fractions become independent on the chain length of the fatty acid with a solvent added in a sufficient high concentration. Both reactions, with saturated and unsaturated C(18) fatty acids, lead to the same equilibrium position. The program TREP is found to make good predictions of the equilibrium amounts of ester and fatty acid. However, systematic deviations arise between measured and calculated amounts of water and glycerol in the organic phase. The calculated water and glycerol amounts are always lower than the measured ones. These deviations seem to be highest in nonpolar media and are probably due to deficiencies in the UNIFAC calculation method. Some preliminary experiments show the effect of the choice of solvent on the reaction rates. In polar solvents, the monoester production rate is enhances by a factor of 1.5 as compared to the reaction rate in a system without solvent. (c) 1993 John Wiley & Sons, Inc.     

Lipase-catalyzed synthesis of glycerol carbonate from renewable glycerol and dimethyl carbonate through transesterification

Glycerol carbonate is a key multifunctional compound employed as solvent, additive, monomer, and chemical intermediate. Enzymatic synthesis of glycerol carbonate from renewable starting materials (glycerol and dimethyl carbonate) was successfully achieved by immobilized lipase from Candida antarctica (CALB, Novozym 435). Addition of molecular sieves as scavenger for the removal of methanol, which was generated from dimethyl carbonate during the reaction, accelerated a reaction rate. After the optimization, the equimolar use of glycerol and dimethyl carbonate in the Novozym 435-catalyzed reaction yielded a glycerol carbonate with almost quantitative yield. The resulting glycerol carbonate from 60 °C reaction has shown the low enantiomeric excess (13% ee) as configuration of (R)-enantiomer.     

Lipase-catalyzed synthesis of glycerol carbonate from renewable glycerol and dimethyl carbonate through transesterification

Glycerol carbonate is a key multifunctional compound employed as solvent, additive, monomer, and chemical intermediate. Enzymatic synthesis of glycerol carbonate from renewable starting materials (glycerol and dimethyl carbonate) was successfully achieved by immobilized lipase from Candida antarctica (CALB, Novozym 435). Addition of molecular sieves as scavenger for the removal of methanol, which was generated from dimethyl carbonate during the reaction, accelerated a reaction rate. After the optimization, the equimolar use of glycerol and dimethyl carbonate in the Novozym 435-catalyzed reaction yielded a glycerol carbonate with almost quantitative yield. The resulting glycerol carbonate from 60 °C reaction has shown the low enantiomeric excess (13% ee) as configuration of (R)-enantiomer.     

Inhibition of the glycerol 3-phosphate oxidation by free fatty acids

Oleate inhibits oxidation of glycerol 3-phosphate, but has no effect on glycerol 3-phosphate dehydrogenase. The inhibitory effect may be completely reversed by bovine serum albumin or menadione. Lysophosphatidylcholine has a quite similar inhibitory effect. In this case, however, the inhibitory effect is reversed rather by menadione only than by serum albumin. The results presented indicate that free fatty acids reversibly block transport of hydrogen between glycerol 3-phosphate dehydrogenase and CoQ and may be considered as physiological regulators of the glycerolphosphate cycle.     

Mutagenicity of glycerol chlorohydrines and of their esters with higher fatty acids present in protein hydrolysates

3-Chloro-1,2-propanediol and 1,3-dichloro-2-propanol caused base substitutions in Salmonella typhimurium TA1535 both with and without metabolic activation. Metabolic activation seemed to act mainly by decreasing the toxicity of these compounds. A difference in the growth of the wild-type and repair-deficient strains of Escherichia coli was observed only for 1,3-dichloro-2-propanol with S9 mix. Esters of both chlorohydrines with fatty acids had smaller mutagenic effects than unesterified compounds.     

Lipase catalyzed production of monoacylglycerols by the esterification of fish oil fatty acids with glycerol

In this study, we attempted the efficient production of monoacylglycerols (MAG) via the lipase-catalyzed esterification of glycerol with fatty acids obtained from sardine oil. The reaction factors that influenced MAG synthesis were the glycerol to fatty acid mole ratio, amount of enzyme, organic solvent, temperature, and the type of lipase used. Porcine pancreas lipase was selected to catalyze this reaction. The optimum conditions we determined for MAG synthesis were a glycerol to fatty acid mole ratio of 1∶6, 100 mg/mL of lipase, and 30°C in dioxane. Under these conditions, the MAG content was 68% (w/w) after 72 h of reaction. The MAGs synthesized via the lipase-catalyzed esterification of glycerol with fatty acids included monomyristin, monopamiltin, and monoolein, as identified by GCMS.     

Comparative fatty acid selectivity of lipases in esterification reactions with glycerol and diol analogues in organic media

Reaction selectivity of Pseudomonas cepacia, Rhizomucor miehei, and Candida antarctica B lipases was assessed in multicompetitive esterification reaction mixtures containing an homologous series of n-chain even carbon number fatty acid (FA; C4-C18) substrates and a single alcohol cosubstrate (glycerol, 1,2-propanediol (1,2-PD), or 1, 3-propanediol (1,3-PD)) in tert-butyl methyl ether at water activity of 0.69 or 0.90 and a reaction temperature of 35 degrees C. For P. cepacia lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C10, C6, C16 > other FA; with 1,2-PD and 1, 3-PD, C16 > C8 > C14 > other FA. For R. miehei lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C12 > C10, C14 > other FA; with 1,2-PD and 1,3-PD, C8 > C12 > other FA. For C. antarctica B lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C10, C6, C12 > other FA; with 1, 2-PD, C8 > C10, C6 > other FA; and with 1,3-PD, C8 > C10 > C6 > other FA. The differences in selectivity among FA ranged up to 16-fold, depending upon the lipase and alcohol cosubstrate used. These findings represent intrinsic and substrate-modulated features of FA selectivities that are of particular relevance to the use of lipases for acylglycerol synthesis reactions.     

Lipase-catalyzed ester exchange reactions in organic media with controlled humidity

Immobilized lipase activity is studied in organic solvent systems of controlled water content under the influence of a variety of reaction parameters, such as temperature, relative humidity, substrate concentrations, and type of fatty acid used. Control of the amount of water in the reaction system was found to be a valuable tool for the orientation of the reaction process and for the determination of the final reaction products. The properties of the immobilized lipase were studied using the interesterification of triolein and palmitic acid as the model system.     

Continuous enzymatic esterification of glycerol with (poly)unsaturated fatty acids in a packed-bed reactor

Enzymatic synthesis of mono-, di-, and triacyglycerols from (poly)unsaturated fatty acids (linoleic, oleic, and conjugated linoleic acids) has been studied as a solvent-free reaction in a packed-bed reactor containing an immobilized lipase from Mucor miehei. The extents of the esterification reactions of interest are primarily determined by the molar ratio of glycerol to fatty acid because the presence of excess glycerol as a immiscible phase is responsible for reducing the activity of the water produced by the esterification reactions. For molar ratios of fatty acid to glycerol of less than 1.5, the percentage of the fatty acid esterified decreases quasi-linearly with an increase in this molar ratio. By appropriate manipulation of the fluid-residence time, one can control the relative proportions of the various acylglycerols in the effluent stream. At the outlet of the reactor, one observes excellent spontaneous separation of the glycerol and acylglycerol/fatty acid phases. At 50 degrees C and a fluid residence time of 1 hour, as much as 90% of the fatty acid can be esterified when the molar ratio of fatty acid to glycerol is 0.33 or less.     

Rapid enzymatic production of acylglycerols from conjugated linoleic acid and glycerol in a solvent-free system

Rates and product distributions have been determined for the consecutive esterification reactions of conjugated linoleic acid (CLA) with glycerol in the presence of an immobilized Candida antarctica lipase from Novo Nordisk (Lipozyme IM). In a solvent-free environment, both rates and product distributions are affected by the ratio of reactants, temperature, and hydration level. For mole ratios of CLA to glycerol ranging from 1/1 to 3/1, incorporation of at least 95% of the original CLA into the product acylglycerols was achieved in less than 7 h of reaction at 50°C. © Rapid Science Ltd. 1998     

Mitochondrial glycerol phosphate acyltransferase directs the incorporation of exogenous fatty acids into triacylglycerol

The mitochondrial isoform of glycerol-3-phosphate acyltransferase (GPAT), the first step in glycerolipid synthesis, is up-regulated by insulin and by high carbohydrate feeding via SREBP-1c, suggesting that it plays a role in triacylglycerol synthesis. To test this hypothesis, we overexpressed mitochondrial GPAT in Chinese hamster ovary (CHO) cells. When GPAT was overexpressed 3.8-fold, triacylglycerol mass was 2.7-fold higher than in control cells. After incubation with trace [(14)C]oleate ( approximately 3 microm), control cells incorporated 4.7-fold more label into phospholipid than triacylglycerol, but GPAT-overexpressing cells incorporated equal amounts of label into phospholipid and triacylglycerol. In GPAT-overexpressing cells, the incorporation of label into phospholipid, particularly phosphatidylcholine, decreased 30%, despite normal growth rate and phospholipid content, suggesting that exogenous oleate was directed primarily toward triacylglycerol synthesis. Transiently transfected HEK293 cells that expressed a 4.4-fold increase in GPAT activity incorporated 9.7-fold more [(14)C]oleate into triacylglycerol compared with control cells, showing that the effect of GPAT overexpression was similar in two different cell types that had been transfected by different methods. When the stable, GPAT-overexpressing CHO cells were incubated with 100 microm oleate to stimulate triacylglycerol synthesis, they incorporated 1.9-fold more fatty acid into triacylglycerol than did the control cells. Confocal microscopy of CHO and HEK293 cells transfected with the GPAT-FLAG construct showed that GPAT was located correctly in mitochondria and was not present elsewhere in the cell. These studies indicate that overexpressed mitochondrial GPAT directs incorporation of exogenous fatty acid into triacylglycerol rather than phospholipid and imply that (a) mitochondrial GPAT and lysophosphatidic acid acyltransferase produce a separate pool of lysophosphatidic acid and phosphatidic acid that must be transported to the endoplasmic reticulum where the terminal enzymes of triacylglycerol synthesis are located, and (b) this pool remains relatively separate from the pool of lysophosphatidic acid and phosphatidic acid that contributes to the synthesis of the major phospholipid species.     

Lipase-catalyzed reactions in organic media: competition and applications

Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) have been used in organic media for the catalysis of reactions such as hydrolysis, esterification and transesterification. In these conditions it was confirmed that all reactions proceed through an acyl enzyme intermediate in two successive steps: acyl enzyme formation and solvolysis. The competition between two acyl acceptors (acyl donors) for reaction with a donor (acceptor) is described for the first time. A kinetic model is proposed using a competitive factor which is in good accordance with experimental results. The model was used successfully for the prediction of alcohol (acid) separations and resolutions by lipases.     

Effect of parturition on serum glycerol and non-esterified fatty acids in obese and normal weight women

Serum glycerol and NEFA content variations are examined before and after labor in obese and normal weighing women (35 subjects). Blood glycerol and NEFA are shown to increase before delivery. Glycerol values are shown to drop to normal immediately after delivery, while NEFA values diminish to a lesser extent. Statistical analysis shown that blood glycerol increase could be pregnancy-dependent in both normal weighing and obese women, but that NEFA increase could be pregnancy-dependent in normal weighing women only. Obesity increases blood glycerol and NEFA concentration considerably, thus masking the effects of pregnancy.