Synthesis of monoacylglycerol containing pinolenic acid via stepwise esterification using a cold active lipase

High purity monoacylglycerol (MAG) containing pinolenic acid was synthesized via stepwise esterification of glycerol and fatty acids from pine nut oil using a cold active lipase from Penicillium camembertii as a biocatalyst. Effects of temperature, molar ratio, water content, enzyme loading, and vacuum on the synthesis of MAG by lipase‐catalyzed esterification of glycerol and fatty acid from pine nut oil were investigated. Diacylglycerol (DAG) as well as MAG increased significantly when temperature was increased from 20 to 40°C. At a molar ratio of 1:1, MAG content decreased because of the significant increase in DAG content. Water has a profound influence on both MAG and DAG content through the entire course of reaction. The reaction rate increased significantly as enzyme loading increased up to 600 units. Vacuum was an effective method to reduce DAG content. The optimum temperature, molar ratio, water content, enzyme loading, vacuum, and reaction time were 20°C, 1:5 (fatty acid to glycerol), 2%, 600 units, 5 torr, and 24 h, respectively. MAG content further increased via lipase‐catalyzed second step esterification at subzero temperature. P. camembertii lipase exhibited esterification activity up to ?30°C. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Production of monoacylglycerol of conjugated linoleic acid by esterification followed by dehydration at low temperature using Penicillium camembertii lipase

Conjugated linoleic acid (CLA) has various physiological activities, and a commercial product is a mixture of free fatty acids (named FFA-CLA) which contains almost equal amounts of 9-cis, 11-trans (9c,11t) and 10t,12c isomers. We attempted to efficiently produce monoacylglycerol (MAG) of CLA by lipase-catalyzed esterification. Study on effect of reaction temperature clarified that synthesis of diacylglycerols (DAGs) from MAGs was repressed at low temperature. When FFA-CLA was esterified at 5 °C with 5 molar equivalents of glycerol using 200 U/g mixture of Penicillium camembertii lipase in the presence of 2% water, the degree of esterification reached 89.6% after 45 h and the contents of MAGs and DAGs were 87.0 and 4.5 wt.%, respectively. Triacylglycerols were not synthesized in this Penicillium lipase-catalyzed esterification. After the esterification was conducted for 20 h (the degree of esterification, 80.8%), dehydration was started by evaporation at 5 mmHg using a vacuum pump. The degree of esterification increased concomitantly with dehydration and reached 94.5% after 16 h (36 h in total). The contents of MAGs (main components, 1(3)-isomers) and DAGs were 92.7 and 2.9 wt.%, respectively. Fatty acid compositions in MAGs synthesized with and without dehydration were the same as that in FFA-CLA. These results showed that the esterification system with dehydration is effective for producing MAGs in a high yield.     

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.     

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.     

Sn-2-monoacylglycerol, not glycerol, is preferentially utilised for triacylglycerol and phosphatidylcholine biosynthesis in Atlantic salmon (Salmo salar L.) intestine

Pathways of lipid resynthesis in the intestine of fish are relatively unknown. Various reports have suggested the existence of both sn-1,3-specific (pancreatic) and non-specific (bile salt-activated) lipase activity operating on dietary triacylglycerol (TAG) in the intestinal lumen of fish during digestion. Thus, sn-2-monoacylglycerol (2-MAG) and glycerol, respective hydrolytic products of each lipase, are absorbed and utilised for glycerolipid synthesis in enterocytes via two alternative routes: monoacylglycerol (MAG) and glycerol-3-phosphate (G3P) pathways. Despite different precursors, both pathways converge at the production of sn-1,2-diacylglycerol (1,2-DAG) where TAG or phosphatidylcholine (PC) synthesis can occur. To elucidate the relative activities of MAG and G3P pathways in Atlantic salmon enterocytes, intestinal segments were mounted in Ussing chambers where equimolar mixtures of sn-2-oleoyl-[1,2,3-(3)H]glycerol (2-MAG) and [(14)C(U)]glycerol, plus unlabelled 16:0 and 18:2n-6 as exogenous fatty acid sources, were delivered in bile salt-containing Ringer solution to the mucosa. The MAG pathway predominated, over the G3P pathway, synthesizing ca. 95% of total TAG and ca. 80% of total PC after a 3 h incubation period at 10 degrees C. Further, the 1,2-DAG branch point into TAG or PC was polarised towards TAG synthesis (6:1) via the MAG pathway but more evenly distributed between TAG and PC (1:1) via the G3P pathway. Effect of long-chain saturated, monounsaturated and polyunsaturated fatty acids on the synthesized TAG/PC ratio was assessed by individually exchanging 16:0, 18:1n-9 or 18:2n-6, for 16:0+18:2n-6, in mucosal solutions. TAG synthesis was influenced considerably more than PC synthesis, via either pathway, by exogenous fatty acids utilised. 18:1n-9 significantly stimulated TAG synthesis via the MAG pathway yielding a TAG/PC ratio of 12:1. Alternatively, 18:2n-6 stimulated TAG synthesis the most via the G3P pathway (TAG/PC=4:1). 16:0 significantly attenuated TAG synthesis via either pathway. Micellar fatty acid species also significantly affected intestinal active transport mechanisms as shown by decreasing transepithelial potential (TEP) and short-circuit current (SSC) with increasing fatty acid unsaturation. The epithelial integrity was, however, not compromised after 3 h of exposure to any of the fatty acids. The implications of these findings on dietary fatty acid composition and enterocytic lipid droplet accumulation are discussed.     

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.     

Esterification reactions of lipase in reverse micelles

The activities of lipase from Candida cylindracea and Rhizopus delemar have been investigated in water/AOT/iso-octane reverse micellar media through the use of two esterification reactions: fatty acid-alcohol esterification and glyceride synthesis. Such media promotes the occurrence of these two lipase-catalyzed reactions due to its low water content. The effect of various parameters on the activity of lipase from C. cylindracea in reverse micelles was determined and compared to results where alternate media were employed. It was observed that the structure of the media, as dictated by the type and concentration of the substrates and products and by the water/AOT ratio, w(0), had a strong impact on enzyme activity. Strong deactivation of both typase types occurred in reverse micelles, especially in the absence of substrates and for w(0) values greater than 3.0. Glyceride synthesis was realized with lipase from R. delemar, but not with that from C. cylindracea; the temperature and concentration of substrates and water strongly dictated the reaction rate and the percent conversion.     

1-Monoglyceride production from lipase-catalyzed esterification of glycerol and fatty acid in reverse micelles

Glycerol-fatty acid esterification has been conducted with lipase from R. delemar in water/AOT/isooctane reverse micellar media, with the major product being 1-monoglyceride, a useful food-emulsifier. 1,3-diglyceride was also synthesized, but to a much lesser extent. For a given set of initial conditions, the reaction productivity, measured in terms of the initial product formation rate, V(0), and the final or equilibrium concentration of product, is optimal for a particular concentration of each surfactant, fatty acid, glycerol, and water. Many of these optimal values correlate well with a "critical" region on the phase diagram. Also, results indicate lipase-catalyzed esterification stops due to the achievement of kinetic equilibrium expect for a few cases where enzyme deactivation is severe. Dynamic light scattering was employed to examine the influence of water, glycerol, and fatty acid on micellar and interfacial structure. Results from this technique indicate enzyme kinetic are linked to interfacial phenomena and the presence of substrates at the interfacial region.     

Optimization of Candida sp. 99-125 lipase catalyzed esterification for synthesis of monoglyceride and diglyceride in solvent-free system

Esterification of glycerol and oleic acid catalyzed by lipase Candida sp. 99-125 was carried out to synthesize monoglyceride (MAG) and diglyceride (DAG) in solvent-free system. Beta-cyclodextrin as an assistant was mixed with the lipase powder. Six reaction variables, initial water content (0–14 wt% of the substrate mass), the glycerol/oleic acid molar ratio (1:1–6:1), catalyst load (3–15 wt% of the substrate mass), reaction temperature (30–60 °C), agitator speed (130–250 r/min) and beta-cyclodextrin/lipase mass ratio (0–2) were optimized. The optimal conditions to the synthesis of MAG and DAG were different: the optimal glycerol/oleic acid molar ratio, beta-cyclodextrin/lipase mass ratio, catalyst load and reaction temperature were 6:1, 0, 5%, 50 °C for MAG, and 5:1, 1.5, 10%, 40 °C for DAG, respectively. The optimal water content and agitator speed for both MAG and DAG were 10% and 190 r/min, respectively. Under the optimal conditions, 49.6% MAG and 54.3% DAG were obtained after 8 h and 4 h, respectively, and the maximum of 81.4% MAG plus DAG (28.1% MAG and 53.3% DAG) was obtained after 2 h under the DAG optimal condition. Above 90% purity of MAG and DAG can be obtained by silica column separation.     

Effect of alcohol chain length on the optimum conditions for lipase-catalyzed synthesis of adipate esters

Immobilized Candida antarctica lipase B, Novozym® 435, was used in the esterification of adipic acid and alcohols with different chain lengths (C₁–C₁₈). Optimum conditions for the synthesis of adipate esters were obtained using response surface methodology (RSM) with respect to important reaction parameters including time, temperature, substrate molar ratio and amount of enzyme. Alcohol chain length specificity of the enzyme in the synthesis of adipate esters was also determined. Minimum reaction time (215 min) for achieving maximum ester yield was obtained for butyl alcohol. Methanol required an increased time (358 min) and enzyme amount (10.2%, w/w) for attaining maximum yield. The maximum required temperature and time of 65°C and 523 min, respectively, were obtained for the synthesis of dioctadecyl adipate. The results demonstrate that alcohol chain length is a determining parameter in optimization of the lipase-catalyzed synthesis of adipate esters. Reactions under optimized conditions yielded a high percentage of esterification (>97%). The optimum conditions can be used to scale up the process.     

Response surface modeling of 1-stearoyl-3(2)-oleoyl glycerol production in a pilot packed-bed immobilized Rhizomucor miehei lipase reactor

A dual response approach using diacylglycerol (DAG) and triacylglycerol (TAG) as responses for optimization of 1-stearoyl-3(2)-oleoyl glycerol-enriched DAG synthesis using response surface methodology (RSM) was investigated. Four variables from a lipase-catalyzed esterification reaction were optimized using a central composite rotatable design. The following optimized conditions yielded 51 wt.% DAG and 22 wt.% TAG: reaction temperature of 55 °C, enzyme dosage of 9.5 wt.%, fatty acid/glycerol molar ratio of 2.1 and reaction time of 3 h. Results were repeatable at 10 kg production scale in a pilot packed-bed enzyme reactor. No significant losses in enzyme activity or changes in fatty acid selectivity on DAG synthesis were observed during the five pilot productions. Lipozyme RM IM showed selectivity towards the production of stearic acid enriched DAG. The purity of DAG oil after purification was 90 wt.%.     

Characteristics of immobilized lipase on hydrophobic superparamagnetic microspheres to catalyze esterification

A novel immobilized lipase (from Candida rugosa) on hydrophobic and superparamagnetic microspheres was prepared and used as a biocatalyst to catalyze esterification reactions in diverse solvents and reaction systems. The results showed that the immobilized lipase had over 2-fold higher activities in higher log P value solvents. An exponential increase of lipase activity against log P of two miscible solvent mixtures was observed for the first time. Both free and immobilized lipase achieved its maximum activity at the range of water activity (a(w)) 0.5-0.8 or higher. At a(w) 0.6, the immobilized lipase exhibited markedly higher activities in heptane and a solvent-free system than did the native lipase. In multicompetitive reactions, the alcohol specificity of the lipase showed a strong chain-length dependency, and the immobilized enzyme exhibited more preference for a longer-chain alcohol, which is different from previous reports. The immobilized lipase showed higher specificities for butyric acid and the medium-chain-length fatty acids (C(8)-C(12)). Then, the immobilized lipase was extended to solvent-free synthesis of glycerides from glycerol and fatty acids. Recovered by magnetic separation, the immobilized lipase exhibited good reusability in repeated batch reaction, indicating its promising feature for biotechnology application.     

Phyllosilicate sol-gel immobilized lipases for the formation of partial acylglycerides**

Lipase PS-30 (pseudomonas cepacia) and Lipase F (Rhizopus oryzae), immobilized within a phyllosilicate sol-gel matrix, catalyzed the esterification of glycerol with short, medium and long-chain fatty acids to produce mono (MAG), di (DAG) and tri (TAG) acylglycerols. The results from the above esterification reactions were compared to reactions using a commercially available immobilized lipase, Lipozyme IM-60. Time course studies showed that free Lipase PS-30 or Lipase F enhanced esterification reactions with the use of silica-supported glycerol. In contrast, immobilized Lipase PS-30-catalyzed reactions occurred at the same conversion rate when using either free or silica-supported glycerol. For immobilized Lipase F and Lipozyme IM-60 reactions, the use of silica-supported glycerol favored the production of DAG and TAG over MAG. All three immobilized lipases could be reused for acylglycerol production.     

Lipase-catalyzed synthesis of monoacylglycerol in a homogeneous system

The 1,3-regiospecifique lipase, Lipozyme IM, catalyzed the esterification of lauric acid and glycerol in a homogeneous system. To overcome the drawback of the insolubility of glycerol in hexane, which is extensively used in enzymatic synthesis, a mixture of n-hexane/tert-butanol (1:1, v/v) was used leading to a monophasic system. The conversion of lauric acid into monolaurin was 65% in 8 h, when a molar ratio of glycerol to fatty acid (5:1) was used with the fatty acid at 0.1 M, and the phenomenon of acyl migration was minimized.     

Lipase-catalyzed synthesis of chlorogenate fatty esters in solvent-free medium

Chlorogenic acid (5-caffeoylquinic acid or 5-CQA) is an hydrophilic phenolic compound with antioxidant properties. Because of its high polarity, its antioxidant properties may be altered when formulated in oil based food or cosmetic preparations. Therefore, there is an interest in trying to enhance its hydrophobicity by grafting of an aliphatic chain. Such lipophilization reactions can be generally achieved through enzymatic catalysis. Our study consisted in synthesizing fatty cholorogenate esters in a two steps reaction. Firstly, 5-CQA was chemically esterified by methanol using an Amberlite IR120 H resin to obtain methyl chlorogenate that is more soluble in the fatty alcohols than 5-CQA. Secondly, this chlorogenate intermediate was transesterified with fatty alcohols of various chain lengths (C₄, C₈, C₁₂, or C₁₆) in the presence of Candida antarctica B lipase. Under optimal reaction conditions (a_w = 0.05; 5% (w/w) of biocatalyst), the transesterification rates were until two-fold higher than in the direct lipase-catalyzed esterification of chlorogenic acid by the same alcohols. The two-step reaction overall yield was between 61 and 93% depending on the alcohol chain length, whereas it was 40–60% for the direct esterification with the same alcohols.     

Lipase-catalyzed enantioselective esterification of (S)-naproxen hydroxyalkyl ester in organic media

A lipase-catalyzed, enantioselective esterification process in organic solvents was developed for the synthesis of (S)-naproxen hydroxyalkyl ester. With the selection of lipase (Candida rugosa lipase) and reaction medium (isooctane and cyclohexane), a high enantiomeric ratio of <100 for the enzyme was obtained. 1,4-Butanediol was the best acyl acceptor. The carbon chain length of the alcohol had a major effect on the enzyme activity and enantioselectivity of lipase-catalyzed esterification.     

Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of >90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30–40°C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.     

Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of >90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30–40°C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.     

Enzymatic synthesis of 2-ethylhexyl esters of fatty acids by immobilized lipase from Candida sp. 99–125

The 2-ethylhexyl esters of fatty acids were synthesized by immobilized lipase from Candida sp. 99–125. The reuse stability of immobilized lipase was at least four batches. The conditions of enzymatic synthesis of 2-ethylhexyl palmitate were optimized. In the system of petroleum ether, 10% (w/w) immobilized lipase was used in the esterfication of 2-ethyl hexanol (7.8 mmol) and palmitic acid (7.8 mmol) at 40 °C with silica gel as the water absorbent. The esterification degree was 91% under these conditions. The purity of 2-ethylhexyl palmitate was 98% after purification consisting washing by water and evaporation to remove the organic solvent.     

Integrated enzymatic production of specific structured lipid and phytosterol ester compositions

Mixtures of specific structured lipids and phytosterol esters, valuable food components, were synthesized by an enzymatic one-pot process in organic-solvent-free medium starting from a mixture of phytosterol, caprylic acid and sunflower oil. Nine biocatalysts, seven commercially available lipases and two air-dried solid state (SSF) fermentation preparations of Aspergillus oryzae NRRL 6270 (AoSSF) and Aspergillus sojae NRRL 6271 (AsSSF), were screened for lipase activity in the transesterification reactions of sunflower oil with caprylic acid and for sterol esterase activity in the direct esterification of phytosterols with free fatty acids. The best process variant using a sequence of sterol esterase (AoSSF)-catalyzed esterification reaction of the free fatty acids and phytosterols, followed by water removal in vacuum and lipase-catalyzed transesterification with immobilized lipase from Rhizomucor miehei (Lipozyme) resulted in 92.1% conversion to phytosterol esters and 44.1% conversion to triacylglycerols containing two caprylic esters.