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.     

Studies on localization and regulation of lipase production by Aspergillus niger

The lipase from strain BTL of Aspergillus niger was studied. The enzyme, which was mainly extracellular, was produced at elevated activity levels under optimum growth conditions. De novo biosynthesis of lipase occurred only in the presence of lipids and was completely repressed by glucose and glycerol. The reaction products, oleic acid and glycerol, showed differed inhibition patterns during triolein hydrolysis. The enzyme exhibited high specificity towards middle chain triglycerides and was possibly activated by double bonds in the fatty acid chain. It exhibited a marked stability against organic solvents.     

Synthesis of monoglyceride containing omega-3 fatty acids by microbial lipase in organic solvent

Summary An enzymatic method for synthesis of monoglyceride from 1,2-isopropylidene glycerol and n-3 polyunsaturated fatty acid concentrate was investigated in organic solvent. Optimal reaction conditions for monoglyceride synthesis by lipase were established. Lipase IM-60 fromMucor miehei produced yields of monoglyceride of up to 80% in this system. The resultant monoglyceride contained 76.2% n-3 polyunsaturated fatty acid (eicosapentaenoic acid 43.3%; docosahexaenoic acid, 32.7%). Isooctane and hexane were suitable organic solvents for monoglyceride synthesis and optimal initial water content was 2.5%. Lipase IM-60 was relatively stable in organic solvent and is easily recovered for reuse.     

Differential effects of eicosapentaenoic acid and oleic acid on lipid synthesis and secretion by HepG2 cells

The effects of eicosapentaenoic acid and oleic acid on lipid synthesis and secretion by HepG2 cells were examined to identify fatty acid specific changes in lipid metabolism that might indicate a basis for the hypolipidemic effect attributed to eicosapentaenoic acid and related n-3 fatty acids. Cellular glycerolipid synthesis, as determined by [3H]glycerol incorporation, increased in a concentration-dependent manner in cells incubated 4 h with either eicosapentaenoic acid or oleic acid at concentrations between 10 and 300 microM. [3H]Glycerol-labeled triglyceride was the principal lipid formed and increased approximately fourfold with the addition of 300 microM oleic acid or eicosapentaenoic acid. Both fatty acids also produced a 20-40% increase in the total cellular triglyceride mass. Although both fatty acids increased triglyceride synthesis to similar extents, eicosapentaenoic acid-treated cells secreted 40% less [3H]glycerol-labeled triglyceride than cells fed oleic acid. Cellular synthesis of [3H]glycerol-labeled phosphatidylethanolamine and phosphatidylcholine was also reduced by 40% and 30%, respectively, in cells given eicosapentaenoic acid versus cells given oleic acid. Similar results were obtained in determinations of radiolabeled oleic acid and eicosapentaenoic acid incorporation. At a fatty acid concentration of 300 microM, incorporation of radiolabeled eicosapentaenoic acid into cellular triglycerides was greater than the incorporation obtained with radiolabeled oleic acid, while the reverse relationship was observed for the formation of phosphatidylcholine from the same fatty acids. Eicosapentaenoic acid is as potent as oleic acid in inducing triglyceride synthesis but eicosapentaenoic acid is a poorer substrate than oleic acid for phospholipid synthesis. The intracellular rise in de novo-synthesized triglyceride in eicosapentaenoic acid-treated cells without corresponding increases in triglyceride secretion suggests that eicosapentaenoic acid is less effective than oleic acid in promoting the transfer of de novo-synthesized triglyceride to nascent very low density lipoproteins.     

Enzymatic synthesis of trieicosapentaenoylglycerol in a solvent-free medium

The enzymatic synthesis of trieicosapentaenoylglycerol from glycerol and eicosapentaenoic ethyl ester in a solvent-free medium is studied here. Novozym SP 435 (immobilized lipase from Candida antarctica) has appeared as a very efficient biocatalyst for this transesterification. A nitrogen bubbling has allowed a good mixing and also the shifting of the reaction toward synthesis by eliminating the ethanol formed. The effect of temperature and of the quantity of lipase has been studied. In the optimal conditions (T=80°C, 5% (w/w) of lipase, 1 mol glycerol for 3 mol ethyl ester), pure triglyceride has been obtained after 10 h.     

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.     

Improvement of a process for purification of tocopherols and sterols from soybean oil deodorizer distillate

Soybean oil deodorizer distillate (SODD) is a useful material for purification of tocopherols and phytosterols (referred to as sterols). The SODD was first distilled, and the two substances were enriched. The preparation, which mainly contained free fatty acids (FFAs), sterols, and tocopherols, was named SODD tocopherols/sterols concentrate (SODDTSC). If sterols are converted to steryl esters and FFAs are converted to fatty acid methyl esters (FAMEs), relatively easy purification of tocopherols and steryl esters can be achieved because the boiling points of FAMEs, tocopherols, and steryl esters are different significantly. Hence, the development of a new two-step in situ reaction system was tried out for esterification of sterols with FFAs (first step) and esterification of FFAs with methanol (MeOH) (second step). A mixture of SODDTSC/water (95:5, w/w) and 250 units (U)/g-mixture of Candida rugosa lipase was prepared beforehand for the first-step reaction, and was agitated at 40 °C for 24 h with dehydration at 20 mmHg. Sterols were efficiently esterified, and the degree of esterification reached 95%. To the reaction mixture were added 7 M amounts of MeOH against unreacted FFAs, 20 wt.% water, and 25 U/g-mixture of Alcaligenes sp. lipase. The second-step reaction was then conducted at 30 °C for 20 h. Consequently, 95% FFAs were converted to FAME, and steryl esters synthesized by the first-step reaction were not reconverted to free sterols. Finally, SODDTSC (1.5 kg) was subjected to this two-step in situ reaction, and tocopherols and steryl esters were purified from the reaction mixture by short-path distillation. Tocopherols were purified to 72% (yield, 88%) and steryl esters were purified to 97% (yield, 97%).     

Response surface modelling of the production of ω-3 polyunsaturated fatty acids-enriched fats by a commercial immobilized lipase

The aim of this study was to model the production of fats, enriched with ω-3 polyunsaturated fatty acids (ω-3 PUFA) for nutraceutical purposes, via the response surface methodology. These fats were obtained by transesterification of palm oil stearin (POS) with a concentrate (EPAX 2050TG) of triglycerides enriched with ω-3 PUFA and soybean oil, catalysed by a commercial immobilized Candida antarctica lipase (“Novozym 435”).

The initial water activity (a_w) of the biocatalyst, POS and EPAX 2050TG concentrations, time and temperature showed a significant effect on the transesterification reaction, as well as on the competing reactions of hydrolysis and lipid oxidation.

Depending on the factors included, the transesterification reaction was described either by first- or second-order models.

The production of free fatty acids, which is ascribed both to the hydrolytic reaction and the mechanism of lipase-catalysed transesterification, showed a second-order dependence on the initial a_w of the biocatalyst.     

Enrichment of polyunsaturated fatty acids from tuna oil using immobilized Pseudomonas fluorescens lipase

Immobilized Pseudomonas fluorescens lipase enzyme was used to enrich the important polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) from tuna oil. Hydrolysis, esterification, and transesterification reactions were studied in detail to find out the fractionation pattern of DHA and EPA during these processes due to preferential selectivity for or against these PUFA. Hydrolysis with P. fluorescens biotype I lipase with stoichiometric amount of water content gave more than 80% of DHA and EPA in the free fatty acid (FFA) form after around 60% of hydrolysis. After some preferential specificity during the early stages of hydrolysis, P. fluorescens lipase exhibits nonselective characteristics on extended hydrolysis. Esterification of FFA extracted from the completely hydrolyzed mixture of tuna oil was found to be better with long chain fatty alcohol like octanol which lead to good enrichment (44.5% for DHA and 11.3% for EPA) and yields of the PUFA in the FFA form. Transesterification (ethanolysis) with immobilized P. fluorescens lipase enzyme resulted in good enrichment and recovery of DHA and EPA in the glyceride mixture. After around 60% of ester synthesis, 74% of (DHA + EPA) enrichment was achieved with yields of more than 90% in the glyceride mixture.     

Lipase mediated simultaneous esterification and epoxidation of oleic acid for the production of alkylepoxystearates

Epoxy alkylstearates were synthesized by lipase catalysed esterification and perhydrolysis followed by epoxidation of oleic acid in a one-pot process. Immobilized Candida antarctica lipase (Novozym^®435) was used as the catalyst. The esterification reaction occurred relatively quickly and was followed by epoxidation of the alkyl ester and the remaining fatty acid. Higher degree of esterification was achieved with n-octanol, n-hexanol and n-butanol as compared to that with ethanol and iso-propanol. The rate and yield of epoxidation was enhanced with iso-propanol but was lowered with the other alcohols. The lipase suffered significant loss in activity during the reaction primarily due to hydrogen peroxide. The presence of alcohols, in particular ethanol, further contributed to the enzyme inactivation. The epoxidation reaction could be improved by step-wise addition of the lipase.     

Synthesis of phosphatidylcholine with defined fatty acid in the sn-1 position by lipase-catalyzed esterification and transesterification reaction

The incorporation of caproic acid in the sn-1 position of phosphatidylcholine (PC) catalyzed by lipase from Rhizopus oryzae was investigated in a water activity-controlled organic medium. The reaction was carried out either as esterification or transesterification. A comparison between these two reaction modes was made with regard to product yield, product purity, reaction time, and byproduct formation as a consequence of acyl migration. The yield in the esterification and transesterification reaction was the same under identical conditions. The highest yield (78%) was obtained at a water activity (a(w)) of 0.11 and a caproic acid concentration of 0.8 M. The reaction time was shorter in the esterification reaction than in the transesterification reaction. The difference in reaction time was especially pronounced at low water activities and high fatty acid concentrations. The loss in yield due to acyl migration and consequent enzymatic side reactions was around 16% under a wide range of conditions. The incorporation of a fatty acid in the sn-1 position of PC proved to be thermodynamically much more favorable than the incorporation of a fatty acid in the sn-2 position.     

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     

Transesterification reaction between medium- and long-chain fatty acid triglycerides using surfactant-modified lipase

Transesterification between medium-chain fatty acid triglycerides (MCT) and long-chain fatty acid triglycerides (LCT) in a nonsolvent system was investigated using surfactant modified lipase which is a complex of lipase, Rhizopus japonicus and surfactant, sorbitan monostearate. 74% conversion of was obtained after a 48-h reaction period, and the triglyceride composition was well described by the 1, 3-random 2-random stochastic model. The transesterification reaction between MCT and LCT closely followed the simple kinetic model, and the change in MCT and LCT contents could be simulated using one parameter. The effects of the water activity (A(w)) of modified lipase, the water content of the reaction system and the reaction temperature on the reaction rate were studied. A modified lipase A(w) of 0.35 and a water content of the reaction system at 0.09 wt % showed the highest activity. Inactivation did not occur below 60 degrees C, however, the activity decreased at temperatures over 70 degrees C.     

Enzymatic fractionation of conjugated linoleic acid isomers by selective esterification

Attempt was done to prepare food supplements with high content of c9, t11-CLA or t10, c12-CLA. A free acid mixture containing CLA isomers was esterified with ethanol by enzyme catalysis. Novozyme 435 and Lipase AY30 were screened, and Lipase AY30 was employed to catalyze esterification reaction because of its high fractionation efficiency. Effect of reaction conditions on total esterification was investigated, and the optimal reaction conditions were: 140 U of lipase amount, reaction temperature at 50 °C, a pH of 6.5, and molar ratio of FFA–CLA to ethanol at 1:1. Based on the studies above, experiments of esterification and purification were done, and the best fractionation efficiency was obtained when the total esterification was 37%, and the corresponding purity and recovery of c9, t11-CLA were 75.50 and 49.85%, and that of t10, c12-CLA were 72.02 and 62.32%.     

Triglyceride synthesis by small-intestinal epithelium of the pig, sheep and chicken

1. A comparative study was made of triglyceride synthesis by the intestinal epithelium of pigs, sheep and chickens. In pig and chicken tissue both the glycerol 3-phosphate and the monoglyceride pathway of triglyceride synthesis were operative, but the former pathway predominated in sheep tissue. 2. The fatty acid specificity of the glycerol 3-phosphate pathway was studied in pig and sheep total-homogenate preparations. Maximum incorporation was obtained with myristic acid and palmitic acid under optimum conditions for each fatty acid. Lauric acid, myristic acid, oleic acid, linoleic acid and linolenic acid were inhibitory at concentrations above their optimum, but octanoic acid, decanoic acid, palmitic acid and stearic acid did not show this effect. 3. Subcellular fractionation located the glycerol 3-phosphate and monoglyceride pathways of triglyceride synthesis in the microsomes in all instances. Phosphatidate phosphohydrolase was associated with both the microsomes and the particle-free supernatant. 4. Glycerol 1-mono-oleate was incorporated into triglycerides to a greater extent than glycerol 1-mono-palmitate or glycerol 1-monostearate by microsomal preparations from pig and chicken. 5. A lipase specific for monoglycerides was detected in the particle-free supernatant of all the species examined.     

Regio- and stereoselective enzymatic esterification of glycerol and its derivatives.

A methodology for regio- and stereoselective preparation of acyl glycerol derivatives is presented. It offers easy access to specific 1,2-, 1,3-diglycerides and triglycerides as well as alkyl glycerol esters, phospholipids and glycolipids. These compounds are prepared by esterification of the corresponding glycerol derivatives such as 2-monoglycerides, alkyl glycerols, glyceryl glycosides, glyceryl phosphate esters, or unsubstituted glycerol. The regio- and stereoselectivity in the esterification is achieved by using fatty acid anhydrides and an enzymatic catalyst, 1,3-specific lipase. NMR methods for determining the regio- and stereoselectivity of esterification are discussed.     

Conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel by immobilized Candida antarctica lipase

Acid oil, which is a by-product in vegetable oil refining, mainly contains free fatty acids (FFAs) and acylglycerols, and is a candidate of materials for production of biodiesel fuel. A mixture (acid oil model) of refined FFAs and vegetable oil was recently reported to be converted to fatty acid methyl esters (FAMEs) at >98% conversion by a two-step reaction system comprising methyl esterification of FFAs and methanolysis of acylglycerols using immobilized Candida antarctica lipase. The two-step system was thus applied to conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel. Under similar conditions that were determined by using acid oil model, however, the lipase was unstable and was not durable for repeated use. The inactivation of the lipase was successfully avoided by addition of excess amounts of methanol (MeOH) in the first-step reaction, and by addition of vegetable oil and glycerol in the second-step reaction. Hence, the first-step reaction was conducted by shaking a mixture of 66 wt% acid oil (77.9 wt% FFAs, 10.8 wt% acylglycerols) and 34 wt% MeOH with 1 wt% immobilized lipase, to convert FFAs to their methyl esters. The second-step reaction was performed by shaking a mixture of 52.3 wt% dehydrated first-step product (79.7 wt% FAMEs, 9.7 wt% acylglycerols), 42.2 wt% rapeseed oil, and 5.5 wt% MeOH using 6 wt% immobilized lipase in the presence of additional 10 wt% glycerol, to convert acylglycerols to FAMEs. The resulting product was composed of 91.1 wt% FAMEs, 0.6 wt% FFAs, 0.8 wt% triacylglycerols, 2.3 wt% diacylglycerols, and 5.2 wt% other compounds. Even though each step of reaction was repeated every 24 h by transferring the immobilized lipase to the fresh substrate mixture, the composition was maintained for >100 cycles.     

Lipase-catalysed synthesis of ester oils from biodiesel by-products

Ester oils obtained from natural long-chain fatty acids and alcohols are versatile substitutes for many petroleum-based products. Their efficient synthesis with the solvent-free esterification of free fatty acids (FFA) from by-products of biodiesel fabrication and 2-ethyl-1-hexanol with immobilised lipase from Thermomyces lanuginosa was investigated. The immobilisation of the biocatalyst in static emulsion yielded a specific esterification activity that was higher by a factor of 4.9-9.4 than the activity of the native enzyme. Favourable properties of the silicone-based immobilisation matrix in terms of stability and immobilisation yield were observed. In biodiesel by-products, the immobilised lipase catalysed the esterification of FFA as well as the transesterification of residual fatty acid methyl esters (FAME) to the desired ester oils. A conversion of 90% FFA and 35% FAME gave a total yield of 60%. The inactivation coefficients during repeated use in a stirred-tank reactor with intermittent pressure reduction were exceptionally low.     

Synthesis of triglycerides of phenylalkanoic acids by lipase-catalyzed esterification in a solvent-free system

Immobilized Candida antarctica lipase B catalyzed the synthesis of triglycerides from glycerol and phenylalkanoic acids in a solvent-free system. 4-Phenylbutyric acid was the best acyl donor and displayed the highest synthetic rate of triphenylbutyrin (glyceryl triphenylbutyrate) at 65 degrees C among various phenylalkanoic acids with straight alkyl chains. The external mass transfer between the immobilized lipase and the bulk reaction mixture was limited. Different methods of removing water during the lipase-catalyzed esterification including spontaneous evaporation, the use of saturated salts solutions, and the use of molecular sieves were studied. The highest yield of triphenylbutyrin at 65 degrees C was 98%, by the elimination of water using molecular sieves in a solvent-free system. The glycerol was almost completely esterified to triphenylbutyrin in excess phenylbutyric acid with various substrate molar ratios.     

The Influence of Fatty Acid and Glycerol on the Kinetics of Fat Hydrolysis by Candida Rugosa Lipase in a Membrane Reactor

The kinetics of lipid-hydrolysis by Candida rugosa lipase was investigated in a membrane reactor and in an emulsion system. Two models were chosen to describe the kinetics of the enzyme:

(1) The hydrolysis of triglycerides to fatty acids was considered to be a chain reaction with the intermediary products di- and mono-glyceride; each step was assumed to be a reversible second-order reaction. The reaction rate constants were determined from batch experiments. The experimental results could be described with this model.

(2) For process optimization and control, a model based on the power law was developed. For this model, the rate of hydrolysis was measured as a function of fatty acid and glycerol concentrations. Relations for the initial rate and equilibrium ester fraction as a function of the glycerol concentration were determined. Further, the reaction rate could be described with the power-law model with a power of 1.75 in the hydrolyzable ester fraction for a wide range of glycerol concentrations. The model with power 1.75 gave much better results when compared to a similar first order model. Although simpler, the first order model can not be used. The power law model was applied in the simulation of a reactor composed of three modules. The fatty acid production rate was calculated for this reactor system as a function of the outgoing glycerol concentration at different conditions.