Substrate preferences for lipase-mediated acyl-exchange reactions with butteroil are concentration-dependent

Substrate preferences for pancreatic lipase-mediated acyl-exchange reactions with butteroil were concentration-dependent for the series of acyl donors and alcohol acceptors evaluated. For acidolysis reactions, the initial reaction rates and percent reaction yields after 18 h at 50 μmol acyl donor per gram substrate mixture were similar forn-fatty acids and their methyl and glycerol esters. At 400–500 μmol g⁻¹ (and greater), order of initial reaction rates and percent reaction yield was fatty acid glycerol esters > fatty acid methyl esters > fatty acids. At concentrations above 300–500 μmol g⁻¹, reaction inhibition was observed for fatty acid substrates, and inhibition took place at lower concentrations for the shorter-chainlength fatty acids of those evaluated (5–17 carbons). Inhibition was primarily attributed to acidification of the microaqueous environment of the lipase. Desorption of water by the fatty acid substrate may be a secondary mode of inhibition. The concentration dependence of initial reaction rates and percent reaction yield was similar for then-alcohol substrates evaluated (2–15 carbons) for alcoholysis reactions with butteroil. Optimum alcohol concentration was 375–500 μmol g⁻¹ (except for butanol, which was 1 mmol g⁻¹, above which reaction inhibition was observed. Inhibition was attributed to desorption of water from the enzyme by the alcohol substrate. Relative reactivity of classes of alcohols for this reaction system was primary alcohols > secondary alcohols > tertiary alcohols. Generally, alcoholysis reactions were faster than acidolysis reactions, and triacylglycerols were the best substrates for acidolysis reactions with butteroil at high levels (up to 2 mmol g⁻¹) of acyl donor substrate.     

Production of FAME from acid oil model using immobilized <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase

Acid oil is a by-product in the neutralization step of vegetable oil refining and is an alternative source of biodiesel fuel. A model substrate of acid oil, which is composed of TAG and FFA, was used in experiments on the conversion to FAME by immobilized Candida antarctica lipase. FFA in the mixture of TAG/FFA were efficiently esterified with methanol (MeOH), but the water generated by the esterification significantly inhibited methanolysis of TAG. We thus attempted to convert a mixture of TAG/FFA to FAME by a two-step process comprising methyl esterification of FFA and methanolysis of TAG by immobilized C. antarctica lipase. The first reaction was conducted at 30°C in a mixture of TAG/FFA (1∶1, wt/wt) and 10 wt% MeOH using 0.5 wt% immobilized lipase, resulting in efficient esterification of FFA. The reaction mixture after 24 h was composed of 49.1 wt% TAG, 1.3 wt% FFA, 49.1 wt% FAME, and negligible amounts of DAG and MAG (<0.5 wt%). The reaction mixture was then dehydrated and used as a substrate for the second reaction, which was conducted at 30°C in a solution of the dehydrated mixture and 5.5 wt% MeOH using 6 wt% immobilized lipase. The activity of the lipase increased gradually when the reaction was repeated by transferring the enzyme to a fresh substrate mixture. The activity reached a maximum after 6 cycles, and the content of FAME achieved was >98.5 wt% after a 24-h reaction. The immobilized lipase was very stable in the first-and second-step reactions and could be used for >100 d without significant loss of activity.     

The Acrylation of Glycerol: A Precursor to Functionalized Lipids

With the objective of expanding the number of functionalized lipids available, the reactive vinyl group of acrylic acid was introduced to triacylglycerol by esterification of glycerol. Didecanoylacryloylglycerol was synthesized from decanoic and acrylic acids and glycerol using K₂O as catalyst. 2 g (21.7 mmol) glycerol, 11.3 g (65.4 mmol) decanoic acid, 6.2 g (86.1 mmol) acrylic acid, 60 ml hexane, and 400 mg K₂O were added to 300-ml closed stainless steel reactor and maintained at 200 °C for 5 h. The resulting product, designated DDA, was isolated at about 40% yield based on the acylglycerol products. The other products included tridecanoylglycerol, diacryloyldecanoylglycerol, and the diacylglycerols of these acids. DDA was then converted to functionalized lipids by the Michael addition and Heck reaction. The Michael addition of thiophenol and 4-bromothiophenol yielded the corresponding linear thioethers whereas and Heck reaction products from bromobenzene and bromoanisole yielded triacylglycerols containing trans-cinnamic acid and trans-(4-methoxy)cinnamic acid, respectively.     

Production of ethyl ester from crude palm oil by two-step reaction with a microwave system

The production of ethyl esters of fatty acids from a feed material of crude palm oil (CPO) with a high free fatty acid (FFA) content under microwave assistance has been investigated. Parametric studies have been carried out to investigate the optimum conditions for the esterification process (amount of ethanol, amount of catalyst, reaction time, and microwave power). As a result, a molar ratio of FFA to ethanol of 1:24 with 4% wt./wt. of H₂SO₄/FFA, a microwave power of 70 W, and a reaction time of 60 min have been identified as optimum reaction parameters for the esterification process aided by microwave heating. At the end of the esterification process, the amount of FFA had been reduced from 7.5 wt.% to less than 2 wt.%. Similar results were obtained following conventional heating at 70 °C, but only after a reaction time of 240 min. Transesterification of the esterified palm oil has been accomplished with a molar ratio of CPO to ethanol of 1:4, 1.5 wt.% KOH as a catalyst, a microwave power of 70 W, and a reaction time of 5 min. This two-step esterification and transesterification process provided a yield of 80 wt.% with an ester content of 97.4 wt.%. The final ethyl ester product met with the specifications stipulated by ASTM D6751-02.     

Reduction of high content of free fatty acid in sludge palm oil via acid catalyst for biodiesel production

In this study, sulphuric acid (H₂SO₄) was used in the pretreatment of sludge palm oil for biodiesel production by an esterification process, followed by the basic catalyzed transesterification process. The purpose of the pretreatment process was to reduce the free fatty acids (FFA) content from high content FFA (> 23%) of sludge palm oil (SPO) to a minimum level for biodiesel production (> 2%). An acid catalyzed esterification process was carried out to evaluate the low content of FFA in the treated SPO with the effects of other parameters such as molar ratio of methanol to SPO (6:1-14:1), temperature (40-80 °C), reaction time (30-120 min) and stirrer speed (200-800 rpm). The results showed that the FFA of SPO was reduced from 23.2% to less than 2% FFA using 0.75% wt/wt of sulphuric acid with the molar ratio of methanol to oil of 8:1 for 60 min reaction time at 60 °C. The results on the transesterification with esterified SPO showed that the yield (ester) of biodiesel was 83.72% with the process conditions of molar ratio of methanol to SPO 10:1, reaction temperature 60 °C, reaction time 60 min, stirrer speed 400 rpm and KOH 1% (wt/wt). The biodiesel produced from the SPO was favorable as compared to the EN 14214 and ASTM D 6751 standard.     

Enzymatic esterification of glycerol II. Lipase-catalyzed synthesis of regioisomerically pure 1(3)-rac-monoacylglycerols

Regioisomerically pure 1(3)-rac-monoacylglycerols are conveniently prepared in high yields (>75%) and in multigram quantities by enzymatic esterification of glycerol in the presence of various lipases(Chromobacterium viscosum, Rhizopus delemar, Rhizomucor miehei) with a variety of different acyl donors, such as free fatty acids, fatty acid alkyl esters, vinyl esters and triacylglycerols, as well as natural fats and oils. All reactions are carried out in aprotic organic solvents with low water content, namelyn-hexane, diethyl ether, tBuOMe or mixtures of these solvents. Essential for the success of these transformations were the following two factors. First, the creation of an artificial interphase between the solvent-immiscible hydrophilic glycerol and the hydrophobic reaction medium by its adsorption onto a solid support. Second, a facile system for the separation of the desired monoacylglycerol from the reaction mixture, coupled with the continuous recycling of acyl donor and undesirable by-products.     

Kinetics of the catalytic esterification of castor oil with lauric acid using <Emphasis Type="Italic">n</Emphasis>-butyl benzene as a water entrainer

The esterification of castor oil with lauric acid was investigated using tetra n-butyl titanate (TBT), SnCl₂·2H₂O (stannous chloride), CoCl₂·6H₂O (cobalt chloride), and (CH₃COO)₂Zn·2H₂O (zinc acetate dihydrate) as catalysts. Effects of catalyst concentration and reaction temperature on the progress of the reaction were investigated. TBT was the best catalyst for the esterification of castor oil with lauric acid at temperatures lower than 200°C. The reaction was first order with respect to each reactant. The activation energy for the esterification reaction of castor oil with lauric acid using TBT was 26.69 kcal/mol. The rate constants obtained for the esterification of castor oil with decanoic, lauric, palmitic, and stearic acids were nearly the same (15.80, 15.44, 15.06, and 14.67 mL mol⁻¹ min⁻¹), as were the rate constants obtained for the reaction of castor oil and hydrogenated castor oil.     

Fatty acid and product selectivities of potato tuber lipid acyl hydrolase in esterification reactions with glycerol in organic media

Fatty acid [FA; butanoic (C₄); octanoic (C₈); tetradecanoic (C₁₄); and cis-9,12-octadecadienoic (C_18:2) acids] reaction selectivity and the corresponding acyl profiles in differentially accumulating acylglycerol (AG) products (mono-, di-, and triacylglycerols; MAG, DAG, TAG, respectively) were evaluated for Celite™-immobilized potato tuber lipid acyl hydrolase (LAH)-mediated esterification reactions in isooctane at 35°C and water activity of 0.19. The ordinal pattern of FA selectivities was C₈>C₁₄>C_18:2>C₄, and the AG products accumulating were α-MAG>DAG>β-MAG>TAG. A dimensionless expression for fatty acid partitioning coefficient (FAPC) was contrived to represent the partitioning patterns of specific FA into specific AG pools on the basis of an equivalent extent of FA reaction. These FAPC values indicated that preferential partitioning of FA was as follows: C₄ was preferentially partitioned into TAG, DAG, and β-MAG; C₈ was preferentially partitioned into DAG; C₁₄ was preferentially partitioned into α,β-MAG; C_18:2 was preferentially partitioned into α,β-MAG and TAG. These findings infer that the tendency for LAH-mediated esterifications to accumulate MAG is based, in part, on a constraint in reactivity of α-MAG of ≥10 acyl carbon groups to serve as acceptors for further esterification events. The general approach taken in this study may assist in identifying the discrete steps in assembling structured glycerides where different biocatalysts exhibit the greatest degree or control of reaction selectivity.     

Enzymatic esterification of glycerol I. Lipase-catalyzed synthesis of regioisomerically pure 1,3-sn-diacylglycerols

Regioisomerically pure 1,3-sn-diacylglycerols are conveniently prepared in high yields (>80%) and in large quantities by enzymatic esterification of glycerol in the presence of various 1,3-selective lipases(Chromobacterium viscosum, Rhizopus delemar, Rhizomucor miehei) and a variety of different acyl donors like free fatty acids, fatty acid alkyl esters and vinyl esters. All reactions are carried out in aprotic organic solvents of low water content, namelyn-hexane, diethyl ether or tBuOMe. The creation of an artificial interphase between the solvent-immiscible hydrophilic glycerol and the hydrophobic reaction media by the adsorption of glycerol onto a solid support prior to use was essential for the success of these transformations. The effects of reaction conditions and the regioselectivities of the lipases on the product yields are described in detail.     

Positional distribution of decanoic acid: Effect on chylomicron and VLDL TAG structures and postprandial lipemia

Although medium-chain FA (MCFA) are mainly absorbed via the portal venous system, they are also incorporated into chylomicron TAG; therefore, the positional distribution of MCFA in TAG is likely to affect their metabolic fate. We studied chylomicron and VLDL TAG structures, as well as the magnitude of postprandial lipemia, after two oral fat loads containing decanoic acid (10∶0) predominantly at the sn-1(3),2 (MML) or at the sn-1,3 positions (MLM) of TAG in a randomized, double-blind, crossover clinical trial with 10 healthy, normal-weight volunteers. An MS-MS method was used to analyze TAG regioisomers. The position of decanoic acid in chylomicron TAG reflected its position in the TAG ingested, and TAG with none, one, two, or three decanoic acid residues were detected after ingestion of both fats. More (P<0.05) 30∶0 and 38∶1 TAG (acyl carbons:double bonds) and fewer 46∶5, 54∶5 and 54∶4 TAG were found in chylomicrons after ingestion of MML than after MLM. The VLDL TAG composition did not differ between the fat loads but did change (P<0.05) 2 to 6 h after ingestion of both fats. No statistical differences were seen between the fat loads in areas under the plasma, chylomicron, or VLDL TAG response curves or in FFA concentrations. Thus, the positional distribution of MCFA in TAG affects their metabolic, fate, but the magnitude of postprandial lipemia does not seem to be dependent on the positional distribution of MCFA in the ingested fat.     

Kinetic Resolution of 1‐Biaryl‐ and 1‐(Pyridylphenyl)alkan‐1‐ols Catalysed by the Lipase B from Candida antarctica

Lipase B from Candida antarctica (CAL‐B) catalyses the highly enantioselective (E>200) transesterification of some 1‐biaryl‐2‐yl‐, ‐3‐yl‐, and ‐4‐ylethanols and ‐propan‐1‐ols, as well as 1‐(o‐, m‐, and p‐pyridylphenyl)ethanols, 6 , with vinyl acetate, Kazlauskas' rule being obeyed in all cases. meta and para‐Substituted substrates were transformed within several hours (conversion degree ranging from 23–50%), reaction rates for propan‐1‐ol derivatives being slower than those for ethanol derivatives. Transesterifications of ortho‐substituted alcohols took several days and were accompanied by a chemoenzymatic side reaction: the formation of another acetate derived from the hemiacetal between 6 and acetaldehyde coming from vinyl acetate. This side reaction was suppressed in the presence of isopropenyl acetate as acyl donor, conversion degrees for transesterification ranging from 20–40% after ten days (E>200). The usefulness of (R)‐ 6p as ligand in the asymmetric addition of diethylzinc to benzaldehyde was also demonstrated.     

Enzymatic preparation of enantiomerically pure sn-2,3-diacylglycerols: A stereoselective ethanolysis approach

Stereoselective ethanolysis of monoacid TAG by immobilized Rhizomucor miehei lipase (RML) was studied for preparation of optically pure sn-2,3-DAG. Trioctanoylglycerol (TO) was used as a model substrate. The enantiomeric purity of the product, sn-2,3-dioctanoylglycerol (sn-2,3-DO), was very high (percent enantiomeric excess >99%) when an excess of ethanol was used. The result indicated that RML was highly stereoselective toward the sn-1 position of TO under conditions of excess ethanol. The stereoselectivity of RML depended on the amount of ethanol. The larger the amount of ethanol was, the higher the stereoselectivity became. After optimizing the parameters such as reactant molar ratio, water content, and temperature, (ethanol/TO molar ratio =31∶1 and water content =7.5 wt% of the reactants at 25°C), optically pure sn-2,3-DO was obtained at 61.1 mol% in the glyceride fraction in 20 min. The above conditions were further applied for ethanolysis of monoacid TAG with different acyl groups such as tridecanoylglycerol (C10∶0), tridodecanoylglycerol (C12∶0), tritetradecanoylglycerol (C14∶0) and trioctadecenoylglycerol [triolein, (C18∶1)]. The yields and enantiomeric purities of 1,2(2,3)-DAG were dramatically reduced when TAG with FA longer than decanoic acid were used.     

Purification of docosahexaenoic acid from tuna oil by a two-step enzymatic method: Hydrolysis and selective esterification

Purification of docosahexaenoic acid (DHA) was attempted by a two-step enzymatic method that consisted of hydrolysis of tuna oil and selective esterification of the resulting free fatty acids (FFA). When more than 60% of tuna oil was hydrolyzed with Pseudomonas sp. lipase (Lipase-AK), the DHA content in the FFA fraction coincided with its content in the original tuna oil. This lipase showed stronger activity on the DHA ester than on the eicosapentaenoic acid ester and was suitable for preparation of FFA rich in DHA. When a mixture of 2.5 g tuna oil, 2.5 g water, and 500 units (U) of Lipase-AK per 1 g of the reaction mixture was stirred at 40°C for 48 h, 83% of DHA in tuna oil was recovered in the FFA fraction at 79% hydrolysis. These fatty acids were named tuna-FFA-Ps. Selective esterification was then conducted at 30°C for 20 h by stirring a mixture of 4.0 g of tuna-FFA-Ps/lauryl alcohol (1:2, mol/mol), 1.0 g water, and 1,000 U of Rhizopus delemar lipase. As a result, the DHA content in the unesterified FFA fraction could be raised from 24 to 72 wt% in an 83% yield. To elevate the DHA content further, the FFA were extracted from the reaction mixture with n-hexane and esterified again under the same conditions. The DHA content was raised to 91 wt% in 88% yield by the repeated esterification. Because selective esterification of fatty acids with lauryl alcohol proceeded most efficiently in a mixture that contained 20% water, simultaneous reactions during the esterification were analyzed qualitatively. The fatty acid lauryl esters (L-FA) generated by the esterification were not hydrolyzed. In addition, L-FA were acidolyzed with linoleic acid, but not with DHA. These results suggest that lauryl DHA was generated only by esterification.     

Esterification of Free Fatty Acids in <Emphasis Type="Italic">Zanthoxylum bungeanum</Emphasis> Seed Oil for Biodiesel Production by Stannic Chloride

In this study, SnCl₄ was chosen as a solid catalyst for esterification of free fatty acids (FFA) in Zanthoxylum bungeanum seed oil (ZSO). A central composite rotatable design was used to investigate the effect of the methanol-to-oil molar ratio, catalyst amount and reaction time on the SnCl₄-catalyzed esterification of FFA. The methanol-to-oil molar ratio and reaction time clearly affected the conversion efficiency of FFA in the test ranges. Response surface methodology was used to optimize the conditions for SnCl₄-catalyzed esterification. A quadratic polynomial equation was obtained for conversion efficiency of FFA by multiple regression analysis and verification experiments confirmed the validity of the predicted model. Under the optimum conditions, the conversion efficiency of FFA in vegetable oil reached above 96 %. This study demonstrates the effectiveness of SnCl₄ as an acid catalyst for the reduction of high FFA content in vegetable oils to a low level by one-step esterification.     

In situ esterification of rice bran oil with methanol and ethanol

In situ esterifications of high-acidity rice bran oil with methanol and ethanol and with sulfuric acid as catalyst were investigated. In the esterification with methanol, all free fatty acids (FFA) dissolved in methanol were interesterified within 15 min, and it was possible to obtain nearly pure methyl esters. The amount of methyl esters obtained from a given rice bran was dependent on the FFA content of the rice bran oil. In the esterification with ethanol, it was not possible to obtain pure esters as in methanol esterification, because the solubilities of oil components in ethanol were much higher than those in methanol.     

Enzymatic Synthesis of Glucose- and Xylose Laurate Esters Using Different Acyl Donors,Higher Substrate Concentrations,and Membrane Assisted Solvent Recovery

Glucose- and xylose laurate esters are enzymatically synthesized using equimolar substrate concentrations in 2-methyl-2-butanol, comparing free lauric acid with methyl- and vinyl-laurate as acyl donors. All reactions result in ≥70% acyl donor conversions after 72 h but the activated donors are also partially hydrolyzed to lauric acid, highlighting the difficulty in controlling water presence in this particular reaction system. The esterification of xylose generates a complex product profile, with several regioisomers of monoesters and diesters. The esterification of glucose is quite selective, forming mainly the 6-O monoester (≥96%) with a small presence of two diester isomers (4%). Increasing substrate concentration up to 800 millimoles kg⁻¹ results in lower conversion values (down to 58%) but shows that the reaction proceeds successfully even in the presence of high amounts of insoluble glucose. However, the reaction is less selective and the proportion of diester increases, becoming up to 46% (molar fraction) of the final product. Solvent recovery after esterification can be achieved by organic solvent nanofiltration through a polymeric membrane able to retain ≥80% of all reaction substrates and products. Practical Applications: The use of high substrate concentrations during the enzymatic synthesis of sugar ester biosurfactants leads to product titers that are more industrially appealing, without the need to find a solvent that can solubilize all initial substrate. The sustainability of the enzymatic conversion at mild temperatures can be enhanced by recycling of the reaction solvent through organic solvent nanofiltration, an energy efficient alternative to other traditional methods like distillation.     

Kinetics of the formation of symmetrical wax esters from the corresponding alcohols with the use of hydrobromic acid and hydrogen peroxide

The primary aliphatic alcohols n-octanol, n-decanol, and n-dodecanol have been converted to their corresponding symmetrical esters by using HBr and H₂O₂ in the absence of a solvent. The reaction was carried out at 30, 40, and 50°C and at mole ratios of alcohol to HBr of 1∶0.1, 1∶0.2, 1∶0.3, and 1∶0.5. The rate of the reaction was found to increase with increase in the reaction temperature and concentration of HBr. The maximal conversion of n-octanol was 72% at 40°C and a mole ratio of n-octanol to HBr of 1∶0.5. The kinetics of the reaction have been established, and the reaction was found to be first-order with respect to alcohol and bromine concentration in the organic phase, and second-order with respect to both. The second-order rate constants for n-octanol, n-decanol, and n-dodecanol are 27.08, 32.58, and 37.42 mL mol⁻¹ min⁻¹, respectively, at 40°C. The activation energy for the esterification reaction of n-octanol was found to be 16.32 kcal mol⁻¹.     

Enzymatic Production of Fatty Acid Methyl Esters by Hydrolysis of Acid Oil Followed by Esterification

Acid oil, a by-product of vegetable oil refining, was enzymatically converted to fatty acid methyl esters (FAME). Acid oil contained free fatty acids (FFA), acylglycerols, and lipophilic compounds. First, acylglycerols (11 wt%) were hydrolyzed at 30 °C by 20 units Candida rugosa lipase/g-mixture with 40 wt% water. The resulting oil layer containing 92 wt% FFA was used for the next reaction, methyl esterification of FFA to FAME by immobilized Candida antarctica lipase. A mixture of 66 wt% oil layer and 34 wt% methanol (5 mol for FFA) were shaken at 30 °C with 1.0 wt% lipase. The degree of esterification reached 96% after 24 h. The resulting reaction mixture was then dehydrated and subjected to the second esterification that was conducted with 2.2 wt% methanol (5 mol for residual FFA) and 1.0 wt% immobilized lipase. The degree of esterification of residual FFA reached 44%. The degree increased successfully to 72% (total degree of esterification 99%) by conducting the reaction in the presence of 10 wt% glycerol, because water in the oil layer was attracted to the glycerol layer. Over 98% of total esterification was maintained, even though the first and the second esterification reactions were repeated every 24 h for 40 days. The enzymatic process comprising hydrolysis and methyl esterification produced an oil containing 91 wt% FAME, 1 wt% FFA, 1 wt% acylglycerols, and 7 wt% lipophilic compounds.     

Effect of temperature programming on the performance of urea inclusion compound-based free fatty acid fractionation

The effect of cooling rate on the degree of removal of saturated acyl groups from FFA derived from canola oil and the isolation of di- and polyunsaturated acyl groups from FFA derived from vegetable and fish oil, respectively, during urea inclusion compound (UIC)-based fractionation was investigated. Traditionally, slow cooling has been used (ca.−1°C min⁻¹). A more rapid cooling rate (−47°C min⁻¹) produced UIC crystals of similar morphology and thermodynamic properties, but of a size an order of magnitude smaller than the UIC formed during slow cooling. Fractionations used only renewable materials (urea, FFA, and 95% ethanol as solvent) and benign operating conditions (ambient pressure, 25–75°C, and neutral pH). When the recovery of FFA (in the solvent-rich phase) was relatively high (>60%), the selectivity of UIC-based fractionation toward the inclusion of saturated FFA and against polyunsaturated FFA was not affected by the cooling rate. In contrast, when the FFA recovery was low, representing cases in which an increase of the PUFA purity is a more important economic goal, a slower cooling rate resulted in a significantly greater discrimination against PUFA groups, hence to a FFA product with a measurably greater purity. This paper was presented at the 96th AOCS Anual Meeting and Expo, Salt Lake City, Utah, on May 4, 2005.     

Concentration of Stearidonic Acid in Free Fatty Acids Form from Modified Soybean Oil by Selective Esterification with Dodecanol

The concentration of stearidonic acid (SDA, 18:4 n-3) in free fatty acids (FFA) formed by selective esterification with dodecanol (lauryl alcohol) was studied. For this purpose, modified soybean oil (initial SDA content, ~23 %) was converted into its corresponding FFA by chemical hydrolysis. In a second step, the resulting FFA were esterified with dodecanol. Process variables such as the type of biocatalyst (lipase), substrate molar ratio and amount of lipase were evaluated. The best SDA concentration (58 %) and recovery (94 %) were attained by performing the esterification reaction for 4 h, with 1:1 molar ratio (dodecanol:FFA), and 5 % (w/w) Candida rugosa lipase as biocatalyst. It was observed that SDA was concentrated in the unesterified fraction.