Characterization of Terebinth Fruit Oil and Optimization of Acidolysis Reaction with Caprylic and Stearic Acids

Characterization of the fatty acid and triacylglycerol composition of terebinth fruit oil and the synthesis of structured lipids (SL) were performed in this study. Interesterification reaction of terebinth fruits oil (Pistacia terebinthus L.) with caprylic acid (CA) and stearic acid (SA) to produce a SL was performed in n-hexane using immobilized sn-1,3 specific lipase from Mucor miehei. The effect of reaction conditions and relationship among them were analyzed by response surface methodology (RSM) with a four-factors five-level central composite rotatable experimental design. The four major factors chosen were enzyme load (10–30 wt% based on substrates), reaction time (7–18 h), reaction temperature (40–60 °C) and substrate mole ratio (terebinth oil:SA:CA 1:1:1–1:1:3). The best fitting quadratic model was determined by regression and backward elimination. Based on the fitted model, the optimal reaction conditions for the incorporation of CA and SA were found to be temperature 50 °C; time 18 h; enzyme load 30 wt%; substrate ratio 1:1:3. Under these optimum conditions, the incorporation of SA and CA could be obtained as 19 and 14%, respectively.     

Typoselectivity of Crude Geobacillus sp. T1 Lipase Fused with a Cellulose-Binding Domain and Its Use in the Synthesis of Structured Lipids

Typoselectivity of crude CBD-T1 lipase (Geobacillus sp. T1 lipase fused with a cellulose binding domain) was investigated. Multi-competitive reaction mixtures including a set of n-chain fatty acids (C8:0, C10:0, C12:0, C14:0, C18:1 n-9, C18:2 n-6 and C18:3 n-3) and tripalmitin-enriched triacylglycerols were studied in hexane. The crude CBD-T1 lipase discriminated strongly against C18:1 n-9 [competitive factor (α) = 0.23] and showed the highest preference for C8:0 (α = 1). Utilizing the catalytic properties of crude CBD-T1 lipase, acidolysis of soybean oil with C8:0 was selected as a model reaction to investigate the ability of the lipase to produce MLM-type (medium-long-medium) structured lipids. Several reaction parameters (added water amount, reaction temperature, substrate molar ratio and reaction time) examined for incorporating C8:0 into soybean oil, the optimum conditions were: 1:3 (soybean oil/C8:0) of molar ratio, 3 mL of hexane, 50 °C of temperature, 48 h of reaction time, 20 % of crude CBD-T1 lipase (w/w total substrates), and 7.5 % of water (w/w enzyme). Under these conditions, the incorporation of C8:0 was 29.6 mol%. The results suggest that crude CBD-T1 lipase, which showed different fatty acid specificity profiles, is a potential biocatalyst for the modification of fats and oils.     

Synthesis of Wax Esters from Crude Fish Fat by Lipase of Burkholderia sp. EQ3 and Commercial Lipases

The lipase from Burkholderia sp. EQ3 was used to synthesize wax esters in comparison with commercial lipases. The supernatant of Burkholderia sp. EQ3 was collected from a liquid basal medium with 1 % fish oil after 12 h cultivation (1.90 U/ml of lipase activity). The crude lipase was prepared by acetone precipitation of the culture supernatant (4.70 U/mg and 9.40 purification folds). The crude fish fat obtained by hexane extraction of waste fat from the wastewater pond of a canned tuna factory and cetyl alcohol were used for wax esters synthesis. Five commercial lipases were screened in comparison with crude lipase from Burkholderia sp. EQ3 in wax esters synthesis. The optimum conditions for wax esters synthesis from crude fish fat using Novozyme 435 were enzyme 1 U, substrate molar ratio of crude fish fat to cetyl alcohol 1:2 (115.30 mg of crude fish fat and 66.67 mg of cetyl alcohol) in hexane at 37 °C and 200 rpm with 90.81 % (TLC–FID peak area) after one h of reaction. The optimum conditions for the synthesis by crude lipase from Burkholderia sp. EQ3 were crude lipase 40 U, substrate molar ratio of crude fish fat and cetyl alcohol 1:2 in isooctane at 30 °C and 200 rpm with 95.07 % (TLC–FID peak area) after 6 h of reaction. The synthesized wax esters were mainly composed of cetyl palmitate and cetyl oleate.     

Effects of Particle Size of Sucrose Suspensions and Pre-incubation of Enzymes on Lipase-Catalyzed Synthesis of Sucrose Oleic Acid Esters

The effects of high-speed homogenization, high-intensity ultrasound, and their combination were evaluated for the reduction of the particle size of sucrose crystals to enhance solvent-free lipase-catalyzed synthesis of sucrose oleate at 65 °C. The combination of homogenization and ultrasound greatly decreased the particle size of suspended sucrose crystals in mixtures of oleic acid/sucrose oleate (86 wt% monoester and 14 wt% diester at a ratio of 90/10 w/w) from 88 to 18 μm. The suspension-based medium was charged to a stirred tank bioreactor that also contained immobilized lipase from Rhizomucor miehei or Candida antarctica (Lipozyme^®IM and Novozym^® 435, respectively; Novozymes, Franklinton, NC, USA), that was pre-incubated in oleic acid for several different temperatures (23–60 °C), durations (4–24 h), and stir rates (50–400 rpm, radius of 3 cm), prior to use. The optimal performance was achieved using C. antarctica lipase (83.3 wt% ester, consisting of 46 wt% monoester) in the presence of molecular sieves (18 wt%). The low water concentration (~0.12 wt%) did not affect the activity of C. antarctica lipase.     

Optimization of Lipase-Catalyzed Fractionation of Two Conjugated Linoleic Acid (CLA) Isomers

The separation of two isomers of conjugated linoleic acid is highly significant since each exhibits different biochemical properties. The aim of this study was to investigate and optimize several factors affecting the esterification of l-menthol with the c9,t11-CLA isomer in an organic solvent-free system using lipase from Candida rugosa (Lipase AY-30). D-optimal design with 5 factors and 3 levels were employed to evaluate the effects of synthesis parameters; reaction time (8–24 h), temperature (30–50 °C), enzyme content (2–20 U/ml), substrate molar ratio of conjugated linoleic acid oil to l-menthol (2:1–1:2) and pH (6–8) on esterification of c9,t11-CLA with l-menthol. Based on the analysis of the residual amount of c9,t11-CLA in the free fatty acid fraction after just one-step esterification, the optimum synthesis conditions were as follows: reaction time 23.12 h, temperature 32.65 °C, enzyme amount 135.40 U, molar ratio of CLA oil to l-menthol at 1:1.7 and pH at 7.7; the lowest purity of c9,t11-CLA in free fatty acid fraction based on the total content of c9,t11 and t10,c12-CLA isomers was 8.6 %.     

Production of Diacylglycerol-Mixture of Regioisomers with High Purity by Two-Step Enzymatic Reactions Combined with Molecular Distillation

Pure diacylglycerol (DAG) is of vital importance for the biomedical and dietetic properties research of DAG. In this study, we aimed to develop an effective process to produce DAG-mixture of regioisomers with high purity. Firstly, DAGs and monoacylglycerols (MAGs) were synthesized by enzymatic esterification of glycerol and free fatty acids (FFAs) from camellia oil with catalysis of Penicillium camembertii lipase, and the obtained reaction mixture was composed of 49.9 % DAG [33.4 % for 1,3-DAG and 16.5 % for 1,2 (2,3)-DAG], 31.6 % MAG and 18.5 % FFA. Secondly, a monoacylglycerol lipase (lipase CBD-MGLP), which was produced by recombinant Escherichia coli in our laboratory, was employed to hydrolyze MAG in the above reaction mixture, and the MAG content decreased to 1.9 % under the optimal conditions with 375 U/g (U/w, with respect to the mass of MAG in the mixture) of CBD-MGLP loading, temperature of 45 °C, mass ratio of esterification mixture to Tris–HCl buffer (w/w) 10:10, and pH of Tris–HCl buffer 9.0. Then, the hydrolytic products were further purified by molecular distillation at low temperature of 130 °C under a pressure of 10 Pa [equivalent to 377 °C at 101.325 kPa (1 atm)], and the DAG purity was up to 98.0 % (66.6 % for 1,3-DAG and 31.4 % for 1,2-DAG) in the final products. This indicated that two-step enzymatic reactions combined with molecular distillation at low temperature could be a feasible and prospective process to produce DAG-mixture of regioisomers with high purity.     

Enrichment of DHA from Tuna Oil in a Packed Bed Reactor via Lipase-Catalyzed Esterification

A packed-bed reactor (length 6.5 cm; id 4.65 mm) has been used to enrich docosahexaenoic acid (DHA) via the lipase-catalyzed esterification of the fatty acid from tuna oil with ethanol. Lipozyme RM IM (from Rhizomucor miehei) was used for the esterification reaction because of its ability to discriminate between different fatty acids, and several reaction parameters, including the temperature, molar ratio of substrates, and water content were explored as a function of residence time. In this way, the optimum conditions for the enrichment process were determined to be a temperature of 20 °C, a molar ratio of 1:5 (i.e., fatty acid to ethanol), and a water content of 1.0 % (based on the total substrate weight). Under these conditions, a residence time of 90 min gave a DHA concentration of 70 wt% and a DHA recovery yield of 87 wt% in the residual fatty acid fraction.     

Lipase-Catalysed Enrichment of γ-Linolenic Acid from Evening Primrose Oil in a Solvent-Free System

The enrichment of γ-linolenic acid (GLA) was carried out in a solvent-free system by lipase-catalysed esterification of free fatty acids from evening primrose oil (EPO-FA) and 1-butanol (BtOH). The lipase employed to conduct this study was a free preparation of Candida rugosa. Variables evaluated were: substrate molar ratio (1:4, 1:6, 1:8, 1:10 and 1:12, EPO-FA:BtOH), temperature (10, 20, 30, 40, 50 and 60 °C), and enzyme loading (5, 10, 15 and 20 %, based on the total weight of substrates). GLA was highly enriched in the non-esterified fatty acid fraction since C. rugosa showed very low selectivity for this fatty acid. We were able to increase the content of GLA to ca. 70 wt.% under the following optimal conditions: 30 °C, 10 % enzyme loading and a 1:10 molar ratio (EPO-FA:BtOH), after 24 h. An additional set of experiments was conducted whereby the amount of water was controlled by addition of molecular sieves to the reaction mixture. The latter experiments produced a higher GLA concentrate (83.74 wt.%), under the optimal conditions described above and by adding 10 % molecular sieves (based on the total weight of substrates) after 36 h.     

Synthesis of Ascorbyl Palmitate with Immobilized Lipase from Pseudomonas stutzeri

Synthesis of ascorbyl palmitate by enzymatic esterification of palmitic acid and ascorbic acid was conducted in an organic medium with Pseudomonas stutzeri lipase TL immobilized in different supports and its performance was compared with commercial Novozym 435 lipase used as a reference. The enzyme was immobilized in different supports and the best catalyst was selected in terms of immobilization yield and mass specific activity to perform the reactions of synthesis. Synthesis of ascorbyl palmitate was optimized considering temperature, substrate molar ratio and enzyme to limiting substrate mass ratio as variables, and substrate conversion and specific productivity as evaluation parameters. The best reaction conditions for immobilized lipase TL were 55 °C, 1:5 ascorbic to palmitic acid molar ratio, and 1:10 lipase to ascorbic acid mass ratio, obtaining 57 % substrate conversion and a specific productivity of 0.013 [g ascorbic acid/(g enzyme × min)]; the best conditions for Novozym 435 were 70 °C, ascorbic to palmitic acid molar ratio 1:10, and 1:10 lipase to ascorbic acid mass ratio, obtaining 51 % substrate conversion and a specific productivity of 0.016 [g ascorbic acid/(g enzyme × min)].     

OPTIMIZATION OF LIPASE-CATALYZED SYNTHESIS OF N-trans-FERULOYLTYRAMINE USING RESPONSE SURFACE METHODOLOGY (RSM)

Enzymatic synthesis of N-trans-feruloyltyramine amide was optimized by response surface methodology (RSM) using 4-hydroxy-3-methoxycinnamic acid and tyramine hydrochloride in a one-step lipase catalyzed reaction using Lipozyme TL IM. Response surface methodology (RSM) based on five-level, four-variable central composite rotatable design (CCRD) was used to evaluate the interaction of synthesis, reaction time (24–96 h), temperature (30°–50°C), amount of enzyme (50–500 mg, 12.5–125.0 IUN), and substrate molar ratio (cinnamic acid:tyramine HCl) 1:1–8:1 mmol on the percentage yield of N-trans-feruloyltyramine amide. The optimum conditions derived via RSM were: reaction time 52 h, temperature 43°C, amount of enzyme 260 mg (65.0 IUN), and substrate molar ratio (cinnamic acid:tyramine HCl) 6.2:1. The actual experimental yield was 96.3% under optimum conditions, which compared well to the maximum predicted value of 97.2%.     

Modification of Stearidonic Acid Soybean Oil by Immobilized Rhizomucor miehei Lipase to Incorporate Caprylic Acid

Immobilized sn-1,3 specific Rhizomucor miehei lipase (RML) was used to catalyze the incorporation of caprylic acid (C8:0) into high stearidonic acid (SDA, C18:4ω3) soybean oil (SDASO) to form structured lipids (SL). The effects of type of biocatalyst (Celite-, octyl-Sepharose-, and Duolite-immobilized RML) and reaction temperature (30, 40, 50, and 60 °C) on acidolysis and acyl migration were studied. Celite-immobilized RML (C-RML) at 50 °C maximized C8:0 incorporation and minimized acyl migration compared to other treatments. Optimal levels of substrate molar ratio (C8:0 to SDASO), incubation time, and enzyme load for SL synthesis by C-RML at 50 °C was determined by response surface methodology to be 6:1, 24 h, and 20 % weight of substrates, respectively. This optimum treatment was scaled-up in hexane or solvent-free reaction media using SDASO or an SDA-enriched acylglycerol mixture as substrate. This yielded various SL with C8:0 contents ranging from 17.0 to 32.5 mol% and SDA contents ranging from 20.6 to 42.3 mol%. When digested, these SL may deliver C8:0 for quick energy and SDA for heart health making them potentially valuable for medical and nutraceutical applications.     

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.     

Lipase-Catalyzed Interesterification of Schizochytrium sp. Oil and Medium-Chain Triacylglycerols for Preparation of DHA-Rich Medium and Long-Chain Structured Lipids

DHA-rich medium and long-chain structured lipids (MLSL) were successfully synthesized by lipase-catalyzed interesterification of microbial oil from Schizochytrium sp. with medium-chain triacylglycerols (MCT) containing 99% of caprylic acid. Parameters that affected the reaction process were investigated and the conditions were selected as follows: lipase from Aspergillus oryzae, NS40086; reaction time, 8 hours; substrate molar ratio (MCT/microbial oil), 1:1; lipase load, 8 wt%; reaction temperature, 60 °C. Under these conditions, the proportions of MCT, MLSL, and long-chain triacylglycerols (TAG) in the final products were 12.5%, 62.8%, and 24.6%, respectively. The final product was then subjected to UPLC-MS/MS. Eighty-three types of TAG were identified, in which 54 types contained MCFA and MLSL species with relatively high contents were 22:6–8:0–8:0 (6.8%), 8:0–8:0–16:0 (7.5%), and 16:0–16:0–8:0 (7.5%). This product rich in MLSL with DHA and MCFA in the same TAG molecule is beneficial for fat digestion and absorption in infant and thus can increase the bioavailability of DHA at the molecular level.     

Lipase-catalyzed glycerolysis of olive oil in organic solvent medium: Optimization using response surface methodology

Enzymatic glycerolysis of olive oil for mono- (MG) and diglycerides (DG) synthesis was investigated. Several pure organic solvents and co-solvent mixtures were screened in a batch reaction system. The yields of MG and DG in co-solvent mixtures exceeded those of the corresponding pure organic solvents. Batch reaction conditions of the glycerolysis reaction, the lipase amount, the glycerol to oil molar ratio, the reaction time, and temperature, were studied. In these systems, the high content of reaction products, especially MG (55.8 wt%) and DG (16.4wt%) was achieved at 40 °C temperature and 0.025 g of lipase with relatively low glycerol to oil molar ratio (2: 1) within 4 h of reaction time in isopropanol/tert-butanol (1: 3) solvent mixture. Glycerolysis reaction was optimized with the assistance of response surface methodology (RSM). Optimal condition for reaction conversion was recommended as lipase amount 0.025 g, glycerol to oil molar ratio 2: 1, reaction time 4 h and temperature 40 °C.     

Concentration of Docosahexaenoic Acid (DHA) by Selective Alcoholysis Catalyzed by Lipases

The aim of this work was to obtain acylglycerols from tuna oil (23 % weight DHA) rich in docosahexaenoic acid (DHA) by selective ethanolysis, catalyzed by lipases. First, seven immobilized lipases were tested and the best DHA concentration and recovery in the acylglycerol fraction were attained with Lipozyme TL IM^® from Thermomyces lanuginosus, Lipozyme RM IM from Rhizomucor miehei, and lipase from Thermomyces lanuginosus immobilized on Immobead 150. As it is the cheapest, Lipozyme TL IM^® was selected to optimize the reaction conditions. The influence of temperature, reaction time, and ethanol/oil and lipase/oil ratios were studied. Under the optimized conditions (35 °C, ethanol/oil molar ratio 2.3, lipase/oil ratio 5 % weight and 48 h) and for a 56 % conversion, acylglycerols were obtained with a 45 % DHA concentration and 90 % recovery. In these optimized conditions the reaction was scaled up to 766 g of tuna oil and carried out in a batch stirred tank reactor, with the lipase contained in a cartridge filter attached to the stirring rod. The results were similar to those obtained on the smaller scale. The DHA-enriched acylglycerols were separated from the ethyl esters by evaporation of the latter in a short-path vacuum distiller, where the influence of distillation temperature was studied. At 170 °C DHA-rich acylglycerols (44 % DHA) were recovered in the residue with 94.5 % purity and 72 % recovery.     

Preparation of Docosahexaenoic Acid-Rich Diacylglycerol-Rich Oil by Lipase-Catalyzed Glycerolysis of Microbial Oil from Schizochytrium sp. in a Solvent-Free System

Docosahexaenoic acid (DHA)-rich diacylglycerol (DAG)-rich oil was prepared by lipase-catalyzed glycerolysis of microbial oil from Schizochytrium sp. in a solvent-free system. The reaction parameters including lipase type, substrate molar ratio, temperature, lipase concentration, and reaction time were screened. The selected conditions were determined as follows: Novozym® 435 (Novozymes A/S, Bagsvaerd, Denmark) as biocatalyst at 8 wt%, substrate ratio (DHA-rich microbial TAG/glycerol) of 1:1 mol/mol, temperature of 50 °C, and reaction time of 12 hours. Under these conditions, the triacylglycerol (TAG), DAG, and monoacylglycerol (MAG) contents in the product were 36.4%, 48.2%, and 15.4%, respectively. The lipase was reused successively for 18 cycles without significant loss of activity under the conditions given above. Fatty acid composition analysis of the final product showed that the contents of DHA in TAG, DAG, and MAG were 53.9%, 44.9%, and 34.8%, respectively. DHA-rich DAG has the potential to be used as an ingredient in infant formula to increase the bioavailability of DHA.     

Application of Taguchi Method in the Enzymatic Modification of Menhaden Oil to Incorporate Capric Acid

Structured lipids (SL) were produced using menhaden oil and capric acid or ethyl caprate as the substrate. Enzymatic reaction conditions were optimized using the Taguchi method L9 orthogonal array with three substrate molar ratio levels of capric acid or ethyl caprate to menhaden oil (1:1, 2:1, and 3:1), three enzyme load levels (5, 10, and 15% [w/w]), three temperature levels (40, 50, and 60 °C), and three reaction times (12, 24, 36 hours). Recombinant lipase from Candida antarctica, Lipozyme^® 435, and sn‐1,3 specific Rhizomucor miehei lipase, Lipozyme^® RM IM (Novozymes North America, Inc., Franklinton, NC, USA), were used as biocatalysts in both acidolysis and interesterification reactions. Total and sn‐2 fatty acid compositions, triacylglycerol (TAG) molecular species, thermal behavior, and oxidative stability were compared. Optimal conditions for all reactions were 3:1 substrate molar ratio, 10% [w/w] enzyme load, 60 °C, and 16 hours reaction time. Reactions with ethyl caprate incorporated significantly more C10:0, at 30.76 ± 1.15 and 28.63 ± 2.37 mol% versus 19.50 ± 1.06 and 9.81 ± 1.51 mol%, respectively, for both Lipozyme^® 435 and Lipozyme^® RM IM, respectively. Reactions with ethyl caprate as substrate and Lipozyme^® 435 as biocatalyst produced more of the desired medium‐long‐medium (MLM)‐type TAGs with polyunsaturated fatty acids (PUFA) at sn‐2 and C10:0 at sn‐1,3 positions.     

Hydrolytic Activity of Castor Bean Powder: Effect of Gum Arabic,Lipase and Oil Concentrations

Lipase activity from castor bean seed powders was evaluated in hydrolysis reactions at 37 °C. The effects of different concentrations of lipase powder (LP), substrate (high oleic sunflower oil, O) and surfactant (gum arabic, A) on lipase activity (R) were assessed using experimental designs. Considered variable bounds were: 0.05–0.15 g_LP, 0.07–0.20 oil:aqueous phase (w/w) and 0–0.025 g gum arabic/mL. All variables had significant effects on the transformed response, R ^1/2. The most important result was the negative effect of gum arabic in lipase activity, even when high oil concentrations were used. Experimental lipase activities involved in this work were within 0.32–16.90 mmol_FFA/g_oil·g_LP·h. Using 0.05 g_LP and 0.20 oil:aqueous phase (w/w) without gum arabic, the activity of 20.47 ± 7.19 mmol_FFA/g_oil·g_LP·h was reached.     

Enzymatic synthesis of monoacylglycerol citrate optimized by response surface methodology

The ability of immobilized lipase B from Candida antarctica (Novozym 435) to catalyze the direct esterification of citric acid (CA) and monoglyceride (MG) for citrate esters of monoacylglycerols (CITREM) preparation was investigated. The effects of substrate concentration, molecular‐sieve amount, substrate molar ratio, reaction temperature, time, and enzyme load on the conversion of CA in the reaction were investigated. Enzyme screening and the effect of solvent on the esterification were also investigated. RSM was used to optimize the effects of the reaction temperature (45–55°C), the enzyme load (6–10%; relative to the weight of total substrates), and the reaction time (24–48 h) on the conversion of CA. Validation of the RSM model was verified by the good agreement between the experimental and the predicted values of CA conversion. The optimum preparation conditions were as follows: CA concentration 0.12 mol/L, molecular‐sieve 120 g/L, molar ratio of MG/CA 2:1, temperature 54.18°C, enzyme load 9.0% (relative to the weight of total substrates), and reaction time 47.5 h. Under the suggested conditions, the conversion of CA was 77.4%. Repeated reaction tests indicated that Novozym 435 could be used eight times under the optimum conditions with 92% of its original catalytic activity still retained.     

Lipase-Mediated Synthesis of Fatty Acid Esters Using a Blending Alcohol Consisting of Methanol and 1-Butanol

Lipase-mediated transesterification of soybean oil with a blending alcohol consisting of methanol and 1-butanol for synthesis of fatty acid esters was carried out. Lipase from Thermomyces lanuginosa (Lipozyme TL IM) was used as a biocatalyst. The lipase was purchased from Novozymes (Seoul, Republic of Korea). The effects of the molar proportions of methanol and 1-butanol in the blending alcohol, reaction temperature, enzyme loading and water content were investigated, for reaction optimization. The relative consuming rates of methanol and 1-butanol during the reaction were also explored. Among seven different ratios of alcohol blends employed in this study, that containing 80 mol% methanol gave the highest yield of fatty acid esters. Optimum reaction temperature, enzyme loading, and water content were 30 °C, 15% (based on the substrate weight), and 0.3% (based on the substrate weight), respectively. Water influenced significantly the reaction rate and yield. On the transesterification, the degree of reaction of methanol was higher than that of 1-butanol and the presence of 1-butanol contributed to increase of the reaction rate as well as yield. The maximum yield of ca. 98 wt% was achieved under the optimized condition.