Optimization of the synthesis of lower glycerides rich in unsaturated fatty acid residues obtained via enzymatic ethanolysis of sesame oil

The lipases Novozym 435, Lipozyme TL IM and Lipozyme RM IM were employed in the production of lower acylglycerols (LG), i.e. mono‐ (MAG) and diacylglycerols (DAG), rich in unsaturated fatty acids from sesame oil in batch reactors. The effect of the molar ratio of ethanol to fatty acids on the reusability of these immobilized lipases was studied in detail. The effects of pretreatment on lipase activity for ethanolysis were investigated. Glycerol had a strong product inhibition effect on the ethanolysis reaction, and a relatively large excess of ethanol was necessary to remove the glycerol adsorbed on these biocatalysts. The enzymatic activity was drastically reduced by addition of water to the reaction medium. The presence of organic solvents (hexane and acetone) did not favor the production of LG. For the Novozym 435‐catalyzed reaction, optimum conditions were a molar ratio of ethanol to fatty acid residues of 5 : 1, 15 wt‐% lipase and 50 °C. For Lipozyme TL IM, the optimum conditions were a molar ratio of ethanol to fatty acid residues of 5 : 1, 20 wt‐% biocatalyst, and 30 °C. Novozym 435 and Lipozyme TL IM produced LG with molar ratios of unsaturated to saturated fatty acids of 20.4 in 1 h and 25.3 in 5 h, respectively. In the original oil, this ratio was 5. For trials conducted under optimum conditions, the products from the Novozym 435 trials contained 21.8 wt‐% triacylglycerols (TAG), 24 wt‐% DAG and 54.2 wt‐% MAG. The products of the Lipozyme TL IM trials consisted of 12.9 wt‐% DAG and 87.1 wt‐% MAG. No TAG species were detected.     

Influence of silica gel in production of diacylglycerol via enzymatic glycerolysis of palm olein

Enzymatic glycerolysis was explored in this paper for the production of diacylglycerol (DAG) oils from palm olein. Three commercial enzymes, Lipozyme TL IM, Lipozyme RM IM and Novozym 435 were used for their ability to synthesize DAG in a solvent‐free system. Novozym 435 was found to be the more effective enzyme, resulting in a high DAG production even in the absence of an adsorbent such as silica gel. The yields of DAG were between 43 and 50 wt‐%. Lipozyme TL IM and RM IM, being supported on hydrophilic materials, require an adsorbent to allow slow release of glycerol for reaction with the enzyme and oil. In the absence of silica, no reaction was observed. The success of the reaction is therefore very dependent on the amount of silica used. The yields of DAG using Lipozyme TL IM and RM IM were 52 and 45 wt‐%, respectively. In addition, the degree of reduction in tocopherols and tocotrienols appeared correlated with the efficacy of the glycerolysis reaction. Changes in the slip melting points and solid fat contents of the products are indicative of the reaction occurring.     

Lipase-catalyzed acidolysis of canola oil with caprylic acid to produce medium-, long- and medium-chain-type structured lipids

Lipase-catalyzed acidolysis of canola oil with caprylic acid was performed to produce structured lipids (SLs) containing medium-chain fatty acid (M) at position sn-1,3 and long-chain fatty acid (L) at the sn-2 position in a solvent-free system. Six commercial lipases from different sources were screened for their ability to incorporate caprylic acid into the canola oil. The sn-1,3 regiospecificity toward the glycerol backbone of canola oil of the lipases with relatively higher acidolysis activity was compared by investigating the fatty acid profiles of the products. The results showed that Lipozyme RM IM from Rhizomucor miehei resulted in the highest caprylic acid incorporation ability and the lowest acyl migration. The reaction parameters including substrate mole ratio, enzyme load, reaction time and temperature of Lipozyme RM IM were investigated. Incorporation of caprylic acid was higher when reactions were carried with 10% lipase of the total weight of substrates with a 1:4 mole ratio of oil and caprylic acid. The optimal time course and temperature for synthesis SLs were 15 h and 50–60 °C. Possible triacylglycerol species and physical properties of the SLs product obtained at relative optimal conditions were characterized.     

Optimization of Reaction Conditions in the Enzymatic Interesterification of Soybean Oil and Fully Hydrogenated Soybean Oil to Produce Plastic Fats

Semisolid fats obtained from oils and fats through enzymatic interesterification have interesting applications. The effect of certain reaction parameters (enzyme concentration, moisture content, reaction time, substrate ratio, temperature, and agitation level) over the enzymatic interesterification of fully hydrogenated soybean oil (FHSO) and refined soybean oil (SO) using two immobilized enzyme types (Lipozyme RM IM and Lipozyme TL IM), was studied with a fractional factorial design (FFD). The reaction products were analyzed with respect to melting point (mp), by-products content and triacylglycerols (TAG) composition. It was found that substrate ratio, reaction time, and their interaction presented the most significant contributions to mp, varying this from 43.4 to 61.5 °C. The highest contributions to by-product content were presented by time and its interaction with the amount of molecular sieves, mainly for Lipozyme TL IM. Through the models obtained, theoretical conditions to achieve minimal by-product generation and mp were found, being 5.0 % (w/w_subst.) of any of both lipases, 24 h, 70:30 (oil:fat,  % w/w), 65 °C, 230 rpm, and absence of molecular sieves. Regression models for TAG groups as a function of significant factors and interactions were constructed, offering useful information to establish the reaction conditions for obtaining a product with a target mp or chemical composition.     

Lipase-catalyzed acidolysis of perilla oil with caprylic acid to produce structured lipids

Structured lipids were synthesized by acidolysis of perilla oil and caprylic acid using two lipases, Lipozyme RM IM from Rhizomucor miehei and Lipozyme TL IM from Thermomyces lanuginosa. Effects of molar ratio, reaction time, reaction temperature, enzyme load, and solvent content on acidolysis reactions were studied. The solvent content ranged from 0.0 (solvent-free) to 85.3%. The results showed that the incorporation increased in parallel with solvent content to 49.0% with Lipozyme RM IM and to 63.8% with Lipozyme TL IM. After 24 h incubation in n-hexane, caprylic acids were incorporated to 48.5 mol% with Lipozyme RM IM and to 51.4 mol% with Lipozyme TL IM, respectively, whereas linolenic acid content was reduced from 61.4 to 31.5 mol% with Lipozyme RM IM and to 28.4 mol% with Lipozyme TL IM, respectively. Lipozyme TL IM showed a higher acyl migration rate than Lipozyme RM IM when acidolysis was performed in the reaction system containing n-hexane as a solvent, whereas the difference in acyl migration between the two lipases in the solvent-free system was negligible.     

Synthesis of Fatty Acid Ethyl Ester from Acid Oil in a Continuous Reactor via an Enzymatic Transesterification

Synthesis of a fatty acid ethyl ester via the lipase‐catalyzed transesterification of acid oil and ethanol was investigated in a continuous reactor. Lipozyme TL IM was employed as the immobilized lipase. This immobilized lipase derived from Thermomyces lanuginosus was purchased from Novozymes (Seoul, Korea). The acid oil was prepared by the acidification of soapstock formed as a by‐product during the refining of rice bran oil. The parameters investigated were water content, temperature, and molar ratio of substrates. The relative activity of Lipozyme TL IM was assessed during the repeated use of the immobilized lipase. The water content of the substrate had a considerable effect on the yield and the optimum water content was 4 %. The optimum temperature and molar ratio of acid oil to ethanol were 20 °C and 1:4, respectively. The maximum yield of approximately 92 % was achieved under the optimum conditions. The corresponding compositions were 92 % fatty acid ethyl esters, 3 % fatty acids, and 5 % acylglycerols. When glycerol formed during the reaction was removed by intermittent washing with ethanol, the relative activity of lipase was maintained over 82 % for a total usage of 27 cycles. For a mean residence time of 4 h, the half‐life times of Lipozyme TL IM on the control (unwashed) and treatment (washed) were 39 and 45 cycles, respectively.     

Fast Production of Diacylglycerol in a Solvent Free System via Lipase Catalyzed Esterification Using a Bubble Column Reactor

In this study, diacylglycerols (DAG) were synthesized rapidly (~30 min) in a solvent‐free system via esterification of glycerol with fatty acids (FA, the mixture of 60 wt% palm oil deodorizer distillate and 40 wt% oleic acid) catalyzed by Lipozyme 435 (Novozymes A/S, Copenhagen, Denmark) using a bubble column reactor. The content of DAG, monoacylglycerols (MAG), triacylglycerols (TAG) and free fatty acids (FFA) in the crude product were 57.94 ± 1.60 wt%, 24.68 ± 2.08 wt%, 2.67 ± 1.72 wt% and 14.69 ± 1.22 wt%, respectively under the selected conditions, which were enzyme load of 5.0 wt%, glycerol/FA mole ratio of 7.5, initial water content of 2.5 wt%, reaction temperature of 60 °C, reaction time of 30 min and N₂ gas flow of 10.6 cm min^?1. The final product containing 91.30 ± 1.10 wt% of DAG was obtained by one‐step molecular distillation at 200 °C. The reusability of Lipozyme 435 was investigated by evaluating the esterification degree (ED) and the DAG content in the crude products in 30 successive runs. The enzyme retained 95.10 % of its original activity during 30 successive runs according to comparison of the ED. The new process showed a very high efficiency in production of DAG with a high purity. The ratio of positional isomers 1,3‐DAG to 1,2 ‐DAG was 2:1 in the final product. The certain plasticity (melting point of 44 °C) and content of unsaturated fatty acids made the product a valuable food ingredient.     

玉米油酶法合成甘油二酯工艺优化研究

以玉米油、甘油为原料,在无溶剂体系中用固定化脂肪酶Lipozyme TL IM催化合成甘油二酯,通过单因素试验和响应面试验研究底物摩尔比(亚油酸/甘油)、反应温度、反应时间、酶质量分数和初始水质量分数等因素对亚油酸转化率的影响。得出:反应温度、底物摩尔比、反应时间、酶质量分数对亚油酸转化率影响较大;影响酯化反应中亚油酸转化率的主次因素依次为反应温度、底物摩尔比、反应时间、酶质量分数;最佳工艺条件为亚油酸和甘油的摩尔比为2.10∶1,反应温度为61.16℃,反应时间为12.17 h,酶质量分数为20.09%,亚油酸转化率达到75.69%。Lipozyme TL IM连续反应3批次,其相对酶活仍有65.7%。     

Production of Human Milk Fat Substitutes Catalyzed by a Heterologous Rhizopus oryzae Lipase and Commercial Lipases

This study aims to produce human milk fat substitutes by an acidolysis reaction between lard and the free fatty acids (FFA) from a fish oil concentrate rich in docosahexaenoic acid, in solvent-free media. The immobilized commercial lipases from (1) Rhizomucor miehei (Lipozyme RM IM), (2) Thermomyces lanuginosa (Lipozyme TL IM) and (3) Candida antarctica (Novozym 435) were tested as biocatalyst. Also, the heterologous Rhizopus oryzae lipase (rROL), immobilized in Accurel^® MP 1000, was tested as a feasible alternative to the commercial lipases. After 24 h of reaction at 50 °C, similar incorporations of polyunsaturated fatty acids (c.a. 17 mol%) were attained with Novozym 435, Lipozyme RM IM and rROL. The lowest incorporation was achieved with Lipozyme TL IM (7.2 mol%). Modeling acidolysis catalyzed by rROL and optimization of reaction conditions were performed by response surface methodology, as a function of the molar ratio FFA/lard and the temperature. The highest acidolysis activity was achieved at 40 °C at a molar ratio of 3:1, decreasing with both temperature and molar ratio. Operational stability studies for rROL in seven consecutive 24-h batches were carried out. After the fourth batch, the biocatalyst retained about 55 % of the original activity (half-life of 112 h).     

Lipase-Catalyzed Synthesis of Medium-Long-Medium Type Structured Lipids Using Tricaprylin and Trilinolenin as Substrate Models

The synthesis of medium-long-medium type structured lipids (SL) by the interesterification of tricaprylin (TC) and trilinolenin (TLN), using selected commercial lipases from Rhizomucor miehei (Lipozyme RM IM) and Candida antarctica (Novozym 435) was investigated. Although the bioconversion yield (BY) for Lipozyme RM IM (24.7 %) was close to that for Novozym 435 (24.0 %), the initial enzyme activity was 6.3 μmol CLnC/g enzyme/min and 1.6 μmol CLnC/g enzyme/min, respectively. Lipozyme RM IM was subsequently selected for further investigation. The structural analyses of SL indicated that the major products were 1,3-dicapryl-2-linolenyl glycerol (CLnC) and 1(3)-capryl-2,3(1)-dilinolenyl glycerol (CLnLn). In order to optimize the BY, selected parameters were investigated. The experimental results showed that using hexane as the reaction medium, at an initial water activity (a _w ) of 0.06, 10 mg solid enzyme/mL, substrate molar ratio of TC to TLN of 6:1 and a reaction time of 9 h, resulted in the highest BY (73.2 %). Using the optimized conditions, the effects of TLN concentration and other selective parameters, including the denaturation of the enzyme, controlling the a _w and the addition of silica gel, on the mass productivity (P _M), enzymatic productivity (P _E) and volumetric productivity (P _V ) of the interesterification reaction, were also investigated.     

Preparation of 1,3‐Diolein by Irreversible Acylation

Earlier findings on the nutritional benefits of diacylglycerols (DAGs) have attracted much attention on the synthesis of DAGs. In this study, we reported an improved method for the lipase‐catalyzed synthesis of 1,3‐diolein by the irreversible glycerolysis of vinyl oleate with glycerol. The effects of reaction system, lipase loading, molar ratio of vinyl oleate to glycerol, reaction temperature and time on 1,3‐diolein content in crude reaction mixture were investigated. When the reaction was conducted in a solvent‐free system at 30 °C for 8 h by reacting 2 mmol vinyl oleate with 1 mmol glycerol with 8 % (w/w, relative to total reactants) Lipozyme RM IM (Novozymes, Beijing, China) as catalyst, there were 90.5 ± 2.9 % (area/area) 1,3‐diolein and (3.3 ± 0.3) % 1,2‐diolein produced. After purification, 1,3‐diolein was obtained at 81.4 % yield with 98.2 % purity. The lipase‐catalyzed synthesis of 1,3‐diolein using vinyl oleate as acyl donor by glycerolysis was also conducted using a medium with 50 mmol of glycerol and 100 mmol vinyl oleate. Compared to enzymatic esterification in a solvent, enzymatic glycerolysis for the synthesis 1,3‐diolein is more effective due to the irreversible reaction, mild due to the low reaction temperature, and environmentally benign due to the use of solvent‐free reaction system.     

Fast synthesis of 1,3‐DAG by Lecitase® Ultra‐catalyzed esterification in solvent‐free system

Lecitase® Ultra, a phospholipase, was explored as an effective biocatalyst for direct esterification of glycerol with oleic acid to produce 1,3‐DAG. Experiments were carried out in batch mode, and optimal reaction conditions were evaluated. In comparison with several organic solvent mediums, the solvent‐free system was found to be more beneficial for this esterification reaction, which was further studied to investigate the reaction conditions including oleic acid/glycerol mole ratio, temperature, initial water content, enzyme load, and operating time. The results showed that Lecitase® Ultra catalyzed a fast synthesis of 1,3‐DAG by direct esterification in a solvent‐free medium. Under the optimal reaction conditions, a short reaction time 1.5 h was found to achieve the fatty acid esterification efficiency of 80.3 ± 1.2% and 1,3‐DAG content of 54.8 ± 1.6 wt% (lipid layer of reaction mixture mass). The reusability of Lecitase® Ultra was evaluated via recycling the excess glycerol layer in the reaction system. DAG in the upper lipid layer of reaction mixture was purified by molecular distillation and the 1,3‐DAG‐enriched oil with a purity of about 75 wt% was obtained. Practical applications: The new Lecitase® Ultra catalyzed process for production of 1,3‐DAG from glycerol and oleic acid described in this study provides several advantages over conventional methods including short reaction time, the absence of a solvents and a high product yield.     

Enzymatic synthesis of phenyl fatty hydroxamic acids from canola and palm oils

Phenyl fatty hydroxamic acids (PFHAs) were synthesized from canola or palm oils and phenyl hydroxylamine (FHA) catalyzed by Lipozyme TL IM or RM IM. The reaction was carried out by shaking the reaction mixture at 120 rpm. The optimization was carried out by changing the reaction parameters, namely; temperature, organic solvent, amount and kind of enzyme, period of reaction and the mol ratio of reactants. The highest conversion was obtained when the reaction was carried out under the following conditions: temperature, 39°C; solvent, petroleum ether; kind and amount of lipase, 80 mg Lipozyme TL IM/mmol oil; reaction period, 72 h and FHA-oil ratio, 7.3 mmol FHA/ mmol oil. The highest conversion percentage of phenyl hydroxylaminolysis of the Ladan and Kristal brands commercial canola oils, palm stearin and palm kernel oils were 55.6, 52.2, 51.4 and 49.7 %, respectively.     

Lipase‐catalyzed production of medium‐chain triacylglycerols from palm kernel oil distillate: Optimization using response surface methodology

Optimization of lipase‐catalyzed esterification for the production of medium‐chain triacylglycerols (MCT) from palm kernel oil distillate and glycerol was carried out in order to determine the factors that have significant effects on the reaction system and MCT yield. Novozyme 435 from Candida antarctica lipase was found to have the highest activity at 52.87 ± 0.03 U/g. This lipase also produced the highest MCT yield, which is 56.67%. The effect of different variables on MCT synthesis was studied with a two‐level five‐factor fractional factorial design. The various variables include (1) reaction temperature, (2) enzyme load, (3) molecular sieves concentration, (4) reaction time and (5) molar substrate ratio. Reaction temperature, reaction time and molar substrate ratio strongly affect MCT synthesis (p <0.05). However, enzyme load and molecular sieve concentration did not have a significant (p >0.05) influence on MCT yield. Therefore, the significant variables such as reaction temperature, reaction time and molar substrate ratio were further optimized through central composite rotatable design (CCRD). Comparisons between predicted and experimental values from the CCRD optimization procedures revealed good correlation, implying that the quadric response model satisfactorily expressed the percentage yield of MCT in the lipase‐catalyzed esterification. The optimum MCT yield is 73.3% by using 2 wt‐% enzyme dosage, a molecular sieves concentration of 1 wt‐%, a reaction temperature of 90 °C, a reaction time of 10 h and a molar substrate ratio of 4 : 1 (medium‐chain fatty acid/glycerol). Experiments to confirm the predicted results using the optimal parameters were conducted and an MCT yield of 70.21 ± 0.18% (n = 3) was obtained.     

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.     

Chemical and enzymatic interesterification of a beef tallow and rapeseed oil equal‐weight blend

A mixture of beef tallow and rapeseed oil (1:1, wt/wt) was interesterified using sodium methoxide or immobilized lipases from Rhizomucor miehei (Lipozyme IM) and Candida antarctica (Novozym 435) as catalysts. Chemical interesterifications were carried out at 60 and 90 °C for 0.5 and 1.5 h using 0.4, 0.6 and 1.0 wt‐% CH₃ONa. Enzymatic interesterifications were carried out at 60 °C for 8 h with Lipozyme IM or at 80 °C for 4 h with Novozym 435. The biocatalyst doses were kept constant (8 wt‐%), but the water content was varied from 2 to 10 wt‐%. The starting mixture and the interesterified products were separated by column chromatography into a pure triacylglycerol fraction and a nontriacylglycerol fraction, which contained free fatty acids, mono‐, and diacylglycerols. It was found that the concentration of free fatty acids and partial acylglycerols increased after interesterification. The slip melting points and solid fat contents of the triacylglycerol fractions isolated from interesterified fats were lower compared with the nonesterified blends. The sn‐2 and sn‐1,3 distribution of fatty acids in the TAG fractions before and after interesterification were determined. These distributions were random after chemical interesterification and near random when Novozym 435 was used. When Lipozyme IM was used, the fatty acid composition at the sn‐2 position remained practically unchanged, compared with the starting blend. The interesterified fats and isolated triacylglycerols had reduced oxidative stabilities, as assessed by Rancimat induction times. Addition of 0.02% BHA and BHT to the interesterified fats improved their stabilities.     

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.     

Sn-1,3-specific Interesterification of Soybean Oil with Medium-chain Triacylglycerol Catalyzed by Lipozyme TL IM

abstract The structured lipids are produced through sn-1,3-specific interesterification of soybean oil with medium-chain triacylglycerol (MCT) in continuous reactions catalyzed by Thermomyces lanuginos...     

Enzymatic production of human milk fat substitutes containing γ-linolenic acid: Optimization of reactions by response surface methodology

Structured lipids resembling human milk fat and containing GLA were synthesized by an enzymatic interesterification between tripalmitin, hazelnut oil FA, and GLA in n-hexane. Commercially immobilized 1,3-specific lipases, lipozyme^® RM IM and Lipozyme^® TL IM, were used as the biocatalysts. The effect of these enzymes on the incorporation levels was investigated. A central composite design with five levels and three factors—substrate ratio, reaction temperature, and time—were used to model and optimize the reaction conditions via response surface methodology. Good quadratic models were obtained for the incorporation of GLA (response 1) and oleic acid (response 2) by multiple regression and backward elimination. The determination coefficient (R ²) values for the models were found to be 0.92 and 0.94 for the reactions catalyzed by Lipozyme RM IM, and 0.92 and 0.88 for the reactions catalyzed by Lipozyme TL IM, respecitively. The optimal conditions generated from the models for the targeted GLA (10%) and oleic acid (45%) incorporation were 14.8 mol/mol, 55°C, and 24 h; 14 mol/mol, 55°C, and 24 h for substrate ratio (moles total FA/mol tripalmitin), temperature and time for the reactions catalyzed by Lipozyme RM IM and Lipozyme TL IM, respectively. Human milk fat substitutes containing GLA that can be included in infant formulas were success-fully produced using both Lipozyme RM IM and Lipozyme TL IM enzymes. The effect of the two enzymes on the incorporation of GLA and oleic acid were found to be similar.     

Enzymatic Interesterification of Palm Stearin and Coconut Oil by a Dual Lipase System

Enzymatic interesterification of palm stearin with coconut oil was conducted by applying a dual lipase system in comparison with individual lipase-catalyzed reactions. The results indicated that a synergistic effect occurred for many lipase combinations, but largely depending on the lipase species mixed and their ratios. The combination of Lipozyme TL IM and RM IM was found to generate a positive synergistic action at all test mixing ratios. Only equivalent amount mixtures of Lipozyme TL IM with Novozym 435 or Lipozyme RM IM with Novozym 435 produced a significant synergistic effect as well as the enhanced degree of interesterification. The interesterification catalyzed by Lipozyme TL IM mixed with thermally inactivated immobilized lipase preparations indicated that the carrier property may play an important role in affecting the interaction of two mixed lipases and the subsequent reactions. A dual enzyme system, consisting of immobilized lipases and a non-immobilized one (Lipase AK), in most cases apparently endows the free lipase with a considerably enhanced activity. 70% Lipase AK mixed with 30% immobilized lipase (Lipozyme TL IM, RM IM and Novozym 435) can achieve an increase in activity greater than 100% over the theoretical value when the reaction proceeds for 2 h. The co-immobilization action of the carrier of the immobilized lipases towards the free lipase was proposed as being one of the reasons leading to the synergistic effect and this has been experimentally verified by a reaction catalyzed by a Lipase AK-inactivated preparation. No apparently synergistic effect of the combinations of Lipozyme TL IM and RM IM was observed when the dual enzyme systems applied to the continuous reaction performed in a packed bed reactor. In brief, this work demonstrated the possibility of increasing the reaction rate or enhancing the degree of conversion by employing a dual lipase system as a biocatalyst.