Parameter optimization for the enzymatic hydrolysis of sunflower oil in high-pressure reactors

This study is concerned with the hydrolysis of sunflower oil in the presence of lipase preparation Lipolase 100T (Aspergillus niger lipase). Supercritical carbon dioxide was used as a solvent for this reaction. In a high-pressure stirred tank reactor operated in a batch mode, the effects of various process parameters (temperature, pressure, enzyme/substrate ratio, pH, and oil/buffer ratio) were investigated to determine the optimal reaction rate and conversion for the hydrolysis process. The optimal concentration of lipase was 0.0714 g/mL of CO₂-free reaction mixture, and the highest conversions of oleic acid (0.193 g/g of oil phase) and linoleic acid (0.586 g/g of oil phase) were obtained at 50°C, 200 bar, pH=7, and an oil/buffer ratio of 1∶1 (w/w).     

Impact of Lipase-Mediated Hydrolysis of Castor Oil on γ-Decalactone Production by Yarrowia lipolytica

γ‐Decalactone is an industrially interesting peach‐like aroma compound that can be produced biotechnologically through the biotransformation of ricinoleic acid. Castor oil (CO) is the raw material most used as the ricinoleic acid source. The effect of different CO concentrations on the γ‐decalactone production by Yarrowia lipolytica was investigated in batch processing, and 30 g L^?1 was found to be the optimal oil concentration. Under these conditions, cells were able to produce lipase but at low activity levels, which might limit ricinoleic acid release and consequently, the γ‐decalactone production rate. Thus, the enzymatic hydrolysis of CO by commercial lipases was studied under different operating conditions. Lipozyme TL IM was found to be the most efficient and the optimal hydrolysis conditions were pH 8 and 27 °C. The use of hydrolyzed CO in the aroma production allowed a decrease in the lag phase for γ‐decalactone secretion.     

Preparation of diacylglycerol‐enriched palm olein by phospholipase A1‐catalyzed partial hydrolysis

Partial hydrolysis of palm olein catalyzed by phospholipase A₁ (Lecitase Ultra) in a solvent‐free system was carried out to produce diacylglycerol (DAG)‐enriched palm olein (DEPO). Four reaction parameters, namely, reaction time (2–10 h), water content (20–60 wt‐% of the oil mass), enzyme load (10–50 U/g of the oil mass), and reaction temperature (30–60 °C), were investigated. The optimal conditions for partial hydrolysis of palm olein catalyzed by Lecitase Ultra were obtained by an orthogonal experiment as follows: 45 °C reaction temperature, 44 wt‐% water content, 8 h reaction time, and an enzyme load of 34 U/g. The upper oil layer of the reaction mixture with an acid value of 54.26 ± 0.86 mg KOH/g was first molecularly distilled at 150 °C to yield a DEPO with 35.51 wt‐% of DAG. The DEPO was distilled again at 250 °C to obtain a DAG oil with 74.52 wt‐% of DAG. The composition of the acylglycerols of palm olein and the DEPO were analyzed and identified by high‐performance liquid chromatography (HPLC) and HPLC/electrospray ionization/mass spectrometry. The released fatty acids from the partial hydrolysis of palm olein catalyzed by phospholipase A₁ showed a higher saturated fatty acid content than that of the raw material.     

Features of the enzymatic hydrolysis of castor oil

Ricinoleic acid, which is used in medicine and veterinary science and is the initial material in the organic synthesis of various valuable products, is obtained via the hydrolysis of castor oil. The enzymatic hydrolysis of castor oil, which allows us to conduct the process under mild conditions (in the temperature range of 35–45°C and without high pressures) is a promising method for obtaining ricinoleic acid. This work demonstrates the feasibility of the enzymatic hydrolysis of castor oil with lipase from Candida rugosa in oil-water systems without an emulsifier. We propose a method for hydrolysis without emulsifiers, simplifying the process of isolating the target product (a mixture of free fatty acids in which ricinoleic acid predominates) and thus the relevant technology. The catalyst that we use ensures the environmental friendliness of the process. Our selection of the experimental conditions for hydrolysis resulted in a 47% yield of fatty acids.     

One‐Pot Lipase Entrapment Within Silica Particles to Prepare a Stable and Reusable Biocatalyst for Transesterification

In order to enhance the reusability, Rhizomucor miehei lipase was entrapped in a single step within silica particles having an oleic acid core (RML@SiO₂). Characterization of RML@SiO₂ by scanning and transmission electron microscopy and Fourier transform infrared studies supported the lipase immobilization within silica particles. The immobilized enzyme was employed for transesterification of cottonseed oil with methanol and ethanol. Under the optimum reaction conditions of a methanol‐to‐oil molar ratio of 12:1 or ethanol‐to‐oil molar ratio of 15:1, stirring speed of 250 revolutions/min (flask radius = 3 cm), reaction temperature of 40 °C, and biocatalyst concentration of 5 wt% (with respect to oil), more than 98 % alkyl ester yield was achieved in 16 and 24 h of reaction duration in case of methanolysis and ethanolysis, respectively. The immobilized enzyme did not require any buffer solution or organic solvent for optimum activity; hence, the produced biodiesel and glycerol were free from metal ion or organic molecule contamination. The activation energies for the immobilized enzyme‐catalyzed ethanolysis and methanolysis were found to be 34.9 ± 1.6 and 19.7 ± 1.8 kJ mol^?1, respectively. The immobilized enzyme was recovered from the reaction mixture and reused in 12 successive runs without significant loss of activity. Additionally, RML@SiO₂ demonstrated better reusability as well as stability in comparison to the native enzyme as the former did not lose the activity even upon storage at room temperature (25–30 °C) over an 8‐month period.     

Investigation of enzymatic biodiesel production using ionic liquid as a co‐solvent

In this study, the lipase catalysed esterification reaction for biodiesel production was investigated in the presence of the ionic liquid [BMIM][PF₆]. Unlike regular organic solvents, many ionic liquids have no vapour pressure, and are therefore considered non‐volatile. When used in systems with enzyme catalysts, ionic liquids may enhance their activity, selectivity, and stability. The use of an enzyme (lipase) as a catalyst, and the ionic liquid as a solvent/immobilization agent also represents an environmentally friendly, “green” technology. Methyl acetate was used as the acyl acceptor as opposed to the more commonly used methanol due to the negative effects methanol and the glycerol by‐product has on lipase enzyme activity. The results of this research indicate that methyl oleate (i.e., biodiesel) was successfully produced, with an 80% overall biodiesel yield in the presence of ionic liquid, at a 1:1 ratio (v/v) to the amount of oil. This verified that the presence of an ionic liquid, at a specified amount, improved the activity of the lipase and the overall biodiesel yield. Results also indicate the addition of ionic liquid facilitated the separation of the methyl esters from the triacetylglycerol by‐product. The best conditions investigated was found to be: 14:1 molar ratio between oil and acyl acceptor; 20% (w immobilised lipase/w of oil; and a temperature in the range of 48–55°C. However, additional purification is required in order for the produced biodiesel to meet ASTM standards.     

Lipase immobilization on ionic liquid‐modified magnetic nanoparticles: Ionic liquids controlled esters hydrolysis at oil–water interface

Ester hydrolysis at oil–water interface by lipase covalently immobilized on ionic liquid‐modified magnetic nanoparticles was investigated. Magnetic supports with a diameter of 10–15 nm were synthesized by covalent binding of ionic liquids (chain length C₄ and C₈ and anions Cl^?, BF₄^?, and PF₆^?) on the surface of Fe₃O₄ nanoparticles. Lipase was covalently immobilized on Fe₃O₄ nanoparticles using ionic liquids as the coupling reagent. Ionic liquid‐modified magnetic nanoparticle‐grafted lipase preferentially located at the oil–water interface. It has higher catalytic activity than its native counterpart. A modified Michaelis–Menten model was used to elucidate the effect of stirring rate, aqueous–organic phase ratio, total amount of enzyme, and ester chain length. The influences of these conditions on esters hydrolysis at oil–water interface were consistent with the introduction of the ionic liquids interlayer. Ionic liquids could be used to control the oil–water interfacial characteristics during lipase catalyzed hydrolysis, and thus control the behavior of immobilized lipase. © 2011 American Institute of Chemical Engineers AIChE J, 2012     

Selectivity of lipases: isolation of fatty acids from castor,coriander, and meadowfoam oils

The lipase‐catalyzed hydrolysis of castor, coriander, and meadowfoam oils was studied in a two‐phase water/oil system. The lipases from Candida rugosa and Pseudomonas cepacia released all fatty acids from the triglycerides randomly, with the exception of castor oil. In the latter case, the P. cepacia lipase discriminated against ricinoleic acid. The lipase from Geotrichum candidum discriminated against unsaturated acids having the double bond located at the Δ‐6 (petroselinic acid in coriander oil) and Δ‐5 (meadowfoam oil) position or with a hydroxy substituent (ricinoleic acid). The expression of the selectivities of the G. candidum lipase was most pronounced in lipase‐catalyzed esterification reactions, which was exploited as part of a two‐step process to prepare highly concentrated fractions of the acids. In the first step the oils were hydrolyzed to their respective free fatty acids, in the second step a selective lipase was used to catalyze esterification of the acids with 1‐butanol. This resulted in an enrichment of the targeted acids to approximately 95—98% in the unesterified acid fractions compared to the 70—90% content in the starting acid fractions.     

Purification of Free DHA by Selective Esterification of Fatty Acids from Tuna Oil Catalyzed by Rhizopus oryzae Lipase

Tuna fish oil contains 25–30 % docosahexaenoic acid (DHA) and is one of the richest sources of DHA. The present paper investigates the enrichment of DHA by selective esterification of fatty acids obtained from hydrolysis of tuna fish oil catalyzed by Rhizopus oryzae lipase (ROL). The fatty acid mixture obtained after hydrolysis of tuna fish oil, referred to as tuna-FFA contained 26 % DHA. For purification/concentration of DHA in free fatty acids, selective esterification of the fatty acid mixtures with butanol was carried out using ROL in a water-organic solvent system. The best reaction parameters found in this study were pH 7, temperature 35 °C, agitation speed 800 rpm and a fatty acid to solvent (iso-octane) ratio of 1:1.32 (w/v). Also, the effects of other parameters such as type of alcohol, type of enzyme, alcohol to fatty acid ratio, enzyme to fatty acid ratio were studied to determine the most suitable reaction conditions. Exactly 76.2 % of tuna-FFA was esterified in 24 h, under the most suitable reaction conditions and the DHA content in the fatty acid fraction rose from 26 to 86.9 % with 80 % recovery of DHA, after selective esterification. The DHA content of fatty acids in butyl esters was found to be 13.6 %.     

Acid lipase of the castor bean

The acid lipase of the castor bean is present in the dormant seed. It is extracted from the fat pad obtained by centrifuging a macerate of the seed in pH 7.0 buffer containing cysteine and ethylene diaminetetraacetic acid. The pH optimum of the enzyme is 4.2; it is rather heat-stable, and is inhibited by mercurials and sulfhydryl reagents. Maximum hydrolysis of saturated triglycerides occurs with fatty acids of chain length C₄ to C₈; unsaturated C₁₈ triglycerides are hydrolyzed at a slightly lower rate. This lipase is a three-component system consisting of the apoenzyme, a lipid cofactor (a cyclic tetramer of ricinoleic acid), and a protein activator (a small, heat-stable glycoprotein which appears to be related to some of the castor allergens). Maximum lipolysis requires all three components. Lipase activity is associated with the spherosomes, the subcellular site of oil storage in the endosperm. Presented at the AOCS-AACC Joint Meeting, Washington, D.C., April 1968. So. Utiliz. Res. Dev. Div., ARS, USDA.     

Pigment removal from canola oil using chlorophyllase

Frost-damaged or prematurely harvested canola seed (rapeseed) may yield oil with a high chlorophyll content (50–60 μg/ml). Enzymatic hydrolysis of chlorophyll, added to buffer/surfactant, buffer/acetone or buffer/acetone/canola oil, to produce water-soluble chlorophyllide (green pigment) was studied using a crude chlorophyllase preparation (acetone-dried chloroplasts) from 15 to 20-day-old sugar beet seedlings. In buffer/surfactant, the optimum pH for enzyme activity was temperature dependent. At 30 C and 0.24% Triton X-100 (or 30% acetone), chlorophyllase showed maximum activity toward a crude chlorophyll preparation over the range of pH 8–10. At 60 C, the activity was more than twofold higher, with a sharp maximum at ∼pH 8. Mg²⁺ enhanced the activity with an optimal concentration of 50 mM. At pH 7.5, 50 C and in the presence of only 6% acetone, the enzyme showed high affinity for chlorophyll (Km=15μM or 13.5 μg/ml), suggesting that the natural chlorophyll concentrations found in green canola oils might facilitate high enzymatic efficiencies. The crude enzyme was stable in buffer/acetone at pH 7.5 and 50 C for at least two hr. With acetone concentrations as low as 6%, maximum enzyme activities in buffer and buffer/canola oil required intensive mixing (homogenization) of the various substrate, enzyme and liquid phases. In general, the rate and extent of chlorophyll hydrolysis were greater in buffer than in buffer/oil. In both reaction systems, chlorophyll hydrolysis slowed down with time due to accumulation of phytol, which proved to be a competitive inhibitor (K_i=11 μM or 3.3 μg/ml). The other hydrolysis product, chlorophyllide, did not affect enzymatic activity. Crude canola oil used in the reconstitution of green oil did not support enzymatic chlorophyll hydrolysis without prior degumming and desoaping. The optimum buffer/oil ratio of the reaction mixtures was above 2/1 (v/v).     

Oil and fat hydrolysis with lipase fromAspergillus sp.

Hydrolysis of olive oil, soybean oil, mink fat, lard, palm oil, coconut oil, and a hydrogenated, hardened oil with lipase from anAspergillus sp. has been studied. The lipase had high specific activity (60,000 U/g) and did not show any positional specificity. The lipase proved to be a more effective catalyst than Lipolase fromA. oryzae, with an optimal activity at 37°C and pH 6.5–7.0. It was activated by Ca²⁺ but inactivated by organic solvents such as isopropanol and propanone. All substrates examined could be hydrolyzed to corresponding fatty acids with this enzyme at concentrations of 5–30 U/meq with yields of 90–99% in 2–24 h. The degree of hydrolysis was almost logarithmically linear with reaction time and occurred in two stages. The lipase was stable and could be repeatedly recycled for hydrolysis.     

Lipase made active in hydrophobic media

Enzymes are distinguished from other catalysts by their high substrate specificity. This is a great asset when one wants to apply them for syntheses of various compounds. Their usage, however, generally is limited to hydrophilic reaction media, because they usually are not soluble and active in hydrophobic media. Recently, we have been able to make various enzymes soluble and active in highly hydrophobic organic solvents. The key to this success is the chemical modification of enzymes with an amphipathic synthetic polymer, polyethylene glycol. The activated polymers can be attached to enzymes in aqueous buffer solutions, and once enzymes are modified they become soluble and active in various organic solvents such as benzene, toluene and cholorinated hydrocarbons and exhibit high enzymic activities in these organic solvents. Modified hydrolytic enzymes catalyzed the reverse reaction of hydrolysis in organic solvents. The modified lipase catalyzed various ester synthesis reactions. Because the reactions were conducted in the pure solvent system, it also was possible to study the kinetics and the substrate specificity for ester synthesis reaction. It also catalyzed the polymerization of a hydroxy group containing carboxylic acid due to the bifunctional nature. The modified lipase catalyzed ester exchange between an ester and an alcohol, between an ester and a carboxylic acid and between two esters in organic solvents. When the two substrates for ester exchange were liquid, the reaction could take place without organic solvents. The modified lipase catalyzed an ester exchange reaction between trilaurin and triolein when dissolved in these substrates. Dilauroyl-monooleoylglycerol and monolauroyl-dioleoyl-glycerol were formed from these two substrates in the presence of the modified lipase. The modified enzyme was extremely thermostable in its substrates. In the ester synthesis and ester exchange reactions, a trace amount of water was necessary for expression of the enzymic activity. It is suggested that the amphipathic polymer molecules retained water in close proximity to the enzyme. Presented at the symposium “The Biology, Biochemistry and Technology of Lipase” at the 78th annual meeting of the American Oil Chemists’ Society held May 17–21, 1987, in New Orleans, Louisiana.     

Hydrolysis of Fish Oil by Lipases Immobilized Inside Porous Supports

A new assay was designed to measure the release of omega-3 acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] from the hydrolysis of sardine oil by lipases immobilized inside porous supports. A biphasic system was used containing the fish oil dissolved in the organic phase and the immobilized lipase suspended in the aqueous phase. The assay was optimized by using a very active derivative of Rhizomucor miehei lipase (RML) adsorbed onto octyl-Sepharose. Standard reaction conditions were: (a) an organic phase composed by 30/70 (v:v) of oil in cyclohexane, (b) an aqueous phase containing 50 mM methyl-cyclodextrin in 10 mM Tris buffer at pH 7.0. The whole reaction system was incubated at 25 °C. Under these conditions, up to 2% of the oil is partitioned into the aqueous phase and most of the 95% of released acids were partitioned into the organic phase. The organic phase was analyzed by RP-HPLC (UV detection at 215 nm) and even very low concentrations (e.g., 0.05 mM) of released omega-3 fatty acid could be detected with a precision higher than 99%. Three different lipases adsorbed on octyl-Sepharose were compared: Candida antarctica lipase-fraction B (CALB), Thermomyces lanuginosa lipase (TLL) and RML. The three enzyme derivatives were very active. However, most active and selective towards polyunsaturated fatty acids (PUFA) versus oleic plus palmitic acids (a fourfold factor) was CALB. On the other hand, the most selective derivatives towards EPA versus DHA (a 4.5-fold factor) were TLL and RML derivatives.     

Extraction of Candida rugosa lipase from aqueous solutions into organic solvents by forming an ion‐paired complex with AOT

Candida rugosa lipase was extracted from aqueous solutions into organic solvents by forming an ion‐paired complex with sodium bis(2‐ethylhexyl)sulfosuccinate (AOT). The optimal aqueous pH for lipase recovery was 4.5 and the optimal CaCl₂ concentration was 10 mmol dm^?3. The lipase recovery decreased with increasing aqueous enzyme concentration but increased with increasing AOT concentration in the organic phase. The presence of polar co‐solvents in the aqueous phase did not obviously improve the lipase recovery, which was also little influenced by the type of hydrophobic organic solvent used for solubilising AOT. Surprisingly, no detectable activity of the ion‐paired C. rugosa lipase was observed for both the esterification of lauric acid with 1‐propanol in isooctane and the hydrolysis of olive oil in isooctane containing an appropriate amount of water. The ion‐paired C. rugosa lipase mediated the enantioselective crystallisation of racemic ketoprofen in isooctane, indicating the feasibility of using it as a chiral mediator for the enantioseparation of hydrophobic racemic compounds in organic systems. Copyright © 2006 Society of Chemical Industry     

Continuous hydrolysis of olive oil by lipase in microporous hydrophobic membrane bioreactor

Continuous hydrolysis of olive oil byCandida cylindracea’s lipase was studied in a microporous hydrophobic membrane bioreactor. Olive oil and buffer solution, fed continuously through two compartments partitioned by membrane, caused reaction at the interface of lipase-adsorbed membrane and buffer solution. Fatty acid was obtained in a single phase without being mixed with components of other phases. At all mean residence times, countercurrent flow mode was superior to cocurrent one. The lipase was adsorbed onto the membrane, and its adsorption was suggested to be partially specific from the experiments with enzymes having various levels of purity. The percent hydrolysis depended hyperbolically on the interfacial enzyme concentration. The hydrolysis seemed to be limited by diffusion of fat or fatty acid through the micropores of the membrane at higher interfacial enzyme concentrations. The lipase was stabilized significantly by glycerol added to the buffer solution. Satisfactory performance of the membrane bioreactor was obtained in a longterm continuous operation which lasted for 24 days by feeding buffer-glycerol (18.0%) solution over the adsorbed lipase. The operational half-life of the adsorbed enzyme was 15 days at 40 C.     

Increased activity of Chromobacterium viscosum lipase in AOT reverse micelles in the presence of short chain methoxypolyethylene glycol

The activity of Chromobacterium viscosum lipase (glycerol‐ester hydrolase, EC 3.1.1.3) entrapped in AOT/isooctane and AOT/Tween 85/isooctane reverse micelles was significantly increased by the addition of short chain methoxypolyethylene glycols (MPEGs), taking the hydrolysis of olive oil as a model reaction. The molecular weight of MPEG had a strong effect on the lipase activity, and MPEG of nominal molecular weight 550 was found to be the most effective. To optimize the factors affecting enzymatic hydrolysis of olive oil in reverse micellar systems containing MPEG 550, the effect of various parameters, such as W_o (molar ratio of water to surfactant), pH, ionic strength, surfactant concentration and temperature were investigated. A kinetic model considering the substrate adsorption equilibrium between the bulk phase of organic solvent and the micellar phase was also successfully used to understand the enzyme activity in the presence of MPEG 550. Both the Michaelis constant and the substrate adsorption equilibrium constant were obviously reduced as compared with those obtained in the simple AOT reverse micellar system. © 2001 Society of Chemical Industry     

Lipase-Catalyzed Production of Biodiesel by Hydrolysis of Waste Cooking Oil Followed by Esterification of Free Fatty Acids

Biodiesel is conventionally produced by alkaline‐catalyzed transesterification, which requires high‐purity oils. However, low‐quality oils can be used as feedstocks for the production of biodiesel by enzyme‐catalyzed reactions. The use of enzymes has several advantages, such as the absence of saponification side reactions, production of high‐purity glycerol co‐product, and low‐cost downstream processing. In this work, biodiesel was produced from lipase‐catalyzed hydrolysis of waste cooking oil (WCO) followed by esterification of the hydrolyzed WCO (HWCO). The hydrolysis of acylglycerols was carried out at 30 °C in salt‐free water (WCO/water ratio of 1:4, v/v) and the esterification of HWCO was carried out at 40 °C with ethanol in a solvent‐free medium (HWCO/ethanol molar ratio of 1:7). The hydrolysis and esterification steps were carried out using immobilized Thermomyces lanuginosus lipase (TLL/WCO ratio of 1:5.6, w/w) and immobilized Candida antarctica lipase B (10 wt%, CALB/HWCO) as biocatalysts, respectively. The hydrolysis of acylglycerols was almost complete after 12 h (ca. 94 %), and in the esterification step, the conversion was around 90 % after 6 h. The purified biodiesel had 91.8 wt% of fatty acid ethyl esters, 0.53 wt% of acylglycerols, 0.003 wt% of free glycerol, viscosity of 4.59 cP, and acid value of 10.88 mg KOH/g. Reuse hydrolysis and esterification assays showed that the immobilized enzymes could be recycled five times in 10‐h batches, under the conditions described above. TLL was greatly inactivated under the assay conditions, whereas CALB remained fully active. The results showed that WCO is a promising feedstock for use in the production of biodiesel.     

Partial purification of <Emphasis Type="Italic">nigella sativa</Emphasis> L. Seed lipase and its application in hydrolytic reactions. Enrichment of γ-linolenic acid from borage oil

Selective hydrolysis of borage (Borago officinalis L.) oil was catalyzed by two lipase preparations of Nigella sativa L. seeds at 40°C in a mixture of borage oil, water, and hexane. Ammonium sulfate-precipitated lipase (Nigella PL) and lipase partially purified by DEAE-ion exchange chromatography (Nigella CPL) exhibited a negative specificity toward γ-linolenic acid (GLA). Best results were obtained in the experiments conducted with 330 U/g oil of Nigella PL and 200 U/g oil of nigella CPL. When 330 U/g oil of Nigella PL was used, after 8 h the GLA level rose from 21.9% in the starting oil to 29.6 and 41.8% in TAG and DAG fractions of the product mixtures, respectively (1.5-fold enrichment of GLA in the total unhydrolyzed acylglycerol fraction). At 200 U/g oil enzyme concentration of Nigella CPL, after 77 h maximum GLA enrichment was observed in the DAG fraction. The GLA content of the DAG increased to 34.6%, corresponding to almost 1.6-fold enrichment. The relative inability of Nigella sativa lipase(s) to hydrolyze γ-linolenoyl moieties of TAG can be used for the enrichment of this acid in the unhydrolyzed acylglycerol fractions of GLA-containing oils.     

Supported Ionic Liquid Enzymatic Catalysis for the Production of Biodiesel

Pseudomonas cepacia lipase supported in the 1‐n‐butyl‐3‐methylimidazolium bis(trifluoromethylsulfonyl)imide ionic liquid is an alternative “green” method for the production of biodiesel from the alcoholysis of soybean oil. The transesterification reaction catalyzed by this ionic liquid‐supported enzyme can be performed at room temperature, in the presence of water and without the use of organic solvents. It is also compatible with various alcohols (including isoamyl alcohol). The biodiesel is separated by simple decantation and the recovered ionic liquid/enzyme catalytic system can be re‐used at least four times without loss of catalytic activity and selectivity.