Lipase‐catalyzed synthesis of butyl esters by direct esterification in solvent‐free system

BACKGROUND: Reactions performed under solvent‐free conditions give processes that are environmentally friendly, since most solvents are polluting agents. In this work, the performance of Candida rugosa lipae (CRL) immobilized on styrene‐divinylbenzene (STY‐DVB) or controlled pore silica (CPS), and the commercial lipase Novozym 435, was evaluated for the synthesis of butyl esters in solvent–free systems (SFS). A 2² full factorial design was used to study the influence of the organic acid chain length and the biocatalyst concentration on the esterification performance. RESULTS: When CRL on STY‐DVB was used, the ester formation was influenced by both variables and their interaction. The reaction conversion was higher (63%) using 10% of immobilized system and lauric acid, corresponding to a productivity of 3.62 g L^?1 h^?1 For CRL on CPS, only the effect of biocatalyst concentration was significant, and the highest yield was attained using 20% of immobilized system and caprilic acid. In the case of Novozym 435, the highest yield (49%) was obtained using butyric acid as acyl donor at 15% of immobilized lipase. CONCLUSION: The results allowed better understanding of the influence of important parameters in this environmentally friendly process, which also has the process advantage of a higher volumetric productivity when compared with a solvent system. Copyright © 2007 Society of Chemical Industry     

Lipase biocatalysis in the production of esters

Lipase biocatalysis was investigated as a tool for the production of butyl oleate and rapeseed oil 2-ethyl-1-hexyl ester by esterification and transesterification, respectively. We screened 25 commercially available lipases and found that butyl oleate was produced at high yields from oleic acid and 1-butanol by lipases fromCandida rugosa, Chromobacterium viscosum, Rhizomucor miehei, and Pseudomonas fluorescens. The initial water content of the system, lipase quantity, and the molar ratio of 1-butanol to oleic acid were important factors in influencing the ester yield. In general, no ester was formed without the addition of water. The exception wasCh. viscosum lipase, which yielded 98% of ester in 12 h with 1-butanol excess without additional water. The addition of 3.2% water increased the initial rate of reaction. With an oleic acid excess and only 0.3% lipase,C. rugosa andR. miehei lipases yielded 94 and 100% esters with initial water contents of 3.2 and 14%, respectively. Lipase-catalyzed alcoholysis of low-erucic acid rapeseed oil and 2-ethyl-1-hexanol without additional organic solvent also was studied in stirred batch reactors. In this case,C. rugosa lipase was the best biocatalyst with an optimal 2-ethyl-1-hexanol to rapeseed oil molar ratio of 2.8, a minimum of 1.0% added water, and 37°C. An increase in temperature up to 55°C increased the rate of reaction but did not affect the final ester yield. The enzyme was inactivated at 60°C. Under optimal conditions, the ester yield increased from 88% in 7 h to nearly complete conversion in 1 h when the lipase content was increased from 0.3 to 14.6%. In a 2-kg small pilot scale, up to 90% conversion (97% of theoretical) was obtained in 8 h at 37°C with 3.4% lipase in the presence of Amberlite XAD-7 resin with 3% added water.     

Synthesis of Oleyl Oleate with Immobilized Lipase from Mucor miehei

The synthesis of oleyl oleate with immobilized 1,3-specific lipase from Mucor miehei is presented in this article. Oleyl alcohol was esterified with oleic acid in the presence of a Mucor miehei lipase (lipozyme^IM) to obtain oleyl oleate. The effects of various temperatures and various enzyme/substrate ratios have been investigated to determine optimal conditions for the esterification process. The highest conversion of oleic acid 86.9 % was obtained at 50°C. The optimal addition of lipase to substrates was determined to be 0.1 g per gram of reaction mixture. The esterification can be modeled successfully as a reverse second-order reaction. Thermodynamic properties of the reaction system at 50°C were also determined. Activation energy was 14.65 kJ/mol, entropy of activation –0.8 J/mol·K and free energy of activation was 98.568 kJ/mol.     

Synthesis of 2‐monoglycerides by alcoholysis of palm oil and tuna oil using immobilized lipases

Commercial immobilized lipases were used for the synthesis of 2‐monoglycerides (2‐MG) by alcoholysis of palm and tuna oils with ethanol in organic solvents. Several parameters were studied, i.e., the type of immobilized lipases, water activity, type of solvents and temperatures. The optimum conditions for alcoholysis of tuna oil were at a water activity of 0.43 and a temperature of 60 °C in methyl‐tert‐butyl ether for ～12 h. Although immobilized lipase preparations from Pseudomonas sp. and Candida antarctica fraction B are not 1, 3‐regiospecific enzymes, they were considered to be more suitable for the production of 2‐MG by the alcoholysis of tuna oil than the 1, 3‐regiospecific lipases (Lipozyme RM IM from Rhizomucor miehei and lipase D from Rhizopus delemar). With Pseudomonas sp. lipase a yield of up to 81% 2‐MG containing 80% PUFA (poly‐unsaturated fatty acids) from tuna oil was achieved. The optimum conditions for alcoholysis of palm oil were similar as these of tuna oil alcoholysis. However, lipase D immobilized on Accurel EP100 was used as catalyst at 40 °C with shorter reaction times (<12 h). This lead to a yield of ～60% 2‐MG containing 55.0‐55.7% oleic acid and 18.7‐21.0% linoleic acid.     

Towards continuous sustainable processes for enzymatic synthesis of biodiesel in hydrophobic ionic liquids/supercritical carbon dioxide biphasic systems

The excellent suitability of immobilized Candida antarctica lipase B (Novozym 435) catalyst to carry out the synthesis of methyl oleate (biodiesel) by methanolysis of triolein in ILs based on imidazolium cations with large alkyl side chain (from C₁₂ to C₁₈) has been demonstrated at 60 and 85 °C. The phase behaviour of IL/triolein/methanol and IL/methyl oleate mixtures were studied at different concentrations and temperatures, the best results (up to 98.6% biodiesel yield after 6 h) being obtained for ILs able to provide monophasic reaction systems, i.e. 1-methyl-3-octadecylimidazolium bis(trifluoromethylsulfonyl)imide). A continuous enzymatic reactor, based on biocatalysts particles coated with hydrophobic ILs, for biodiesel synthesis in supercritical carbon dioxide was studied at 60 °C and 180 bar. The operational stability of the immobilized lipase was improved by its coating with ILs, i.e. 1-methyl-3-octadecylimidazolium hexafluorophosphate, leading to a two-phase systems with respect to the biodiesel product, which showed an excellent catalytic behaviour in continuous operation under supercritical conditions (up to 82% biodiesel yield after 12 cycles of 4 h).     

Sustainable production of the emulsifier methyl oleate by Candida rugosa lipase nanoconjugates

Acid functionalization of multi-walled carbon nanotubes (F-MWCNTs) using a mixture of HNO₃ and H₂SO₄ (1:3, v:v) was used as support materials for the adsorption of Candida rugosa lipase (CRL) as nanoconjugates (CRL-MWCNTs) for producing methyl oleate. To evaluate the competency of the CRL-MWCNTs nanoconjugates, parameters viz. reaction time, surfactant as well as thermostability and reusability were investigated. The characterization of CRL-MWCNTs nanoconjugates using Fourier transform infrared spectroscopy, Field Scanning Electron Microscopy and Transmission Electron Microscopy revealed successful attachment of CRL onto the F-MWCNTs. Utilization of CRL-MWCNTs nanoconjugates resulted in a higher acid conversion in the synthesis of methyl oleate (79.85% at 11 h of reaction time) when compared with the free CRL i.e. an approximately 1.5-fold improvement over the free CRL. The highest percentage of esterification (83.62%) was observed following the use of nonionic surfactant when compared with the anionic and cationic ones. The CRL-MWCNTs nanoconjugates could be used up to 5 cycles, retaining 50% of its residual activity. Since the preparation of the CRL-MWCNTs nanoconjugates was facile and cheap while producing reasonable yield, the CRL-MWCNTs nanoconjugates developed here were found as promising biocatalysts for the production of methyl oleate.     

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.     

Protein engineering and applications of <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase isoforms

Commercial preparations of Candida rugosa lipase (CRL) are mixtures of lipase isoforms used for the hydrolysis and synthesis of various esters. The presence of variable isoforms and the amount of lipolytic protein in the crude lipase preparations lead to a lack of reproducibility of biocatalytic reactions. Purification of crude CRL improve their substrate specificity, enantioselectivity, stability, and specific activities. The expression of the isoforms is governed by culture or fermentation conditions. Unfortunately, the nonsporogenic yeast C. rugosa does not utilize the universal codon CTG for leucine; therefore, most of the CTG codons were converted to universal serine triplets by site-directed mutagenesis to gain expression of functional lipase in heterologous hosts. Recombinant expressions by multiple-site mutagenesis or complete synthesis of the lipase gene are other possible ways of obtaining pure and different CRL isoforms, in addition to culture engineering. Protein engineering of purified CRL isoforms allows the tailoring of enzyme function. This involves computer modeling based on available 3-D structures of lipase isoforms. Lid swapping and DNA shuffling techniques can be used to improve the enantioselectivity, thermostability, and substrate specificity of CRL isoforms and increase their biotechnological applications. Lid swapping can result in chimera proteins with new functions. The sequence of the lid can affect the activity and specificity of recombinant CRL isoforms. Candida rugosa lipase is toxicologically safe for food applications. Protein engineering through lid swapping and rationally designed site-directed mutagenesis will continue to lead to the production of CRL isoforms with improved catalytic power, thermostability, enantioselectivity, and substrate specificity, while providing evidence for the mechanisms of actions of the various isoforms.     

Immobilization of lipase from Candida rugosa on synthetic polymer beads for use in the synthesis of fatty esters

Lipase from Candida rugosa was immobilized on three different supports, i.e. Amberlite XAD7, poly(methylmethacrylate) (PMMA) and celite. With the conditions tested, maximum adsorption can be achieved after 30 min. The activities of the immobilized lipases were determined by the esterification reaction of oleic acid and butanol. The immobilized lipases were found to be very effective in the esterification reaction. The immobilized activities generally were high in apolar organic solvents with log P values from 2·0 to 4·0. The preference for fatty acids as acyl donors differed in all cases of immobilized lipases. Lipase immobilized on XAD7 and PMMA exhibited high preference of acyl donors (fatty acids) with chain lengths 12–18 and 8–18, respectively. Lipase immobilized on celite, however, showed high activity in all cases of fatty acids. The nucleophile (alcohol) selectivity studies showed that lipase immobilized on XAD7 and celite was more accessible to alcohols of chain lengths 3–12. However, lipase immobilized on PMMA showed a significant preference towards alcohols of chain lengths from 3 to 10.     

Enzymatic alcoholysis of triolein to produce wax ester

An alcoholysis reaction between triolein and oleyl alcohol catalyzed by Lipozyme and Novozyme was carried out to produce oleyl oleate, a wax ester. The effects of various reaction parameters such as time, reaction temperature, amount of enzyme, molar ratio of substrates (oleyl alcohol/triolein), various organic solvents used and the initial water activity, a_w of the reaction system were studied. The best conditions tested to produce wax ester were respectively, incubation time, 5 h; temperature, 50 °C for Lipozyme and 60 °C for Novozyme; weight of enzyme, 0.30 g and molar ratio of oleyl alcohol to triolein, 6:1. The use of organic solvents greatly influenced the activity of lipase. Generally, the activity of lipase was high in nonpolar solvents with log P values greater than 2.50. Heptane and hexane were the best solvents tested. The enzymatic synthesis of oleyl oleate was best carried out at a_w 0.32. Analysis of the yield of the products of the reaction at optimized reaction condition using Lipozyme showed that 75.66% oleyl oleate was produced. © 2001 Society of Chemical Industry     

Enzymatic synthesis of acetylated glucose fatty acid esters in organic solvent

Two immobilized lipases fromCandida antarctica (SP 382) andC. cylindraceae, nowrugosa (2001), catalyzed the synthesis of novel acetylated glucose fatty acid esters with glucose pentaacetate (GP) and Trisun 80 (80% oleic) vegetable oil or methyl oleate as substrates in organic solvents. The relative yield was between 6.4–52%, and the incorporation of oleic acid onto the glucose was between 31–100%. In addition, these enzymes were able to catalyze the synthesis of glucose fatty acid esters with free glucose as the sugar substrate. The highest oleic acid incorporation (100%) was obtained in benzene with SP 382 lipase and Trisun 80 as the acyl donor. With methyl oleate as the acyl donor, greater incorporation was obtained in benzene (90.5%) compared to 75% in isooctane. The 2001 lipase was better in benzene/pyridine (2∶1 vol/vol) 74%) and chloroform (61%) compared to benzene and isooctane. However, with free glucose and Trisun 80 as substrates, both enzymes gave acceptable levels of oleic acid incorporation (82–100%) in benzene, benzene/pyridine and pyridine. The best conditions for the ester interchange reaction reported are: lipase (10% by weight of substrate); incubation time 48 h; molar ratio of Trisun/GP 1∶2; 3 mL solvent and 3% added water. These glucose esters have potential applications as emulsifiers in food, cosmetics and pharmaceutical formulations.     

Synthesis and Properties of Ascorbyl Esters Catalyzed by Lipozyme TL IM using Triglycerides as Acyl Donors

Esters of l-ascorbic acid with long-chain fatty acids (E-304) are employed as antioxidants in foods rich in lipids. Although their enzymatic synthesis offers some advantages compared with the current chemical processes, most of the reported methods employ the immobilized lipase from Candida antarctica as biocatalyst and free fatty acids or activated esters as acyl donors. In order to diminish the cost of the process, we have investigated the synthesis of ascorbyl oleate and ascorbyl palmitate esters with the immobilized Thermomyces lanuginosus lipase Lipozyme TL IM—which is significantly less expensive than Novozym 435—and triglycerides as source of fatty acids. Lipozyme TL IM gave rise to a lower yield of 6-O-ascorbyl oleate than Novozym 435 when using triolein (64 vs. 84%) and olive oil (27 vs. 33%) as acyl donors. Both 6-O-ascorbyl oleate and 6-O-ascorbyl palmitate displayed excellent surfactant and antioxidant properties. The Trolox Equivalent Antioxidant Capability values for the oleate and palmitate were 71 and 84%, respectively, of those obtained with l-ascorbic acid; however, both derivatives were able to stabilize soybean oil towards peroxide formation.     

Efficient esterification of sorbitan oleate by lipase in a solvent-free system

The lipase-catalyzed esterification of sorbitan with oleic acid in a solvent-free system to form sorbitan oleate (commercial name Span80) was studied as a feasible approach aimed at meeting the demand for sugar alcohol-based surfactants. Screened results obtained from enzymatic synthesis of sorbitan oleate indicated that Novozym 435 had its highest catalytic activity in a solvent-free system. The introduction of a reduced-pressure system increased the production of sorbitan oleate to a maximum of 95% of theoretical, obtained from 0.2 mol sorbitan, 0.1 mol oleic acid, and 2.0 g lipase (6 wt% of sorbitan) in a solvent-free reaction mixture at optimal reaction conditions. Results obtained from lipase-catalyzed batch esterification reactions showed that more than 90% conversion of sorbitan oleate was maintained after 10 batches of esterification reactions, indicating excellent enzyme stability. Subsequent analysis by HPLC indicated that the product of enzyme-catalyzed esterification by the immobilized lipase contained a significantly greater amount of monoester (about 80%) compared to the composition obtained by chemical synthesis (about 50%).     

Fatty Acid Alkyl Esters as Feedstocks for the Enzymatic Synthesis of Alkyl Methacrylates and Polystyrene-<Emphasis Type="Italic">co</Emphasis>-alkyl Methacrylates for use as Cold Flow Improvers in Diesel Fuels

The enzymatic transesterifications of fatty acid methyl esters (FAME) with hydroxyethyl methacrylate (HEMA) were carried out using the Candida antarctica lipase B immobilized within a porous polymethacrylate resin. The enzymatic activity in the transesterification reaction of FAME with HEMA depended on the polarity of the solvent and the highest yield was obtained in toluene (non-polar). The molar ratio of 1:4 (for methyl laurate:HEMA) and 1:2 (for methyl oleate:HEMA) was most favorable for the transesterification yield. The reaction condition (at 60 °C/24 h), and the enzyme concentration of 5% (w/w) for methyl laurate with HEMA, 2% (w/w) for methyl oleate with HEMA resulted in the highest final yield. Under these conditions, the maximum yields for the transesterification of methyl laurate with HEMA, methyl oleate with HEMA were 97 ± 5.4% and 91 ± 4.7%, respectively. After ten batches of transesterification of FAME with HEMA, enzyme activity was retained at the level of 88 ± 2.6% and 76 ± 2.3%, respectively, compared with their initial activity. Also, alkyl methacrylate/styrene copolymers were synthesized by radical polymerization of HEMA-LMA (or HEMA-OMA) and styrene. The prepared copolymers have average molecular weights from 2.6 × 10⁴ to 5.5 × 10⁴. Especially, the poly(styrene-co-alkyl methacrylate)s (PStmHAMAn) led to a reduction in the pour point in ultra low sulfur diesel (ULSD) treated with 200–1,000 ppm of poly(styrene-co-alkyl methacrylate). Diesel fuel containing 1,000 ppm of the copolymer (PSt2HLMA8) showed a 15 ± 1.25 °C reduction in its pour point.     

The influence of water on the synthesis of n-butyl oleate by immobilizedMucor miehei lipase

The influence of initial water concentration on the synthesis of n-butyl oleate was investigated. The synthesis was done with immobilizedMucor miehei lipase—Lypozyme^™—at various reaction conditions. The activity of the enzyme is lower at higher amounts of water. Initial reaction rates, as well as equilibrium conversion, increase at low initial water concentration. Optimal water concentration for the activity of immobilized lipase is temperature dependent at the pressure of 1 bar. Low initial water concentration barely effects equilibrium esterification at 0.032 bar. At high initial water concentrations equilibrium conversion, as well as initial reaction rates, decrease at both pressures.     

Preparation of trehalose and sorbitol fatty acid polyesters by interesterification

Trehalose octaoleate and sorbitol hexaoleate were prepared by a one-stage solvent-free interesterification of methyl oleate with trehalose octaacetate (TOAC) and sorbitol hexaacetate (SOHA), respectively, in the presence of 1–2.5% sodium metal as catalyst. The greatest yield of trehalose octaoleate, 97.5%, and sorbitol hexaoleate, 96.0%, were obtained at a mole ratio of methyl oleate: SOHA of 6:1 and methyl oleate:TOAC of 8:1 at a synthesis time of 2.5 hr, and temperatures of 105–115°C. The structure of both trehalose octaoleate and sorbitol hexaoleate was confirmed by infrared (IR) and nuclear magnetic resonance (C-13 NMR) spectroscopy. Physical properties such as viscosity, HLB, solubility, color, refractive index, specific gravity and density approximated the physical properties of sucrose polyesters (SPE) and vegetable oils. Trehalose octaoleate and sorbitol hexaoleate, as well as sucrose octaoleate, were not susceptible toin vitro lipolysis, suggesting potential application as low calorie oils. Presented at American Oil Chemists’ Society Annual Meeting in New Orleans, LA, in May, 1987.     

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)].     

Further improvements in the yield of monoglycerides during enzymatic glycerolysis of fats and oils

Three approaches were used in an effort to increase the yield of monoglycerides (MG) during the lipase catalyzed reaction of glycerol with triglyceride fats and oils: i) various commercially available lipases were screened for ability to catalyze MG synthesis; ii) mixtures of lipases were compared with single lipases; and iii) two-step temperature programming was applied during the reaction. Of these, temperature programming was found to be the most effective. With an initial temperature of 42°C for 8–16 hr followed by incubation at 5°C for up to 4 days, a yield of approximately 90 wt% MG was obtained from beef tallow, palm oil and palm stearin. When the second incubation temperature was greater than 5°C, the yield of MG was progressively lower with increasing temperature. In the case of screening of newly available commercial lipase preparations, lipases fromPseudomonas sp. were found to be most effective, giving a yield of approximately 70 wt% MG at 42°C from tallow. Lipases fromGeotrichum candidum, Penicillium camembertii (lipase G) andCandida rugosa were inactive. A mixture of lipases fromPenicillium camembertii andHumicola lanuginosa was found to be more effective than either enzyme alone, giving a yield of approximately 70 wt% MG using beef tallow or palm oil. A mixture ofPenicillium camembertii lipase with eitherPseudomonas fluorescens lipase orMucor miehei lipase was not more effective thanPseudomonas fluorescens orMucor miehei lipase alone.     

Zwitterionic polymer-coated porous poly(vinyl acetate–divinyl benzene)microsphere: A new support for enhanced performance of immobilized lipase

Enzyme immobilization has attracted great attention for improving the performance of enzymes in industrial applications. This work was designed to create a new support for Candida rugosa lipase(CRL) immobilization.A porous poly(vinyl acetate–divinyl benzene) microsphere coated by a zwitterionic polymer, poly(maleic anhydride-alt-1-octadecene) and N,N-dimethylethylenediamine derivative, was developed for CRL immobilization via hydrophobic binding. The catalytic activity, reaction kinetics, stabilities and reusability of the immobilized CRL were investigated. It demonstrated the success of the zwitterionic polymer coating and subsequent CRL immobilization on the porous microsphere. The immobilized lipase(p2-MS-CRL) reached27.6 mg·g~(-1) dry carrier and displayed a specific activity 1.5 times higher than free CRL. The increase of Vmax and decrease of Kmwere also observed, indicating the improvement of catalytic activity and enzyme-substrate affinity of the immobilized lipase. Besides, p2-MS-CRL exhibited significantly enhanced thermal stability and p H tolerance. The improved performance was considered due to the interfacial activation regulated by the hydrophobic interaction and stabilization effect arisen by the zwitterionic polymer coating. This study has thus proved the advantages of the zwitterionic polymer-coated porous carrier for lipase immobilization and its potential for further development in various enzyme immobilizations.     

Pseudomonas sp. lipase-catalyzed synthesis of geranyl esters by transesterification

Pseudomonas sp. lipase was immobilized by adsorption onto five supports and tested for its ability to synthesize geranyl esters by transesterification using short-chain triacylglycerols as acyl donors. Reaction mixtures were prepared in 2 mL ofn-hexane, 0.1 M geraniol, 0.03M triacylglycerol, and 200 units of lipase, and incubated at 30°C and 200 rpm for 24 h. Overall, glass beads were the best support. Geranyl acetate and caproate performed best with Duolite (77.5 and 95.3%, respectively). Geranyl butyrate and caprylate performed best with polyvinylpyrrolidone, (80.2 and 95.5%, respectively). Values for nonimmobilized enzyme also were obtained. Immobilization improved yields, with geranyl caproate exhibiting best results.