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656 DOI 10.1002/ejlt.200300871 Eur. J. Lipid Sci. Technol. 105 (2003) 656660

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1 IntroductionOils and fats are essential constituents of all plant and an-imal life. They are produced world-wide at a level of ap-proximately 67 Mt per year, more than 90% of which isused in edible products [1-2]. In addition to this nutritionalfunction, more than 2 Mt is used as starting material forsynthesis of valuable intermediates in oleochemistrywhich are used as lubricants, plasticizers, biodiesel, incosmetics etc. [3-4]. To achieve the synthesis of suchproducts, high energy consuming processes such as hy-drolysis, glycerolysis and alcoholysis are used. Alcoholy-sis is widely employed to convert lipids directly to esterwithout prior isolation of free fatty acids.

For methanolysis, the fat or oil is dissolved in an excess ofmethanol in a continuous process using an alkaline cata-lyst at 240 C and 10 MPa. A procedure for convertingcrude vegetable oils (up to 30% free fatty acid) to methylesters on a pilot-plant scale (500 kg/h or 3000 t/year) hasbeen described [5]. However, the resulting products areoften impure and require re-distillation to remove degra-dation by-products, resulting from the severe reactionconditions. In addition, highly unsaturated oils cannot beused without prior hydrogenation.

Alternatively, a biocatalytic process can be used to re-place the conventional chemical process, thus saving en-ergy and minimizing thermal degradation. Consequently,the application of lipases in the oleochemical industry hasbecome very attractive [6]. In spite of their potential in-dustrial application, however, alcoholysis of vegetable oilsusing lipases has not been extensively investigated.

Among various studies on enzymatic alcoholysis of veg-etable oils, two main difficulties have been reported. First,the insolubility of free glycerol in oil or organic solvent caninhibit the reaction by limiting substrate and product diffu-sion. To solve this problem silica gel and other adsorbentshave been used, which allow glycerol extraction from thealcoholysis reaction mixture, leading to near quantitativeconversion of various fats or oils to fatty alkyl esters [7]. Asecond problem is inactivation of the enzymes by short-chain alcohols such as methanol [8-9]. Using a Michaelis-Menten type equation for substrate inhibition, variation ofthe initial reaction rate with methanol has been analyzed[10]. Based on this equation, a procedure for the stepwiseaddition of methanol has been shown to protect sensitivelipases such as Candida antarctica lipase against inacti-vation [9].

In this paper, we report the enzymatic alcoholysis of vari-ous vegetable oils in n-hexane and a solvent-free system.In view of industrial processing, stability of immobilized li-pases has also been investigated.

Mohamed M. Soumanoua,b, Uwe T. Bornscheuera

a Department of TechnicalChemistry and Biotechno-logy, Institute of Chemistryand Biochemistry, Greifs-wald University, Greifswald,Germany

b Polytechnic College Univer-sity, Research Laboratoryfor Applied Chemistry andBiology (LARECBA),Abomey-Calavi University,Cotonou, Benin

Lipase-catalyzed alcoholysis of vegetable oilsFatty acid alkyl esters were produced from various vegetable oils by transesterificationwith different alcohols using immobilized lipases. Using n-hexane as organic solvent,all immobilized lipases tested were found to be active during methanolysis. Highestconversion (97%) was observed with Thermomyces lanuginosa lipase after 24 h. Incontrast, this lipase was almost inactive in a solvent-free reaction medium usingmethanol or 2-propanol as alcohol substrates. This could be overcome by a three-stepaddition of methanol, which works efficiently for a range of vegetable oils (e.g. cotton-seed, peanut, sunflower, palm olein, coconut and palm kernel) using immobilized lipases from Pseudomonas fluorescens (AK lipase) and Rhizomucor miehei (RM lipase). Repeated batch reactions showed that Rhizomucor miehei lipase was verystable over 120 h. AK and RM lipases also showed acceptable conversion levels forcottonseed oil with ethanol, 1-propanol, 1-butanol and isobutanol (50-65% conversionafter 24 h) in solvent-free conditions. Methyl and isopropyl fatty acid esters obtained byenzymatic alcoholysis of natural vegetable oils can find application in biodiesel fuelsand cosmetics industry, respectively.

Keywords: Immobilized lipase, alcoholysis, vegetable oils, fatty acid alkyl esters, stability, organic solvent.

Correspondence: Uwe T. Bornscheuer, Department of Techni-cal Chemistry and Biotechnology, Institute of Chemistry and Bio-chemistry, Greifswald University, Soldmannstrae 16, D-17487Greifswald, Germany. Phone: +49-3834-86-4367, Fax: +49-3834-86-4346; e-mail: uwe.bornscheuer@uni-greifswald.de,web site: http://www.chemie.uni-greifswald.de/ ~biotech

2 Material and methods2.1 Chemicals and lipasesAll chemicals and solvents used for alcoholysis andanalysis of reaction products were of reagent grade andpurchased from common commercial suppliers. The car-rier material polypropylene EP 100 (Akzo Nobel FaserAG, Obernburg, Germany) was used for lipase immobi-lization. Vegetable oils were obtained from IBCG industry(Cotonou, Benin), except sunflower oil which was pur-chased from ATI industry (Cotonou, Benin). Lipase fromPseudomonas fluorescens (AK) was a gift from AmanoPharmaceutical Co. Ltd. (Nagoya, Japan). Lipases fromRhizomucor miehei (Lipozyme RM, IM) immobilized onan anion exchange resin, and Thermomyces lanuginosa(Lipozyme TL, IM) immobilized on silica gel were giftsfrom Novozymes (Bagsvaerd, Denmark).

2.2 Lipase immobilizationLipase from Pseudomonas fluorescens was immobilizedby an adsorption method. Before immobilization, thepolypropylene carrier EP100 (0.5 g) was soaked with 2 mlabsolute ethanol. Lipase (0.5 g) was dissolved in 20 mlphosphate buffer (pH 7.0, 0.1 M). This solution was addedto the wet support and stirred slowly overnight at roomtemperature. The immobilized lipase preparation was col-lected by filtration, washed twice with 20 ml phosphatebuffer followed by distilled water (20 ml) and driedovernight under vacuum.

2.3 Alcoholysis reaction2.3.1 Reactions in organic solventReactions were performed as batch process in 2 ml n-hexane in glass tubes containing vegetable oil(0.2 mmol), methanol (0.6 mmol) and 10% (w/w veg-etable oil) immobilized lipase. The reaction mixture wasincubated in a water bath at 40 C and agitated with amagnetic stirrer at 200 rpm. For time course studies, 5 laliquots of reaction medium were taken at various time in-tervals and diluted in n-heptane for gas chromatography(GC)-analysis.

2.3.2 Reactions in a solvent free systemReactions were carried out in eppendorf tubes (2 ml) con-taining a mixture of 0.2 mmol vegetable oil and alcohol(0.6 mmol) using 10% (w/w vegetable oil) immobilized li-pase. The reaction mixture was incubated at 40 C andshaken in an eppendorf thermomixer. The stability ofcommercially immobilized lipases in batch alcoholysis re-actions with stepwise addition of methanol was investi-

gated in the reaction media. After each batch reaction, im-mobilized lipase was recovered by filtration, washed threetimes with chilled acetone and dried at room temperature.Dried immobilized lipase was used in the next batch reac-tion with new substrates. Residual activity was expressedin terms of conversion achieved after each 24 h reactioncycle.

2.4 GC-analysis

Samples from organic solvent medium and solvent-freesystems were analyzed by injecting 1 l of reaction mix-ture and an internal standard (methyl pentadecanoate),into a gas chromatograph equipped with a polar column(25 0.25 mm id., Macherey & Nagel, Dren, Germany).Analysis was carried out with a temperature program run-ning from 150 C to 250 C at 5 C/min. Injection and de-tection temperature were fixed at 250 C using hydrogenas carrier gas. The conversion level was calculated fromthe amount of fatty acid alkyl ester formed.

3 Results and discussion

3.1 Alcoholysis in organic solvent

Unsaturated vegetable oils such as cottonseed oil andpalm olein were used for methanolysis, in n-hexane as or-ganic solvent, with three immobilized lipases (two com-mercially immobilized lipases RM and TL, and lipase fromAK immobilized on EP100). All lipases tested were activein this reaction medium (Fig. 1). As can be seen,methanolysis using both vegetable oils, progressed fasterwith immobilized lipase from TL. The highest conversionin this reaction medium after 24 h was observed with TL li-pase using palm olein (97%). For cottonseed oil, howev-er, AK lipase showed the best performance (82% conver-sion). Thus, in n-hexane, palm olein appeared to be thebest substrate for lipases TL and RM, whereas AK lipasewas most effective in converting cottonseed oil to methylesters. This may be due to the different specificities dis-played by lipases towards certain fatty acids and their po-sition along the glycerol backbone in vegetable oils. In an-other interesterification reaction reported in the literature,TL and RM showed experimentally similar activity profiles[11]. Despite these high activities displayed by lipases inorganic media, some important disadvantages of organicsolvents remain, namely higher costs, toxicity and flam-mability, which can limit enzymatic processes in industry.Therefore, a solvent-free system [12] which offers greatersafety, increased reactant concentrations and high volu-metric productivity was investigated.

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3.2 Alcoholysis in solvent free media

Alcoholysis in solvent free medium using various alcoholswith cottonseed oil as substrates was investigated at40 C. Results in Fig. 2 show alkyl ester production fromcottonseed oil after 24 h reaction time in a solvent freesystem. Low conversion levels were observed with theshort chain alcohol methanol, especially with TL lipase.This low performance in alkyl ester production from veg-etable oils and methanol is in agreement with the work ofMittelbach [8]. The low yield of methyl esters can be at-tributed to unfavorable viscosity conditions which affectsmixing of substrates with the lipase [13-14]. However, AKand RM lipases displayed high catalytic activity in this re-

action medium with most alcohol substrates (Fig. 2). Low-est conversion was observed with TL lipase with all alco-hol substrates, which is in striking contrast to its perfor-mance in organic solvent but in agreement with our previ-ous work [15]. Using secondary alcohols as substrate forester production, conversion was found to be lower with2-propanol than with isobutanol. In all cases, better con-version levels were obtained when cottonseed oil wastransesterified with 1-propanol and isobutanol, as demon-strated in Fig. 2.

To increase conversion levels with methanol as substrate,a stepwise addition was used. The reaction was started ata 1:1 molar equivalent methanol: cottonseed, oil followed

658 Soumanou and Bornscheuer Eur. J. Lipid Sci. Technol. 105 (2003) 656660

2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.de

Fig. 1. Time course of methano-lysis reaction of cottonseed oil(open symbols) or palm olein (fullsymbols) in n-hexane using im-mobilized lipases. Symbols: , li-pase AK; , lipase RM; , lipaseTL. See Materials and methodsfor details.

Fig. 2. Effect of alcohol type oncottonseed oil alcoholysis, in sol-vent-free system using immobi-lized lipases. See Materials andmethods for details.

by addition of 1 molar equivalent methanol after 4 and 8 h.With this strategy, over 90% conversion was possible after30 h using AK and RM lipases (Fig. 3). A lower conversionlevel was observed with TL lipase, which was neverthelessconsiderably higher than a one-step methanol addition withthis enzyme (10% conversion after 24 h, Fig. 2). This is inagreement with results reported by Shimada et al. [9] whodemonstrated that stepwise addition of methanol is ex-tremely effective in converting vegetable oil fatty acids totheir respective alkyl esters, by reducing lipase inactivation.Particularly with water-miscible alcohol substrates such asmethanol, water required to maintain lipase structure can

be stripped leading to lower activity and eventual biocata-lyst inactivation. Step-wise addition of methanol is thoughtto reduce this effect, explaining the higher stabilities andconversion levels observed.

The stability of immobilized lipases during consecutivebatch transesterification of cottonseed oil was studied underthese conditions. As shown in Fig. 4, both immobilized TLand RM lipases exhibited high stability (above 75%) whenreused during the first five batch reactions (each 24 h). Ad-ditional reactions lead to further loss of enzyme activity,which was particularly significant for TL lipase. This enzyme

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Fig. 3. Effect of stepwisemethanol addition on methanoly-sis of cottonseed oil in a solvent-free system, using immobilized li-pases. Symbols: , lipase AK; ,lipase RM; , lipase TL. See Ma-terials and methods for details.

Fig. 4. Stability of commerciallyimmobilized lipases, RM and TLduring eight consecutive batchreactions. For conditions see Fig.3 and Materials and methods.

retained only 35% residual activity after 8 batch reactions.This can be attributed to different factors such as, inactiva-tion of the biocatalyst in the oil phase, type of carrier usedfor immobilization or enzyme sensitivity to long-termmethanol exposure. In contrast, the RM lipase preparationappears to be very suitable for alkyl esters production.

Finally, RM and immobilized AK lipase activities werecompared for alcoholysis of various vegetable oils usingstepwise addition of methanol or 2-propanol. Data in Tab.1 demonstrates that both lipases can achieve conversionlevels between 63 and 84% after 24 h methanolysis.

Using the secondary alcohol 2-propanol, lower conver-sions were found with all vegetable oils. In these experi-ments RM lipase was found to be the most suitable en-zyme with 69% conversion using palm kernel oil. Theseconversion levels are nevertheless higher than those re-ported for one step esterification of 2-propanol with lauricoils [14].

AcknowledgementsWe gratefully acknowledge financial support for Dr. M. M.Soumanou by the German Academic Exchange Service(DAAD, Bonn, Germany). The gifts of lipases fromNovozymes, Bagsvaerd, Denmark and Amano Pharma-ceuticals Inc., Nagoya, Japan are gratefully appreciated.

References[1] R. OBrien: Food service use of fats and oils. Inform 4

(1993) 913-921.[2] F. Gunstone: Yields of oilseeds and of oils and fats. Inform

12 (2001) 1093-1096.[3] K. Harrigton, C.A. dArcy-Evans: Comparison of conven-

tional and in situ methods of transesterification of seed oilfrom a series of sunflower cultivars. J. Am. Oil Chem. Soc.16 (1994) 109-1013.

[4] D. C. Drown, K. Harper, E. Frame: Screening vegetable oilalcohol esters as fuel lubricity enhancers. J. Am. Oil Chem.Soc. 78 (2001) 579-584.

[5] Y. M. Choo, K. Y. Cheah, A. N. Ma, A. Halim, A. S. H. Ong:Conversion of crude palm kernel oil into its methyl esters ona pilot plant scale. In: World Conference on Oleochemicals.Ed. T.H. Applewhite, AOCS-Press, Champaign, IL (USA)1990, p. 292-295.

[6] R. D. Schmid, R. Verger: Lipases-interfacial enzymes with at-tractive applications. Angew. Chem. 110 (1998) 1694-1720.

[7] D. E. Stevenson, R. A. Stanley, R. H. Furneaux: Near-quanti-tative production of fatty acid alkyl esters by lipase-catalyzedalcoholysis of fats and oils with adsorption of glycerol by sili-ca gel. Enzyme Microb. Technol. 16 (1994) 478-484.

[8] M. Mittelbach: Lipase catalyzed alcoholysis of sunflower oil.J. Am. Oil Chem. Soc. 67 (1990) 168-170.

[9] Y. Shimada, Y, Watanabe, T. Samukawa, A. Sugihara, H.Noda, H. Fukuda, Y. Tominaga: Conversion of vegetable oilto biodiesel using immobilized Candida antarctica lipase. J.Am. Oil Chem. Soc. 76 (1999) 789-793.

[10] T. Samukawa, M. Kaeida, T. Matsumoto, K. Ban, A. Kondo,Y. Shimada, H. Noda, H. Fukuda: Pretreatment of immobi-lized Candida antarctica lipase for biodiesel fuel productionfrom plant oil. J. Biosci. Bioeng. 90 (2000) 180-183.

[11] H. Zhang, X. Xu , J. Nilsson, H. Mu, J. Adler-Nissen, C.-E.Hy: Production of margarine fats by enzymatic interesteri-fication with silica-granulated Thermomyces lanuginosa li-pase in a large-scale study. J. Am. Oil Chem. Soc. 78(2001) 57-64.

[12] V. Dossat, D. Combes, A. Marty: Lipase-catalyzed transes-terification of high oleic sunflower oil. Enzyme Microb. Tech-nol. 30 (2002) 90-94.

[13] L. A. Nelson, T. A. Foglia, W. N. Marmer: Lipase-catalyzedproduction of biodiesel. J. Am. Oil Chem. Soc. 73 (1996)1191-1195.

[14] R. D. Abigor, P. O. Uadia, T. A. Foglia, M. J. Haas, K. C.Jones, E. Okpefa, J. U. Obibuzor, M. E. Bafor: Lipase-cat-alyzed production of biodiesel fuel from some nigerian lau-ric oils. Biochem. Soc. Trans. 28 (2000) 979-981.

[15] M. M. Soumanou, U. T. Bornscheuer: Improvement in li-pase-catalyzed synthesis of fatty acid methyl esters fromsunflower oil. Enzyme Microb. Technol. 33 (2003) 97-104.

[Received: July 8, 2003; accepted: July 29, 2003]

660 Soumanou and Bornscheuer Eur. J. Lipid Sci. Technol. 105 (2003) 656660

2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.de

Tab. 1. Alcoholysis of vegetable oils by stepwise addition of methanol or 2-propanol using immobilized lipase. Alcoholysis re-action was carried out for 4 h in a mixture of 0.2 mmol vegetable oil/alcohol (1:1, mol/mol) using 10% immobilized lipase. Af-ter 4 h and 8 h, 0.2 mmol alcohol was added to the reaction mixture. RM: immobilized lipase from Rhizomucor miehei; AK: li-pase from Pseudomonas fluorescens immobilized on polypropylene EP100. Conversion levels were determined after 24 h.

Conversion [%]Vegetable oil RM AK

Methanol 2-Propanol Methanol 2-Propanol

Cottonseed oil 83 41 82 33Peanut oil 63 42 73 37Sunflower oil 84 35 75 36Palm olein 72 50 82 32Coconut oil 64 56 66 34Palmkernel oil 72 69 74 35