IntroductionThe interest in the production of biodegradable, envi-ronmentally acceptable esters for biodiesel, lubricants,solvents, surface active agents, etc., from vegetable oilsby lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) bio-catalysis has markedly increased during the last few years(Linko et al., 1995b; Linko and Seppl, 1996). For exam-ple, butyl oleate (Linko and Wu, 1996) may be used asbiodiesel additive, PVC plastisizer, water resisting agent,and in hydraulic fluids. Rapeseed oil fatty acid esters of2-ethyl-1-hexanol (Linko et al., 1994) can be employedto replace conventional organic solvents in a number ofdetergent applications such as in car shampoos, and as asolvent for printing ink. Biodegradable esters (Linko etal., 1995a) are of interest for example as surgicalimplants and agricultural plastic films.

The interest in environmentally acceptable biodegrad-able lubricating base oils has recently rapidly increased(Mang, 1994). Biodegradable lubricants were first devel-oped for two-stroke outboard engines in the beginningof 1980s, with the main base fluid composed ofneopentylpolyol esters of branched chain fatty acids.Eychenne et al. (1996) have recently reviewed the devel-opments in environmentally friendly lubricating oilsbased on neopentylpolyols such as neopentyl glycol,pentaerythritol, and trimethylolpropane. In the middleof 1980s, biodegradable chain-saw oils based on naturalesters of rapeseed oil were introduced on the market.Biodegradable trimethylolpropane esters of fatty acidsfrom sunflower oil (Bongardt et al., 1996) or rapeseedoil (Lms, 1995) fatty acids can be used for examplein the production of hydraulic fluids. Further, trimethy-lolpropane esters have been developed as lubricants forjet turbine (Cooley and Slovinsky, 1961), motor-car

(Leleu et al., 1977), and gas turbine engines (Carr andDeGeorge, 1989).

Lipase catalyzed transesterification has been previouslyproposed for example for the modification of food fatsand oils (Coleman and Macrae, 1977; Yokozeki et al.,1982), and production of biodegradable solvents (Linkoet al., 1994) and polymers (Linko and Seppl, 1996).Osada et al. (1987) and Monot et al. (1990) have demon-strated that also hydrophilic polyols can be esterified bylipase in the presence of an organic solvent such as di-n-butyl ether or tetrahydrofuran. However, organicsolvents are undesirable from the point of view of prac-tical applications. Preliminary results have shown,however, that rapeseed oil based trimethylolpropaneesters can also be synthesized by lipase biocatalysiswithout an additional organic solvent (Lms et al.,1995; Linko et al. 1996). We describe now for the firsttime the enzymic transesterification between trimethy-lolpropane and rapeseed oil fatty acid methyl esters inhigh trimethylolpropane tri-ester yields using immobi-lized lipases.

Materials and methodsMaterialsFinnish rapeseed oil was obtained from Raisio Group,Oil Milling Divion. The average fatty acid compositionof the low erucic acid rape seed oil was: oleic acid 57%,linoleic acid 22%, linolenic acid 12%, palmitic acid4%, eicosaenoic acid 2%, stearic acid 1%, erucic acid< 1%, others 1%. The carriers tested for lipase immo-bilization were: Amberlite XAD-7 and Amberlite IRA-94s (Rohm and Haas, Philadelphia, USA), Celite R-630(Manville, UK), Dowex 66, MWA-1WGR-2 and XUS40339.01 (Dow Chemical Company, Midland, USA),

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Production of trimethylolpropane estersof rapeseed oil fatty acids byimmobilized lipaseY.-Y. Linko,1* T. Tervakangas,1 M. Lms2 and P. Linko11Laboratory of Bioprocess Engineering, Helsinki University of Technology, P.O.Box 6100, FIN-02015 HUT, Finland.Fax +3589462373, E-mail: yu-yen.linko@hut.fi. 2Raisio Chemicals, P.O.Box 101, FIN-21201, Raisio, Finland

The polyol, trimethylolpropane (2-ethyl-2-hydroxymethyl-1,3-propanediol), and a mixture of rapeseed oil fatty acidmethyl esters were transesterified by immobilized lipases without additional organic solvent. The conversion to thepolyol tri-ester with immobilized Rhizomucor miehei lipase Lipozyme IM 20 was about 75% after 24 h at 58 C, 5.3 kPa,with no added water, and the highest conversion of about 90% was reached in 66 h.

1997 Chapman & Hall

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Duolites ES-561 and ES-762 (neutral adsorption resin)(Dia-Prosim, Vitry Chauny, France), GCC and GDC200 (weak alkaline anion exchange resin) (Cultor,Finland), HPA 25 (strong alkaline anion exchange resin)(Mitsubishi Kasei, Japan), Kieselgel 60 (Merck, Darm-stadt, Germany), WA 30 (weak anion exchange resin)(Mitsubishi Kasei, Japan) and Whatman DE 52 (W &R Balston, Maidstone, UK).

ChemicalsTrimethylolpropane, 2-ethyl-2-(hydroxymethyl)-1,3-propanediol, was obtained from E. Merck (Darmstadt,Germany). All other reagents were of analytical grade,unless otherwise indicated.

EnzymesLipase preparations used were from Candida rugosa(powder, hydrolytic activity 8000 U g1) (Biocatalyst,Pontypridd, UK) and immobilized lipases from Rhizo-mucor miehei Lipozyme IM 20 (hydrolytic activity830 U g1 and Candida antarctica Novozym 435 (hydro-lytic activity 7400 U g1) (Novo Nordisk, Bagsvaerd,Denmark).

Enzyme immobilizationCarriers were first washed with boiling, deionized water.A suitable quantity of the solid lipase preparation wasmixed for 2 h in 0.05 M sodium phosphate buffer (pH5.8), filtered, and 60 ml of the enzyme solution wasmixed with 40 g buffered carrier in a 250 ml conicalflask for 6 h at 28 C. The mixture was filtered, and theprecipitate was washed six times with 60 ml of de-ionized water and freeze-dried for 30 h to a dry solidscontent of at least 99%.

Synthesis of rapeseed oil methyl esterRapeseed oil methyl ester was synthesized chemically asfollows: 264 g rapeseed oil was weighed into a 1000 ml3-necked flask, equipped with a thermometer, con-denser, stirrer and sample adapter, and 34 g methanolwas added under stirring. The reaction mixture washeated to 60 C and 0.5% (w/w) alkaline catalyst wasadded. After the reaction was completed in 4 h as deter-mined by TLC, the reaction mixture was washed byacidic water. Glycerol formed was separated and theexcess alcohol was distilled off. The rapeseed oil methylester (melting range 56 to 59 C) content of the productwas 96.6%, as determined by HPLC.

Enzymic synthesis of rapeseed oil trimethylolpropane esterTransesterification between trimethylolpropane andrapeseedoil methyl ester was carried out at a reduced

pressure (2.0 to 13.3 kPa) in 25 ml round bottomedflasks equipped with a 20 cm vertical condenser (coolingwater temperature 6 C) and a magnetic stirrer typicallyas follows: trimethylolpropane (0.607 g) was firstdissolved in 0.7 ml (15%, w/w of total mass of thesubstrates) of water, after which rapeseed oil methylester (4.00 g) and either solid lipase preparation (40%w/w) or immobilized lipase (50% w/w) were added.Reaction was usually carried out either at 37 C or 47 Cat 5.3 kPa (40 mmHg) with magnetic stirring at 150rev min1), and the average trimethylolpropane to rape-seed oil methyl ester molar ratio was either 1:3.5 or1:4.5. The condenser was flushed with about 2 mlacetone after which the total sample was extracted 5 times with 4 ml acetone. The biocatalyst residue wasremoved by centrifuging (1900 g). The supernatantcontaining the product trimethylolpropane tri-esters ofrapeseed oil fatty acids was transferred into a 1.5 mlEppendorf tubes and stored at 20 C for later analyses.

AnalyticalLipase activity (hydrolytic) was determined as follows.The lipase sample, 2.5 ml of deionized water, and1.0 ml of McIlvane buffer of pH 7.0 were kept at 37 Cfor 5 min in a conical flask equipped with a magneticstirrer, after which a mixture of 3.0 ml of olive oilsubstrate and 2.0 ml of deionized water was added, andthe flask was incubated for 30 min at 37 C. The reaction was stopped by adding 3.0 ml 95% (v/v)ethanol and titrated immediately with 0.05 M sodiumhydroxide using phenolphthalein as an indicator. Oneunit (U) of lipase releases one micromol of fatty acid inone minute under the specified conditions, and theactivities were reported as Ug1).

The reaction was monitored by semiquantitative thinlayer chromatography (TLC), using Kieselgel 60 F254TLC plates and ethyl acetate-n-heptane (4:96 v/v) assolvent. The plates were developed for 45 min, sprayedwith a mixture of acetic acid-sulfuric acid-anisaldehyde(100:2:1 v/v), dried for 10 min at room temperatureand heated at 105 C for 5 min.

Quantitative analyses were carried out with high perfor-mance gel permeation chromatography, using an RI-detector and Ultrastyragel 500 and 100 columns(Waters, Milford, USA) with a 0.45 mm filter in thefront of the detector. Acetone was evaporated off from1000 ml samples in 4 h in a vacuum oven at 2.6 kPa,after which 1000 ml HPLC-grade tetrahydrofuran wasadded. The chromatograms were developed with HPLC-grade tetrahydrofuran. The components were eluted onthe basis of their molar mass in descending order. The

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rapeseed oil conversion was reported as % trimethylol-propane tri-ester or as a total conversion in % totrimethylolpropane mono-, di- and tri-esters.

Results and discussionBecause preliminary experiments had suggested that theconversion of rapeseed oil methyl ester to the desiredtrimethylolpropane tri-esters might increase with theuse of an immobilized lipase, several carrier materialsfor the C. rugosa lipase employed were investigated. Thehighest total conversions of about 95% to trimethylol-propane esters were obtained only in 24 h (47 C,5.3 kPa, 13% water) with the lipase immobilized onDuolite ES-561 (40%, w/w, biocatalyst). The highestyield of about 70% trimethylolpropane tri-esters wasreached in 78 h (Fig. 1). When the carriers Duolite ES-762, GDC 200, GCC and HPA were used relativelyhigh conversions were also obtained, while the othercarrier systems yielded inferior results.

With the commercial immobilized lipase, Lipozyme IM20 (20% w/w) a conversion to trimethylolpropane tri-esterss was about 75% in 24 h (58 C, 5.3 kPa, no addedwater), while total conversions of as high as 92.5% couldbe obtained. When the temperature was decreased to47 C again with no water addition, at best about 50%conversion to trimethylolpropane tri-esters was obtainedalready in 24 h, 84% in 48 h and 90% in 66 h with40% (w/w) of the biocatalyst. The water content of thereaction mixture had little influence to the result. Fig.2 shows a typical time course of the transesterificationwith Lipozyme IM 20 as the biocatalyst.

A stepwise addition of lipase did not markedly improvethe result. the transesterification between trimethylol-propane and rapeseed oil methyl ester, and withLipozyme IM 20 as the biocatalyst the product esterprofile was little affected by water content of the reac-tion mixture up to 15.9% water. The immobilizedlipase Novozyme 435 from C. antarctica behaved quitedifferently. It was much more sensitive to the watercontent than Lipozyme IM 20 from R. miehei, and typi-cally only mono- and di-esters were formed even in 66 h(58 C, 5.3 kPa, 40%, w/w biocatalyst), with at mosttraces of tri-esters. In all cases the conversion to the tri-ester in 66 h with Lipozyme IM 20 was in excess of80%. It should be noted here that according to a recentreport of Wehtje and Adlercreutz (1997) lipase activityprofiles as the function of water activity vary withdifferent lipases. This agrees well with our results. Theresults also clearly showed that different immobilizedlipases behaved quite differently in the transesterifica-tion of trimethylolpropane.

ConclusionsTotal conversions to trimethylolpropane esters in theexcess of 90%, and to trimethylolpropane tri-ester ofabout 75% were obtained under a reduced pressure in24 h (58 C, 5.3 kPa, no added water) with the commer-cial immobilized lipase Lipozyme IM 20 (20% w/w). In66 h a conversion to trimethylolpropane tri-esters ofhigher than 90% was reached. With the Candida rugosalipase immobilized on Duolite 561 an about 70%conversion was obtained in 78 h at 47 C, 5.3 kPa. Theoptimal water content was in this case about 13%.Relatively high conversions were also obtained whenDuolite ES-762, GDC 200, GCC and HPA 25 wereused as carriers, while with the other carriers tested infe-rior results were obtained. With the commercial immo-bilized Candida antarctica lipase Novozyme 435 onlymono- and di-esters were obtained, with traces of thetri-ester at low water levels.

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Figure 1 Time course of transesterification betweentrimethylolpropane and rapeseed oil methyl ester, catalyzedby C. rugosa lipase immobilized in Duolite ES 561 (47 C,5.3 kPa, 40%, w/w biocatalyst, 15% water of substrates; rtrimethylolpropane tri-ester, u trimethylolpropane di-ester, Dtrimethylolpropane mono-ester, 7 rapeseed oil methylester; s unidentified compound).

Figure 2 Time course of transesterification between tri-methylolpropane and rapeseed oil methyl ester, catalyzedby immobilized R. miehei lipase Lipozyme IM 20 (58 C,5.3 kPa, 40%, w/w biocatalyst, no added water; r trimethy-lolpropane tri-ester, u trimethylolpropane di-ester, D tri-methylolpropane mono-ester, rapeseed oil methyl ester;sunidentified compound).

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Received 20 August 1997;Revisions requested 29 August 1997 and 25 September 1997;

Final Revisions received 10 October 1997;Accepted 13 October 1997

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