Lipase-catalyzed solid-phase synthesis of sugar fatty acid esters

Lipase-catalyzed solid-phase synthesis of sugar fatty acid estersRemoval of byproducts by azeotropic distillationYouchun Yan a ,Uwe T.Bornscheuer a ,Linqiu Cao b ,Rolf D.Schmid a,*aInstitute of Technical Biochemistry,University of Stuttgart,Allmandring 31,D-70569,Stuttgart,GermanybLaboratory of Organic Chemistry and Catalysis,Delft University of Technology,Julianalaan 136,NL-2628BL Delft,NetherlandsReceived 13April 1999;received in revised form 5July 1999;accepted 14July 1999Abstract6-O -␤-D (ϩ)-Glucose fatty acid monoesters were synthesized from non-activated ␤-D(ϩ)-glucose and fatty acids or fatty acid methyl esters (C 8,C 16,C 18)with lipase from Candida antarctica B immobilized on polypropylene EP 100.Highest yields (up to 90%)were achieved in ethyl methylketone or acetone as solvent by conducting the reactions under reduced pressure at 60°C in order to remove the byproducts water (produced in the esterification of free fatty acids)or methanol (produced in the transesterification of fatty acid methyl esters)by creating an azeotropic mixture.Both solvents could be easily removed from the reaction mixture and are regarded as biocompatible in the preparation of food additives.In case of caprylic acid,highest conversion (76%)was achieved at 25°C.Published by Elsevier Science Inc.Keywords:Lipase;Solid phase synthesis;Fatty acid;Azeotrope;Glucose ester;Surfactant1.IntroductionSugar fatty acid esters are widely used as surfactants and emulsifiers in the pharmaceutical,cosmetic,and food indus-try.At present,mono-,di-and triesters of sucrose and different esters of sorbitan are commercially available.However,most of the emulsifiers manufactured by chemical methods using unprotected sugar moieties and fatty acids may not be used in food applications because toxic organic solvents such as dimethyl sulfoxide,tetrahydrofuran and dimethylformamide are required for the solubilization of sugar components and their removal is laborious.Enzy-matic processes offer an alternative access to these surfac-tants.Especially the use of lipases [1,2]enables the prepa-ration of a wide range of monosaccaride fatty acid esters often as single regioisomers and with no requirement for laborious regioselective protection [3,4].In previous articles we reported a method for the syn-thesis of sugar fatty acid esters,which was based on a mainly solid-phase system in which the acylation of a solid sugar with a fatty acid was performed via lipase-catalysis inthe presence of a small amount of organic solvent (e.g.,t-butanol,acetone)serving mainly as adjuvant.This circum-vents problems that arise from the different solubilities of sugars and fatty acids in organic solvents[5–7].Water gen-erated during the esterification was removed by addition of activated molecular sieves.Unfortunately,this is not prac-tical on a larger scale,because this increases the reactor volume and mass transfer limitations can occur due to difficult stirring.On the other hand,water removal is critical to achieve high yields in the sugar ester synthesis and enzyme activity and/or stability are negatively affected by higher concentrations of water [8].To date,water removal in solvent-free systems has been achieved by performing reactions in open test tubes,evac-uation in vacuo,pervaporation using special membranes,and dry gas bubbling [9–13].However,when organic sol-vents are present in the reaction medium,the exclusive removal of water becomes difficult.In the solid-phase sugar ester synthesis developed in our laboratory,low boiling solvents like acetone,or t-butanol have been most appro-priate;however,reactions under reduced pressure would also remove the solvent from the reaction mixture.We now report on a reaction system in which the by-products (water or methanol)from lipase-catalyzed sugar ester synthesis are removed by azeotropic distillation with*Corresponding author.Tel.:ϩ49-711-685-3192;fax:ϩ49-711-685-3196.E-mail address:rolf.d.schmid@rus.uni-stuttgart.de (R.D.Schmid)Enzyme and Microbial Technology 25(1999)725–7280141-0229/99/$–see front matter Published by Elsevier Science Inc.PII:S0141-0229(99)00106-4the intention to develop a process that is practical on a largescale.2.Materials and methods2.1.Enzymes and chemicalsNovozyme SP525(free lipase B from Candida antarc-tica)was a gift from Novo Nordisk A/S,(Bagsvaerd,Den-mark).Accurel EP100(macroporous polypropylene,parti-cle size200–400␮m)was obtained from Akzo Nobel (Obernburg,Germany).␤-D(ϩ)-Glucose was from Sigma, Acetone,acetonitrile,methanol,dichloromethane,and tet-rahydrofuran were purchased from Riedel de Haen(Seelze,Germany).Caprylic acid,ethyl methylketone,and molecu-lar sieves(3or5Å)were from Fluka(Buchs,Switzerland).Palmitic acid and stearic acid were gifts from Henkel(Du¨s-seldorf,Germany).2.2.General procedure for enzymatic reactionsThe reaction mixture consisted of equimolar amounts offree glucose and fatty acid(or fatty acid methyl ester)andan organic solvent in the amounts stated in the legends ofFigs.2and3.The reaction mixture was incubated in a50-ml two-necked round-bottomflask equipped with aSoxhlet extractor on top of a condenser,which was con-nected to a vacuum controller.Activated molecular sieve(activated by heating overnight to250°C under reducedpressure)was placed in the Soxhlet extractor for the re-moval of byproducts(water,3Å;methanol,5Å).Thecondensed solvent was dried by passing through activatedmolecular sieves before returning to the reaction system.This provides constant removal of water or methanol gen-erated in the reaction and drives the equilibrium towardssugar ester synthesis.It should be noted,that onlyϳ10%ofthe solvent is present in the round-bottomflask,whereas ϳ90%are circulating through the condenser and the Soxhlet extractor.The apparatus was placed in an oil bath,thermostated to60°C(also25and40°C for the reactionwith caprylic acid),and stirred by a magnetic bar(250rev./min)under reduced pressure.The reaction was startedby addition of CAL-B,which was immobilized on EP-100as described previously[7].Samples from the suspensionwere taken at intervals,centrifuged,and the supernatantswere analyzed by thin-layer chromatography and high-per-formance liquid chromatography as described previously[6].At the end of the reaction,the mixture was extractedwith warm acetone(50°C)andfiltered to remove lipase andunreacted glucose.The sugar fatty acid ester solution wascooled toϪ10°C and white crystals formed were collectedbyfiltration.The purity of the products was determined byhigh-performance liquid chromatography,1H-,and13C-nu-clear magnetic resonance-spectroscopy to beϾ99%.Data for azeotropic mixtures of acetone/methanol and ethyl methylketone/water were taken from[14].2.3.Determination of solubilities of glucose and glucose fatty acid estersSaturated solutions of glucose or glucose fatty acid esters were prepared in2ml of acetone or ethyl methylketone, mixed for30min at the temperatures given in Fig.1,and centrifugated at10000rev./min for10min.From the su-pernatant,the concentration of each compound was then determined by high-performance liquid chromatography.3.Results and discussion3.1.Selection of suitable organic solventsA suitable organic solvent should dissolve enough sub-strate to allow the lipase-catalyzed esterification but,at the same time,the product solubility should be low enough to facilitate crystallization necessary to achieve a favored equi-librium for ester formation.Furthermore,the solvent should not affect lipase activity and stability.Previously,we found that t-butanol and acetone are suitable for the solid-phase synthesis of sugar fatty acid esters by using free fatty acids as acyl donors[5,6].However,continuous removal of water from the reaction mixture containing these solvents is rather difficult because their boiling point is lower than that of water.In addition,t-butanol is not permitted for the manu-facture of food additives.In order tofind an appropriate solvent,we determined the solubility of glucose and glucose palmitate in various sol-vents(hexane,acetone,ethyl methylketone,methanol,eth-anol),which are biocompatible for the production of food additives.It turned out that only acetone and ethyl methyl-ketone(EMK)fulfill this requirement and at the same time dissolve sufficient amounts of glucose(0.08mg/ml in EMK, 0.35mg/ml in acetone)and only limited amounts of glucose palmitate(2.5mg/ml in EMK,4.6mg/ml in acetone).In addition,both solvents form suitable azeotropic mixtures allowing the removal of the byproducts generated during the enzymatic reaction.Acetone can be used in the removal of methanol in a transesterification with palmitic acid methyl ester(azeotrope of86%acetone and14%methanol at a boiling point of54.6°C).EMK is suitable for the removal of water by using free fatty acids as acyl donors(azeotrope of 67%EMK and33%water at a boiling point of73.5°C).3.2.Determination of an optimal reaction temperatureThe reaction temperature has a great influence on both, the rate of esterification and the solubility of the glucose fatty acid esters.The influence of reaction temperature on the solubilities of glucose palmitate,stearate,and caprylate in acetone or EMK are shown in Fig.1.The solubility of726Y.Yan et al./Enzyme and Microbial Technology25(1999)725–728glucose is not affected by temperature,but the solubility of the sugar fatty acid esters significantly increases at temper-atures above 50–60°C.Glucose caprylate has an approxi-mately 10-fold higher solubility than the esters of long-chain fatty acids,which also explains the lower yields obtained in the solid-phase synthesis reported previously [5].The solubilities of all products were slightly lower in EMK compared to acetone.According to the manufactur-er’s information,the highest activity of lipase from C.antarctica B is around 60°C.3.3.Synthesis of sugar fatty acid esters with continuous byproduct removalThe synthesis of 6-O -palmitoyl-␤-D (ϩ)-glucose with continuous byproduct removal was investigated using either palmitic acid as acyl donor and EMK as solvent or palmitic acid methyl ester and acetone (Fig.2).Water or methanol produced during the reaction were removed by azeotropic distillation as given in Section 2.Highest yields (81%),of sugar fatty acid esters were obtained with free palmitic acid,which almost exactly corresponds to the 82%yield obtained in a solid-phase system without azeotropic distillation[6].Similar results were found for 6-O -stearoyl-␤-D (ϩ)-glu-cose,where 90%yield were achieved in both reaction sys-tems.Considerably lower yields (66%for palmitate,20%for stearate)were possible with the corresponding methyl esters,although the boiling point of the azeotrope with acetone/methanol is much lower (54.6°C)compared to EMK/water (73.5°C).One explanation might be that the direct esterification of the free fatty acid is faster compared to the transesterification with the methyl ester.In the case of caprylic acid,reactions were also per-formed at lower temperatures,because the solubility of 6-O -caproyl-␤-D (ϩ)-glucose was very high at 60°C (see Fig.2).It turned that the highest conversion (ϳ76%)was achieved at 25°C,whereas 62%were obtained at 40°C and 40%at 60°C (Fig.3).Without azeotropic distillation,the highest conversion was 50%[5].AcknowledgmentsWe thank Novo Nordisk A/S (Bagsvaerd,Denmark)and Henkel KGaA (Du ¨sseldorf,Germany)for their generous gift of lipase and chemicals and the KAAD (Bonn,Ger-many)for their stipend to Youchun Yan.References[1]Schmid RD,Verger R.Lipases—interfacial enzymes with attractiveapplications.Angew Chem Int Ed Engl 1998;37:1608–33.[2]Bornscheuer UT,Kazlauskas RJ.Hydrolases in organic synthesis—regio-and stereoselective biotransformations.Weinheim;Wiley-VCH,1999.[3]Bjo ¨rkling F,Godtfredsen SE,Kirk O.The future impact of industriallipases.Trends Biotechnol 1991;9:360–3.[4]Mutua LN,Akoh CC.Synthesis of alkyl glycoside fatty acid esters innon-aqueous media by Candida 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