Formation of Polyol-Fatty Acid Esters by Lipases in Reverse Micellar Media Douglas G. Hayes* and Erdogan Gulari’ Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 48109-2 136 Received July 19, 1991Mccepted January 7, 1992 The synthesis of polyol-fatty acid esters has strong implica- content to shift thermodynamic equilibrium in favor of tions in such industries as foods, cosmetics, and polymers. esterification over hydrolysis, and be heterogeneous or We have investigated these esterification reactions employ- biphasic in nature to accommodate all media compo- ing the polyols ethylene glycol, 2-monoglyceride, and sugars and their derivatives with the biocatalyst lipase in water/ nents and provide lipases with an interface, which in AOT/isooctane reverse micellar media. For the first reaction, most cases enhances the enzymes’ performance. We 50-60% conversion was achieved and product selectivity have employed reverse micellar media composed of toward the monoester over the diester shown possible by nanometer-sized dispersions of aqueous or polar mate- employing lipase from Rhizopus delemar. A simple kinetic rial in a lipophilic organic continuum formed by the ac- model based on the formation of an acyl-enzyme interme- diate accurately predicted the effect of polyol concentra- tion of surfactants/cosurfactants to fulfill these criteria. tion but not the effect of fatty acid or water concentration In addition, compared to alternate types of reaction me- probably due to the model exclusion of partitioning effects. dia, reverse micellar media provide excellent enzyme- The success of this reaction in reverse micellar media is substrate contact due to its dynamic nature, is easy to due greatly to its capacity to solubilize large quantities of prepare, and has a large amount of interfacial area. The glycol despite the media’s overall hydrophobicity. The sec- ond reaction, investigated for its potential for production of properties of reverse micelles and their application in “mixed“ glycerides, also achieved about 50% conversion enzymology and biotechnology have been thoroughly but had only a small portion of triglyceride in its product reviewed. l5 distribution. Also, isomerization of the 2-monoglyceride to In the first two articles of this series we have investi- l-monoglyceride,followed by hydrolysis of the latter, unfor- gated the esterification of fatty acid and monohydroxyl tunately occurred to a significant extent. Attempts at esteri- fication with hexoses and their derivatives such as glucose alkyl alcohols for production of flavor esters’ and esteri- and mannitol produced no conversion. fication with glycerol and a positional-specific lipase Key words: lipases reverse micelles ethylene glycol-fatty to produce monoglycerides, which are important food acid esters “mixed” glycerides emulsifiers.” Our goal here is to extend the work of the latter article to esterification with other polyols. Besides INTRODUCTION producing emulsifiers and texturizers, such reactions have importance in polymerization, e.g., as monomer Esters of alcohols and long-chained carboxylic or “fatty” units for crosslinking6 and in production of optically acids have a variety of applications in industry, such as active polymers which can be employed in chiral separa- in food” and cosmeticz”*emulsifiers and lubricants, in tions.16 Fatty acid esters of sugars are of particular im- flavors and fragrances, and as components of or interme- portance as low-calorie fat substitutes, biodegradable diates for food products (e.g., cooking oils).2oCommon detergents, and components of paints and varnishes.23’8.20 industrial practice for producing esters involves catalytic The positional and regioselectivity of lipases is espe- hydrolysis or esterification directed by alkali or other cially desirable for this reaction since the ester’s physical metals at high temperatures (e.g., 100-300°C) and/or properties are quite dependent on its molecular struc- extremes in pressure.2vzoThis leads to high operating ture. Here, fatty acid esterification will be explored with costs. Also, reactions are slow due to poor mass trans- the polyol substrates ethylene glycol (HOCH2CH20H), fer,’ reaction selectivity is poor, and undersirable side sugars, and their derivatives, such as glucose and manni- reactions often occur which produce, e.g., discoloration to1 and 2-monoglycerides. The latter reaction is of par- or undesirable odors.” Thus, in recent years, employ- ticular interest in synthesizing “mixed” triglycerides, ment of lipases (EC 3.1.1.3) as biocatalysts has achieved which are important components of synthetic oils that much attention since they operate at mild conditions and are difficult to produce selectively by conventional dictate the product distribution through their stereo-, means. The latter reaction also represents a step of the regio-, and positional specificities. For lipase-catalyzed lipase-catalyzed interesterification reaction between, esterification, the medium employed has to be primarily e.g., palm oil and stearic acid, to produce a cocoa butter hydrophobic to solubilize substrates, contain low water substitute through inexpensive means. It may also help explain why yields of triglycerides for the reaction be- * Present address: U.S. Department of Agriculture, NCAUR, 1815 N. University St., Peoria, IL 61604. tween glycerol and lauric acid in reverse micelles are ’ To whom all correspondence should be addressed. low, as obtained by Fletcher et aI?*l9 Biotechnology and Bioengineering, Vol. 40, Pp. 110-118 (1992) 8 1992 John Wiley 81 Sons, Inc. CCC 0006-3592/92/010110-09$0400 MATERIALS AND METHODS [lauric acid] = [AOT] = 100 mM, [n-butanol] = 250 mM, temperature 23.5 ?1.5"C, and aqueous pH Materials of 6.9. Kinetic trends and results reported here are re- peatable, with errors being within 5% for those cases The surfactant sodium bis(2-ethylhexyl) sulfosuccinate checked. (AOT) was purchased from Aldrich (Milwaukee, WI) Phase diagrams for water/AOT/isooctane media con- and used without further purification. Moreover, its high taining fatty acid and ethylene glycol were determined degree of purity (reported to be above 99%) was veri- experimentally through titrations of water (PBS) and gly- fied by ultraviolet spectroscopy. In addition, calibrations col into organic solvent containing AOT and fatty acid. from infrared spectroscopy indicate the AOT is nearly The titration endpoints were noted by visual observation anhydrous, with water content being at water-surfactant of the cloudiness-clarity transition. Phase boundaries molar ratios, or values, between 0.2 and 0.3. Two wo for a given lauric acid/AOT/organic solvent ratio and lipase types were employed in these reactions and used temperature are plotted in terms of wo and go, the without further purification: the first, from Rhizopus (ethylene) glycol/AOT ratio. Thus, the procedure and delemar, was purchased from Fluka (Ronkokoma, NY); means of displaying results is similar to that contained the second, from Candida cylindracea, was purchased in our previous article for glycerol-containing media." from Sigma (St. Louis, MO). All other reagents used were of high purity, including 2-monopalmitin (pur- chased from Sigma) and glycerol (from U.S. Biochemical RESULTS AND DISCUSSION of Cleveland, OH); for fatty acids, glycerides, and poly- ols, their high degree of purity was verified by gas chro- Ethylene Glycol-Lauric Acid Esterification matography. Deionized water was employed throughout. The reaction investigated in this section is the esterifi- cation of ethylene glycol and lauric (dodecanoic) acid in Methods isooctane-based reverse micellar media formed by the All reactions were carried out similarly to that described surfactant AOT. To better understand how the two sub- previously by us.8s1oFirst, a solution of surfactant (AOT strates interact with the micellar media and to demark was employed exclusively) and substrates in isooctane the quantities of glycol and fatty acid permitted by the was prepared. Typical concentration ranges for AOT and media for micelle formation, phase diagrams were deter- fatty acid substrate each were from 100 to 200 mM. A mined. As was performed for glycerol-containing reverse brief period of shaking was required for incorporation of micellar media," phase boundaries separating monopha- ethylene glycol into reverse micelles. Next, a small ali- sic reverse micellar media from two-phase media were quot of a lipase solution in 50 mM phosphate buffer estimated for constant ratios of surfactant-fatty acid- solution (PBS) at a pH of 6.89 20.05 was injected into organic solvent and temperatures. An example of experi- the media to initiate the reaction; gentle agitation for mentally determined phase boundaries is depicted in durations of a few seconds to 5 min was required for Figure 1. Here, the boundaries are plotted in terms of their incorporation into reverse micelles, as indicated the water-AOT molar ratio wo,and the (ethylene) gly- by the cloudiness-clarity transition. (As a note concern- ing aqueous pH values, the addition of lipase from R. delemar slightly increased the pH of PBS by 0.1 unit at the concentrations employed while the addition of li- pase from C. cylindracea had no such effect.) All reac- &n tions reported here were operated at room temperature: .s 23.5 +0.5"C. The procedure for operating sugar esterifi- 3 cation reactions is similar to that just described, except i2 51 10 the sugar substrate was added to the medium as a com- t ponent of