575 Enzymatic Synthesis of Geranyl Acetate in n-Hexane with Candida antarctica Lipases Paul A. Claon and Casimir C. Akoh* Department of Food Science and Technology, The University of Georgia, Athens, Georgia 30602-7610 Geranyl acetate is an important flavor and fragrance com- pound. Two immobilized Candida antarctica lipases, SP382 and SP435, were investigated for their use in the synthesis of geranyl acetate by direct esterification. Yields between 95 and 99% molar conversion were obtained with 2 and 15% (w/w reactants) of SP435 and SP382 lipases, respec- tively. Optimum yields were obtained at 0.1M acetic acid and 0.12M geraniol after l~h incubation. No inhibitory ef- fect was observed at increasing concentrations of geraniol. Addition of 60% (w/w reactants) water led to 50 and 60% reduction in the esterification activity of SP382 and SP435 lipuses, respectively. The best yields were obtained at added water contents between 0-5% (w/w reactants). Solvents with a log P value of 0.85 or more gave reaction yields of more than 80% molar conversion. Higher log P values did not necessarily lead to higher conversion yields. The immobilized lipase SP382 was still active after reus- ing ten times in the direct esterification reaction. KEYS WORDS: Candidaantarctica, direct esterification, enzymatic synthesis, geranyl acetate, lipases, organic solvents. Esters of geraniol, especially the acetate, are valuable flavor and fragrance compounds. Geranyl acetate is a major con- stituent of numerous essential oils, such as lime oil, which has considerable economic importance (1). Recent trends in consumer preference toward natural products indicates that biocatalysts may have an advantage over their chemical counterparts, and products of biocatalytic processes may be termed "natural" (2). In addition, biocatalysts have lower energy requirements than chemical catalysts and can be used to synthesize products of better quality (3). Lipases (triacylglycerol acylhydrolase EC 3.1.1.3) can be used as biocatalysts for the production of flavor esters by trans- esterification (3-7) and direct esteriflcation reactions (8-15}. The use of acetic acid as an acyl donor in transesterifica- tion and direct esterification reactions was previously at- tempted with relatively little or no success (4,5,8,9,12,16-19}. Acetic acid has an inhibitory effect on most lipases. It lowers the pH of their microenvironment and interferes with their aqueous layer (4,9,13,17). Recently, Claon and Akoh (20) reported the synthesis of terpene esters of acetic acid by direct esterification with yields of more than 90% molar con- version. Here we report the effect of various reaction para- meters (i~, enzyme and substrate concentration, time cours~ temperature water, solvents and enzyme reuse) on the direct esterification of geraniol with acetic acid by means of two immobilized lipases from Candida antarctica. MATERIALS AND METHODS Materials. Nonspecific lipases from C antarctica, immobi- lized on acrylic resin, SP382 (40 BIU/g) and SP435 (7000 BLU/g; cloned into Aspergillus oryzae), were obtained from Novo Nordisk Bioindustrials, Inc. (Danbury, CT). Geraniol (95% pure) was purchased from Sigma Chemical Co. (St. Louis, MO). Acetic acid and all solvents were high- *To whom correspondenceshould be addressed. performance liquid chromatography-grade and obtained from Fisher Scientific (Norcross, GA). Molecular sieve 4~, was purchased from Davison Chemical (Baltimore, MD). The reagent for coulometric determination of water (Hy- dranal-Coulomat AG) was purchased from Crescent Chemical Co., Inc. (Hauppauge, NY). Esterification method. Ester synthesis was carried out in screw-capped test tubes. Unless otherwise specified, 0.12M geraniol, 0.10M acetic acid and 15% (w/w reactants) of SP382 or 2% (w/w reactants) of SP435 lipase were suc- cessively added to 2 mL of dry n-hexane. Molecular sieves were added to remove water formed during the reaction. All samples were prepared in duplicate and incubated in an orbital shaking water bath at 30~ for 24 h at 200 rpm. A control with no enzyme was incubated under the same conditions. Extraction and analysis. At the end of the incubation period, the reaction mixtures were cooled in ice and passed through an anhydrous sodium sulfate (Na2SO4) column to remove the enzyme and any residual water. Internal standard (DL-menthol; 200 gg) was added to each sam- ple. A 1-~L aliquot was analyzed by gas-liquid chroma- tography with a Hewlett-Packard HP5890 Series II gas chromatograph (Hewlett-Packard, Avondale~ PA) equip- ped with a flame-ionization detector. A DB-5 fused-silica capillary column (30 m • 0.25 mm i.d.; J&W Scientific. Folsom, CA) was used and operated isothermally at 150~ Injector and detector temperatures were set at 250 and 260~ respectively. Helium was used as the carrier gas at a total flow rate of 24 mL/min. The extent of syn- thesis was determined from the amount of geraniol con- sumed in the reaction and quantitated by an on-line computer. For enzyme reuse, the reaction product was removed and passed through an anhydrous sodium sulfate column while the enzyme in the test tube was rinsed with hexane and subsequently dried in a desiccator. RESULTS AND DISCUSSION The amount of lipase used in a given bioprocess is a crucial economic factor. The concentrations of lipases reported are often too high for any industrial application (21). En- zyme concentrations of 38, 37 and 93% (w/w reactants) have been reported {4,15,17}. The effect of varying enzyme concentration on the yield of geranyl acetate by direct esterification is presented in Figure 1. The minimum con- centration necessary to achieve maximum yield {96%}was 15% for SP382 and 2% for SP435 lipase. The concentra- tion of SP435 reported here is well below the recom- mended concentrations for this lipase (5-10%) (19}. The effect of substrate concentrations on the synthesis of flavor esters with Mucor miehei (7,14,21,22) and C cylin- draceae lipases has been reported (11). Lipases from M. miehei were inhibited by increasing concentrations of ethanol (14), butanol (21) and geraniol (7,22). Candida cylindraceae lipases were also inhibited by increasing con- centrations of ethanol, with an optimum at 0.4M (11). The effects of geraniol and acetic acid concentrations on the Copyright 9 1994 by AOCS Press JAOCS, Vol. 71, no. 6 (June 1994)