ARTICLE Miniemulsion as Efficient System for Enzymatic Synthesis of Acid Alkyl Esters Dragana P.C. de Barros, 1 Luı´s P. Fonseca, 1 Joaquim M. S. Cabral, 1 Eugen M. Aschenbrenner, 2 Clemens K. Weiss, 2 Katharina Landfester 2 1 Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Te´cnico, Av Rovisco Pais, 1049-001 Lisbon, Portugal; telephone: þ351-218419065; fax: þ351-218419062; e-mail: dragana@ist.utl.pt 2 Max Planck Institute for Polymer Research, Mainz, Germany Received 3 December 2009; revision received 7 February 2010; accepted 23 February 2010 Published online 26 March 2010 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.22726 ABSTRACT: The aim of this work is to devise an efficient enzymatic process for the production of linear alkyl esters in aqueous miniemulsion systems. The esterification reactions of linear alcohols and carboxylic acids were performed with three different enzymes, commercial Amano lipase PS from Pseudomonas cepacia, Lipase type VII from Candida rugosa, and lyophilized Fusarium solani pisi cutinase expressed in Saccharomyces cerevisiae SU50. The miniemulsion system shows a high potential for the synthesis of linear alkyl esters, for example, hexyl octanoate, which could be synthesized with an ester yield of 94% using Amano lipase PS. Even with hydrophilic alcohols as ethanol, ethyl decanoate could be obtained with a concentration of 0.45 M and a yield of 62% using F. s. pisi cutinase as catalyst. High esterification rates for ethyl- and hexyloleate in miniemulsion showed a sig- nificant shift in cutinase selectivity towards longer chain length carboxylic acids. The stepwise addition of the alcohol led to an increase of the esterification yield. Moreover, increasing the amount of dispersed organic phase, mainly consisting of the substrates, led to a significant increase of the final ester concentration (e.g., concentration of 1.4 M for ethyl decanoate for the esterification with Amano Lipase PS). Biotechnol. Bioeng. 2010;106: 507–515. ß 2010 Wiley Periodicals, Inc. KEYWORDS: miniemulsion; alkyl esters; lipase; cutinase; esterification Introduction Linear alkyl acid esters are a large group of flavors, and very important ingredients of natural aromas in various food, beverage, cosmetic and pharmaceutical products (Hari- Krishna and Karanth, 2002). Many of them are responsible for fruity type flavors (derivatives of short and medium length chain of alkyl acids) or are of great importance as emollient in cosmetic industry (derivates of medium chain length alkyl acids; Chang et al., 2005; Schrader et al., 2004). Esters are most commonly produced by chemical reactions between an alcohol and an organic acid in the presence of catalyst or by extraction from natural sources. Natural aroma products obtained by extraction are expensive and the yields are very low. Although, the chemical synthesis of flavor products is cheap, their production requires high temperatures and pressures, the use of organic solvents, and strong acids or alkali as catalyst, possibly leaving remaining traces as contaminants in the final products or requiring additional and complex purification steps (Longo and Sanroma´n, 2006). The biotechnological production of flavor esters using enzymes can be an interesting and important alternative to catalytic manufacturing. These biotechnological processes benefit from the use of mild reaction conditions, high quality and purity of the final products due to less by-product formation and the regio- and stereo-specificity of enzymes. Using biocatalysts the waste production such as the generation and use of hazardous organic reactants and the use of solvents could be significantly reduced (Serra et al., 2005). Lipases and cutinases can be employed for esterification reactions and show high potential for applications in industry (Hills, 2003; Jaeger and Reetz, 1998; Welsh et al., 1990). The industrial use and potential of lipases to produce fatty acid esters was reported as significant, but there are only few examples of the successful production processes in practice (Serra et al., 2005). Cutinases belong to a group of enzymes that can be considered as a link between esterases and lipases. Contrary to most lipases, no interfacial activation is observed due to the missing lid to cover the active site. Fusarium solani pisi cutinase was first cloned in E. coli, although its expression in Saccharomyces cerevisiae is more efficient and safe for the production of food and pharmaceutical products (Carvalho et al., 1999). Correspondence to: Dragana P.C. de Barros ß 2010 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 106, No. 4, July 1, 2010 507