Research Article Received: 19 August 2011 Revised: 15 November 2011 Accepted: 16 December 2011 Published online in Wiley Online Library: (wileyonlinelibrary.com) DOI 10.1002/jsfa.5599 Enzymatic synthesis of rose aromatic ester (2-phenylethyl acetate) by lipase Chia-Hung Kuo, a† Shu-Hua Chiang, b† Hen-Yi Ju, a Yu-Min Chen, c Ming-Yuan Liao, c Yung-Chuan Liu d and Chwen-Jen Shieh a∗ Abstract BACKGROUND: 2-Phenylethyl acetate (2-PEAc) is a highly valued natural volatile ester with a rose-like odour that is widely used to add scent or flavour to cosmetics, soaps, foods and drinks. In this study, 2-PEAc was synthesised enzymatically by transesterification of vinyl acetate with 2-phenethyl alcohol catalysed by immobilised lipase (Novozym  435) from Candida antarctica. RESULTS: Response surface methodology and a three-level/three-factor Box – Behnken design were used to evaluate the effects of time, temperature and enzyme amount on the molar conversion % of 2-PEAc. The results showed that temperature was the most important variable. Based on the ridge max analysis results, optimum enzymatic synthesis conditions were predicted as a reaction time of 79 min, a temperature of 57.8 ◦ C and an enzyme amount of 122.5 mg. The predicted and experimental yields were 86.4 and 85.4% respectively. CONCLUSION: Three immobilised lipases were screened and 15 reaction conditions were tested in order to find the combination for maximum yield. The optimisation of 2-PEAc synthesis catalysed by Novozym  435 was successfully developed. The kinetic study of this transesterification reaction showed that it followed an ordered ping-pong bi-bi mechanism without any inhibition by reactants. c  2012 Society of Chemical Industry Keywords: lipase; 2-phenylethyl acetate; kinetics; acetylation; transesterification INTRODUCTION Volatile esters such as 2-phenylethyl acetate (2-PEAc) and geranyl acetate are important contributors to the fragrance of roses and many other flowers. 1 In plants the production of scent volatile acetate esters results mainly from the acetylation of aroma alcohols catalysed by alcohol acetyltransferase. 2–4 When a rose flower has fully opened, the emission of acetate esters reaches its maximum level. The emitted scent is mainly contributed by 2-PEAc, cis-3- hexenyl acetate, geranyl acetate and citronellyl acetate; of these, 2-PEAc is the major emitted volatile ester. 5 Owing to the labour- intensive production and purification process involved, natural products containing aroma volatile esters, such as rose essential oil, command a high price. Therefore the synthesis of aromatic chemicals that reproduce rose odour and the flavour of natural products has become commonplace. 2-PEAc, the major aromatic volatile ester emitted from rose flowers, is a transparent, colourless oily liquid. 2-PEAc (GRAS 2857) is also a valuable flavour and fragrance compound. 6 Generally, it is used in perfumes, cosmetics, soaps and other products as well as to add flavour to food and drinks and scent to incense and household cleaning products. 2-PEAc is traditionally produced by chemical acetylation of 2-phenethyl alcohol (2-PE). 7 Generally, chemical esterification of 2-PE occurs at high temperature in the presence of mineral acids (H 2 SO 4 , H 3 PO 4 ) or Lewis acids (ZnCl 2 , FeCl 3 ) employed as catalysts. 8,9 These catalysts have inherent drawbacks, such as requiring a high reaction temperature (>100 ◦ C), high pressure and long reaction time; difficulty is also experienced in catalyst recovery, and the product poses environmental hazards. Alternative methods for obtaining natural flavours to meet the growing consumer demand include production by microbial fermentation or biocatalysis. Micro-organisms, especially yeasts, are capable of producing 2- PE by normal metabolism. The production of 2-PE by various yeasts has been relatively well studied, 10,11 but only one strain has been reported to produce 2-PEAc. 12 Over the years the use of enzymes, especially lipases in low-water media, has emerged as a powerful method for catalysing synthetic reactions. Enzymatic ∗ Correspondence to: Chwen-Jen Shieh, Biotechnology Center, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 402, Taiwan. E-mail: cjshieh@nchu.edu.tw † These authors contributed equally to this work. a Biotechnology Center, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 402, Taiwan b Department of Food and Beverage Management, Taiwan Hospitality and Tourism College, 268 Chung-Hsing Street, Feng-Shan Village, Shou-Feng County, Hualien 974, Taiwan c Department of Chemistry, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 402, Taiwan d Department of Chemical Engineering, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 402, Taiwan J Sci Food Agric (2012) www.soci.org c  2012 Society of Chemical Industry