Synthesis of Geranyl Butyrate with the Poly(acrylic acid-co-hydroxy propyl methacrylate-cl-ethylene glycol dimethacrylate) Hydrogel Immobilized Lipase of Pseudomonas aeruginosa MTCC-4713 Shamsher S. Kanwar, 1 Satyendra Gehlot, 1 Madan Lal Verma, 1 Reena Gupta, 1 Yogesh Kumar, 2 Ghansham S. Chauhan 2 1 Department of Biotechnology, Himachal Pradesh University, Shimla 171 005, India 2 Department of Chemistry, Himachal Pradesh University, Shimla 171 005, India Received 9 September 2006; accepted 10 January 2008 DOI 10.1002/app.28241 Published online 26 August 2008 in Wiley InterScience (www.interscience.wiley.com). ABSTRACT: Microbial lipases (E.C. 3.1.1.3) are the pre- ferred biocatalysts for the synthesis of various fragrance compounds, such as linalool acetate, citronellal acetate, and geranyl acetate, in organic solvents over chemical synthesis. In this study, a purified alkaline extracellu- lar lipase of Pseudomonas aeruginosa MTCC-4713 was effi- ciently immobilized onto a synthetic poly(AAc-co-HPMA- cl-EGDMA) hydrogel by surface adsorption, and the bound lipase was evaluated for its hydrolytic potential toward various p-nitrophenyl acyl esters, which differed in their C-chain length. Among four series of hydrogels prepared by the variation of the concentrations of mono- mer and crosslinker, two hydrogels, namely, I 5d and I 20d , that exhibited relatively higher protein (lipase activity) bindings were selected to perform hydrolytic and synthetic (geranyl butyrate) reactions in aqueous and organic sol- vents. The hydrogel-bound lipase was highly hydrolytic toward p-nitrophenyl ester (C: 16; p-nitrophenyl palmitate). The hydrogel-immobilized lipase was quite stable and retained approximately 57.6% of its original hydrolytic ac- tivity after the fifth cycle of reuse under optimized condi- tions (pH 8.5, 658C). The hydrogel-immobilized lipase when used to perform the esterification of geraniol/butyric acid (400 : 100 mM) in n-heptane resulted in 98.8 mM geranyl butyrate at 658C under shaking (120 rpm) after 15 h of reaction time. The addition of a molecular sieve (3 A ˚ 3 1.5 mm) to the reaction system at a concentration of 100 mg per reaction volume (1 mL) resulted in the complete conversion of the reactants into geranyl butyrate. Ó 2008 Wiley Periodicals, Inc. J Appl Polym Sci 110: 2681–2692, 2008 Key words: enzymes; esterification; hydrogels INTRODUCTION Lipases (E.C. 3.1.1.3) are versatile biocatalysts that have been used to catalyze a range of reactions, such as the esterification, amidation, and transesterifica- tion of esters and organic carbonates. 1 They are highly stable under adverse conditions, such as organic solvents and high temperatures. The appli- cations of lipase include the production of food additives, chiral intermediates, and pharmaceutical products. 2 Lipase-catalyzed condensation reactions in organic solvents are useful for the synthesis of esters. 3–8 In the last few years, there has been in- creasing interest in the use of enzymes for the biosynthesis of molecules in organic media. 9–11 At present, many esters are industrially manufactured by chemical methods. However, chemical methods involve high temperatures or high pressures; it is difficult in many cases to esterify unstable substan- ces, such as polyunsaturated fatty acid, ascorbic acid, and polyols. 12 Furthermore, the regiospecific acylation of alcohol requires protection and depro- tection steps. 13 Such steps are likely to cause a rise in the manufacturing costs of the esters. In case of esters derived for use in food as additives, chemical impurities are not desired. Many lipase-catalyzed esterification or condensa- tion reactions have been developed by the use of a variety of lipases of microbial origin. 14–19 Such reac- tions are possible in organic solvents. However, in a lipase-catalyzed reversible reaction (esterification), the direction and equilibrium of the reaction are determined by the concentration or activities of the substrates and products, the temperature, the pres- sure, and so on. 13 The conversion in the transesterifi- cation reaction with another ester as a substrate is quite high compared to that achievable in a conden- sation reaction. 20,21 However, acetaldehyde forms a Schiff base with the lipase. 22 In contrast, a condensa- Correspondence to: S. S. Kanwar (kanwarss2000@yahoo. com). Contract grant sponsor: Department of Biotechnology, New Delhi, India (to M.L.V. through a senior research fel- lowship). Journal of Applied Polymer Science, Vol. 110, 2681–2692 (2008) V V C 2008 Wiley Periodicals, Inc.