Z)-3-Hexen-1-yl butyrate is an important flavor and fragrance compound as it represents the model of a natural herbaceous (green) note. Two immobilized lipases from Mucor miehei (Lipozym IM) and from Candida antarctica (Novozym 435) were investigated for their use in the synthesis of (Z)-3-hexen-1-yl butyrate by direct esterification in n-hexane. To determine optimal conditions for esterification, we examined the following parameters: temperature, amount of lipase, acid/alcohol ratio, and absence of solvent. In n-hexane, bioconversion yields reached 95 (after 4 h) and 92% (after 6 h) for, respectively, Lipozym IM [17 (w/w reactants)] and Novozym 435 [2% (w/w reactants)]. In the absence of solvent, at 60°C, Novozym 435-catalyzed esterification afforded the title compound in 80% yield. Up to 250 g (in hexane) and 160 g (without solvent) of ester were easily prepared, in a single operation, at a laboratory scale, in few hours, using 2% (w/w reactants) lipase.Low-molecular-weight esters derived from short-chain carboxylic acids (acetates, propionates, butyrates, …) represent an important class of flavoring materials. They are responsible for the fruity odors of many foods and fragrances (1,2). In foodstuff industries, flavor and fragrance loss due to various reasons including manufacturing processes must be compensated for by the addition of aromas. Moreover, there is a growing demand for natural flavors containing green notes represented by C-6 alcohols derivatives. Hence, the biological production of such esters is of much current commercial interest. In this context, the use of immobilized lipases working in nonaqueous media (3-14) has potential since esters produced from natural acids and alcohols may be considered as natural. This mode of production is of great interest, as it would make food industry less dependent on seasonal, climatic, and geographical variations.The present work deals with the biosynthesis of (Z)-3-hexen-1-yl butyrate as a model compound catalyzed by two commercial immobilized lipases from Mucor miehei and from Candida antarctica. Our purpose was to determine best experimental conditions and procedure to produce rather large amounts of C 6 alcohol-derived esters (150 to 250 g), at a laboratory scale, in a single operation as an intermediate step to pilot scale, our ultimate goal being the industrial production from natural acids and alcohols of esters considered to be natural (15)(16)(17). The impact of several parameters on the bioreaction was carefully investigated and optimized, i.e., the nature and the amount of enzyme, the substrate concentration, the temperature, and the acid/alcohol ratio. We also investigated C. antarctica-catalyzed synthesis of the title compound in the absence of solvent.
MATERIALS AND METHODS
Materials.Lipases from M. miehei immobilized on macroporous anion exchange resin Lipozym IM [5-6 Batch Acidolysis Units NOVO (BAUN)·g −1 , cloned into Aspergillus oryzae] and C. antarctica immobilized on acrylic resin Novozym 435 [7000 Propyl Laurate Units (PLU)·g −1 , cloned...
