Lipae-catalyzed synthesis of isoamyl isobutyrate — optimization using a central composite rotatable design
Introduction
Low molecular weight esters are responsible for the aroma of many fruits and constitute mainly short-chain fatty acid derivatives like acetate, propionate, butyrate and isobutyrate. For example, ethyl butyrate and isoamyl isobutyrate are present in the aroma of strawberry and banana, respectively. Isoamyl esters are valuable and high-demand flavour compounds of commercial importance, and are widely used in the food, beverage, cosmetic and pharmaceutical industries [1]. Natural flavour esters extracted from plant sources are often in short supply or expensive. Moreover, flavour quality and quantity varies from region to region. Most of these esters can also be produced chemically by acid- or base-catalyzed condensation of an alcohol and an acid at elevated temperatures. However, recent trends in consumer preference toward ‘natural’ products indicate that biocatalysis may have an advantage over chemical synthesis as products of biocatalysis may obtain a ‘natural’ label [2]. In this context, it was also observed that fatty acid esters, synthesized by enzymes such as lipase, often have better odour and flavour characteristics compared to similar esters produced by conventional means [3], [4].
Lipases (triacylglycerol hydrolases; EC 3.1.1.3) are extensively used for several types of synthetic reactions due to their regio-, stereo- and substrate-specificities, milder reaction conditions and relatively lower energy requirement. Lipase-catalyzed esterification reactions have been carried out in suitable organic solvents or under solvent-free conditions to produce esters of glycerol [5], aliphatic alcohols [6], [7], [8], hydroxy fatty acids [9] and terpene alcohols [10]. However, most of the work on lipase-catalyzed esterification reported in the literature have been based on reactions using longer-chain length substrates. The esterification of short-chain fatty acids and alcohols has received little attention. Short-chain (<C5) substrates are more water soluble and thus react differently than long-chain substrates in nonaqueous systems.
Initial work in this laboratory has indicated that enzyme/substrate (E/S) ratio, substrate concentration, reaction time and temperature significantly affected the esterification reaction. The present investigation was aimed to enhance knowledge about the reaction parameters affecting lipase-catalyzed synthesis of isoamyl isobutyrate and to optimize the process for economical enzymatic synthesis. Response surface methodology (RSM) is an efficient statistical tool for optimization of multiple variables to predict best performance conditions using a minimum number of experiments [11]. It is superior to the traditional approach in which optimization studies are carried out by varying one parameter at a time while keeping others constant. The present study employs a RSM technique based on a five-level, four-variable central composite rotatable design (CCRD).
Section snippets
Enzyme
Immobilized lipase (triacylglycerol hydrolase, EC 3.1.1.3; Lipozyme IM-20, 25 BIU/g) from Rhizomucor miehei (earlier termed as Mucor miehei) supported on macroporous weak anionic resin beads, was a kind gift from Novo Nordisk (Bagsvaerd, Denmark).
Solvent and substrates
n-Hexane obtained from S.D. Fine Chemicals (Mumbai, India) was used as the organic solvent. Isoamyl alcohol was purchased from Fluka Chemie AG (Buchs, Switzerland). Isobutyric acid, methanol and sodium hydroxide were procured from S.D. Fine Chemicals.
Model development
The coefficients of the model were evaluated for significance with a Student t-test. All the linear coefficients and two quadratic terms (E/S ratio and temperature) were highly significant (P<0.02). All the cross-product coefficients were eliminated in the refined equation as the P values of these coefficients are very insignificant (P>0.5). The values of the coefficients and the analysis of variance (ANOVA) are presented in Table 3. The ANOVA indicates that the model is highly significant as
Discussion
The present study has clearly indicated that ester synthesis can be effected even at high substrate concentrations (>2.0 M; i.e., 176 g/litre) using low E/S ratios (<10 g/mol; i.e., 20 g/litre at 2.0 M substrate). Earlier works [2], [13] on esterification employed low substrate (<0.5 M) and high enzyme (25–50 g/litre; i.e., E/S=50–100 g/mol) concentrations. This study shows that high conversions can be achieved in reasonably short reaction times (<24 h) at ambient temperatures (∼30°C) using
Acknowledgements
The authors wish to thank the Director, CFTRI, Mysore for providing facilities and encouragement. S. Hari Krishna is thankful to the Council of Scientific and Industrial Research, New Delhi, India for the award of Senior Research Fellowship.
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