JOURNAL OF FERMENTA~ON ANDBIOENGINEERING Vol. 81, No. 2, 143-147. 1996 Characteristics and Kinetics of Lipase-Catalyzed Hydrolysis of Olive Oil in a Reverse Micellar System KOICHIRO SHIOMORI,‘* MAKOTO ISHIMURA,’ YOSHINARI BABA,’ YOSHINOBU KAWANO,’ RYOICHI KUBOI,2 AND ISA0 KOMASAWA2 zyxwvutsrqponmlkjihgfedcbaZYXWVUT Department of M aterials Science, M iy azaki University , M iy azaki 889- 21 1 and Department of Chemical Engineering, Osaka University, Toyonaka, Osaka 560,2 Japan Received 11 September 199YAccepted 24 November 199.5 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR The hydrolysis rates of olive oil catalyzed by lipase were measured in a reverse micellar system using sodium bis(Z-ethylhexyl) sulfosuccinate (AOT) under various conditions. The maximum activity of lipase in the reverse micelles was obtained between pH 6.3 and 7.3. The dependence of the activity on the water content, WO,was influenced by the concentrations of both AOT and lipase. The activity at W , = 7 decreased with increasing lipase concentration when the AOT concentration was below 100 mol/m3. The stability of the lipase in the micelles decreased with increases in the value of W, and in the concentration of AOT. The hydrolysis reaction catalyzed by lipase in the reverse micelles was interpreted by a reaction model based on the interfaciol reaction between lipase adsorbed at the interface and the substrate in the organic phase. The rate is controlled by the desorption step of free fatty acids into the organic solution. The desorption rate constants of fatty acids produced were independent of the AOT concentration at a 6zed W ,, and were of nearly the same order of magnitude as that obtained in the emulsion system. [Key words: reverse micelles, lipase, hydrolysis, AOT, enzyme kinetics] Lipase-catalyzed hydrolysis of fats and oils is seen as a promising process for the production of fatty acids and glycerol because the reaction can be carried out under mild conditions. Since fats and oils as substrates are in- soluble in the aqueous phase, in which enzymes are solu- ble, enzymatic reactions proceed at the interface between the aqueous and the organic phases. Hence, such reac- tions are often carried out in a heterogeneous system. Various types of reactors and reaction systems have been proposed for lipase reactions, including the stirred tank, membrane, and immobilized enzyme reactors, and the emulsion, and reverse micellar reaction systems. Reverse micelles can solubilize enzymes in an organic solvent holding their activity (1). Many studies on en- zyme reactions in reverse micellar systems have been reported (2, 3, 10, 17). The enzyme activity in reverse micelles is influenced by several factors, especially the water content, W ,, which controls the diameter of the water pool of the micelles (2, 5, 9, 12). The optimum values of W, for enzyme activity have been correlated with the effective diameter of the solubilized enzyme (2). Reaction and kinetic models have been proposed to inter- pret the characteristics of enzyme reaction in reverse micelles (4-8). The location of the enzyme, the substrate and the reaction field are important in studying enzyme reaction kinetics in reverse micellar systems. A few stu- dies on reverse micellar systems have focused on the loca- tion of the substrate and enzyme (5, 7, 8, 10). Analysis of the enzyme reaction kinetics, such as that of lipase, proceeding at the interface, requires a consideration of the characteristics of the interface, the interaction be- tween the enzyme and the interface, and the behavior of the enzyme activity at the interface. It is, however, difficult to reveal these factors separately in a reverse micellar system. The aim of the present study is to elucidate the charac- * Corresponding author. teristics and kinetics of the hydrolysis of olive oil cata- lyzed by lipase from Candida cy lindracea in a reverse micellar system. The kinetic data were analyzed by the in- terfacial reaction model proposed previously (14). The results were compared with the those for a Lewis-type transfer cell (14) and the emulsion system (15). MATERIALS AND METHODS Reagents Lipase from C. cy lindracea (905 units/mg- solid) was purchased from Sigma Chemical Co., USA. Guaranteed reagents of olive oil from Wako Pure Chemical Co. and sodium bis(2-ethylhexyl)sulfosuccinate (AOT) from Nacalai Tesque Co. were used without fur- ther purification. The buffers used were acetic acid-sodium acetate (pH 4-5.6), phosphate (pH 6-7), and Tris-HCl (pH 7.2-g), at a concentration of 100mol/m3. Other chemicals used were of reagent grade. Preparation of reverse micellar solutions Isooctane solution in which AOT was dissolved was used as the reverse micellar solution. A small volume of buffer solu- tion in which lipase was dissolved was injected into the reverse micellar solution, and the mixed solution was then vigorously shaken until it became transparent. Im- mediately after solubilization, the initial activity of the lipase in the micelles was determined. To determine the stability of the lipase in the micelles, the micellar solu- tion containing lipase was incubated in capped sample tubes in a thermostated bath at 25°C with mild stirring. After a certain period of incubation, the residual activity of the lipase was measured. The reverse micellar solution containing olive oil without lipase was used as the substrate micellar solu- tion. A small volume of buffer solution without lipase was injected into the substrate micellar solution to ob- tain the same water content as that of the micellar solu- tion containing lipase. The W, value of the organic phase was defined as the molar ratio of water to AOT 143