To enhance the Chromobacterium viscosum lipase (glycerol-ester hydrolase; EC 3.1.1.3) activity for the reaction of water-insoluble substrates, the AOT/isooctane reverse micellar interface was modi®ed by co-adsorption of a non-ionic surfactant. Polyoxyethylene sorbitan trioleate (Tween 85) was used as the non-ionic surfactant and olive oil as a water-insoluble substrate. An appreciable increase of lipase activity was observed and at higher W o values (where W o = molar ratio of water to total surfactants of the micellar system) there was no sharp fall of the enzyme activity such as a typical bell-shaped pro®le. The kinetic model for the lipase-catalysed hydrolysis of olive oil in AOT/isooctane reverse micellar system was applied to the enzymatic reaction in this mixed reverse micellar system. It was found that the predictions of the model agree well with the experimental kinetic results and that the adsorption equilibrium constant of olive oil molecules between the micellar phase and the bulk phase of the organic solvent is smaller in AOT/Tween 85 mixed reverse micellar systems than in simple AOT reverse micellar systems.

The activity of Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) entrapped in AOT/isooctane and AOT/Tween 85/isooctane reverse micelles was signi®cantly increased by the addition of short chain methoxypolyethylene glycols (MPEGs), taking the hydrolysis of olive oil as a model reaction. The molecular weight of MPEG had a strong effect on the lipase activity, and MPEG of nominal molecular weight 550 was found to be the most effective. To optimize the factors affecting enzymatic hydrolysis of olive oil in reverse micellar systems containing MPEG 550, the effect of various parameters, such as W o (molar ratio of water to surfactant), pH, ionic strength, surfactant concentration and temperature were investigated. A kinetic model considering the substrate adsorption equilibrium between the bulk phase of organic solvent and the micellar phase was also successfully used to understand the enzyme activity in the presence of MPEG 550. Both the Michaelis constant and the substrate adsorption equilibrium constant were obviously reduced as compared with those obtained in the simple AOT reverse micellar system.

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The activity and stability of Chromobacterium viscosum lipase (glycerolester hydrolase, EC 3.1.1.3)-catalyzed olive oil hydrolysis in sodium bis (2-ethyl-l-hexyl)sulfosuccinate (AOT)/isooctane reverse micelles is increased appreciably when low molecular weight polyethylene glycol (PEG 400) is added to the reverse micelles. To understand the effect of PEG 400 on the phase behavior of the reverse micellar system, the phase diagram of AOT/ PEG 400/water/isooctane system was studied. The influences of relevant parameters on the catalytic activity in AOT/PEG 400 reverse micelles were investigated and compared with the results in the simple AOT reverse micelles. In the presence of PEG 400, the linear decreasing trend of the lipase activity with AOT concentration, which is observed in the simple AOT reverse micelles, disappeared. Enzyme entrapped in AOT/PEG reverse micelles was very stable, retaining >75% of its initial activity after 60 d, whereas the half-life in simple AOT reverse micelles was 38 d. The kinetics parameter maximum velocity (Vmax) exhibiting the temperature dependence and the activation energy obtained by Arrhenius plot was suppressed significantly by the addition of PEG 400.

The activity of Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) entrapped in AOT/isooctane reverse micelles was significantly increased by the addition of short chain polyethylene glycols (PEGs) or methoxypolyethylene glycols (MPEGs) for the hydrolysis of olive oil. To understand enzyme activity in the presence of PEG 400 or MPEG 550 molecules, a kinetic model was proposed. The validity of this model was verified by experimental data on the lipase-catalyzed hydrolysis of olive oil in AOT/isooctane reverse micellar systems, in which PEG 400 or MPEG 550 had been added. The large value of the equilibrium constant (k D ) for enzyme activation indicated that the affinity between C viscosum lipase and PEG 400 or MPEG 550 molecules was very strong. The Michaelis constant (K m ) predicted by the proposed model explained enzymatic reactions more exactly than that by the previously published model.