Enzyme kinetics in biphasic aqueous-organic systems

Although the equilibrium position of reactions may be shifted in biphasic systems, this is of no practical consequence unless the reaction actually occurs. The enzyme must be able to convert the reactants to products. For this to proceed, even in the presence of fully active enzymes, there must be some reactant present in the aqueous microenvironment surrounding the enzyme. Reactants may have fairly low aqueous solubilities under the normal conditions associated with such biphasic processes. The aqueous phase remains effectively unstirred, even when the organic phase is well-stirred, during the reaction so that the thickness of the 'unstirred layer' (d) is equal to the thickness of the aqueous layer surrounding the enzyme. Where d is large, the rate of reaction is likely to be controlled by the passage of reactants through this aqueous layer as the concentration gradients will be very shallow due to the low solubilities of the reactants in water. However, d is often very thin, however, especially where the enzyme is freely soluble, causing the reactions to be effectively diffusionally controlled by the passage of the reactants from the more concentrated organic phase through the interphase boundary to very low concentrations within the microenvironment of the enzyme. Clearly, the thickness of the aqueous layer around an immobilised enzyme will be of great importance in determining the degree of diffusional resistance involved, and hence the lowering of the reaction rate. As with all immobilised enzymes, the volumetric surface area (A/V) is an important parameter governing the overall flux of the substrate to the biocatalytic surface, the effectiveness of the enzyme and the productivity. Whereas in reactions involving a single liquid phase this surface area is the area presented by the exterior of the particles to the bulk of the liquid, in most practical biphasic liquid systems it is the interfacial area between the two liquid phases that is relevant. Under normal operating conditions this interfacial area will depend upon the volume of the aqueous droplet surrounding and enclosing the enzymic biocatalyst. As the relative volume of the organic phase increases the volumetric surface area will decrease, so reducing the maximum volumetric productivity. This will be of particular importance where a needs to be very high in order to shift the reaction equilibrium. Clearly this will reduce the reaction rate due to the reduction in the amount of enzyme that can be present, due to its associated water, and the associated reduction in the interfacial area. Where the catalysed reaction generates water, this should not generally be allowed to accumulate as it not only has a detrimental effect on the equilibrium constant but will also slow down the rate of reaction.

There are some rules which allow the optimisation of the two-phase system for enzyme-catalysed reactions. They depend upon the concept of LogP being extended to cover the substrates, products and the interphase (see Figure 7.1).

1. The difference between the LogP values of the substrate(s) and the interphase should be as small as possible whereas that of the substrate(s) should be much lower than that of the organic phase. These conditions encourage high concentrations of substrate(s) within the interphase and, hence, the transfer of the substrate from the organic phase into the aqueous phase. 

2. The difference between the LogP values of the product(s) and the organic phase should be as small as possible whereas that of the product(s) should be much greater than that of the interphase. These conditions encourage the transfer of the product from the aqueous phase through the interphase into the organic phase, after reaction.

The LogP of the interphase can be varied independently of the organic phase by suitable choice of surfactants and cosurfactants. The precise structure of this interphase may be much more complex than outlined in Figure 7.1. In particular, when there is excess water and surfactant present, the surfactant may form multiple concentric membranes, consisting of surfactant bilayers, around the aqueous core. This presents another reason for the removal of the water produced by use of hydrolases (used as synthetases) in biphasic systems. Control over the amount of water surrounding the enzyme is often possible by means of molecular sieves (e.g., potassium aluminium silicate), which absorb water. Removal of water dissolved in the organic phase can easily be achieved by their use and this leads to the depletion of water from the aqueous biocatalytic pools by its partition. If the organic phase has a high boiling point the removal of water may be simply achieved by vacuum distillation; e.g., in the esterification of fatty acids and fatty alcohols catalysed by lipase and used in wax manufacture.