Apart from being a substrate, water also has a vital function in maintaining the three-dimensional conformation of the enzyme and hence its intrinsic activity (24, 25). This effect is most conveniently expressed in terms of the thermodynamic water activity maintained in the reaction medium. It allows comparison of results of enzymatic reactions in various organic solvents, even though absolute water concentrations are significantly different (26).
It has been shown that the form of this relationship is rather lipase specific. For example, Rhizomucor mie-hei lipase has an optimum water activity ~ 0.5 and still retains 40% of its maximum activity at water activities close to zero (27, 28). On the other hand, the lipases from Candida rugosa, Humicola,* and Pseudomonas cepacia not really showed a maximum, their activity
* Humicola lanuginosa, currently known as Thermomyces lanuginosus.
continuously increasing with the water activity up to saturation level (29). The relationship is rather independent of the type of reaction catalyzed by the lipase (30), the carrier material of the lipase (28) as well as the type of solvent applied (27). However, in polar solvents interactions with the solvent molecule itself may affect the enzyme activity (31), which may explain the low activity of certain lipases in such solvents (32).
The existence of an optimum water activity urges proper control of the water activity throughout a reaction. In fact several methods have been developed which allow accurate control of the water activity in batch as well as continuous reactor systems: e.g., vacuum, controlled air sparging (33), membrane per-vaporation (34), and equilibration with concentrated salt systems (35, 36). These techniques have been shown adequate to obtain optimum performance of biocatalysts in ester synthesis.
In some cases water activity control during the process is not relevant at all. For example, in the synthesis of esters from hydrophobic fatty acids and hydropho-bic alcohols degrees of synthesis in the range of 90% can easily be reached, even in the presence of free water (13, 14). Moreover, this is independent of the type of lipase applied (13).
A more general case is the interesterification or acid-olysis of oils and fats. Starting with a pure triglyceride (acylglycerol) mixture, the thermodynamics of the system will cause nearly all water initially present to be used for hydrolysis (26). Though this reaction does create the diglyceride intermediates required for the interesterification reaction to proceed, it simultaneously results in a significant reduction of the water activity. Moreover, the latter may easily drop below the optimum of the lipase applied. Adding extra water to the system to increase the aw is no solution as it will only result in further hydrolysis and hence decreased product (modified triglyceride) yield (37).
Thus, quickly losing water by hydrolysis, the major part of the interesterification reaction will proceed at a very low equilibrium water activity (38-42). As a result of this drying process, the initial water activity of the catalyst itself is of less importance, especially in a continuous reactor system. Instead, the supply of water via the feed stream to a reactor is essential in order to establish a low but constant water activity in the system, thereby preventing exhaustive drying of the catalyst and providing for fresh intermediates to keep the interesterification reaction going. It is therefore generally accepted that in interesterification reactions, the water content in the feed steam should be close to saturation (41, 43, 44).
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