Thought must be given to the initial hydration state of every component of the reaction mixture. If only some are pre-equilibrated, then water redistribution after they are combined will give a final water activity perhaps quite different from the intention. For example, if a biocatalyst brought to a desired water activity is suspended in a dried solvent, it will probably rapidly lose water. The equilibrium water activity eventually reached may be significantly reduced. Such combination of a biocatalyst and organic phase of different water activity may, in fact, be desirable to obtain high catalytic activity because of hysteresis effects (15). However, if conditions are to be reproducible, all components should still be pre-equilibrated to a defined hydration level. This holds even if different values are chosen for the different phases that will eventually be combined, in order to exploit these hysteresis effects. If the aim is to
pre-equilibrate the entire reaction mixture to the same water activity, a decision must be made as to what to mix at what stage. It is difficult to give clear recommendations here on what combinations of components should be mixed before water pre-equilibration. In principle, mixing afterward may disturb the previously equilibrated hydration conditions. To obtain a system in complete water-distribution equilibrium, the entire reaction mixture should ideally be pre-equilibrated together. However, something must be kept separate until the reaction is to be started, of course. In practice, we have used two options for the choice of the two combinations to be separately pre-equilibrated:
1. (i) The biocatalyst particles and, separately, (ii) an organic solution of all the reactants. Problems may result from the effect of organic solvent on water binding by the biocatalyst. Displacement of water may lead to an increased water activity and aggregation of the biocatalyst (13).
2. (i) The biocatalyst suspended in an organic phase and (ii) one separate reactant. Dissolution or mixing of the missing reactant to start the reaction may alter the pre-equilibrated water activity. To minimize this, the chosen reactant should have a small final concentration. For example, we often include 1 M alcohol nucleo-phile in the pre-equilibrated organic phase. The reaction will then be started by adding about 20 mM ester.
In all cases, achieving the desired final water activity is easier when the organic solvent used is polar and dissolves a lot of water itself. This will act to buffer the water activity of the whole system.
In general, brief exposure to the laboratory atmosphere during transfer and mixing of pre-equilibrated components does not seem to have major effects. In critical cases, however, especially where the total water content is very small, all such operations can be performed inside a glove box. The interior of the glove box is pre-equilibrated to the desired water activity, by recirculating the air inside it through or over the appropriate salt solution or drying agent; the progress of the glove box interior to the desired water activity can be monitored using one of the sensors described in Subheading 4.3.
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