The method was first described in ref. 3. In the small-scale laboratory setup (Fig. 1), the saturated salt solution (containing excess salt) is kept in a glass vessel. If the reaction to be carried out produces water, there is a need for more excess salt compared to other cases. However, it is important to have enough solution so that it can be pumped through the system. When a water-producing reaction is carried out, the amount of remaining salt crystals in the vessels is a good indication of the remaining water-uptake capacity. The solution returning to the salt vessel (with a slightly changed water activity) is led into the salt-crystal region in order to be equilibrated with the salt (see Note 2).
The reaction vessel contains the reaction medium, including the substrates and the enzyme preparation. In most cases, the enzyme is immobilized on a
Fig. 1. Small-scale setup for control of water activity by equilibration through silicone tubing. (1) silicone tubing; (2) reaction medium; (3) immobilized enzyme; (4) organic solvent (to keep the salt solution solvent saturated); (5) saturated salt solution; (6) salt crystals.
support material (e.g., Celite or EP-100) prior to use. Mixing in the reaction vessel is normally carried out by magnetic stirring.
Both vessels are equipped with screw caps and rubber septa through which the silicone tubing passes. The salt solution is pumped through the silicone tubing by a peristaltic pump. The flow rate of salt solution is normally not crucial and a moderate rate can be used. Ideally the pump speed should be high enough to ensure that the water activity does not change too much during one pass through the reactor. This is easily established. It is more critical in water-consuming reactions, as there is the risk of salt crystallization in the tubing. (See Notes 3-7.)
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