Water Activity Control Using Saturated Salt Solutions

1 Water Activity Control Using Saturated Salt Solutions 

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Berberich et al. used salt hydrate pairs to control water activity in [BMIM][PF6]. The results were in good agreement with that obtained for water activity control using saturated salt solutions. The advantage of pre-equilibration is that the contact of the enzyme with the used salt and thus enzyme deactivation can be avoided. On the other hand it is only applicable for initial rate measurements. This disadvantage can be overcome by controlling water activity with salt hydrate pairs. Berberich et al. measured initial rate - water activities for the transesterification reaction of methyl methacrylate with 2-ethylhexanol in either hexane or [BMIM][PF6]. Both reaction systems gave similar profiles [72],... 

As already mentioned, control of vrater content is of great importance in enzyme catalysis. Studies on Pseudomonas sp. lipase have also revealed a strong influence of the vrater content of the reaction medium [70]. In order to compare the enzyme activity and selectivity as a fonction of the vrater present in solvents of different polarities, it is necessary to use the vrater activity a in these solvents. We used the method of vrater activity equilibration over saturated salt solutions [71] and could demonstrate that, in contrast to MTBE, which is commonly used for this type of reaction, the enantiosdectivity of the lipase is less influenced either by the water content or the temperature when the reaction is performed in [BMIM][(CFjS02)2N]. 

Samples (1.0 tol.5 g) of the freeze-dried product were equilibrated over saturated salt solutions at 25°C, in order to achieve water activities between 0.11 and 0.90 (Spiess and Wolf, 1983). After equilibration (about 2 to 3 weeks), small samples were taken for DSC analyses and the remaining material was used to determine equilibrium moisture content. Phase transitions were determined by differential scanning calorimetry using a DSC TA2010 controlled by a TA5000 module (TA Instruments, Newcastle, USA). Samples of about 10 mg ( 0.01), conditioned in TA aluminum pans... 

One of the major advantages of employing control is the improvement of precision and repeatability of kinetic results because the water content of solvents and protein preparations can vary from batch to batch and with time. In addition, control of water activity is quite simple. It is achieved by storing solvents (of low volatility), substrates, and enzymes over saturated salt solutions. The thermodynamic properties of the salt dictate the observed water activity. Several different salt types used to control for nonaqueous enzymology are listed in Table 8.5. - - - The storage of saturated salt solutions is achieved by the following procedure ... 

Using mainly water non-misdble solvents several approaches are possible. In most cases, the organic solvent has to be saturated with water in order not to remove the boundary water surrounding the enzyme, which otherwise results in deactivation. In such microaqueous systems the pH of this tiny amount of water should be carefully chosen for optimal enzyme activity. The control of water activity can be achieved by addition of salts or utilization of saturated salt solutions I81, 821. The simplest way of using an enzyme in organic solvents is to suspend the insoluble enzyme in the required solvent. This technique was first reported in 1900 [83] and has been extended over the last few years to encompass many systems (mainly proteases and lipases) [75, 84, 85L Organic solvents may be replaced by supercritical liquid carbon dioxide, which exhibits similar properties to hexane[86, 146]. 

There have been some cases of confusion in the control of water activity between saturated salt solutions (see above) and salt hydrate pairs. These can both be useful methods, but the principles and recommended applications are quite different. Avoid phrases like control of water activity using salts , which do not make it clear which method is being used. 

Fig. 8. Effect of the water content of the system on the rate of S5mthesis of dodecyl decanoate catalyzed hy the lipase from Rhizomucor miehei (formerly known as Mucor miehei). 0.5 M of each substrate was dissolved in the appropriate solvent (hexane (V), toluene (A), trichloroethylene (o), isopropyl ether ( ), pentane-3-one (0)). In the case of pure reactants (+), an equimolar amount was used (2.4 Af). Water activity is defined as a- = p/po, where p is the vapor pressure of water in the system and po is the vapor pressure of pure water at the same temperature. Water activity was controlled through the vapor phase by equilibration with various saturated salt solutions. Reprinted from Ref (70), cop5Tight 1992, with kind permission from Elsevier Science).

The water activity of pre-equilibrated substrates and enzymes may change when they are put in contact with the SCF for example, SCCO2 may take up water from the enzyme. Furthermore, if water is consumed or produced in the reaction, the water activity of the reaction system will change. Putting salt hydrates in contact with the reaction mixture should buffer the water activity. Suitable pairs of solid salt hydrates may be used to control the water activity more accurately. Examples of salt hydrates and saturated solutions are given in Table 4.9-5. 

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