Water Effects on Activity

The catalytic activity of an enzyme in an organic medium often varies by several orders of magnitude depending on the degree of enzyme hydration [9,19]. Control of enzyme hydration or water activity is thus a key issue when optimizing enzymatic conversions in organic solvents. 

All enzymes to be used in organic media have at a previous stage been in an aqueous phase. They are then transferred to the organic medium, and this transfer process involves removal of water. This can be achieved by lyophilization or just drying of the enzyme solution, possibly in the presence of a support material or other additives. Another possibility is to dilute the aqueous enzyme solution with a water-miscible organic solvent which dissolves the water and causes the enzyme to precipitate. In one version, the enzyme solution also contains a crystal-forming solute such as an inorganic salt or an amino acid [20]. In this case, crystals are formed and the enzyme covers the crystals. 

Lyophilization is a very common method to prepare enzymes for use in organic media, but the procedure often results in preparations having low catalytic activity. The method can cause inactivation of the enzyme both in the freezing step and in the drying step [7]. FT-IR spectroscopy has been used to study secondary structure in enzyme preparations, and lyophilization has been shown to decrease the a-helix content and increase the (i-sheet content compared to native enzyme and 

Figu re 1.4 Normalized reaction rate as function of water activity for the esterification of dodecanol with decanoic acid catalyzed by Rhizopus arrhizus lipase at three different concentrations 20mM (O), 200mM ( ) and 800mM ( ) of each substrate. Data obtained from Ref. [26]. 

It is not known which molecular features of Upases keep them active at low water activity. It is worth pointing out that when they are used to catalyze various reactions, such as hydrolysis, reversed hydrolysis, and transesterification reactions, the water activity dependence is similar in the different reactions [26, 28]. The same is true for phospholipase A2 [29]. This shows that the effect of water on the enzyme is more important than the effect of water as a reactant when determining the reaction rate at different water activities. 

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