The use of enzymes in organic solvents to introduce chirality to a number of relevant organic compounds has been well documented. However, there are still major drawbacks in such applications, in particular the frequently much lower enzyme activity under nonaqueous conditions. In addition, the reaction outcome (substrate enantioselectivity and reaction rates) cannot be accurately predicted. To overcome these limitations, herein we introduce methyl-β-cyclodextrin (MβCD) as a new macrocyclic additive to simultaneously enhance the activity and enantioselectivity of dehydrated subtilisin Carlsberg suspended in neat organic solvents. MβCD was efficient in dramatically increasing the activity and significantly improving the enantioselectivity of subtilisin in co-lyophilizates when compared to the powder lyophilized from buffer alone. The initial rate determined for the transesterification between sec-phenethyl alcohol and vinyl butyrate increased by up to 164-fold and the enantioselectivity could be doubled. In addition, marked solvent effects were noted. To investigate the possible relationship between enzyme structure and these kinetic data, the secondary structure of subtilisin was investigated by Fourier transform infrared (FTIR) spectroscopy under all relevant conditions. Using the α-helix content determined from the amide I vibrational band as the main quantitative parameter, we found that MβCD is partially efficient in ameliorating dehydration-induced structural perturbations. Suspension of the subtilisin−MβCD co-lyophilizate in the various solvents revealed solvent-induced structural perturbations in some of them (e.g., acetonitrile), while no such changes were observed in others (e.g., THF and 1,4-dioxane). For the first time the results demonstrated that enantioselectivity and structural intactness in the various solvents were clearly related. Increase in the enzyme activity in contrast is mainly caused by increased structural mobility of subtilisin in the solvents by MβCD. We conclude that it is important to carefully select the additive and the solvent system to achieve high enantioselectivity and activity in such applications. Simultaneous improvement of both enzyme properties requires careful optimization of the enzyme formulation and proper selection of a suitable solvent. FTIR spectroscopy has proven to be a very valuable methodology to structurally guide such an optimization procedure.

Colyophilization or codrying of subtilisin Carlsberg with the crown ethers 18-crown-6, 15-crown-5, and 12-crown-4 substantially improved enzyme activity in THF, acetonitrile, and 1,4-dioxane in the transesterification reactions of N-acetyl-L-phenylalanine ethylester and 1-propanol and that of (+/-)-1-phenylethanol and vinylbutyrate. The acceleration of the initial rate, V(0), ranged from less than 10-fold to more than 100-fold. All crown ethers activated subtilisin substantially, which excludes a specific macrocyclic effect from being responsible. The secondary structure of subtilisin was studied by Fourier-transform infrared (FTIR) spectroscopy. 18-Crown-6 and 15-crown-5 led to a more nativelike structure of subtilisin in the organic solvents employed when compared with that of the dehydrated enzyme obtained from buffer alone. However, the high level of activation with 12-crown-4 where this effect was not observed excluded overall structural preservation from being the primary cause of the observed enzyme activation. The conformational mobility of subtilisin was investigated by performing thermal denaturation experiments in 1,4-dioxane. Although only a small effect of temperature on subtilisin structure was observed for the samples prepared with or without 12-crown-4, both 18-crown-6 and 15-crown-5 caused the enzyme to denature at quite low temperatures (38 degrees C and 56 degrees C, respectively). No relationship between this property and V(0) was evident, but increased conformational mobility of the protein decreased its storage stability. The possibility of a "molecular imprinting" effect was also tested by removing 18-crown-6 from the subtilisin-18-crown-6 colyophilizate by washing. V(0) was only halved as a result of this procedure, an effect insignificant compared with the ca. 80-fold rate enhancement observed prior to washing in THF. This suggests that molecular imprinting is likely the primary cause of subtilisin activation by crown ethers, as recently suggested.

Colyophilization or codrying of subtilisin Carlsberg with the crown ethers 18-crown-6, 15-crown-5, and 12-crown-4 substantially improved enzyme activity in THF, acetonitrile, and 1,4-dioxane in the transesterification reactions of N-acetyl-L-phenylalanine ethylester and 1-propanol and that of (±)-1-phenylethanol and vinylbutyrate. The acceleration of the initial rate, V 0 , ranged from less than 10-fold to more than 100-fold. All crown ethers activated subtilisin substantially, which excludes a specific macrocyclic effect from being responsible. The secondary structure of subtilisin was studied by Fourier-transform infrared (FTIR) spectroscopy. 18-Crown-6 and 15-crown-5 led to a more nativelike structure of subtilisin in the organic solvents employed when compared with that of the dehydrated enzyme obtained from buffer alone. However, the high level of activation with 12-crown-4 where this effect was not observed excluded overall structural preservation from being the primary cause of the observed enzyme activation. The conformational mobility of subtilisin was investigated by performing thermal denaturation experiments in 1,4-dioxane. Although only a small effect of temperature on subtilisin structure was observed for the samples prepared with or without 12-crown-4, both 18-crown-6 and 15-crown-5 caused the enzyme to denature at quite low temperatures (38°C and 56°C, respectively). No relationship between this property and V 0 was evident, but increased conformational mobility of the protein decreased its storage stability. The possibility of a "molecular imprinting" effect was also tested by removing 18-crown-6 from the subtilisin-18-crown-6 colyophilizate by washing. V 0 was only halved as a result of this procedure, an effect insignificant compared with the ca. 80-fold rate enhancement observed prior to washing in THF. This suggests that molecular imprinting is likely the primary cause of sub-tilisin activation by crown ethers, as recently suggested.