Improved enzyme activity and enantioselectivity in organic solvents by methyl-beta-cyclodextrin

The use of enzymes in organic solvents to introduce chirality to a number o f relevant organic compounds has been well documented. However, there are s till major drawbacks in such applications, in particular the frequently muc h lower enzyme activity under nonaqueous conditions. In addition, the react ion outcome (substrate enantioselectivity and reaction rates) cannot be acc urately predicted. To overcome these limitations, herein we introduce methy l-beta-cyclodextrin (M beta CD) as a new macrocyclic additive to simultaneo usly enhance the activity and enantioselectivity of dehydrated subtilisin C arlsberg suspended in neat organic solvents. M beta CD was efficient in dra matically increasing the activity and significantly improving the enantiose lectivity of subtilisin in co-lyophilizates when compared to the powder lyo philized from buffer alone. The initial rate determined for the transesteri fication between sec-phenethyl alcohol and vinyl butyrate increased by up t o 164-fold and the enantioselectivity could be doubled. In addition, marked solvent effects were noted. To investigate the possible relationship betwe en enzyme structure and these kinetic data, the secondary structure of subt ilisin was investigated by Fourier transform infrared (FTIR) spectroscopy u nder all relevant conditions. Using the alpha-helix content determined from the amide I vibrational band as the main quantitative parameter, we found that M beta CD is partially efficient in ameliorating dehydration-induced s tructural perturbations. Suspension of the subtilisin-M beta CD co-lyophili zate in the various solvents revealed solvent-induced structural perturbati ons in some of them (e.g., acetonitrile), while no such changes were observ ed in others (e.g., THF and 1,4-dioxane). For the first time the results de monstrated 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 sol vents by M beta CD. We conclude that it is important to carefully select th e additive and the solvent system to achieve high enantioselectivity and ac tivity in such applications. Simultaneous improvement of both enzyme proper ties requires careful optimization of the enzyme formulation and proper sel ection of a suitable solvent. FTIR spectroscopy has proven to be a very val uable methodology to structurally guide such an optimization procedure.