Improved Enzyme Activity and Enantioselectivity in Organic Solvents by Methyl-β-cyclodextrin

Abstract

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.