Activation of subtilisin Carlsberg in organic solvents by methyl-beta-cyclodextrin: Lyoprotection versus substrate and product-complex effect

Enzyme catalysis in organic solvents is a valuable tool for the synthesis of a number of important chiral compounds. However, their usually low activity in these applications is a major drawback which hampers their potential. To overcome this, research groups around the world have successfully employed a variety of additives to protect enzyme catalysts from the detrimental effects of dehydration (mostly by freeze-drying, a required step prior to introducing the catalyst to organic solvents) and subsequent exposure to organic media. Modified cyclodextrins are among the most successful additives employed for this purpose. However, to maximize their constructive effect it is necessary to determine how these cyclic macromolecules enhance enzyme catalytic activity as well as the enantioselectivity. Our data suggests that M?CD activates the serine protease subtilisin C. predominantly by preserving the enzyme structure. Supporting data include the facts that: (a) M?CD must be co-dissolved with the enzyme in buffer prior to lyophilization; (b) formation of inclusion complexes is excluded from contributing to the activation because no increase in enantioselectivity or in activity was observed when M?CD was added directly to the enzyme-organic solvent mixture; (c) M?CD works at an optimum concentration, after which a decrease in enzyme activity is observed; (d) removal of the additive by washing steps only causes minor activity decreases; (e) co-lyophilization of a lipase was also found to increase its enantioselectivity towards the opposite enantiomer than subtilisin. These data are irreconcilable with a mechanism of activation that involves the formation of inclusion complexes between the additive, the substrates and the products, which has been proposed to contribute to the activation of a lipase.