Activation of enzymes for nonaqueous biocatalysis by denaturing concentrations of urea

Urea is one of the most commonly used denaturants of proteins. However, her ein we report that enzymes lyophilized from denaturing concentrations of aq ueous urea exhibited much higher activity in organic solvents than their na tive counterparts. Thus, instead of causing deactivation, urea effected une xpected activation of enzymes suspended in organic media. Activation of sub tilisin Carlsberg (SC) in the organic solvents (hexane, tetrahydrofuran, an d acetone) increased with increasing urea concentrations up to 8 M. Active- site titration results and activity assays indicated the presence of partia lly unfolded but catalytically active SC in 8 M urea; however, the urea-mod ified enzyme retained high enantioselectivity and was ca. 80 times more act ive than the native enzyme in anhydrous hexane. Likewise, the activity of h orseradish peroxidase (HRP) lyophilized from 8 M urea was ca. 56 times and 350 times higher in 97% acetone and water-saturated hexane, respectively, t han the activity of HRP lyophilized from aqueous buffer. Compared with the native enzyme, the partially unfolded enzyme may have a more pliant and les s rigid conformation in organic solvents, thus enabling it to retain higher catalytic activity. However, no substantial activation was observed for al pha -chymotrypsin lyophilized from urea solutions in which the enzyme retai ned some activity, illustrating that the activation effect is not completel y general. (C) 2001 EIsevier Science B.V. All rights reserved.