Stability of protease in organic solvent: Structural identification by solid-state NMR of lyophilized papain before and after 1-propanol treatment and the corresponding enzymatic activities

Lyophilized enzyme powder is often used in organic solvents. However, the enzymatic activity decreases during the reaction process. In the present study, the relation between structural stability and enzymatic activity in an organic solvent was investigated. C-13 cross-polarization magic angle spinning NMR spectroscopy was used to determine the secondary structure of Iyophilized papain in the solid-state. Deconvolution of the peaks of the backbone carbonyl carbons suggested that the proportion of beta-sheet conformation increased after Iyophilization from a phosphate buffer solution. The esterification of N-benzyloxycarbonyl phenylalanylalanine amide was attempted using the Iyophilized papain as a catalyst in anhydrous 1-propanol. The yield of ester was 46.1% after 48 h at 50 degrees C, but this reaction slowed remarkably after 48 h. When the Iyophilized papain was suspended in anhydrous 1-propanol for 7 days without the substrate, the proportion of P-sheet conformation was further increased and the suspended papain had no activity. These results suggest that the increase in beta-sheet conformation caused inactivation of papain. The increase in beta-sheet conformation caused by both Iyophilization and suspension in propanol was found, which was related to a decrease in enzymatic activity. (c) 2005 Wiley Periodicals, Inc.