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. 13C cross-polarization magic angle spinning NMR spectroscopy was used to determine the secondary structure of lyophilized papain in the solid-state. Deconvolution of the peaks of the backbone carbonyl carbons suggested that the proportion of beta-sheet conformation increased after lyophilization from a phosphate buffer solution. The esterification of N-benzyloxycarbonyl phenylalanylalanine amide was attempted using the lyophilized 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 lyophilized papain was suspended in anhydrous 1-propanol for 7 days without the substrate, the proportion of beta-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 lyophilization and suspension in propanol was found, which was related to a decrease in enzymatic activity.
Matsubara
Teruhiko
T
Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan. matsubara@bio.keio.ac.jp
Fujita
Risa
R
Sugiyama
Shigeru
S
Kawashiro
Katsuhiro
K
D000433:1-Propanol / Q000737:chemistry*; D000577:Amides / Q000737:chemistry; D002942:Circular Dichroism; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D004951:Esterification; D005612:Freeze Drying; D019906:Nuclear Magnetic Resonance, Biomolecular; D009930:Organic Chemicals / Q000737:chemistry; D010206:Papain / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012997:Solvents / Q000737:chemistry; D013696:Temperature
D000577:Amides; D004151:Dipeptides; D009930:Organic Chemicals; D012997:Solvents; C039553:N-benzyloxycarbonylphenylalanylalanine; D000433:1-Propanol; D010206:Papain