Hydrophobic ion pairing as a method for enhancing structure and activity of lyophilized subtilisin BPN' suspended in isooctane.
The use of enzymes in low water environments permits reactions to occur that are difficult or impossible in aqueous solution. In this manner, proteases can be used to form, rather than hydrolyze, ester and amide linkages. Presumably, the native-like structure of the enzyme must remain intact for catalysis to transpire. However, little is known regarding the integrity of the overall structure of lyophilized proteins suspended in organic media. In this study, the structural changes that occur during the freeze-drying process and those effected by suspension in the organic solvent were examined. Using Fourier-transform infrared spectroscopy, the secondary structure of lyophilized subtilisin BPN' was monitored and correlated to the level of enzymatic activity when suspended in isooctane. In addition, the ability of ionic detergents to stabilize subtilisin BPN' via ion pairing was evaluated. It was found that subtilisin unfolds to some degree during lyophilization, whether it is ion paired or not. Furthermore, there are structural changes observed when the enzyme is placed in isooctane, although the effects are less with ion-paired subtilisin. This higher level of retention of secondary structure results in increased enzymatic activity.
Kendrick
B S
BS
School of Pharmacy, University of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, Colorado 80262, USA. kendrick@york.uchsc.edu
Meyer
J D
JD
Matsuura
J E
JE
Carpenter
J F
JF
Manning
M C
MC
D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation; D004795:Enzyme Stability; D005612:Freeze Drying; D009795:Octanes / Q000494:pharmacology; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012997:Solvents; D017550:Spectroscopy, Fourier Transform Infrared; D013381:Subtilisins / Q000737:chemistry* / Q000378:metabolism*; D014867:Water
D003902:Detergents; D009795:Octanes; D012997:Solvents; D014867:Water; D013381:Subtilisins; C045798:2,2,4-trimethylpentane