부산대학교 도서관

Despite the extensive use and study of enzymes suspended in organic solvents, whether activity differences between different preparations can be accounted for by differences in protein secondary structure is still unknown. To address this issue, in the current study two model enzymes, α-chymotrypsin and subtilisin Carlsberg, were lyophilized and suspended in both polar and nonpolar organic solvents. The secondary structures of the proteins in the initial aqueous solution, in the lyophilized powder, and in the subsequent suspensions in organic solvents were determined using infrared spectroscopy. Lyophilization perturbed the secondary structure of both enzymes. With α-chymotrypsin, lyophilization from buffer followed by suspension in ethanol, hexane, or pyridine did not alter the unfolded structure observed in the dried powder. In contrast, with subtilisin Carlsberg, suspension of the dried enzyme in ethanol led to further perturbation of structure, whereas in hexane, and more so in pyridine, there was some return toward native structure. Lyophilization of the aqueous protein solutions in the presence of either trehalose or sorbitol led to retention of more native-like structure of both enzymes in the dried solid. However, large structural perturbations arose when these samples were suspended in organic solvents. The only exception was the subtilisin–trehalose mixture, which regained some native structure in ethanol and hexane. The greatest changes were noted in samples suspended in pyridine, in which the infrared spectra indicated extensive intermolecular beta-sheet formation from protein aggregates. There was not any consistent correlation between activity in organic solvents and either the initial structure obtained in the dried powders or the final structure when suspended in organic solvents. Nor could differences in residual water contents in dried samples or the total water content in the organic solvent reaction system account for the activity differences.