Activity, stability, and conformation of methoxypoly(ethylene glycol)-subtilisin at different concentrations of water in dioxane.

The transesterification activity, autolysis, thermal stability and conformation of subtilisin Carlsberg, made soluble in dioxane by covalent linking to methoxy-poly(ethylene glycol) (PEG), were investigated as a function of the concentration of water in the medium. Electrospray mass spectrometry showed that the modified enzyme preparation was a mixture of proteins containing from 2 to 5 covalently linked PEG chains per subtilisin molecule. PEG-subtilisin catalyzed transesterification be tween vinyl butyrate and 1-hexanol was optimum at 0.55 M H2O, while hydrolysis prevailed above 2 M H2O. There was a decrease in the overall enzyme activity with increasing water concentration because of autolysis and denaturation of the enzyme. Subtilisin powder and celite-immobilized subtilisin were more stable and less susceptible to autolysis than the PEG-modified enzyme. Circular dichroism and intrinsic protein-fluorescence studies showed that the conformation of PEG-subtilisin did not change as a function of water concentrations between 0 and 9 M. The K-m,K-app value of PEG-subtilisin for 1-hexanol was highly influenced by water, which behaved as a competitive inhibitor in the transesterification reaction with an affinity for the enzyme similar to that of the alcohol. The K-m,K-app for the acylating agent was not significantly modified by water. Lyoprotectants such as sorbitol and free PEG did not influence the activity of PEG-subtilisin but notably increased the activity of subtilisin powder and celite-immobilized subtilisin. The addition of 1.7-5.5 M water, however, rendered enzyme preparations containing no additives as active as those containing the lyoprotectants. (C) 1997 John Wiley & Sons, Inc.