Comparison of theoretical and experimental data to evaluate substrate diffusional limitations for crown ether- and methyl-beta-cyclodextrin-activated serine protease subtilisin Carlsberg in tetrahydrofuran

Simple co-lyophilization of serine protease subtilisin Carlsberg with [12]-crown ether-4 (12-crown-4) or methyl-beta-cyclodextrin (MbetaCD) drastically increases its catalytic activity in organic solvents. We investigated whether the improved activity would cause substrate diffusional limitations. To experimentally assess the issue, the enzyme was inactivated with PMSF. Different amounts of active and inactive subtilisin were codissolved in 10 mM phosphate buffer (pH 7.8) followed by lyophilization with or without 12-crown-4 or MbetaCD. Initial rates for the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester and 1-propanol in anhydrous THF were plotted vs. the amount of active enzyme present in the formulations. For all three enzyme formulations a linear relationship was observed and the results clearly show that activation of subtilisin Carlsberg by crown ethers and MbetaCD did not cause diffusional limitations. This was somewhat surprising because theoretical models predicted such diffusional limitations for the activated formulations. However, investigation of the protein powder particles obtained after co-lyophilization with 12-crown-4 and MbetaCD revealed a drastically reduced particle size for these formulations when suspended in THF. The particle micronization afforded by the excipients prevented substrate diffusional limitations, a factor that should be taken into account when designing improved enzyme formulations for synthetic applications in organic solvents.