Effect of pressure on the catalytic activity of subtilisin Carlsberg suspended in compressed gases.

We studied the effect of pressure up to 300 bar on the catalytic efficiency of subtilisin Carlsberg suspended in compressed propane, near-critical ethane, near-critical carbon dioxide and tert-amyl alcohol, at constant temperature and fixed enzyme hydration. Increasing pressure lowered the catalytic efficiency of the enzyme in all the solvents, resulting in positive activation volumes, delta V#. The delta V# values in compressed propane and in tert-amyl alcohol were similar and larger in magnitude than the value reported in the literature for the same reaction in an aqueous buffer, although within the range of typical delta V# values in aqueous media. In the near-critical fluids, the delta V# were much larger, e.g., an increase in pressure of only 200 bar causing a sixfold decrease in the catalytic efficiency of subtilisin in carbon dioxide. These data should reflect the proximity of ethane and carbon dioxide to the critical point, and the resulting condensation of solvent molecules about the solutes, yielding negative solute partial molar volumes.

Solvent effects on the catalytic activity of subtilisin suspended in organic solvents.

We studied a model transesterification reaction catalyzed by subtilisin Carlsberg suspended in toluene, n-hexane, diisopropyl ether, and mixtures of these solvents. To account for solvent effects due to differences in water partitioning between the enzyme and the bulk solvents, we measured water sorption isotherms for the enzyme in each solvent. We measured catalytic activity as a function of enzyme hydration and obtained bell-shaped curves with maxima at the same enzyme hydration in all the solvents. However, the activity maxima were different in all the media, being the lowest in toluene. Differences in the partitioning of substrates and product between the bulk solvent phase and the enzyme active site were accounted for but could not explain the lower catalytic activity observed in toluene. The fact that toluene is very similar to one of the substrates suggested the possibility of competitive inhibition by this solvent. We derived a model allowing for differences in solvation of the substrates, by using thermodynamic activities instead of concentrations, as well as for competitive inhibition by toluene. The model fit the experimental data well, confirming that toluene had a direct adverse effect on the catalytic activity of the enzyme.

Solvent effects on the catalytic activity of subtilisin suspended in compressed gases.

We studied a model transesterification reaction catalyzed by subtilisin Carlsberg suspended in carbon dioxide, propane, and mixtures of these solvents under pressure. To account for solvent effects due to differences in water partitioning between the enzyme and the bulk solvents, we measured water sorption isotherms for the enzyme in each solvent. We measured catalytic activity as a function of enzyme hydration and obtained bell-shaped curves with maxima at the same enzyme hydration (12%) in all the solvents. However, the activity maxima were different in all media, being much higher in propane than in either CO(2) or the mixtures with 50 and 10% CO(2). Considerations based on the solvation ability of the solvents did not offer an explanation for the differences in catalytic activity observed. Our results suggest that CO(2) has a direct adverse effect on the catalytic activity of subtilisin. (c) 1996 John Wiley & Sons, Inc.

Lipase catalyzed esterification of glycidol in nonaqueous solvents: solvent effects on enzymatic activity.

We studied the effect of organic solvents on the kinetics of porcine pancreatic lipase (pp) for the resolution of racemic glycidol through esterification with butyric acid. We quantified ppl hydration by measuring water sorption isotherms for the enzyme in the solvents/mixtures tested. The determination of initial rates as a function of enzyme hydration revealed that the enzyme exhibits maximum apparent activity in the solvents/mixtures at the same water content (9% to 11% w/w) within the associated experimental error. The maximum initial rates are different in all the media and correlate well with the logarithm of the molar solubility of water in the media, higher initial rates being observed in the solvents/mixtures with lower water solubilities. The data for the mixtures indicate that ppl apparent activity responds to bulk property of the solvent. Measurements of enzyme particle sizes in five of the solvents, as function of enzyme hydration, revealed that mean particle sizes increased with enzyme hydration in all the solvents, differences between solvents being more pronounced at enzyme hydration levels close to 10%. At this hydration level, solvents having a higher water content lead to lower reaction rates; these are the solvents where the mean enzyme particle sizes are greater. Calculation of the observable modulus indicates there are no internal diffusion limitations. The observed correlation between changes in initial rates and changes in external surface area of the enzyme particles suggests that interfacial activation of ppl is only effective at the external surface of the particles. Data obtained for the mixtures indicate that ppl enantioselectivity depends on specific solvent-enzyme interactions. We make reference to ppl hydration and activity in supercritical carbon dioxide.