EFFECT OF POLYOLS ON ALPHA-CHYMOTRYPSIN THERMOSTABILITY - A MECHANISTIC ANALYSIS OF THE ENZYME STABILIZATION

The influence of the synthetic substrate (N-acetyl-L-tyrosine ethyl es ter) and the different polyols (ethylene glycol, glycerol, erythritol, xylitol and sorbitol) on the thermostability of alpha-chymotrypsin at 60 degrees C have been studied. The results obtained showed an import ant stabilizing effect in the presence of both additives. In order to describe the kinetics of enzyme stabilization, the experimental result s were analyzed by a four-parameters deactivation model with excellent agreement. In all cases, alpha-chymotrypsin exhibited non-first-order deactivation kinetics, corresponding to a two-step unimolecular mecha nism, where the main protective effect of polyols was observed in the first-step of the deactivation profile. Thus, the presence of polyols increased the level of activity stabilization (alpha(1)), and dereased the first-order deactivation rate constant (k(1)). Additionally, the experimental results were analyzed as a function of both, the change i n the standard free energy of denaturation (Delta(Delta G degrees)), a nd a protective effect, defined as the ratio of alpha-chymotrypsin hal f-lives (with and without polyols), showing in both cases a clear stab ilizing effect of these polyhydroxylic cosolvents for the enzyme. The overall protective effect of polyols was also simultaneously related t o their concentration and their water-activity depressing power.