Influence of Additives on High Pressure Stability of β-Galactosidase from Kluyveromyces Lactis and Invertase from Saccharomyces Cerevisiae

Abstract

High pressure denaturation of two yeast enzymes, Kluyveromyces lactis β-galactosidase and Saccharomyces cerevisiae invertase in aqueous solutions of salts and polyols was investigated. Polyols (more than salts) were shown to act as very effective agents against pressure denaturation for both enzymes, leading to an increase in half-lives by factors between 1–10,000. NaBr and KBr were essentially destabilizing compounds, leading to protective effects near zero in the case of β-galactosidase. The maximum stability was obtained when KCl was used as additive. The β-galactosidase half-life is increased by a factor of 60 in a 3 m KCl solution and the invertase half-life by a factor of 40. Whereas NaCl had no stabilizing effect on β-galactosidase up to 2 m, this salt was of greater efficiency for invertase whatever the concentration, leading to a protective effect of almost 30 at 3 m. The use of polyols (glycerol, erythritol, xylitol, and sorbitol) allows β-galactosidase and invertase to be stabilized against pressure deactivation. This stabilizing effect increases with the polyol concentration. For invertase in the presence of xylitol, the half-life is increased by a factor of 7 at 1 m and by a factor near 50 at 2 m. The increase in pressure resistance due to the presence of polyols was always greater with β-galactosidase than with invertase. The protective effect of a 2 m xylitol solution is above 10,000 for β-galactosidase whereas it is about 50 for invertase. After the systematic study of the influence of salts and polyols, certain hypotheses are advanced on the possible mechanisms by which additives stabilize proteins to pressure denaturation.