Many organisms that have adapted to stressful conditions, such as high temperature, high salinity and desiccation, accumulate small organic molecules, known as compatible solutes, which protect their cellular components from disrupting environmental conditions [1]. Mannosylglycerate, is widely distributed among thermophilic and hyperthermophilic microrganisms when subjected to osmotic or temperature stresses [2-4]. Recently, it was demonstrated that this solute exerted a strong protecting effect on enzymes against stress imposed by heat or freeze drying, and the usefulness of mannosylglycerate as an enzyme stabilizer in biotechnological applications was suggested [5].

In recent years, a great effort has been devoted to the understanding of the principles involved in protein protection by well known osmolytes (trehalose, sucrose, glycerol, and other polyols), and the preferential hydration of proteins in solutions with high concentrations of osmolytes is generally accepted [6]. Bovine ribonuclease A is one of the most common model-proteins used in these studies. The energetics and mechanism of denaturation of RNase have been investigated in depth, and these studies demonstrated that the protein unfolds reversibly in a process that can be described by a two-state process [7,8]. For these reasons, RNase was the protein selected for this study. Differential scanning calorimetry was used to investigate the effect of mannosylglycerate on the unfolding thermodynamics of bovine ribonuclease A. The stabilizing effect of mannosylglycerate was dependent on the working pH, emphasising the relevance of the negative charge in the protection mechanism. It is worth pointing out that most solutes from hyperthermophiles are negatively charged, in contrast to the non-charged nature of the typical compatible solutes from mesophiles. Although no significant differences were observed on the heat capacity change upon unfolding in the presence of mannosylglycerate, there was a clear increase in stability (Gibbs energy) at high temperatures. Differential UV spectroscopy was also used to monitor RNase unfolding at two different pH values, 4.5 and 7.5. These studies were coupled with measurements of enzyme activity as a function of temperature, with and without mannosylglycerate in order to correlate enzyme structural stabilization and its activity. The solute and protein concentration effects on the irreversible activity loss imposed by high temperatures were studied.

[1] da Costa, M.S., Santos, H. and Galinski, E.A., Adv. Biochem. Eng. Biotechnol., 61, 117-143, 1998.

[2] Nunes, O.C., Manaia, C.M., da Costa, M.S. and Santos, H., Appl. Environ. Microbiol., 61, 2351-2357, 1995.

[3] Martins, L.O. and Santos, H., Appl. Environ. Microbiol., 61, 3299-3303,1995

[4] Martins, L.O., Huber, R., Huber, H., Stetter, K.O., da Costa, M.S. and Santos, H., Appl. Environ. Microbiol., 63, 896-902, 1997.

[5] Ramos, A., Raven, N.D.H., Sharp, R.J., Bartolucci, S., Rossi, M., Cannio, R., Lebbink, J., van der Oost, J., de Vos, W.M., and Santos, H., Appl. Environ. Microbiol., 63, 4020-4025, 1997.

[6] Xie, G. and Timasheff, S.N., Prot. Sci., 6, 211-221, 1997.

[7] del Pino, I.M.P. and Sanchez-Ruiz, J.M., Biochemistry, 34, 8621-8630, 1995.

[8] Knapp, S., Ladenstein, R., Galinski, E.A., Extremophiles, 3, 191-198, 1999.