Microorganisms that have adapted to stressful conditions, such as high temperature, high salinity and desiccation, accumulate small organic molecules, often known as compatible solutes, which protect their cellular components from disrupting environmental conditions [1]. These solutes may well find a technological application as stabilizers in enzyme technology. Trehalose is widely distributed among microorganisms and mannosylglycerate, first detected in species of the algal Ceramiales, has also been found to accumulate in thermophilic or hyperthermophilic microorganisms [2]. Mannosylglycerate was purified from Pyrococcus furiosus biomass. Fusarium solani cutinase expressed in E. coli was used as a model enzyme to study the mechanisms responsible for protein stabilization. Thermal stability of this enzyme was difficult to assess due to protein aggregation; therefore guanidine hydrochloride was used to induce reversible unfolding. The unfolding equilibria were quantified by near UV difference and fluorescence spectroscopy in the presence and absence of solutes. Both techniques probe three-dimensional structure but they show different unfolding curves revealing the presence of a stable intermediate at least, besides the folded and unfolded state. Free energy changes for the unfolding transitions were calculated by considering the guanidine hydrochloride effect on the solvation energy of amino acid side chains which are buried in the folded but exposed in the unfolded state [3]. Trehalose and mannosylglycerate stabilize cutinase by more than 2 kcal/mol. In addition, the kinetics for cutinase unfolding was characterized by stop-flow experiments.

[1] da Costa, M.S., Santos, H. and Galinski, E.A., Adv. Biochem. Eng. Biotechnol., 61, 117-153, 1998.

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

[3] Staniforth, R.A., Burston, S.G., Smith, C.J., Jackson, G.S., Badcoe, I.G., Atkinson, T., Holbrook, J.J. and Clarke, A.R., Biochemistry, 32, 3842-3851, 1993.