Many microorganisms have the ability of surviving and proliferating in highly aggressive environments, such as those characterized by extremes of temperature, salinity, pH and pressure. To keep the turgor pressure and/or cell volume and protect the activity of intracellular enzymes and other macromolecules, halophilic organisms accumulate compatible solutes [1]. In recent years, several new compatible solutes have been identified and characterised in thermophilic and hyperthermophilic organisms [2-4]. Among these, mannosylglycerate has been demonstrated to exert a strong protecting effect on enzymes against stress imposed by heat or freeze drying. The usefulness of this solute as an enzyme stabilizer in biotechnological applications was suggested [5].

The protective effect of mannosylglycerate was compared to that of other compatible solutes isolated from several sources, such as trehalose, di-myo-inositol-phosphate, di-glycerol-phosphate, hydroxyectoine, and ectoine. The thermostability of rabbit muscle lactate dehydrogenase, used as model system in this study, was determined in vitro and found to improve drastically when enzyme solutions were subjected to thermal stress in the presence of mannosylglycerate and hydroxyectoine. Di-glycerol-phosphate and trehalose improved the enzyme thermostability to a smaller extent. Ectoine conferred no protection, and di-myo-inositol-phosphate had a drastic destabilising effect. In an attempt to understand which chemical features of mannosylglycerate have a preponderant role on enzyme thermostabilisation, the effect of related compounds (mannosylglyceramide, mannose, methyl­mannoside, glycerate, glucosylglycerol, glycerol, and glucose) was evaluated. Glucosylglycerol had a protecting effect comparable to that of mannosylglycerate, whereas mannosylglyceramide conferred only a small protection against thermal stress. Glycerol, glucose and methyl-mannoside did not enhance the thermostability of LDH, whereas a strong destabilisation was caused by mannose. Mannosylglycerate was also an efficient thermostabiliser of glucose oxidase from Aspergillus niger, an enzyme with opposite global charge to that of LDH under the experimental conditions used, indicating that stabilisation by mannosylglycerate is not strongly determined by the net charge of the enzymes. Light scattering measurements showed a high effectiveness of mannosylglycerate in preventing LDH aggregation during thermal stress.

Acknowledgement: We thank bitop GmbH for providing samples of ectoine and hydroxyectoine

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