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In the last few years, increased attention has been focused on a class of organisms called psychrophiles. These organisms, hosts of permanently cold habitats, often display metabolic fluxes more or less comparable to those exhibited by mesophilic organisms at moderate temperatures. Psychrophiles have evolved by producing, among other peculiarities, "cold-adapted" enzymes which have the properties to cope with the reduction of chemical reaction rates induced by low temperatures. Thermal compensation in these enzymes is reached, in most cases, through a high catalytic efficiency associated, however, with a low thermal stability. Thanks to recent advances provided by X-ray crystallography, structure modelling, protein engineering and biophysical studies, the adaptation strategies are beginning to be understood. The emerging picture suggests that psychrophilic enzymes are characterized by an improved flexibility of the structural components involved in the catalytic cycle, whereas other protein regions, if not implicated in catalysis, may be even more rigid than their mesophilic counterparts. Due to their attractive properties, i.e., a high specific activity and a low thermal stability, these enzymes constitute a tremendous potential for fundamental research and biotechnological applications.

Cold-adapted, or psychrophilic, organisms are able to thrive at low temperatures in permanently cold environments, which in fact characterize the greatest proportion of our planet. Psychrophiles include both prokaryotic and eukaryotic organisms and thus represent a significant proportion of the living world. These organisms produce cold-evolved enzymes that are partially able to cope with the reduction in chemical reaction rates induced by low temperatures. As a rule, cold-active enzymes display a high catalytic efficiency, associated however, with a low thermal stability. In most cases, the adaptation to cold is achieved through a reduction in the activation energy that possibly originates from an increased flexibility of either a selected area or of the overall protein structure. This enhanced plasticity seems in turn to be induced by the weak thermal stability of psychrophilic enzymes. The adaptation strategies are beginning to be understood thanks to recent advances in the elucidation of the molecular characteristics of coldadapted enzymes derived from X-ray crystallography, protein engineering and biophysical methods. Psychrophilic organisms and their enzymes have, in recent years, increasingly attracted the attention of the scientific community due to their peculiar properties that render them particularly useful in investigating the possible relationship existing between stability, flexibility and specific activity and as valuable tools for biotechnological purposes.

The ␤-galactosidase from the Antarctic gram-negative bacterium Pseudoalteromonas haloplanktis TAE 79 was purified to homogeneity. The nucleotide sequence and the NH 2 -terminal amino acid sequence of the purified enzyme indicate that the ␤-galactosidase subunit is composed of 1,038 amino acids with a calculated M r of 118,068. This ␤-galactosidase shares structural properties with Escherichia coli ␤-galactosidase (comparable subunit mass, 51% amino sequence identity, conservation of amino acid residues involved in catalysis, similar optimal pH value, and requirement for divalent metal ions) but is characterized by a higher catalytic efficiency on synthetic and natural substrates and by a shift of apparent optimum activity toward low temperatures and lower thermal stability. The enzyme also differs by a higher pI (7.8) and by specific thermodynamic activation parameters. P. haloplanktis ␤-galactosidase was expressed in E. coli, and the recombinant enzyme displays properties identical to those of the wild-type enzyme. Heat-induced unfolding monitored by intrinsic fluorescence spectroscopy showed lower melting point values for both P. haloplanktis wild-type and recombinant ␤-galactosidase compared to the mesophilic enzyme. Assays of lactose hydrolysis in milk demonstrate that P. haloplanktis ␤-galactosidase can outperform the current commercial ␤-galactosidase from Kluyveromyces marxianus var. lactis, suggesting that the cold-adapted ␤-galactosidase could be used to hydrolyze lactose in dairy products processed in refrigerated plants.Enzymes from psychrophilic organisms are in general quite efficient in compensating for the reduction of reaction rates induced by low temperatures through improvement of the turnover number (k cat ) or of the physiological efficiency (k cat / K m ). It is thought that optimization of the catalytic parameters originates from a higher flexibility of crucial parts of the molecular edifice, providing an enhanced ability to undergo conformational changes at low energy cost during catalysis. Coldadapted enzymes are also characterized by a thermal instability which is regarded as a consequence of their conformational flexibility (6). The gain in reaction rate which usually covers the temperature range from 0 to 30°C is due to a decrease in the activation energy, induced by a decrease in the activation enthalpy, itself partially compensated by an unfavorable modification of the activation entropy compared to mesophilic enzymes (13). The adaptation of the molecular structure mainly consists in a decrease of the number of strength of intramolecular interactions and in some cases in a better accessibility of the catalytic cavity (7).In the context of the study of protein adaptation to low temperatures, an Antarctic bacterial strain producing a ␤-galactosidase was collected in an environment displaying an average temperature of Ϫ1°C. ␤-D-Galactosidase (␤-D-galactoside galactohydrolase; EC 3.2.1.23) catalyzes the hydrolysis of ␤-1,4-D galactosidic linkages. This enzyme is widely distributed in nat...

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