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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