Cold-adapted beta-galactosidase from the Antarctic psychrophile Pseudoalteromonas haloplanktis.

The beta-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 beta-galactosidase subunit is composed of 1,038 amino acids with a calculated M(r) of 118,068. This beta-galactosidase shares structural properties with Escherichia coli beta-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 beta-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 beta-galactosidase compared to the mesophilic enzyme. Assays of lactose hydrolysis in milk demonstrate that P. haloplanktis beta-galactosidase can outperform the current commercial beta-galactosidase from Kluyveromyces marxianus var. lactis, suggesting that the cold-adapted beta-galactosidase could be used to hydrolyze lactose in dairy products processed in refrigerated plants.

Trypsin and trypsinogen from an Antarctic fish: molecular basis of cold adaptation.

Trypsin from Antarctic fish Paranotothenia magellanica displays molecular and kinetic properties typical of enzymes produced by psychrophilic organisms. The enzyme has a high catalytic efficiency at low and moderate temperatures and is rapidly inactivated at temperatures higher than 30 degrees C. The nucleotide sequence was determined after mRNA extraction and cDNA synthesis. The cDNA encodes a pretrypsinogen which includes a seven residue activation peptide containing only three acidic residues preceeding the 222 amino-acid mature enzyme. A three-dimensional model of the enzyme was built. Structural parameters possibly involved in the adaptation to cold have been derived from comparison with the three-dimensional structure of the bovine enzyme. Among them are the lack of Tyr-151 in the substrate binding pocket, an overall decrease in the number of salt bridges and hydrophobicity and the increase in the surface hydrophilicity.