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.
Laboratory of Biochemistry, Institute of Chemistry, University of Liege, Sart-Tilman, B-4000 Liege, Belgium.
A
A
Hoyoux
I
I
Jennes
P
P
Dubois
S
S
Genicot
F
F
Dubail
J M
JM
François
E
E
Baise
G
G
Feller
C
C
Gerday
D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D000864:Antarctic Regions; D003001:Cloning, Molecular; D003080:Cold Temperature; D003470:Culture Media; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000254:growth & development; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D003470:Culture Media; D001616:beta-Galactosidase