Antarctic marine bacterium Pseudoalteromonas sp. 22b as a source of cold-adapted β-galactosidase

• Published in: Biomolecular engineering Vol. 20; no. 4; pp. 317 - 324
• Main Authors: Turkiewicz, Marianna, Kur, Józef, Białkowska, Aneta, Cieśliński, Hubert, Kalinowska, Halina, Bielecki, Stanisław
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-07-2003
• Subjects: Antarctic Regions; beta-Galactosidase - chemistry; beta-Galactosidase - classification; beta-Galactosidase - isolation & purification; beta-Galactosidase - metabolism; Cold Temperature; Cold-adapted β-galactosidase; Enzyme Activation; Enzyme Stability; Euphausiacea - microbiology; Lactose - metabolism; Lactose hydrolysis; Molecular Weight; Pseudoalteromonas; Pseudoalteromonas - chemistry; Pseudoalteromonas - enzymology; Pseudoalteromonas - genetics; Pseudoalteromonas - isolation & purification; Pseudoalteromonas sp; Seawater - microbiology; Species Specificity; Temperature; Antarctic Regions; Antarctica; Pseudoalteromonas sp; Lactose hydrolysis; Cold-adapted β-galactosidase
• ISSN: 1389-0344; 1878-559X
• DOI: 10.1016/S1389-0344(03)00039-X

The marine, psychrotolerant, rod-shaped and Gram-negative bacterium 22b (the best of 41 β-galactosidase producers out of 107 Antarctic strains subjected to screening), classified as
Pseudoalteromonas sp. based on 16S rRNA gene sequence, isolated from the alimentary tract of Antarctic krill
Thyssanoessa macrura, synthesizes an intracellular cold-adapted β-galactosidase, which efficiently hydrolyzes lactose at 0–20
°C, as indicated by its specific activity of 21–67 U mg
−1 of protein (11–35% of maximum activity) in this temperature range, as well as k
cat of 157 s
−1, and k
cat/K
m of 47.5 mM
−1 s
−1 at 20
°C. The maximum enzyme synthesis (lactose as a sufficient inducer) was observed at 6
°C, thus below the optimum growth temperature of the bacterium (15
°C). The enzyme extracted from cells was purified to homogeneity (25% recovery) by using the fast, three-step procedure, including affinity chromatography on PABTG-Sepharose. The enzyme is a tetramer composed of roughly 115 kDa subunits. It is maximally active at 40
°C (190 U mg
−1 of protein) and pH 6.0–8.0. PNPG is its preferred substrate (50% higher activity than against ONPG). The
Pseudoalteromonas sp. 22b β-galactosidase is activated by thiol compounds (70% rise in activity in the presence of 10 mM dithiotreitol), some metal ions (K
+, Na
+, Mn
2+—40% increase, Mg
2+—15% enhancement), and markedly inactivated by pCMB and heavy metal ions, particularly Cu
2+. Noteworthy, Ca
2+ ions do not affect the enzyme activity, and the homogeneous protein is stable at 4
°C for at least 30 days without any stabilizers.