Antarctic marine bacterium Pseudoalteromonas sp. 22b as a source of cold-adapted beta-galactosidase.
The marine, psychrotolerant, rod-shaped and Gram-negative bacterium 22b (the best of 41 beta-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 beta-galactosidase, which efficiently hydrolyzes lactose at 0-20 degrees 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 degrees C. The maximum enzyme synthesis (lactose as a sufficient inducer) was observed at 6 degrees C, thus below the optimum growth temperature of the bacterium (15 degrees 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 degrees 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 beta-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 degrees C for at least 30 days without any stabilizers.
Turkiewicz
Marianna
M
Institute of Technical Biochemistry, Technical University of Łódź, Stefanowskiego Street 4/10, 90-924 Łódź, Poland. mtur@ck-sg.p.lodz.pl
Kur
Józef
J
Białkowska
Aneta
A
Cieśliński
Hubert
H
Kalinowska
Halina
H
Bielecki
Stanisław
S
D000864:Antarctic Regions; D003080:Cold Temperature; D004789:Enzyme Activation; D004795:Enzyme Stability; D033201:Euphausiacea / Q000382:microbiology; D007785:Lactose / Q000378:metabolism*; D008970:Molecular Weight; D031161:Pseudoalteromonas / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D012623:Seawater / Q000382:microbiology*; D013045:Species Specificity; D013696:Temperature; D001616:beta-Galactosidase / Q000737:chemistry* / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism*
D001616:beta-Galactosidase; D007785:Lactose