Biochemical and phylogenetic analyses of a cold-active beta-galactosidase from the lactic acid bacterium Carnobacterium piscicola BA

We are investigating glycosyl hydrolases from new psychrophilic isolates to examine the adaptations of enzymes to low temperatures. A beta-galactosida se from isolate BA, which we have classified as a strain of the lactic acid bacterium Carnobacterium piscicola, was capable of hydrolyzing the chromog en 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal) at 4 degrees C and possessed higher activity in crude cell lysates at 25 than at 37 deg rees C, Sequence analysis of a cloned DNA fragment encoding this activity r evealed a gene cluster containing three glycosyl hydrolases with homology t o an alpha-galactosidase and two beta-galactosidases. The larger of the two beta-galactosidase genes, bgaB, encoded the 76.8-kDa cold-active enzyme. T his gene was homologous to family 42 glycosyl hydrolases, a group which con tains several thermophilic enzymes but none from lactic acid bacteria, The bgaB gene from isolate BA was subcloned in Escherichia coli, and its enzyme , BgaB, was purified. The purified enzyme was highly unstable and required 10% glycerol to maintain activity, Its optimal temperature for activity was 30 degrees C, and it was inactivated at 40 degrees C in 10 min, The K-m of freshly purified enzyme at 30 degrees C was 1.7 mM, and the V-max was 450 mu mol.min(-1).mg(-1) with o-nitrophenyl beta-D-galactopyranoside. This col d-active enzyme is interesting because it is homologous to a thermophilic e nzyme from Bacillus stearothermophilus, and comparisons could provide infor mation about structural features important for activity at low temperatures .