Biochemical and Phylogenetic Analyses of a Cold-Active β-Galactosidase from the Lactic Acid Bacterium Carnobacterium piscicola BA

Abstract

ABSTRACT We are investigating glycosyl hydrolases from new psychrophilic isolates to examine the adaptations of enzymes to low temperatures. A β-galactosidase from isolate BA, which we have classified as a strain of the lactic acid bacterium Carnobacterium piscicola , was capable of hydrolyzing the chromogen 5-bromo-4-chloro-3-indolyl β- d -galactopyranoside (X-Gal) at 4°C and possessed higher activity in crude cell lysates at 25 than at 37°C. Sequence analysis of a cloned DNA fragment encoding this activity revealed a gene cluster containing three glycosyl hydrolases with homology to an α-galactosidase and two β-galactosidases. The larger of the two β-galactosidase genes, bgaB , encoded the 76.8-kDa cold-active enzyme. This gene was homologous to family 42 glycosyl hydrolases, a group which contains 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°C, and it was inactivated at 40°C in 10 min. The K m of freshly purified enzyme at 30°C was 1.7 mM, and the V max was 450 μmol · min −1 · mg −1 with o -nitrophenyl β- d -galactopyranoside. This cold-active enzyme is interesting because it is homologous to a thermophilic enzyme from Bacillus stearothermophilus , and comparisons could provide information about structural features important for activity at low temperatures.