Characterization of Two New Glycosyl Hydrolases from the Lactic Acid Bacterium Carnobacterium piscicola Strain BA

Glycosyl hydrolases (EC 3.2.1 to 3.2.3) cleave the glycosidic bond(s) between two or more carbohydrates or the bond between a carbohydrate moiety and a noncarbohydrate moiety. Traditionally, glycosyl hydrolases were grouped together based on substrate specificity. For example, all β-galactosidases were combined into one group (EC 3.2.1.23) because of their shared ability to hydrolyze lactose. However, classification based on substrate specificity is complicated by the fact that some enzymes hydrolyze more than one substrate. Some glycosyl hydrolases have activity on both phosphorylated and nonphosphorylated substrates (3, 21) or on β-glucosides and β-galactosides (2) and some β-galactosidases have activity on β-fucosides and β-galacturonides (11, 15, 25). The increase in the number of sequenced glycosyl hydrolases and the availability of new analytical methods has permitted the reorganization of these enzymes into families based on amino acid sequence similarities and hydrophobic cluster analysis (12, 13, 14). There are presently four families containing enzymes with β-galactosidase activity, families 1, 2, 35, and 42, and three families which contain enzymes with α-galactosidase activity, families 4, 27, and 36. New glycosyl hydrolases which have been sequenced can be grouped into a specific family on the basis of DNA or deduced amino acid similarity. In many cases, however, there is no information to verify the substrate specificity of the enzymes within these groups or their possible role(s) in cellular metabolism. The glycosyl hydrolases found in lactic acid bacteria have been of special interest because of their importance to the dairy and food processing industries. In contrast to most other bacteria, nearly all lactic acid bacteria transport and utilize lactose via the phosphoenolpyruvate-dependent phosphotransferase system, which requires the concomitant activity of a phospho-β-galactosidase. β-Galactosidases belonging to a different family, and sharing sequence similarity with the well-characterized Escherichia coli lacZ-encoded enzyme, have also been detected in lactic acid bacteria such as Streptococcus thermophilus or Lactococcus lactis (7). The genus Carnobacterium is a recent taxonomic addition to the lactic acid bacteria group (4, 5). Most Carnobacterium species were isolated from meat or fish (1, 23) and are similar to those in the Lactobacillus genus but do not grow on acetate and have a higher tolerance to oxygen and high pH (24). Research on Carnobacterium species has centered on their ability to produce bacteriocins (8, 19). Recently, during our investigation of psychrophilic organisms, we isolated from soil a new Carnobacterium piscicola strain, BA, which hydrolyzed the β-galactosidase chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) at 4°C. Initial work discovered a gene, bgaB, encoding a family 42 glycosyl hydrolase that had a temperature optimum of 30°C (6). This was the first report of a gene from this family in any lactic acid bacterium. Additional sequencing of this cloned fragment suggested that the bgaB gene is centered between two regions with homology to other glycosyl hydrolases. The gene agaA is located in the region adjacent to the N-terminal end of bgaB, and shared sequence homology with a group of α-galactosidases characterized from other bacteria and some eukaryotes, including a sequence from the lactic acid bacterium Lactobacillus plantarum. Adjacent to the C-terminal end of the bgaB β-galactosidase gene was a second, unrelated β-galactosidase gene, bgaC. Genes similar to bgaC have not been reported in the lactic acid bacteria. This includes L. lactis, for which the sequence of the entire genome is known. In order to explore the functions encoded by these two new putative genes, they were subcloned and their ability to produce enzymes with α- and β-galactosidase activities was tested. The arrangement of these genes on a single fragment suggested that they might function together to degrade saccharides containing both alpha and beta linkages rather than being involved in lactose hydrolysis. We examined the regulation of these enzymes in the native C. piscicola strain BA and found that their activities decreased when the medium was supplemented with either glucose or lactose. In contrast, a phospho-galactosidase activity increased during growth with lactose. These results suggest that a phospho-galactosidase is responsible for lactose utilization and that the unusual cluster of glycosyl hydrolase genes reported here might be involved in the degradation of other polysaccharides.