Characterization of two new glycosyl hydrolases from the lactic acid bacterium Carnobacterium piscicola strain BA

Three genes with homology to glycosyl hydrolases were detected on a DNA fra gment cloned from a psychrophilic lactic acid bacterium isolate, Carnobacte rium piscicola strain BA. A 2.2-kb region corresponding to an a-galactosida se gene, agaA, was followed by two genes in the same orientation, bgaB, enc oding a 2-kb beta -galactosidase, and bgaC, encoding a structurally distinc t 1.76-kb beta -galactosidase. This gene arrangement had not been observed in other lactic acid bacteria, including Lactococcus lactis, for which the genome sequence is known. To determine if these sequences encoded enzymes w ith alpha- and beta -galactosidase activities, we subcloned the genes and e xamined the enzyme properties. The alpha -galactosidase, AgaA, hydrolyzes p ara-nitrophenyl-alpha -D-galactopyranoside and has optimal activity at 32 t o 37 degreesC. The beta -galactosidase, BgaC, has an optimal activity at 40 degreesC and a half-life of 15 min at 45 degreesC. The regulation of these enzymes was tested in C. piscicola strain BA and activity on both alpha- a nd beta -galactoside substrates decreased for cells grown with added glucos e or lactose. Instead, an increase in activity on a phosphorylated beta -ga lactoside substrate was found for the cells supplemented with lactose, sugg esting that a phospho-galactosidase functions during lactose utilization. T hus, the two beta -galactosidases may act synergistically with the alpha -g alactosidase to degrade other polysaccharides available in the environment.