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

Abstract

ABSTRACT Three genes with homology to glycosyl hydrolases were detected on a DNA fragment cloned from a psychrophilic lactic acid bacterium isolate, Carnobacterium piscicola strain BA. A 2.2-kb region corresponding to an α-galactosidase gene, agaA , was followed by two genes in the same orientation, bgaB , encoding a 2-kb β-galactosidase, and bgaC , encoding a structurally distinct 1.76-kb β-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 with α- and β-galactosidase activities, we subcloned the genes and examined the enzyme properties. The α-galactosidase, AgaA, hydrolyzes para -nitrophenyl-α- d -galactopyranoside and has optimal activity at 32 to 37°C. The β-galactosidase, BgaC, has an optimal activity at 40°C and a half-life of 15 min at 45°C. The regulation of these enzymes was tested in C. piscicola strain BA and activity on both α- and β-galactoside substrates decreased for cells grown with added glucose or lactose. Instead, an increase in activity on a phosphorylated β-galactoside substrate was found for the cells supplemented with lactose, suggesting that a phospho-galactosidase functions during lactose utilization. Thus, the two β-galactosidases may act synergistically with the α-galactosidase to degrade other polysaccharides available in the environment.