Expression and Characterization of a β-Galactosidase from the Pacific Oyster, and Evaluation of Strategies for Testing Substrate Specificity.

β-Galactosidases (EC 3.2.1.23) are exoglycosidases that catalyze the cleavage of glycoconjugates with terminal β-D-galactose residues in β1,3-, β1,4- or β1,6-linkage. Although this family of exoglycosidases has been extensively studied in vertebrates, plants, yeast, and bacteria, little information is available for mollusks. Mollusks are a diverse and highly successful group of animals that play many different roles in their ecosystems, including filter feeders and detritivores. Here, the first β-galactosidase from the Pacific oyster, was discovered, biochemically characterized, and compared to our previously characterized slug enzyme from (UniProt Ref. Nr.: A0A0B7AQJ9). Overall, the mussel enzyme showed similar biochemical parameters to the snail enzyme. The enzyme from was most active in an acidic environment (pH 3.5) and at a reaction temperature of 50 °C. Optimal storage conditions were up to 37 °C. In contrast to the enzyme from , the supplementation of cations (Ni, Co, Mn, Mg, Ca, Cu, Ba) increased the activity of the enzyme from . Substrate specificity studies of the β-galactosidases from the mussel, and the slug, , revealed activity towards terminal β1,3- and β1,4-linked galactose residues for both enzymes. Using the same substrates in labeled and unlabeled form, we were able to detect the effect of labeling on the β-galactosidase activity using MALDI-TOF MS, HPTLC, and HPLC. While lactose was cleaved by the enzymes in an unlabeled or labeled state, galacto-N-biose was not cleaved as soon as a 2-amino benzoic acid label was added. In this study we present the biochemical characterization of the first recombinantly expressed β-galactosidase from the Pacific oyster, , and we compare different analytical methods for the determination of β-galactosidase activity using the enzyme from and .