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

beta-Galactosidases (EC 3.2.1.23) are exoglycosidases that catalyze the cleavage of glycoconjugates with terminal beta-D-galactose residues in beta 1,3-, beta 1,4- or beta 1,6-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 beta-galactosidase from the Pacific oyster, Crassostrea gigas was discovered, biochemically characterized, and compared to our previously characterized slug enzyme from Arion vulgaris (UniProt Ref. Nr.: A0A0B7AQJ9). Overall, the mussel enzyme showed similar biochemical parameters to the snail enzyme. The enzyme from C. gigas was most active in an acidic environment (pH 3.5) and at a reaction temperature of 50 degrees C. Optimal storage conditions were up to 37 degrees C. In contrast to the enzyme from A. vulgaris, the supplementation of cations (Ni2+, Co2+, Mn2+, Mg2+, Ca2+, Cu2+, Ba2+) increased the activity of the enzyme from C. gigas. Substrate specificity studies of the fi-galactosidases from the mussel, C. gigas, and the slug, A. vulgaris, revealed activity towards terminal beta 1,3- and beta 1,4-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 beta-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 beta-galactosidase from the Pacific oyster, C. gigas, and we compare different analytical methods for the determination of beta-galactosidase activity using the enzyme from C. gigas and A. vulgaris.