ABSTRACT
BACKGROUND: The diverse nature of carbohydrate structures and linkages requires a variety of enzymes responsible for sugar degradation. The E. coli periplasmic protein encoded by the bglX gene has been assigned to glycoside hydrolase family 3 and is predicted to function as a ß-glucosidase. OBJECTIVES: We investigated the catalytic properties of the E. coli protein BglX and identified two functionally important amino acid residues. METHODS: The bglX gene was cloned into a pET20b(+) vector, and three mutants, D111N, D287G, and E293Q, were generated using site-directed mutagenesis. Kinetic studies were performed on the wild-type and mutant enzymes. RESULTS: Substrate specificity tests indicated that the BglX enzyme hydrolyzes ß-glycosidic bonds in nitrophenyl-ß-glycosides and demonstrates greater activity towards galactose-containing substrates compared to glucose derivatives. Monomeric glucose and galactose inhibit enzyme activity to a different degree in a substrate-dependent manner. In addition, BglX can hydrolyze lactose but not cellobiose, maltose, or laminarin. Subsequently, E. coli cells overexpressing active BglX have a growth advantage on minimal media supplemented with lactose as a carbon source. Mutation of D287 or D111 residues negatively affected the activity of BglX indicating their involvement in catalysis. Overexpression of BglX by E. coli cells did not increase biofilm formation. CONCLUSIONS: The low activity towards glucose-containing substrates and significantly elevated activity towards galactosides suggests that ß-glucosidase activity may not be the primary function of the BglX enzyme.
