Characterization of a Novel Metagenome-Derived 6-Phospho-β-Glucosidase from Black Liquor Sediment

The enzyme 6-phospho-β-glucosidase is an important member of the glycoside hydrolase family 1 (GH1). However, its catalytic mechanisms, especially the key residues determining substrate specificity and affinity, are poorly understood. A metagenome-derived gene sequence, encoding a novel 6-phospho-β-glucosidase designated Pbgl25-217, was isolated and characterized. The optimal conditions for enzymatic activity were 37�C and pH 7; Ca2+ , Mg2+ , and Mn2+ stabilized the activity of Pbgl25-217, whereas Ni2+ , Fe2+ , Zn2+ , Cu2+ , and Fe3+ inhibited its activity. TheKm andV max of Pbgl25-217 were 4.8 mM and 1,987.0 U mg−1 , respectively. Seven conserved residues were recognized by multiple alignments and were tested by site-directed mutagenesis for their functions in substrate recognition and catalytic reaction. The results suggest that residues S427, Lys435, and Tyr437 act as “gatekeepers” in a phosphate-binding loop and play important roles in phosphate recognition. This functional identification may provide insights into the specificity of 6-phospho-β-glycosidases in GH1 and be useful for designing further directed evolution.