Biochemical Characterization of a Disaccharidase From Enterococcus faecalis CTB

A disaccharidase (GenA) from Enterococcus faecalis CTB was heterologously expressed in Escherichia coli and purified to > 95% homogeneity using chromatographic techniques. The enzyme exhibited a monomeric molecular weight of 54 kDa and demonstrated hydrolytic activity toward maltose, cellobiose, and lactose, but not sucrose. Kinetic analysis revealed maltose as the preferred substrate (Km = 0.27 ± 0.05 mM, V _max = 33.8 ± 2.24 μM/min), followed by lactose (Km = 0.42 ± 0.04 mM, V _max = 42.0 ± 2.91 μM/min) and cellobiose (Km = 0.47 ± 0.06 mM, V _max = 51.0 ± 1.90 μM/min). GenA also hydrolyzed synthetic substrates including PNP-α-D-glucoside and PNP-β-D-galactopyranoside. The enzyme displayed substrate-dependent optimal conditions: 40°C-60°C and pH 7.5-9.0. MgCl₂ enhanced enzymatic activity 2.0-4.0 fold across all substrates, while NiCl₂ and MnCl₂ were generally inhibitory. These findings provide insights into GenA's catalytic mechanisms and highlight its potential applications in biocatalysis and industrial biotechnology.