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.
Yan et al. (Thu,) studied this question.