Biochemical Characterization of a Disaccharidase From Enterococcus faecalis CTB

TL;DR

This study characterizes a new disaccharidase from Enterococcus faecalis, showing its activity on various sugars and optimal conditions for use in biotechnology.

Contribution

The biochemical characterization of GenA, a novel disaccharidase with potential industrial applications.

Findings

GenA hydrolyzes maltose, cellobiose, and lactose but not sucrose, with maltose being the preferred substrate.

MgCl2 enhances GenA activity 2.0–4.0 fold, while NiCl2 and MnCl2 inhibit it.

The enzyme functions optimally at 40°C–60°C and pH 7.5–9.0, depending on the substrate.

Abstract

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.

This study explores GenA, a novel disaccharidase from
Enterococcus faecalis
. Cloned/expressed in
Escherichia coli
and 98% purified, GenA hydrolyzes maltose (optimal), cellobiose, lactose, with substrate‐dependent optimal T/pH. MgCl2 boosts activity; NiCl2/MnCl2 inhibit. It aids catalytic mechanism studies and industrial biocatalysis.

Figures