Characterization of Recombinant GH 43 β-Xylosidases and a GH 3 β-Glucosidase from <i>Levilactobacillus brevis</i> for the Hydrolysis of Lignocellulose-Derived Substrates

Lactic acid bacteria (LAB) harbor diverse enzymes that facilitate carbohydrate metabolism and have immense biotechnological potential. This study examined the genome of a LAB strain isolated from kimchi, <i>Levilactobacillus brevis</i> strain B3A1, for the identification of glycoside hydrolase (GH) enzymes that have the potential to hydrolyze lignocellulose-derived materials. Whole-genome sequencing revealed that B3A1 harbors a genome of 2.47 Mbp and two plasmids. Phylogenetic analysis showed that it is closely related to other food-associated <i>Lv. brevis</i> strains. Survey of the B3A1 genome revealed 28 putative GH genes belonging to 15 families. To examine the potential of B3A1 for saccharification, three GH enzymes were chosen and recombinantly expressed in <i>Escherichia coli</i>: two GH 43 β-xylosidases (LbXyl43A and LbXyl43B) and a GH 3 β-glucosidase (LbBgl3). The recombinant enzymes were purified and characterized. The LbXyl43A and LbXyl43B showed substrate preference towards <i>p</i>-nitrophenyl-β-D-xylopyranoside and <i>p</i>-nitrophenyl-β-D-arabinofuranoside. The optimal β-xylosidase activities of these enzymes were achieved at pH 7.0 (both enzymes) and 25°C and 40°C, respectively. Both enzymes demonstrated activity towards xylobiose and xylan hemicelluloses substrates. LbBgl3 exhibited specificity toward <i>p</i>-nitrophenyl-β-D-glucopyranoside, with optimal activity at pH 9.0 and 45°C. The LbBgl3 demonstrated thermal stability at up to 45°C. It showed high activity towards cellobiose. Structural analysis of the three GH enzymes confirmed the presence of hallmark domains in their respective GH families and conserved catalytic residues. This study aims to contribute to the understanding of LAB-derived GH enzymes through genomic surveys and recombinant protein expression and their potential use in industrial applications.