Aug 2026· Metabolites· Vol 16· 0 citations· 37 references
Medicine
TL;DR
This study presents the first characterization of a GH3 β-glucosidase from L. buchneri and reveals a non-classical stabilizing effect of EDTA, offering valuable insights for enzyme engineering and biocatalytic applications.
Abstract
Background/Objectives: β-Glucosidases from lactic acid bacteria are valuable biocatalysts for carbohydrate conversion, flavour enhancement, and bioactive glycoside biotransformation. Although GH3 family enzymes have been characterized from several Lactobacillus species, no systematic study exists for Lentilactobacillus buchneri—a GRAS strain with plant-polysaccharide-degrading potential. This work aimed to clone, heterologously express, and comprehensively characterize a novel GH3 β-glucosidase (LbBgl3) from L. buchneri, with a particular focus on its catalytic properties, substrate profile, stability, and the unexpected EDTA-mediated thermostabilization mechanism. Methods: A novel β-glucosidase gene (LbBgl3) from L. buchneri was successfully expressed in Escherichia coli. Results: Biochemical characterization revealed that LbBgl3 is a cold-adapted, moderately acidophilic enzyme, exhibiting optimal activity at 37 °C and pH 5.0, with a maximum specific activity of 799.67 U·mg−1. Under optimal conditions, the enzyme displayed kinetic parameters toward pNPG with a Km of 1.697 mM, Vmax of 442.5 μmol·mg−1·min−1, kcat of 634.32 s−1, and kcat/Km of 373.79 mM−1·s−1. LbBgl3 exhibited high salt tolerance, with activity peaking at ~140% at 0.5 M NaCl and retaining ~68% at 2.0 M NaCl, while showing sensitivity to glucose inhibition. Notably, EDTA significantly enhanced both the activity and stability of LbBgl3. At 50 mM, the relative activity increased to 161%, and stability at 25 °C was prolonged. Molecular docking simulations suggested that EDTA binds near the substrate-binding pocket, forming hydrogen bonds with Ala57, Gln766, Ser768, and Lys770, thereby stabilizing the local conformation and enhancing thermal resistance. Conclusions: This study presents the first characterization of a GH3 β-glucosidase from L. buchneri and reveals a non-classical stabilizing effect of EDTA, offering valuable insights for enzyme engineering and biocatalytic applications.
Local enrichment of acidic residues on the PlGH3 surface could generate a negative electrostatic potential, which enables adaptation to high-salt and alkaline environments, thereby sustaining the enzyme's catalytic activity under such extreme conditions.
Kaijuan Wu, Ke Guo, Zheng Yu et al.· Applied Biochemistry and Bio...· 0 citations
β-glucosidases belong to the class of glycoside hydrolases (GHs) and can mediate the cleavage of β-1-4 glycosidic bonds in alkyl or aryl-β-D-glucosides, amino, cyanogenic glucosides, and oligo- or disaccharides. The gene encoding β-glucosidase of Anoxybacillus flavithermus DSM2641T was subcloned and expressed in E. coli BL21 Star(DE3) cells with pET100 Directional TOPO Expression Kit. The recombinant AFBGL was purified by a Ni-NTA affinity chromatography. The enzyme was 53 kDa and had 5.13 × 105 µmol/min/mg protein of Vmax and 422.3 µM of Km towards p-nitrophenyl β-D-glucoside as a main substrate. The optimum temperature and pH of the purified enzyme were 65°C and 6.8, respectively. We also determined kinetic parameters of AFBGL against the other artificial substrats, pNPC, pNPX, and oNPG. The enzyme had the Vmax and Km for 5.13 × 105 µmol/min/mg protein and 0.42 mM for pNPG, 0.007 µmol/min/mg protein and 0.19 mM for pNPC, 0.01 µmol/min/mg protein and 11.97 mM for pNPX, 0.06 µmol/min/mg protein and 0.134 mM for oNPG, respectively. AFBGL's substrate affinity was found to be more directed towards oNPG. Although 0.5 mM Mg2+ increases the enzyme's activity 1.6-fold, higher concentrations of Mg2+ and the presence of K+, Fe2+, Mn2+, Zn2+, Cu2+, and Al3+ decreases its activity. AFBGL retained 50% of the initial activity towards pNPG for 25 min and the activity was not detected after 225 min. According to amino acid motifs, AFBGL belongs to family 1 of glycosylhydrolase and 4/7 super family. So, AFBGL is a member of GH1, as the amino acid sequence contained a GH1 specific domain.
Aytül Uzun Akgeyik, Esma Akyıldız, Hakan Karaoglu et al.· Indian Journal of Biochemist...· 0 citations
BSEG2 is highly thermostable, tolerant of high salt and ionic liquids, and notably resistant to cellobiose inhibition up to 200 mM, suggesting potential to reduce freshwater demand in biomass processing and a strong candidate for simplified, cost-effective enzyme formulations for glucose production from lignocellulosic feedstocks.
Debjyoti Ghosh, Aditi Konar, Yi Gao et al.· Biotechnology Journal· 0 citations
Xylan, the principal component of hemicellulose, requires the concerted action of endo- and exo-acting xylanolytic enzymes for its complete depolymerization into fermentable sugars. This study reports the biochemical and thermodynamic characterization of a glycoside hydrolase family 10 endo-β-1,4-xylanase from the thermophilic bacterium Geobacillus stearothermophilus CECT43 (GsXynA), cloned and heterologously expressed in Escherichia coli BL21(DE3) and purified to homogeneity by immobilized metal-ion affinity chromatography. The 338-residue enzyme behaved as a monomer and showed maximal activity on xylan substrates at pH 6.5 and 75 °C, retaining over 90% residual activity between pH 5 and 9 and remaining stable up to 70 °C, after 60 min of incubation. Kinetic and thermodynamic analyses of thermal inactivation monitored by enzymatic activity measurements revealed an enthalpy-driven denaturation process, while circular dichroism indicated that this irreversible unfolding is kinetically, not thermodynamically, controlled. GsXynA hydrolyzed beechwood xylan mainly to xylobiose and xylotetraose, consistent with mixed endo and exo activity, and was uncompetitively inhibited by 4-hydroxybenzoic acid. Combined action with the β-xylosidase GsXynB2 markedly increased D-xylose release, reaching a 53.23% hydrolysis yield and a degree of synergy of 3.79 after 24 h. These results demonstrate that GsXynA is a thermostable, versatile xylanase with promising potential for lignocellulosic biomass bioconversion.
Alba Carrillo-Moreno, María Gabriela Álvarez-Rodríguez, Sergio Martínez-Rodríguez et al.· International Journal of Mol...· 0 citations
The results demonstrate that XynA is a highly efficient thermostable xylanase suitable for mild, eco-friendly functional modification of xylan-based biomaterials.
Weiwei Fu, Qiang Li, Chao Teng et al.· 3 Biotech· 0 citations
