Discovery and characterization of a novel GH6 multifunctional enzyme from soil metagenomic library.

Enzymatic Characterization of a Novel GH3 β-Glucosidase from Lentilactobacillus buchneri: EDTA-Mediated Enhancement of Thermostability

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.

Discovery of a multifunctional chitinase-cellulase from Thermococcus chitonophagus with expanded polysaccharide specificity.

BACKGROUND The discovery of novel biocatalysts for the sustainable valorization of complex biomass feedstocks remains a significant challenge. Domain-centric exploration of characterized CAZyme families offers a promising but underexplored strategy for identifying enzymes with unusual architectures and potentially expanded substrate specificities. RESULTS Systematic analysis of archaeal glycoside hydrolase family 18 (GH18) chitinases using the CANDy domain annotation pipeline led to the identification of TcChi from Thermococcus chitonophagus, a multidomain enzyme combining a GH12 and a GH18 catalytic domain alongside two carbohydrate-binding modules. Given that T. chitonophagus also encodes dedicated standalone cellulases and chitinases, we hypothesized that this multidomain assembly may have evolved a broader functional range than either composing domain alone. Biochemical assays of truncated constructs confirmed this hypothesis: the GH18 domain hydrolyzed chitin, chitosan, and β-1,3-glucan, marking the first report of β-1,3-glucanase activity (EC 3.2.1.58) in a GH18 chitinase, while the GH12 domain exhibited strong cellulase activity alongside unexpected chitosanase activity (EC 3.2.1.132), extending the known functional range of this family. Both domains demonstrated high thermostability consistent with the hyperthermophilic origin of T. chitonophagus. CONCLUSIONS TcChi is a thermostable, multifunctional biocatalyst capable of degrading chitin, chitosan, cellulose, and β-1,3-glucan from a single protein scaffold, making it a promising candidate for consolidated biomass deconstruction and waste valorization. These findings also demonstrate that domain-centric analysis of CAZyme families is an effective strategy for uncovering hidden functional diversity in well-characterized enzyme families.

System reconstruction of Bacillus licheniformis for efficient expression of alkaline protease

Industrial enzymes are widely used in diverse applications, but low productivity limits their further widespread utilization. This research aimed to develop high-performance alkaline protease (AprE) expression strains of Bacillus licheniformis through element optimization and modular engineering. Firstly, the aprE gene expression cassette was systematically optimized through element engineering. To minimize host background interference, five large gene fragments were deleted from the genome of B. licheniformis DW2. This expression cassette and genome-reduced strain resulted in 5.77-, 4.84- and 1.31-fold increases in the activities of alkaline protease, nattokinase and chitinase, respectively. Crucially, metabolomics analysis then served as the pivotal discovery tool, revealing that high expression of AprE was constrained by insufficient precursor amino acids and excessive metabolic overflow. Subsequently, the amino acid biosynthesis, energy metabolism, overflow metabolism, and cell membrane/wall modules of the strain were successively modified. The final AprE expression host DM6E10 achieved a remarkable enzyme activity of 34,343 U/mL, with a maximum activity of 107,100 U/mL in a 5-L bioreactor. This study built an efficient cell factory for AprE production and provided insights for the optimization of other protein expression hosts.

A recombinant xylose reductase enzyme with biotechnological potential for xylitol production

Bioinformatics analysis of extracellular glycosyl hydrolases mined from the genome of Halophilic Meridianimaribacter sp. CL38.

Biological pre-treatment of lignocellulosic waste is crucial for efficient fermentation in second-generation biofuel production. Halophilic bacteria produce glycosyl hydrolases with enhanced stability for industrial application. This study aimed to characterise putative glycosyl hydrolases (cellulases and hemicellulases) encoded in the genome of the halophilic bacterium Meridianimaribacter sp. CL38 using bioinformatic approaches, in order to address the scarcity of structural information for this genus. Four putative glycosyl hydrolases (GH9, GH3, GH144 and GH16) were analysed, focusing on their functional domains and structural properties. Phylogenetic analysis indicated a close relationship with other members of the Flavobacteriaceae family. Structural analyses revealed significant predicted halophilic and thermostable properties, suggesting these putative enzymes may endure industrial conditions. This study provides insights into putative glycosyl hydrolase candidates for efficient lignocellulosic waste pre-treatment.

Characterization of a Novel GH3 β-Glucosidase with Alkali-Tolerant and Halophilic Properties from Pontixanthobacter luteolus SW-109.