In Silico Insights into Carbohydrate-Active Enzymes (CAZymes) of Bacillus subtilis T7: Lignin and Polysaccharide Degradation Mechanisms

Structural insights into xyloglucan recognition by an ABC transporter from a Gram-positive, thermophilic bacterium.

These findings provide the first structural basis for xyloglucan oligosaccharide recognition by an ABC transporter in Gram-positive bacteria.

Deciphering the Function and Structure of PA1216 as an S‐Adenosyl‐l‐Methionine Binding Protein Using Differential Scanning Fluorimetry and Circular Dichroism

Microbes produce bioactive secondary metabolites as toxins, pigments, or virulence factors. These specialized compounds are produced by nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), or hybrid NRPS/PKS pathways. The genes encoding NRPS and PKS reside in biosynthetic gene clusters (BGCs), some of which have no identified metabolite associated with them. Characterization of these orphan BGCs could provide insights into potential bioactive compounds that have yet to be discovered. Here, we characterize PA1216, a putative methyltransferase embedded within an NRPS BGC in Pseudomonas aeruginosa strain PAO1. We cloned, expressed, and purified PA1216, and developed an optimized differential scanning fluorimetry assay to measure its thermal stability, demonstrating concentration‐dependent stabilization in the presence of established methyltransferase cofactors and inhibitors. We then adapted this assay for high‐throughput screening of potential PA1216 substrates, identifying destabilizing compounds, including glycyl‐glycine dipeptides, amino esters with aromatic or basic side chains, and N‐Boc‐protected amino acids. In contrast, sodium salts of organic acids stabilized PA1216. Lastly, we employed AlphaFold to construct a predictive model, revealing that PA1216 contains a Rossmann‐like fold and a glycine‐rich loop, typical of class I methyltransferases, and we corroborated these secondary structural elements using circular dichroism spectroscopy. Overall, these studies illuminate PA1216 function and establish a platform for characterizing cryptic gene clusters within secondary metabolic pathways.

Comparative genomic analysis of hemicellulose-degrading potential in bacterial isolates from the anterior intestine of Eisenia andrei (Bouché, 1972)

Experimental, Genomic, and Structural Evidence Supporting Putative PET‐Hydrolases in Thermophilic Bacteria Isolated From Hot Springs in Cajamarca, Peru

ABSTRACT Polyethylene terephthalate (PET) waste represents a major environmental challenge due to limited recycling solutions. Thermophilic bacteria from geothermal environments harbor diverse enzymatic machinery adapted to extreme conditions, offering promising biocatalysts for plastic degradation; however, biological resources from Peru and other South American countries remain scarce. We characterized four bacterial strains isolated from two geothermal sites in Cajamarca, Peru, screened for PET hydrolysis at 50°C. Whole‐genome sequencing using hybrid assembly achieved near‐complete circular genomes. GTDB‐Tk classification identified three species: Neobacillus thermocopriae (strain 19A), Bacillus licheniformis (strains 16P and BI2), and Brevibacillus agri (strain BI8). Quantitative assays revealed that strain 16P achieved the highest mass loss (0.598%), followed by strain BI8 (0.449%). ATR‐FTIR analysis of the incubated sheets showed a significant reduction of the ester carbonyl index in strains 16P, 19A, and BI8 relative to both non‐incubated PET and an abiotic control, whereas strain BI2 did not differ from the controls, indicating preferential modification of ester bonds at the sheet surface. Genome mining and structure‐based homology searches identified multiple candidate enzymes similar to validated PETases and carboxylesterases, including PETase46‐like homologs in strains BI8 and 16P and a terephthalate‐active carboxylesterase homolog in strain 16P. Molecular docking supported the conservation of catalytic geometry and substrate‐binding sites in these candidates. This work represents one of the first systematic genomic and structural characterizations of putative PET‐hydrolases in Peruvian geothermal bacteria, expanding knowledge of extremophile diversity and advancing thermostable enzymes for sustainable plastic waste management.

Predictive characterization and molecular docking of lignocellulolytic enzymes: comparative analyses of Moroccan fungal species

Lignocellulosic biomass has significant potential as a renewable feedstock for the production of biofuels and bioproducts. However, its structural complexity, particularly the crystalline nature of cellulose and protective lignin matrix, poses considerable challenges for enzymatic degradation. Fungi isolated from diverse lignocellulosic wastes, particularly olive pomace in Morocco, are a significant, yet underutilized resource of lignocellulolytic enzymes with a potential to overcome these challenges. This study investigated the structural and functional properties of lignocellulolytic enzymes derived from 9 filamentous fungi. A total of 80 curated sequences were systematically categorized into cellulases and ligninases. Phylogenetic analysis was conducted on representative endo­glucanases, β-glucosidases, and laccases to assess their diversity. Physicochemical parameters, secon­dary structure content, and thermostability indices were determined using ExPASy ProtParam and SOPMA. Homology models were generated with SWISS-MODEL and validated through PROCHECK, ERRAT, and ProSA. Molecular docking with AutoDock was used to evaluate the interactions of β-glucosidase with cellobiose. Phylogenetic analysis revealed high evolutionary diversity among the examined species. Seve­ral enzymes showed favorable aliphatic indices and GRAVY scores, suggesting thermostability and hydrophilicity. β-Glucosidase from Fusarium equiseti showed the strongest predicted binding energy to cellobiose (–5.32 kcal/mol), with hydrogen bonding mediated by key residues, including Asp88, Arg94, Lys185, Gln197, Asp276, and Gln278, at optimal distances (1.95–2.58 Å). This integrative in silico study highlights the predictive potential of Moroccan fungal β-glucosidases, particularly from F. equiseti, for future applications in lignocellulose bioconversion.

Journal of Integrated Biotechnology Research