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

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.