Bioinformatics-guided discovery and functional verification of fungal polyester hydrolases for polycaprolactone degradation

Polycaprolactone (PCL) is a biodegradable polyester that can degrade slowly under ambient environmental conditions. Here, we explored natural fungal sequence diversity to identify cutinase-like enzymes (CLEs) with PCL-degrading activity. Using a previously reported Cryptococcus CLE as the query, bioinformatics analysis identified 14 fungal homologs that were subsequently expressed in the heterologous hostSaccharomyces cerevisiae and screened using a plate-based assay. Nine homologs exhibited detectable PCL degradation, with three producing larger degradation zones than the Cryptococcus reference enzyme. These three homologs exhibited distinct apparent optimal temperatures of 30°C, 37°C, and 40°C, compared with 45°C for the reference enzyme under the tested conditions. Signal peptide replacement and GFP-based analyses suggested that native signal peptide recognition alone was unlikely to account for the five inactive homologs. Collectively, this study provides a practical bioinformatics-to-experiment strategy for screening natural enzyme diversity and prioritizing promising candidates for further biochemical characterization.