Discovery of two novel cutinases from a gut yeast of plastic-eating mealworm for polyester depolymerization

Identification of novel plastic-degrading enzymes is crucial for developing enzymatic degradation and recycling strategies for plastic waste. Here, we report the discovery of two novel cutinases, SiCut1 and SiCut2, from a yeast strain <i>Sakaguchia</i> sp. BIT-D3 was isolated from the gut of plastic-eating mealworms. Their amino acid sequences share less than 25% identity with all previously described cutinases and reveal a conserved S-D-H catalytic triad with a unique GYSKG motif. Their recombinant proteins were successfully overexpressed in <i>Pichia pastoris</i>. The pH range for both enzymes was 4.0 to 11.0 and the temperature range for SiCut1 and SiCut2 was 10°C to 50°C and 10°C to 70°C, respectively. Both enzymes showed strong activity against apple cutin and short-chain fatty acid esters of <i>p</i>-nitrophenol and glycerol, substantiating their classification as true cutinases. SiCut1 and SiCut2 have been demonstrated to exhibit efficient degradation of polycaprolactone (PCL) film, polybutylene succinate (PBS) film, and polyester-polyurethane (PUR) foam. Molecular docking and molecular dynamics simulations were used to elucidate the underlying mechanisms of the observed catalytic activity and thermal stability. This study shows that SiCut1 and SiCut2 are novel yeast-derived cutinases with the potential for depolymerization and recycling of plastic waste.IMPORTANCEThe identification of novel plastic-degrading enzymes is critical in addressing the pervasive problem of plastic pollution. This study presents two unique cutinases, SiCut1 and SiCut2, derived from the yeast <i>Sakaguchia</i> sp. BIT-D3 isolated from the gut of plastic-feeding mealworms. Despite sharing less than 25% sequence identity with known cutinases, both enzymes exhibit remarkable degradation capabilities against various polyester plastics, including polycaprolactone (PCL) film, polybutylene succinate (PBS) film, and polyester-polyurethane (PUR) foam. Our results elucidate the catalytic mechanisms of SiCut1 and SiCut2 and provide insights into their potential applications in enzymatic degradation and recycling strategies. By harnessing the gut microbiota of plastic-degrading organisms, this research lays the foundation for innovative enzyme-based solutions to reduce plastic waste and promote sustainable practices in waste management.