Abstract
Polyurethane (PU) is one of the most widely used synthetic polymers globally, yet its remarkable chemical durability renders it essentially non-biodegradable under natural conditions, contributing substantially to the plastic pollution crisis. Bioremediation using filamentous fungi offers a promising eco-friendly alternative, with Aspergillus tubingensis having demonstrated notable capacity for polyester-polyurethane degradation through secretion of hydrolytic enzymes. However, the degradation rates of wild-type strains remain insufficient for practical application. This study employed sequential ultraviolet (UV) and ethyl methane sulfonate (EMS) mutagenesis to develop hyper-producing mutant strains of A. tubingensis with significantly enhanced polyurethane biodegradation capability. UV exposure at 254 nm for 15 minutes (achieving 95% kill rate) followed by 1% EMS treatment for 150 minutes (90% kill rate) was applied to generate a library of approximately 5,000 mutants. Primary screening on polyurethane agar plates identified 47 mutants (0.94%) with degradation index (DI) values exceeding 2.0, compared to 1.30 for the wild-type. Secondary screening in liquid mineral salt medium over 28 days identified five hyper-producing mutants (AT-M1 to AT-M5) achieving 34.2–38.4% polyurethane weight loss, representing 1.83–2.05-fold enhancement over the wild-type (18.7%). FTIR-ATR analysis confirmed enhanced ester bond cleavage, with mutant AT-M1 showing 46.7% reduction in the ester carbonyl peak (1728 cm⁻¹) versus 22.8% for the wild-type. Scanning electron microscopy (SEM) revealed extensive surface erosion, deep crack formation, and hyphal penetration in mutant-treated films. Enzyme assays demonstrated significantly elevated esterase specific activity (44.2 U/mg protein for AT-M1 vs. 25.8 U/mg for wild-type; 1.71-fold) and protease activity (1.55-fold), with strong positive correlations between esterase activity and weight loss (R² = 0.96). All five mutants retained >95% of their degradation capacity after five subcultures, confirming genetic stability. These results establish sequential mutagenesis as an effective strategy for enhancing fungal polyurethane biodegradation, with mutant AT-M1 representing a promising biocatalyst for PU waste bioremediation applications