Structure-guided mining of cutinase-like enzyme from compost metagenome for ester-bond hydrolysis of polyester...

The mining of novel plastic-degrading enzymes is imperative for the development of enzymatic degradation and recycling strategies for plastic waste. Here, a cutinase-like enzyme (MhCulp3) was identified for polyester-polyurethane (PU) degradation from a compost metagenome in virtue of protein structure clustering. The recombinant MhCulp3 was expressed in Escherichia coli with the pET-28a vector, possessing optimal activity at 30 °C, pH 8.0 against p-nitrophenyl-hexanoate (pNPH, C₆). The results of Fourier Transform Infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and liquid chromatography-tandem mass spectrometry (HPLC-MS) demonstrated that MhCulp3 exhibited activity towards PU emulsions (Impranil®DLN-SD), PU films (PCL-MDI), and PU foams (PEGA-TDI) by cleaving ester bonds in soft segments rather than urethane bonds in hard segments. Additionally, MhCulp3 could not hydrolyze the natural substrate cutin based on the results of gas chromatography-mass spectrometry (GC-MS). Molecular docking and site-directed mutagenesis of MhCulp3 revealed the substrate binding model and catalytic mechanism. Taken together, this study substantiates the reliability of AI-assisted structure clustering strategy in the mining of plastic-degrading enzymes, and provides a novel biocatalyst for enzymatic degradation and recycling of polyester-PU waste.