Identification and characterization of a fungal cutinase-like enzyme CpCut1 from Cladosporium sp. P7 for polyurethane degradation

Abstract

Plastic degradation by biological systems emerges as a prospective avenue for addressing the pressing global concern of plastic waste accumulation. The intricate chemical compositions and diverse structural facets inherent to polyurethanes (PU) substantially increase the complexity associated with PU waste management. Despite the extensive research endeavors spanning over decades, most known enzymes exhibit a propensity for hydrolyzing waterborne PU dispersion (i.e., the commercial Impranil DLN-SD), with only a limited capacity for the degradation of bulky PU materials. Here, we report a novel cutinase (CpCut1) derived from Cladosporium sp. P7, which demonstrates remarkable efficiency in the degrading of various polyester-PU materials. After 12-h incubation at 55°C, CpCut1 was capable of degrading 40.5% and 20.6% of thermoplastic PU film and post-consumer foam, respectively, while achieving complete depolymerization of Impranil DLN-SD. Further analysis of the degradation intermediates suggested that the activity of CpCut1 primarily targeted the ester bonds within the PU soft segments. The versatile performance of CpCut1 against a spectrum of polyester-PU materials positions it as a promising candidate for the bio-recycling of waste plastics.IMPORTANCEPolyurethane (PU) has a complex chemical composition that frequently incorporates a variety of additives, which poses significant obstacles to biodegradability and recyclability. Recent advances have unveiled microbial degradation and enzymatic depolymerization as promising waste PU disposal strategies. In this study, we identified a gene encoding a cutinase from the PU-degrading fungus Cladosporium sp. P7, which allowed the expression, purification, and characterization of the recombinant enzyme CpCut1. Furthermore, this study identified the products derived from the CpCut1 catalyzed PU degradation and proposed its underlying mechanism. These findings highlight the potential of this newly discovered fungal cutinase as a remarkably efficient tool in the degradation of PU materials.

Keywords: Cladosporium sp.; biodegradation; fungal cutinase; polymer recycling; polyurethane.

Fig 1

Zymographic identification and sequence analysis of the PU depolymerase CpCut1 from the fungus P7. (A) Zymographic analysis of the concentrated crude enzyme, lane M: low-molecular-weight standard protein markers; lane 1: concentrated crude enzyme; lane 2: an agar plate containing 100 mg/L pNPB, the yellow position indicates esterase activity; lane 3: an agar plate containing 2 g/L PBA-PU, the formation of a clear zone indicates polyesterase activity. (B) Multiple sequence alignment of CpCut1 with characterized fungal cutinases: HiC (PDB code 4OYY from Humicola insolens), FsC (PDB code 1CEX from Fusarium solani), McCut (PDB code 5X88 from Malbranchea cinnamomea), Gc (PDB code 3DCN from Glomerella cingulata), and Ac (PDB code 3GBS from Aspergillus oryzae). Other important residues are marked, as explained on the right of the figure. Identical and highly conserved residues are colored in red and white, respectively. The highly conserved motif is depicted by an orange box. Disulfide bridges are numbered in neon green. (C) Neighbor-joining phylogenetic tree of selected fungal and bacterial cutinases constructed by MEGA 10. Bootstrap analysis of 1,000 replicates was conducted, and values above 50% are shown. Bacterial cutinases were used as an outgroup.

Fig 2

SDS-PAGE analysis of the purification of recombinant CpCut1 (A) and optimal pH (B), pH stability (E), optimal temperature (C), thermostability (F), dependency on metal ions (D), and organic reagents (G) of CpCut1. Lane M: low-molecular-weight standard protein markers; lane 1: CpCut1 purified with Ni²⁺-NTA resin. Buffers used were symbolled as follows: citrate buffer (square), pH 4.0–6.0; PBS (circle), pH 6.0–8.0; glycine-NaOH buffer (triangle), pH 8.0–10.0.

Fig 3

Hydrolysis of different PUs by CpCut1 and other polyester hydrolases. (A) Degradation efficiency of waterborne PU dispersions (Impranil DLN-SD) measured by OD₄₀₀ within 150 min at 55°C. (B) Weight loss of thermoplastic PU (PBA-PU film) measured within 48 h at 55°C. (C) Weight loss of thermoset PU (polyester-PU foam) measured within 48 h at 55°C. The images of PU before and after degradation by CpCut1 are shown below. (D) Degradation efficiency of different PUs by CpCut1, LCC, TfCut2, and HiC at 55°C for 12 h. The value bars with ns are not significant; asterisk denotes statistically significant differences: *P < 0.05, **P < 0.01.

Fig 4

SEM images of the thermoplastic PU (PBA-PU film) and thermoset PU (polyester-PU foam) after a 12-h treatment with CpCut1.

Fig 5

Changes in the chemical structure and thermostability of thermoplastic PU (PBA-PU film) and thermoset PU (polyester-PU foam) after treatment with CpCut1. FTIR spectra (A) and GPC analysis (B) of control PBA-PU film and CpCut1-treated PBA-PU film. FTIR spectra (C) and TG/DTG curves (D) of control polyester-PU foam and CpCut1-treated polyester-PU foam.

Fig 6

Identification of the metabolites released during the degradation of thermoplastic PU (PBA-PU film) by CpCut1. (A) The monomer AA was detected through HPLC analysis. (B) The monomer BDO was detected through GC analysis. (C–F) The metabolites released during the degradation of PBA-PU were analyzed by mass spectroscopy. (G) The proposed mechanism of CpCut1-mediated PBA-PU degradation, including endo- and exo-scission of PBA-PU by CpCut1.