Complete bio-degradation of poly(butylene adipate-co-terephthalate) via engineered cutinases

Abstract

Poly(butylene adipate-co-terephthalate) (PBAT), a polyester made of terephthalic acid (TPA), 1,4-butanediol, and adipic acid, is extensively utilized in plastic production and has accumulated globally as environmental waste. Biodegradation is an attractive strategy to manage PBAT, but an effective PBAT-degrading enzyme is required. Here, we demonstrate that cutinases are highly potent enzymes that can completely decompose PBAT films in 48 h. We further show that the engineered cutinases, by applying a double mutation strategy to render a more flexible substrate-binding pocket exhibit higher decomposition rates. Notably, these variants produce TPA as a major end-product, which is beneficial feature for the future recycling economy. The crystal structures of wild type and double mutation of a cutinase from Thermobifida fusca in complex with a substrate analogue are also solved, elucidating their substrate-binding modes. These structural and biochemical analyses enable us to propose the mechanism of cutinase-mediated PBAT degradation.

Fig. 1. Chemical structures of PBAT and PET and the putative ester hydrolysis products.

Similar to PET, biodegradation of PBAT could also yield TPA.

Fig. 2. Degradation of PBAT by TfCut.

a PBAT film hydrolysis catalyzed by TfCut at 70 °C over time. The optimal temperature of TfCut was determined and shown in Supplementary Fig. 1. b HPLC chromatograms of PBAT hydrolytic products at 4 h. c The structures of the intermediates in b were identified based on the mass spectrum. d The time-course of four major PBAT-hydrolytic intermediates produced by TfCut treatment. TPA (or Ta), terephthalic acid; B, 1,4-butanediol; A, adipic acid. Triplicate independent assay was performed in each experiment. Data are presented as mean values +/– standard deviation. The individual data points are shown as black circles. Source data are provided as a Source Data file.

Fig. 3. The MHET-bound TfCut-DM structure.

a The X-ray crystal structure of TfCut-DM. The numbered α-helices, β-sheets, and loop regions are colored in blue, yellow, and white, respectively. Three amino acids that constitute the catalytic triad are shown in green sticks and circled in black. b Superimposed residues in the active site of TfCut-DM (white, PDB ID: 7XTR) with WT TfCut (green, PDB ID: 5ZOA) displayed in sticks. c Key residues surrounding MHET in the active site are shown as green sticks. The alternative residue in LCC is labeled under I218 in blue color. The catalytic triad residues are shown as white sticks. Residue S170 is from the WT TfCut (white stick, PDB ID: 5ZOA). d Comparison of the substrate-binding poses between MHET-bound WT TfCut (orange, PDB ID: 7XTV), LCC-ICCG (blue, PDB ID: 7VVE), TfCut-DM (green, PDB ID: 7XTT), and IsPETase (pink, PDB ID: 7XTW).

Fig. 4. Comparison of binding poses of MHET in WT TfCut and TfCut-DM.

a The superimposed active site clefts of MHET-bound complex of WT TfCut (PDB ID, 7XTV), LCC-ICCG (PDB ID: 7VVE), TfCut-DM (PDB ID, 7XTT), and IsPETase (PDB ID: 7XTW). The MHET-binding pockets are displayed with surface representation, and the dashed circles highlight the location of H224 and F228. b Comparison of binding modes of MHET in WT TfCut (red) and TfCut-DM (green). The selected residues are shown as sticks.

Fig. 5. TfCut-catalyzed PBAT degradation.

a The predicted binding mode of the polymer chain of PBAT in the active site tunnel of TfCut-DM. The polymer chain is drawn as BABTaB (cyan sticks). The catalytic triad, W195, and DM are colored in yellow, magenta, and blue, respectively. The zoom in figure is shown on the right side. The interaction network of BABTaB with surrounding residues in TfCut-DM is shown in magenta sticks and gray dash lines (<3.5 Å). The catalytic triad residues are shown in green sticks and the interactions are shown with orange dash lines. b The proposed method of TfCut-mediated PBAT degradation, including endo- and exo-cleavage. The representative PBAT polymer is drawn as the repeats of BABTaB and other combinations. The major intermediates that have TPA on the termini are depicted.