Fig. 1. Spectrophotometric screening of PET depolymerization activity. (a) Spectrophotometry of TPA. (b) Positive correlation between absorbance at 290 nm (OD290) and TPA concentrations. (c) Absorbance of the LCC reaction mixtures at 290 nm, compared with the control reactions in the absence of LCC enzyme or PET powder substrate (crystallinity 42.0 ± 3.1%). The reactions were carried out at 72 °C for 36 h. (d) Workflow for the directed evolution of WCCG in Escherichia coli.

Fig. 2. Structure-guided directed evolution of WCCG. (a) The interactions between 2-HE(MHET)4 and W243 in WCCG. The 2-HE(MHET)4 is colored in green, W243 is shown in stick representation, the magenta dashed lines indicate π-π stacking interactions. (b) Structural model of 2-HE(MHET)4 docked in ICCG. (c) The identified 24 amino acid residues within 5 ? to the substrate according to the WCCG-2-HE(MHET)4 complex. The 24 amino acid residues were shown as sticks style in magentas. The catalytic residues were shown as sticks style in red. (d) Results of iterative site-saturation mutagenesis of WCCG for improved PET depolymerization. WCCG was shown as black sphere; Mutantions obtained from Round 1, 2, 3, 4, 5, 6 and FireProt-predicting mutagenesis were shown as grey, blue, green, orange, cyan, purple and red spheres, respectively. The order of mutation was indicated by black arrows. The arrows started from WCCG, and pointed to the best mutation in the next round. The mutants with the highest activity obtained from Round 1, 2, 3, 4, 5, 6 and FireProt-predicting mutagenesis were marked as M1, M2, M3, M4, M5, M6 and WCCG-sup1, respectively. And the amino acid substitutions of these mutants were shown in the table. The reaction was carried out at 72 °C for 36 h, with 30 mg PET powder substrate (crystallinity 42.0 ± 3.1%).

Fig. 3. Characterization of PET depolymerization activity of the obtained mutants. Temperature (a) and pH (b) dependence of WCCG and its mutants (enzyme concertration 25 μg mL-1) on PET powder with 42.0 ± 3.1% crystallinity for 5 h. Time courses of total products released from depolymerization of PET powder with 42.0 ± 3.1% crystallinity (c) or gf-PET powder (d) by various mutants (enzyme concertration 0.4 μg mL-1) performed at 72 °C, pH 8.0. (e) Enzymatic degradation of films of commercial PET materials or gf-PET by various depolymerase mutants (enzyme concertration 5.54 μg mL-1) for 36 h, performed at respective optimum conditions. (f) Degradation of different synthetic polymers by various depolymerase mutants (enzyme concertration 5.54 μg mL-1) for 36 h at respective optimum conditions. PET was degraded to TPA and small amounts of MHET. Polybutylene terephthalate (PBT) was degraded to TPA and small amounts of mono(4-hydroxybutyl)-TPA (MHBT). TPEE was degraded to TPA, small amounts of MHET and MHBT. No bis(4-hydroxybutyl)-TPA (BHBT) or BHET was observed. The reactions were performed at 72 °C (for WCCG, ICCG and their mutants), 75 °C (for LCCICCG_I6M) or 50 °C (for FAST-PETase).

Fig. 4. Complete depolymerization of a piece of unpretreated PET container. (a) Time course of degradation of a piece of unpretreated PET container (length 11.7 cm, width 7.8 cm, weight 2.5 g, crystallinity 16.1 ± 0.2%) with 7.5 mg of WCCG-sup1, ICCG, or FAST-PETase, determined with HPLC. (b) Morphological changes and (c) Scanning electron microscope images for depolymerization of a piece of unpretreated PET container following various exposure times in WCCG-sup1 degradation.

Fig. 5. Structure analysis of amino acid substitutions. (a,b) Location of substitutions in the overall structure of WCCG-sup1 docked with 2-HE (MHET)4 (cyan). The WCCG-sup1 structure is shown as cartoon (green), while the Cα atoms of the eight substitutions (G88A, T96S, F125T, S136Q, N239L, S241D, N246M and S247R) are shown as magenta atoms. The key residues are shown in stick representations. (c-i) Structural effects of substitutions of N246M (c), N239L (d), T96S (e), F125T (f), G88A (g), S136Q (h) and S241D/S247R (i) of WCCG-sup1 (green) compared to WCCG (orange). The green dashed lines indicate hydrogen bonds, the yellow dashed lines indicate electrostatic interactions, and the pink dashed lines indicate hydrophobic interactions.

Fig. 6. Structural model of 2-HE(MHET)4 docked in WCCG-sup1 (a), WCCG-sup2 (b), and WCCG (c). The proteins were shown in surface, mutation sites were shown as sticks style in oranges, residue 247 was colored in magentas, and the catalytic residues were shown as sticks style in blue. The 2-HE(MHET)4 was showed as stick model in cyans, W243 was shown in stick model in line.

Fig. 7. Enzyme-PET binding assay. The adsorption of WCCG (a,d), WCCG-sup1 (b,e) and WCCG-sup2 (c,f) to unpretreated PET (42.0 ± 3.1% crystallinity) (a-c) and gf-PET (d-f) monitored by Zeiss Axio Image A2. (g) The adsorption rate of WCCG, WCCG-sup1 and WCCG-sup2 to unpretreated PET and gf-PET monitored by fluorescence analysis.