Fig. 1. Thermostable mutant DuraMHETase obtained via the GRAPE strategy. (a) The GRAPE strategy comprises three sequential steps: generation of a single-point mutation library, clustering of beneficial mutations, and combination of mutations within each cluster through a greedy approach. (b) The accumulation process of the beneficial mutations using the GRAPE strategy. Mutants were selected in each round based on the MHET hydrolytic activity and thermal stability. MHET hydrolysis activity was determined by measuring the TPA production after subtracting chemical hydrolysis background. Reactions were performed with 40 mmol/L MHET, 39 nmol/L enzyme in 100 mmol/L phosphate buffer (pH = 7.0) for 2 h at 58 °C for the mutations in the 1st cluster and at 60 °C for the mutations in the 2nd and 3rd clusters. Data are from one independent experiment. (c) Time-course hydrolysis of MHET by IsMHETase (wild-type), DuraMHETase (M13), and other intermediate-hydrolyzing enzymes at 60 °C. MHET hydrolysis activity was determined by measuring the TPA production after subtracting chemical hydrolysis background. Reactions were performed with 40 mmol/L MHET, 39 nmol/L enzyme in 100 mmol/L phosphate buffer (pH = 7.0) (Fig. S1) for 8 h at 60 °C. Each experiment was performed in triplicate (n = 3).

Fig. 2. Dual- and fusion-enzyme systems combining DuraMHETase with thermostable PET hydrolases on Gf-PET Films (?8 mm, ~15 mg, 7% crystallinity) (a) Time course comparison of total product release (TPA+MHET+BHET) between the dual-enzyme systems and PETase-only controls. Bar colors represent individual degradation products: TPA (medium purple), MHET (light purple), and BHET (black). Circles represent PETase-only reactions and diamonds represent dual-enzyme reactions, respectively. Reactions were conducted in 50 mmol/L phosphate buffer (pH 8.0) with 30 gPET/kg substrate loading and 0.1 μmol/L enzyme loading of PETases. The molar ratio of PETase to DuraMHETase was 1:0.3. DepoPETase and FastPETase were assayed at 50 °C; PES-H1L92F/Q94Y, HotPETase, LCCICCG, and TurboPETase were assayed at 60 °C. Each experiment was performed in triplicate (n = 3). (b) Degradation of PET film using DT at 60 °C. Reaction were performed with an enzyme loading of 1 μmol/L and a substrate concentration of 30 gPET/kg. Each experiment was performed in triplicate (n = 3). (c) SEM images showing the effects of the fusion enzyme on PET degradation.

Fig. 3. Overall structure and key mutations of DuraMHETase. (a) The Q410F mutation may introduce a π-π stacking interaction between W397, F415, and the aromatic ring of MHET. (b) The overall structure of DuraMHETase, with α-helices colored blue, β-sheets colored purple, and key mutations highlighted in pink. (c) The S413N mutation may enhance the hydrogen bond network involving D276, Q278, N407, and S413. (d) The S136E mutation is suggested to strengthen the hydrogen bond between E136 and N168. (e) The homologous protein AoFaeB is shown as a dimer in its crystal structure, whereas IsMHETase exists as a monomer. The extended loop between residues 320 and 340 in IsMHETase and DuraMHETase may interfere dimerization.

Fig. 4. The exo-PETase and endo-PETase activities of MHETase. (a) Time-course analysis of exo-PETase activity at 60 °C by IsMHETase (wild-type) and DuraMHETase. The PET films were pretreated by TurboPETase to allow for preliminary degradation, generating additional PET chain ends. After incubation, the films were removed from the TurboPETase enzyme solution and immersed in water for 3 h, followed by one methanol wash and three water washes. Subsequently, the dried pretreated films were hydrolyzed by 1 μmol/L MHETases in 50 mmol/L phosphate buffer (pH = 8.0) for 8 h. Exo-PETase activity was determined by measuring the total released products (TPA+MHET+BHET) after subtracting background control values. Each experiment was performed in triplicate (n = 3). (b) Schematic representation of exo-PETase and endo-PETase mechanisms. (c) Endo-PETase activity of well-known intermediate-hydrolyzing enzymes over 24 h at 63 °C. The untreated PET films were hydrolyzed by MHETases using an enzyme loading of 1 μmol/L in 50 mmol/L phosphate buffer (pH = 8.0) at 63 °C for 24 h. Each experiment was performed in triplicate (n = 3).