Fig. 3: Substrate selectivity varies across PET morphologies.

A Heat map profiles of pH and temperature screening for hydrolytic activity on 3 PET substrate morphologies, the same amorphous PET film presented in Fig. 2, as well as an amorphous PET powder and a crystalline PET powder, using a subset of 9 candidate enzymes and positive control enzyme, LCC ICCG. The heat map gradient indicates extent of measured product release up to 500 mg/L of total aromatic products after 96 h reaction time, and is reported as the average of reactions performed in triplicate (n = 3). Each heat map displays the reaction conditions utilized (citrate at pH 6.0, NaH₂PO₄ at pH 7.0, NaH₂PO₄ at pH 7.5, HEPES (H) at pH 7.5, bicine at pH 8.0, and glycine at pH 9.0), and reaction temperature (30, 40, 50, 60, or 70 °C). The heat maps for all other enzymes tested on the 3 PET substrate morphologies are shown in Supplementary Fig. 6. Source data are provided the a Source Data file. B Log-plot of the sum of aromatic products measured after 168 h reaction time using amorphous PET film (aFilm, black squares), crystalline PET powder (cryPow, open circles) and amorphous PET powder (aPow, gray circles) as substrates. Reaction conditions used for time course experiments correspond to the pH and temperature resulting in the highest product release observed in amorphous PET film screening reactions, which are listed in Supplementary Table 13. Ratios of product release observed from hydrolysis reactions for each PET substrate morphology pairwise comparison, demonstrating differences in substrate selectivity for each selected enzyme is presented in Supplementary Fig. 9. For all enzymatic reactions shown in A, B, the enzyme loading was 0.7 mg enzyme/g PET and the solids loading was 2.9% (29  g/L). The reaction products were quantified with UHPLC, and the results show the sum of aromatic products, including BHET, MHET, and TPA. All reactions were conducted in triplicate (n = 3). Error bars represent standard deviation and are centered on the average of the three reaction measurements. Source data are provided as a Source Data file.