## 3 Results and Discussion
### Product Profile and Activity of IsPETase and Trx-IsPETase
The apparent inactivation rate constant (\(a_{S}^{app}\)) was determined according to first-order kinetics with the coexistence of a sufficient substrate (20 mg PET per 2 mL) [56]:  
\[\frac{[P]}{[E]}=\int_{0}^{t}k_{S}^{app}dt=\ \frac{k_{S,0}^{app}}{a_{S}^{app}} \left(1-e^{(-a_{S}^{app}t)}\right)\]  
where \([P]\) is the degradation products concentration, \([E]\) is the enzyme concentration, and \(k_{S,0}^{app}\) and \(k_{s}^{app}\) are the initial enzyme activity in a given time (\(t\)). The integrate form describes the apparent saturation curve (Figure 4). Thus, \(a_{S}^{app}\) yielded 0.09 h\({}^{-1}\), and \(k_{S,0}^{app}\) yielded 6.2 mg Degradation products. Figure 3: Analysis of the products of the _Is_PETase and Trx-_Is_PETase activity on PET. (**A**) Both enzymes have the same product profile, where the main product is MHET, followed by TPA and a small fraction of BHET. (**B**) LC-MS analysis confirming the identity of TPA, MHET, and BHET. (**C**) Production of degradation products after 72 h. Trx-_Is_PETase showed higher activity than _Is_PETase for an independent samples student’s \(t\)-test where (*) \(p<0.05\) and (**) \(p<0.01\) (also see Table 2). We noticed that the enzymatic reactions apparently showed an impact on the crystallinity (or re-crystallization rate) of the residual PET, as indicated by the DSC results (Figure 5). The DSC analysis showed a cold crystallization peak at 122 \({}^{\circ}\)C in all three enzymatically treated PET samples, while the control did not (Figure 5). The measured crystallinity of the residual PET after enzymatic reaction (~22%) or the control (30.5%) was higher than that of the initial PET powder sample (~11%). This suggests that PET, prepared by precipitation, could re-crystallize under the reaction conditions. Thus, assuming that the Trx-_Is_PETase primarily attacks amorphous regions, it may retard the re-crystallization rate. We should emphasize that the observed change of crystallinity of the PET was likely a complex consequence including the cold re-crystallization process under the reaction conditions, selectivity of the enzymatic degradation of the crystalline and amorphous regions, and the possible influence of the enzyme on the rate of re-crystallization. More thorough investigations on the crystallization processes will be needed to allow for deeper insight into the impact of enzymes on the crystallization rate and crystallinity of PET. On the other hand, the residual weight of the enzymatically treated PET corresponded to 34 \(\pm\) 5% degradation at 235 h, consistent with the percentage of degradation calculated by the HPLC method at 72 h (Table 2), considering that the reactions reached saturation at the maximum product formation at 72 h.  
Figure 4: Apparent saturation curve of the PET depolymerization activity of Trx-_Is_PETase. Figure 5: DSC diagrams of PET samples treated with Trx-_Is_PETase (R1, R2, and R3 denote three independent reactions) and the control without the enzyme. Notice that all enzymatic reactions produced a cold crystallization peak (T\({}_{\mathrm{c}}\)) at 122 \({}^{\circ}\)C, while the control did not.