Thomsen_2024_Enzyme.Microb.Technol_173_110353

Plastic pollution poses a significant environmental challenge, with poly(ethylene terephthalate) (PET) being a major contributor due to its extensive use in single use applications such as plastic bottles and other packaging material. Enzymatic degradation of PET offers a promising solution for PET recycling, but the enzyme kinetics in relation to the degree of crystallinity (X(C)) of the PET substrate are poorly understood. In this study, we investigated the hypersensitive enzyme kinetic response on PET at X(C) from -8.5-12% at 50 degreesC using the benchmark PET hydrolysing enzyme LCC(ICCG). We observed a substantial reduction in the maximal enzymatic reaction rate ((inv)V(max)) with increasing X(C), corresponding to a 3-fold reduction in (inv)V(max) when the X(C) of PET increased from 8.6% to 12.2%. The kinetic analysis revealed that the level of the Mobile Amorphous Fraction (X(MAF)) was a better descriptor for the enzymatic degradation rate response than X(C (or (100%-XC))). By continuous monitoring of the enzymatic reaction progress, we quantified the lag phase prolongation in addition to the steady-state kinetic rates (v(ss)) of the reactions and found that the duration of the lag phase of a reaction could be predicted from the v(ss) and X(C) by multiple linear regression modeling. The linear correlation between the duration of the lag phase and the v(ss) of the enzymatic PET degradation affirmed that the LCC(ICCG) worked via a random/endo-type enzymatic attack pattern. The longer lag phase at increased X(C) of PET is proposed to be due to increased substrate entanglement density as well as unproductive enzyme binding to the crystalline regions of PET. The findings enhance our understanding of PET enzymatic degradation kinetics and its dependence on substrate composition, i.e., X(MAF) and X(C).