An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films

Increased interest in poly(ethylene terephthalate) (PET)-degrading enzymes (PETases) have generated efforts to find mutants with improved catalytic activity and thermostability. Here, we present a simple and fast method to determine relative enzyme kinetics through bulk absorbance measurements of released products over time. A thermostable variant of PETase from Ideonella sakaiensis was engineered (R280A S121E D186H N233C S282C) with a denaturation temperature of 69.4 +/- 0.3 degrees C. This was used to assess the method's ability to determine relative enzyme kinetics across variants and reveal structure-function relationships. Measurements at 24 and 72 h at 400 nM of enzyme suggest that the mutations improved catalytic rates 5- to 7-fold. On the contrary, kinetic analyses of the thermostable variant and wild-type reveal different reaction trajectories despite similar maximum catalytic rates, resulting in higher product accumulation from the thermostable variant over time. The results of the assay support the necessity for kinetic measurements to determine relationships between sequence and function for IsPETase and other PET hydrolases.

Automated summary

Increased interest in PET-degrading enzymes has led to efforts to find mutants with improved catalytic activity and thermostability. A simple and fast method for determining relative enzyme kinetics was presented in this study, showing that mutations in the thermostable variant of PETase improved catalytic rates 5- to 7-fold. The results support the necessity of kinetic measurements to determine relationships between sequence and function for PET hydrolases like IsPETase.