Evaluating the effectiveness of a lysine mutation and its portability across different Poly(ethylene terephthalate)-hydrolyzing enzymes

This study investigates the impact of a specific mutation, designated as the lysine mutation (N233K), on the hydrolytic activity and thermostability of Poly(ethylene terephthalate)-hydrolyzing enzymes (PHEs). This mutation was originally predicted in our prior study using the wild-type I. sakaiensis PETase as a scaffold as a mutation that potentially increases hydrolytic activity of the enzyme. To demonstrate the lysine mutation portability across different PHEs, six PHEs, each containing a mutation equivalent to N233K in PETase, were tested and exhibited improved terephthalic acid (TPA) and mono-(2-hydroxyethyl) terephthalate acid (MHET) monomers release, ranging from 1.05-fold to 5.88-fold relative to the unmutated PHEs. PHL7^R205K showed a total of 16.3 mM TPA and MHET monomers release from a plastic disc (at 40 ºC, 72 h) which was 7.1-fold to 18.7-fold higher than all other tested lysine mutants. Finally, kinetic modeling was performed under ambient temperature conditions for FAST-PETase and Z1-PETase^C233K, enabling a benchmark for selecting enzymes for environment remediation and in vivo PET biorecycling.