Modulation ofIdeonella sakaiensisPETase active site flexibility and activity on morphologically distinct substrates by surface charge engineering

Enzymatic hydrolysis of polyethylene terephthalate (PET) waste is a compelling strategy for environmentally friendly recycling of a major pollutant. Here, we investigate the effects of surface charge point mutations both proximal and distal to the active site of the mesophilic PET-degrading enzyme from Ideonella sakaienses ( Is PETase) and an engineered thermostable variant with superior activity, STAR PETase. The vicinal K95A mutation significantly inhibited Is PETase activity on mechanically processed PET powder. Conversely, this mutation significantly increased hydrolysis of PET powder in the STAR PETase background. Activity of both enzymes on PET film was inhibited by the K95A mutation, highlighting complex interplay between mutation context and substrate morphology. Further installing the distal R132N and R280A surface charge mutations potentiated activity of STAR on all substrates tested. This variant afforded 100% degradation of bottle-grade PET powder in 3 days at 40°C reaction temperature, a 3-fold improvement over Is PETase. Molecular dynamics simulations reveal modulation of active site flexibility in mutants, which differentially impacts both hydrolysis of morphologically distinct PET substrates and the concentration-dependent inhibition phenomenon observed for PETase.