Abstract
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 IsPETase and the thermostable V3 variant with superior activity. The vicinal K95A mutation significantly inhibited IsPETase activity on mechanically processed PET powder. Conversely, this mutation significantly increased hydrolysis of PET powder in the V3 PETase. 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 V3 on all substrates tested. This variant afforded 100 % degradation of pre-processed bottle-grade PET powder in 3 days at 40°C reaction temperature, a 3-fold improvement over IsPETase. Whilst reduction of positive charge on the PETase surface is known to reduce interaction with PET, molecular dynamics simulations suggest this can be offset by context-dependent modulation of active site flexibility, which differentially impacts both hydrolysis of morphologically distinct PET substrates and the concentration-dependent inhibition phenomenon observed for PETase.
Published Version
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