Counter-intuitive enhancement of degradation of solid plastic through
engineering of lowered enzyme binding to solid plastic
Abstract
Degradation of solid polyethylene terephthalate (PET) by leaf branch
compost cutinase (LCC) produces various PET-derived degradation
intermediates (DIs), in addition to terephthalic acid (TPA), which is
the recyclable terminal product of all PET degradation. Although DIs can
also be converted into TPA, in solution, by LCC, the TPA that is
obtained through enzymatic degradation of PET, in practice, is always
contaminated by DIs. Here, we demonstrate that the primary reason for
non-degradation of DIs into TPA in solution is the efficient binding of
LCC onto the surface of solid PET. Although such binding enhances the
degradation of solid PET, it depletes the surrounding solution of enzyme
that could otherwise have converted DIs into TPA. To retain a
sub-population of enzyme in solution that would mainly degrade DIs, we
introduced mutations to reduce the hydrophobicity of areas surrounding
LCC’s active site, with the express intention of reducing LCC’s binding
to solid PET. Despite the consequent reduction in invasion and
degradation of solid PET, overall levels of production of TPA were
~3.6-fold higher, due to the partitioning of enzyme
between solid PET and the surrounding solution, and the consequent
heightened production of TPA from DIs. Further, synergy between such
mutated LCC (F125L/F243I LCC) and wild-type LCC resulted in even higher
yields, and TPA of nearly ~100% purity.