Thermobifida fusca cutinase (TfCut2) is a carboxylesterase (CE) which degrades polyethylene terephthalate (PET) as well as its degradation intermediates [such as oligoethylene terephthalate (OET), or bis-/mono-hydroxyethyl terephthalate (BHET/MHET)] into terephthalic acid (TPA). Comparisons of the surfaces of certain CEs (including TfCut2) were combined with docking and molecular dynamics simulations involving 2HE-(MHET) 3, a three-terephthalate OET, to support the rational design of 22 variants with potential for improved generation of TPA from PET, comprising 15 single mutants (D12L, E47F,
Thermobifida fusca cutinase ( TfC ) is a
carboxylesterase (CE) that degrades the environmental pollutant,
polyethylene terephthalate (PET). TfC also acts upon PET’s
degradation intermediates (DIs), such as oligoethylene terephthalate
(OET), and bis-/mono-hydroxyethyl terephthalate (BHET/MHET), to convert
these into terephthalic acid (TPA), the terminal product of PET
degradation. We examined TfC’s surface, compared it to that of
other CEs, and performed molecular docking and MD simulations with an
OET, 2HE-(MHET) , to understand interactions between
TfC’s surface and the OET, at TfC’s active site as well as
vicinal regions. We mutated 17 residues on TfC’s surface, mostly
individually, but sometimes using pairs of mutations, to see how these
modulate TfC’s activity. Most mutants/variants showed a decrease
in activity against solid PET. Some killed activity completely. However,
three mutations (L90F, F209I and F249R), made using a background
mutation (G62A) already reported to improve activity by almost
~2.0-fold, yielded increases in activity that were
between ~1.3- and ~2.0-fold higher than
that of G62A TfC (which we found to display a
~1.7-fold increase in activity over TfC, in our
own experiments). TfC variants, G62A/F249R, and G62A/F209I,
exhibit the highest activities yet observed in any TfC
mutants/variants, against PET, and BHET, respectively.
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