posted on 2022-11-29, 18:40authored byGerlis von Haugwitz, Xu Han, Lara Pfaff, Qian Li, Hongli Wei, Jian Gao, Karen Methling, Yufei Ao, Yannik Brack, Jan Mican, Christian G. Feiler, Manfred S. Weiss, David Bednar, Gottfried J. Palm, Michael Lalk, Michael Lammers, Jiri Damborsky, Gert Weber, Weidong Liu, Uwe T. Bornscheuer, Ren Wei
TfCa, a promiscuous carboxylesterase from Thermobifida
fusca, was found to hydrolyze polyethylene terephthalate
(PET) degradation intermediates such as bis(2-hydroxyethyl) terephthalate
(BHET) and mono-(2-hydroxyethyl)-terephthalate (MHET). In this study,
we elucidated the structures of TfCa in its apo form, as well as in
complex with a PET monomer analogue and with BHET. The structure–function
relationship of TfCa was investigated by comparing its hydrolytic
activity on various ortho- and para-phthalate esters of different
lengths. Structure-guided rational engineering of amino acid residues
in the substrate-binding pocket resulted in the TfCa variant I69W/V376A
(WA), which showed 2.6-fold and 3.3-fold higher hydrolytic activity
on MHET and BHET, respectively, than the wild-type enzyme. TfCa or
its WA variant was mixed with a mesophilic PET depolymerizing enzyme
variant [Ideonella sakaiensis PETase
(IsPETase) PM] to degrade PET substrates of various
crystallinity. The dual enzyme system with the wild-type TfCa or its
WA variant produced up to 11-fold and 14-fold more terephthalate (TPA)
than the single IsPETase PM, respectively. In comparison
to the recently published chimeric fusion protein of IsPETase and MHETase, our system requires 10% IsPETase
and one-fourth of the reaction time to yield the same amount of TPA
under similar PET degradation conditions. Our simple dual enzyme system
reveals further advantages in terms of cost-effectiveness and catalytic
efficiency since it does not require time-consuming and expensive
cross-linking and immobilization approaches.