Structural Insights into (Tere)phthalate-Ester Hydrolysis by a Carboxylesterase and Its Role in Promoting PET Depolymerization

Abstract

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.

An archaeal lid-containing feruloyl-esterase degrades polyethylene terephthalate (PET)

“…Together with the previously obtained BHET and MHET degradation activities, and the fact that PET46 is a FAE, we assume hydrolysis happens at the polymer chain end (exo-activity), where 3PET is quickly cleaved to MHET units, which are subsequently converted to TPA and ethylene glycol (EG). This method of action has been proposed and experimentally shown recently for another 3PET-degrading esterase 45 , although their structures differ substantially (Supplementary Fig. S5d).…”

“…Furthermore, PET46 was faster in degradation of the intermediates BHET and MHET. This makes PET46 a good candidate for evolution experiments towards a possible industrial application 45,69 .…”

Molecular insights into the catalytic mechanism of plasticizer degradation by a monoalkyl phthalate hydrolase

“…They also investigate the catalytic activity of TfCa to DEP (Fig. 1 ), and found that TfCa completely converted DEP to MEP, but cannot further hydrolyze MEP to PA 24 , highlighting the significance of developing PAE hydrolases.…”

“…In recent years, enzymatic depolymerization of plastics has emerged as an alternative technology to conventional mechanical and chemical plastic recycling approaches. Huge success has been achieved on the charicaterization and mechanistic study of several enzymes that depolymerize polyethylene terephthalate (PET) [21][22][23][24] . However, in addition to the production of monomers that form the plastics, PAEs are also released during the polymer hydrolysis process as they are not covalently bound in the plastics.…”

“…They also investigate the catalytic activity of TfCa to DEP (Fig. 1), and found that TfCa completely converted DEP to MEP, but cannot further hydrolyze MEP to PA 24 , highlighting the significance of developing PAE hydrolases. One interesting question is whether or not PAE hydrolases could degrade terephthalate-based mono-and di-esters generated during PET degradation.…”