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.

Department of Biotechnology and Enzyme Catalysis Institute of Biochemistry University of Greifswald Felix Hausdorff Str 4 17487 Greifswald Germany

Department of Cellular Biochemistry and Metabolomics Institute of Biochemistry University of Greifswald Felix Hausdorff Str 4 17487 Greifswald Germany

Department of Synthetic and Structural Biochemistry Institute of Biochemistry University of Greifswald Felix Hausdorff Str 4 17487 Greifswald Germany

International Clinical Research Center St Anne's University Hospital Pekarska 53 656 91 Brno Czech Republic

Loschmidt Laboratories Department of Experimental Biology and RECETOX Faculty of Science Masaryk University Kamenice 5 C13 625 00 Brno Czech Republic

Macromolecular Crystallography Helmholtz Zentrum Berlin Alber Einstein Straße 15 12489 Berlin Germany

National Technology Innovation Center of Synthetic Biology 32 West 7th Avenue Tianjin Airport Economic Area Tianjin 300308 China

Tianjin Institute of Industrial Biotechnology Chinese Academy of Sciences 32 West 7th Avenue Tianjin Airport Economic Area Tianjin 300308 China

University of Chinese Academy of Sciences 19A Yuquan Road Beijing 100049 China

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