Cargando…

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

[Image: see text] 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 structur...

Descripción completa

Detalles Bibliográficos
Autores principales: von Haugwitz, Gerlis, Han, Xu, Pfaff, Lara, Li, Qian, Wei, Hongli, Gao, Jian, Methling, Karen, Ao, Yufei, Brack, Yannik, Mican, Jan, Feiler, Christian G., Weiss, Manfred S., Bednar, David, Palm, Gottfried J., Lalk, Michael, Lammers, Michael, Damborsky, Jiri, Weber, Gert, Liu, Weidong, Bornscheuer, Uwe T., Wei, Ren
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2022
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9764356/
https://www.ncbi.nlm.nih.gov/pubmed/36570084
http://dx.doi.org/10.1021/acscatal.2c03772
_version_ 1784853257098625024
author von Haugwitz, Gerlis
Han, Xu
Pfaff, Lara
Li, Qian
Wei, Hongli
Gao, Jian
Methling, Karen
Ao, Yufei
Brack, Yannik
Mican, Jan
Feiler, Christian G.
Weiss, Manfred S.
Bednar, David
Palm, Gottfried J.
Lalk, Michael
Lammers, Michael
Damborsky, Jiri
Weber, Gert
Liu, Weidong
Bornscheuer, Uwe T.
Wei, Ren
author_facet von Haugwitz, Gerlis
Han, Xu
Pfaff, Lara
Li, Qian
Wei, Hongli
Gao, Jian
Methling, Karen
Ao, Yufei
Brack, Yannik
Mican, Jan
Feiler, Christian G.
Weiss, Manfred S.
Bednar, David
Palm, Gottfried J.
Lalk, Michael
Lammers, Michael
Damborsky, Jiri
Weber, Gert
Liu, Weidong
Bornscheuer, Uwe T.
Wei, Ren
author_sort von Haugwitz, Gerlis
collection PubMed
description [Image: see text] 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.
format Online
Article
Text
id pubmed-9764356
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher American Chemical Society
record_format MEDLINE/PubMed
spelling pubmed-97643562022-12-21 Structural Insights into (Tere)phthalate-Ester Hydrolysis by a Carboxylesterase and Its Role in Promoting PET Depolymerization von Haugwitz, Gerlis Han, Xu Pfaff, Lara Li, Qian Wei, Hongli Gao, Jian Methling, Karen Ao, Yufei Brack, Yannik Mican, Jan Feiler, Christian G. Weiss, Manfred S. Bednar, David Palm, Gottfried J. Lalk, Michael Lammers, Michael Damborsky, Jiri Weber, Gert Liu, Weidong Bornscheuer, Uwe T. Wei, Ren ACS Catal [Image: see text] 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. American Chemical Society 2022-11-29 2022-12-16 /pmc/articles/PMC9764356/ /pubmed/36570084 http://dx.doi.org/10.1021/acscatal.2c03772 Text en © 2022 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle von Haugwitz, Gerlis
Han, Xu
Pfaff, Lara
Li, Qian
Wei, Hongli
Gao, Jian
Methling, Karen
Ao, Yufei
Brack, Yannik
Mican, Jan
Feiler, Christian G.
Weiss, Manfred S.
Bednar, David
Palm, Gottfried J.
Lalk, Michael
Lammers, Michael
Damborsky, Jiri
Weber, Gert
Liu, Weidong
Bornscheuer, Uwe T.
Wei, Ren
Structural Insights into (Tere)phthalate-Ester Hydrolysis by a Carboxylesterase and Its Role in Promoting PET Depolymerization
title Structural Insights into (Tere)phthalate-Ester Hydrolysis by a Carboxylesterase and Its Role in Promoting PET Depolymerization
title_full Structural Insights into (Tere)phthalate-Ester Hydrolysis by a Carboxylesterase and Its Role in Promoting PET Depolymerization
title_fullStr Structural Insights into (Tere)phthalate-Ester Hydrolysis by a Carboxylesterase and Its Role in Promoting PET Depolymerization
title_full_unstemmed Structural Insights into (Tere)phthalate-Ester Hydrolysis by a Carboxylesterase and Its Role in Promoting PET Depolymerization
title_short Structural Insights into (Tere)phthalate-Ester Hydrolysis by a Carboxylesterase and Its Role in Promoting PET Depolymerization
title_sort structural insights into (tere)phthalate-ester hydrolysis by a carboxylesterase and its role in promoting pet depolymerization
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9764356/
https://www.ncbi.nlm.nih.gov/pubmed/36570084
http://dx.doi.org/10.1021/acscatal.2c03772
work_keys_str_mv AT vonhaugwitzgerlis structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT hanxu structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT pfafflara structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT liqian structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT weihongli structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT gaojian structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT methlingkaren structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT aoyufei structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT brackyannik structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT micanjan structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT feilerchristiang structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT weissmanfreds structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT bednardavid structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT palmgottfriedj structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT lalkmichael structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT lammersmichael structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT damborskyjiri structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT webergert structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT liuweidong structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT bornscheueruwet structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization
AT weiren structuralinsightsintoterephthalateesterhydrolysisbyacarboxylesteraseanditsroleinpromotingpetdepolymerization