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PreprintCC BY-NC-NDChemRxiv

Stabilization and Functionalization of an Enzyme Complex via Modular In Situ Conjugation

  • Juan Lizandra Pérez1
  • George H. Hutchins1,2
  • Rasmus Klintrot1
  • Pascal Poc1
  • Leonardo Romão Soares1
  • Ivana Drienovska1
  • Sven Hennig1,3
  • Saskia Neubacher2
  • Tom N. Grossmann1,3
  1. 1Department of Chemistry and Pharmaceutical Sciences, VU University Amsterdam, De Boelelaan 1108, Amsterdam, Netherlands, 1081 HZ
  2. 2Incircular B.V., De Boelelaan 1108, Amsterdam, Netherlands, 1081 HZ
  3. 3Institute of Organic and Biomolecular Chemistry, Georg-August-Universität Göttingen, Tammannstr. 2, Göttingen, Germany, 37077
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Abstract

Enzymes are unparalleled catalysts that combine high activity and selectivity with operation under mild conditions. Despite these advantages, their broader use as industrial biocatalysts is limited by insufficient stability under process-relevant conditions and by the difficulty of achieving site-specific chemical functionalization. Simultaneously enhancing protein stability and enabling site-specific functionalization through a single chemical strategy remains a central challenge, particularly, when addressing multimeric enzymes. Here, we show that a four-armed crosslinker with tetrahedral symmetry enables simultaneous stabilization and programmable functionalization of the tetrameric enzyme borneol dehydrogenase. A twostep crosslinking–modification reaction yields a well-defined and stable protein quaternary architecture with an incorporated reactive handle, enabling the installation of diverse functionalities, including bioorthogonal tags and functional groups for immobilization. The chemically engineered bicyclic enzyme exhibits remarkably enhanced thermal stability and resistance to organic cosolvents, which results in exceptional longevity supporting ten weeks of continuous flow operation. Unlike previous methods that decouple stabilization from modification, this strategy unites structural reinforcement and functionalization, thereby expanding the scope of chemical protein engineering.

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Cite

BibTeXRIS
BibTeX
@misc{perez2026stabilization,
  title = {{Stabilization and Functionalization of an Enzyme Complex via Modular In Situ Conjugation}},
  author = {Juan Lizandra Pérez and George H. Hutchins and Rasmus Klintrot and Pascal Poc and Leonardo Romão Soares and Ivana Drienovska and Sven Hennig and Saskia Neubacher and Tom N. Grossmann},
  howpublished = {ChemRxiv},
  year = {2026},
  month = sep,
  doi = {10.26434/chemrxiv.15008156/v1},
  url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15008156/v1},
  note = {CC BY-NC-ND},
}
RIS
TY  - UNPB
TI  - Stabilization and Functionalization of an Enzyme Complex via Modular In Situ Conjugation
AU  - Juan Lizandra Pérez
AU  - George H. Hutchins
AU  - Rasmus Klintrot
AU  - Pascal Poc
AU  - Leonardo Romão Soares
AU  - Ivana Drienovska
AU  - Sven Hennig
AU  - Saskia Neubacher
AU  - Tom N. Grossmann
PY  - 2026
DA  - 2026/09/01
PB  - ChemRxiv
DO  - 10.26434/chemrxiv.15008156/v1
UR  - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15008156/v1
AB  - Enzymes are unparalleled catalysts that combine high activity and selectivity with operation under mild conditions. Despite these advantages, their broader use as industrial biocatalysts is limited by insufficient stability under process-relevant conditions and by the difficulty of achieving site-specific chemical functionalization. Simultaneously enhancing protein stability and enabling site-specific functionalization through a single chemical strategy remains a central challenge, particularly, when addressing multimeric enzymes. Here, we show that a four-armed crosslinker with tetrahedral symmetry enables simultaneous stabilization and programmable functionalization of the tetrameric enzyme borneol dehydrogenase. A twostep crosslinking–modification reaction yields a well-defined and stable protein quaternary architecture with an incorporated reactive handle, enabling the installation of diverse functionalities, including bioorthogonal tags and functional groups for immobilization. The chemically engineered bicyclic enzyme exhibits remarkably enhanced thermal stability and resistance to organic cosolvents, which results in exceptional longevity supporting ten weeks of continuous flow operation. Unlike previous methods that decouple stabilization from modification, this strategy unites structural reinforcement and functionalization, thereby expanding the scope of chemical protein engineering.
ER  -