NMR characterisation of an improved PETase: Backbone thermostability and catalytic histidine pKa
Jury composition:
M. Ewen LESCOP, Reviewer, CNRS
Mme Caroline SMET-NOCCA, Reviewer, Université de Lille
Mme Nadia IZADI-PRUNEYRE, Examiner, CNRS
Mme Magali REMAUD-SIMEON, Examiner, INSA
M. Guy LIPPENS, Thesis director, CNRS
M. Cyril CHARLIER, Thesis co-director, CNRS
Abstract:
The global accumulation of plastic, particularly polyethene terephthalate (PET), poses a great environmental challenge. The very properties that make plastics invaluable in almost all areas of our lives, resilience and durability, also represent the core of their persistence and environmental burden. Common recycling methods themselves can represent additional environmental challenges, thus PET-degrading enzymes (PETases) have emerged as promising candidates. The engineered Leaf-branch compost cutinase variant, LCCI-CCG, developed in collaboration between Carbios and the Toulouse Biotechnology Institute, stands out for its industrial potential due to enhanced thermostability and activity. Despite these advances, the mechanistic understanding of PETase function and stability remains incomplete. This thesis applies nuclear magnetic resonance (NMR) spectroscopy to investigate LCC-ICCG with the aim of providing a residue-level understanding. The research is divided into two main parts. The first focuses on thermostability, one of the key properties for industrial applications where high operating temperatures accelerate PET hydrolysis. Here, NMR-derived parameters (1) peak presence in spectra recorded at increasing temperatures, (2) protection from H/D exchange, and (3) HN temperature coefficients, are combined to characterise the backbone hydrogen bond network of LCC-ICCG. They reflect the intricate interplay between hydrogen bonding strength, structural orientation, and solvent accessibility. The second branch addresses the pH dependence of LCC-ICCG. Most PETases exhibit optimal activity under alkaline conditions, yet the molecular basis of this remains poorly understood. NMR pH titrations of histidine residues, with particular focus on the catalytic histidine, are used to determine its pKa and assess its role in enzymatic activity. This work provides a complementary insight into the molecular determinants of PETase function. The results rationalise key features of LCC-ICCG and provide a framework for detailed residue-level analysis of thermostable proteins. Ultimately, these insights could contribute to the further advancement in the rational design of PETases, bringing enzymatic recycling closer to large-scale, sustainable applications.
