[1S03e] 06. Enzyme Engineering/Enzymology - Part 3

5:25 PM - 5:40 PM JST(8:25 AM - 8:40 AM UTC)

[1ENZ-16] Tuning aromatic polyester specificity of a thermostable PET hydrolase by PETase-inspired mutations

Min Fey Chek¹, Emi Kawano¹, Rikako Sanuki¹, Kengo Nishimura², Shin-ichi Hachisuka¹, Toshio Hakoshima¹, ○Shosuke Yoshida¹ (1. Nara Institute of Science and Technology (Japan), 2. Corporate Research Center, TOYOBO CO., LTD. (Japan))

Keywords:

PET,biodegradation,PETase,LCC,PEF

Enzymatic depolymerization of aromatic polyesters offers a promising route for sustainable plastic recycling, but the molecular determinants governing substrate specificity remain incompletely understood. PETase from Piscinibacter sakaiensis shows high activity and specificity toward poly(ethylene terephthalate) (PET) under mesophilic conditions, whereas the thermostable leaf-branch compost cutinase (LCC) is better suited for reactions at elevated temperatures. Here, we examined whether PETase-specific substrate-recognition features could be introduced into LCC to tune polyester specificity in a thermostable enzyme scaffold.Structure-guided comparison of PETase and LCC identified four non-conserved residues in the active-site region. Introduction of PETase-inspired substitutions into LCC altered substrate preference, and several variants displayed enhanced specificity toward PET, indicating that PETase-type residues contribute directly to aromatic polyester recognition. We further examined hydrolysis of poly(ethylene-2,5-furandicarboxylate) (PEF), a bio-based alternative to PET, and found that PETase shows substantially higher selectivity for PEF over PET than LCC. Remarkably, the LCC mutant H164W exhibited PETase-like behavior, showing greatly enhanced PEF hydrolysis while retaining PET-hydrolyzing activity.To clarify the structural basis of this shift in specificity, we determined the crystal structure of PETase in complex with bis(2-hydroxyethyl) furan-2,5-dicarboxylate (BHEF), a model moiety of PEF. The structure suggested an alternative catalytic mode involving cleavage at subsite IIa, and docking simulations supported that the H164W substitution facilitates favorable binding of the furan-based substrate in the LCC active site.