Truncation of an esterase enhances the hydrolysis on polyesters: Less is more

Polymers, and especially polyesters, frequently used for one-way applications like packaging are preferably biodegradable, albeit non-biodegradable polyesters are mostly used, namely polyethylene terephthalate (PET). Biodegradability of the aliphatic/aromatic copolyester poly(butylene adipate-co- terephthalate) (PBAT) has been investigated, showing biological decomposability under composting conditions. However, little is known about its anaerobic hydrolysis, while large amounts of food packaging end up in biogas plants. Two different enzymes belonging to the carboxylesterase superfamily from Clostridium botulinum (Cbotu_EstA) and from Pelosinus fermentans, were reported to actively hydrolyze PBAT, while they failed to act on PET. Yet, enzymes would allow mild decomposition of widely used PET enabling recycling of the monomeric building blocks. The enhancement of the hydrolase activity with regard to polyester hydrolysis can be achieved by fusion of hydrophobic domains, improving the biocatalyst adsorption on the hydrophobic polymer surface, or by substitution of specific residues, enlarging the active site of the enzyme. Interestingly, analysis of the 3D structure of Cbotu_EstA revealed the presence of an extra domain at the N-terminus of the enzyme which covered the lid structure and a hydrophobic patch. The deletion of the Cbotu_EstA N-terminal domain can satisfactory combine the enlarging of the active site and the presence of hydrophobic domain on the surface of the enzyme for improved sorption properties. Surface engineering successfully produced a highly active Cbotu_EstA variant (del71Cbotu_EstA) which was able to hydrolyze PET. Truncation of the N-terminal domain of Cbotu_EstA improved the adsorption of the enzyme on hydrophobic polyester surfaces and enhanced their hydrolysis eight times more compared to the wild-type enzyme, based on released monomers quantification.