The role of binding modules in enzymatic poly(ethylene terephthalate) hydrolysis at high-solids loadings

Abstract

In nature, enzymes that deconstruct biological polymers, such as cellulose and chitin, often exhibit multi-domain architectures, comprising a catalytic domain and a non-catalytic binding module; the latter serves to increase the enzyme concentration at the substrate surface. This multi-domain architecture has been shown to improve the hydrolysis of poly(ethylene terephthalate) (PET) using engineered cutinase enzymes. Here, we examine the role of accessory binding modules at industrially relevant PET solids loadings necessary for cost-effective enzymatic recycling. Using a thermostable variant of leaf compost cutinase (LCC), we produced synthetic fusion constructs of LCC with five type A carbohydrate-binding modules (CBMs). At solids loadings below 10 wt %, the CBMs improve aromatic monomer yield from PET, but above this threshold, conversion extents up to 97% are reached with no added benefits from the presence of CBM fusions. This suggests that fusion constructs with the herein studied CBMs are not necessary for industrial enzymatic PET recycling. • A new variant of LCC exhibits higher thermostability and greater PET turnover • Specific CBMs stimulate PET hydrolysis at low-solids loading • The advantage of these CBMs is lost at industrially relevant high-solids loading Closed-loop recycling and open-loop upcycling of waste plastics represent a suite of critical technologies to address the plastic-waste pollution crisis. Enzymatic deconstruction of the synthetic polyester poly(ethylene terephthalate) (PET) is among the several options available for chemical recycling of this common plastic. While early demonstrations of enzymatic PET recycling have shown feasibility, the technology is not yet cost competitive with production of virgin petroleum-derived PET. This study analyzes an enzyme engineering approach that takes a lesson from nature to improve the catalytic efficiency of PET-hydrolyzing enzymes and evaluates the impact of this strategy under reaction conditions that simulate a key industrial feature of enzymatic PET recycling. The implications of this study can help focus the resources available to the field on solutions that can enable rapid deployment of viable technologies to realistically address plastics pollution. Enzymatic recycling of PET offers a promising solution to the global plastic problem. Research into accessory domains to enhance PET turnover has so far not been tested at high PET solids loadings. Here, we show that carbohydrate-binding domains do not offer any benefit at high PET solids loadings that are relevant to industrial-scale biorecycling.