A High-Throughput Investigation of the Binding Specificity of Carbohydrate-Binding Modules for Synthetic and Natural...

Carbohydrate-binding modules (CBMs) are noncatalytic domains that enhance enzyme binding to substrates. Type A CBMs show potential for engineering plastic-degrading enzymes due to their affinity for synthetic polymers. This study presents a high-throughput screening pipeline for characterizing the affinity and specificity of type A CBMs towards the synthetic polymers polyethylene terephthalate (PET), polystyrene (PS), and polyethylene (PE), and the polysaccharides cellulose, chitin, and starch. ≈800 CBMs from the families CBM2, CBM3, CBM10, and CBM64 are expressed as green fluorescent protein (GFP)-fusion proteins and tested for binding using a modified holdup assay, which produced up to 10 000 data points per day. The screening identifies ≈150 binders for PET and PE, around 250 for PS, and demonstrates family-specific binding patterns for avicel, chitin, and starch. To demonstrate practical utility, four CBMs with high PET affinity are fused to the PET hydrolase LCC^ICCG, enhancing activity on PET powder by around 5-fold. These CBM-enzyme fusions mitigate competitive binding to plastic impurities, improving performance in mixed plastic assays. This work significantly expands the repertoire of CBMs binding to synthetic polymers, advances our understanding of CBM-substrate interactions, and provides knowledge for engineering enzymes to tackle plastic pollution, particularly where mixed plastics pose significant challenges.