A high throughput investigation of the binding specificity of carbohydrate-binding modules for synthetic and natural polymers

Abstract

Carbohydrate-binding modules (CBMs) are non-catalytic domains that enhance enzyme binding to substrates, crucial for polysaccharide degradation. Type A CBMs also show potential for engineering plastic-degrading enzymes due to their high affinity for synthetic polymers. This study presents a high-throughput screening pipeline for characterizing the affinity and specificity of Type A CBMs to the synthetic polymers polyethylene terephthalate (PET), polystyrene (PS), and polyethylene (PE), and the natural polysaccharides cellulose, chitin, and starch. Approximately 800 CBMs from the families CBM2, CBM3, CBM10, and CBM64 were expressed as enhanced green fluorescent protein (EGFP)-fusion proteins and tested for binding using a modified holdup assay, which could produce up to 10,000 data points per day. The screening identified approximately 150 binders for PET and PE, around 250 for PS, and demonstrated family-specific binding patterns for avicel, chitin, and starch. Distinct patterns of substrate affinity and specificity were observed, which allowed for rationalizations of binding at the structural and phylogenetic levels. To demonstrate practical utility, CBMs with high PET affinity were fused to the PET hydrolase LCCICCG, enhancing enzymatic activity on crystalline PET powder by around 5-fold. Importantly, these CBM-enzyme fusions mitigated competitive binding to inert plastic impurities, improving performance in mixed plastic assays. This work significantly expands the known repertoire of CBMs capable of binding synthetic polymers, advances our understanding of CBM-substrate interactions, and provides knowledge for engineering enzymes to tackle plastic pollution, particularly in municipal solid waste where mixed plastics pose significant challenges. Significance StatementImproper recycling of plastic poses a severe environmental threat, with synthetic polymers persisting in ecosystems for decades. Enzymatic recycling of plastic offers a promising solution, with optimization of the enzymes and processes ongoing. To expand the toolkit for engineering enzymes via substrate-binding module fusion, we developed a high-throughput screening platform to identify carbohydrate-binding modules (CBMs) that selectively bind synthetic plastics such as PET, polystyrene, and polyethylene, as well as natural polysaccharides. By screening 797 CBMs, we discovered binding domains with distinct specificities, enabling the design of enzyme fusions that enhance plastic degradation, even in mixed plastic environments. This approach provides insights into protein-substrate interactions that can be leveraged for both waste management and bioengineering innovations.