Investigating the Impact of Polymer Length, Attachment Site, and Charge on Enzymatic Activity and Stability of Cellulase.

The thermophilic cellulase Cel5a from Fervidobacterium nodosum (FnCel5a) was conjugated with neutral, cationic, and anionic polymers of increasing molecular weights. The enzymatic activity toward an anionic soluble cellulose derivative, thermal stability, and functional chemical stability of these bioconjugates were investigated. The results suggest that increasing polymer chain length for polymers compatible with the substrate enhances the positive impact of polymer conjugation on enzymatic activity. Activity enhancements of nearly 100% were observed for bioconjugates with N,N-dimethyl acrylamide (DMAm) and N,N-dimethyl acrylamide-2-(N,N-dimethylamino)ethyl methacrylate (DMAm/DMAEMA) due to proposed polymer-substrate compatibility enabled by potential noncovalent interactions. Double conjugation of two functionally distinct polymers to wild-type and mutated FnCel5a using two conjugation methods was achieved. These doubly conjugated bioconjugates exhibited similar thermal stability to the unmodified wild-type enzyme, although enzymatic activity initially gained from conjugation was lost, suggesting that chain length may be a better tool for bioconjugate activity modulation than double conjugation.

Hydrolytically Stable Maleimide-End-Functionalized Polymers for Site-Specific Protein Conjugation.

Site-specific conjugation to cysteines of proteins often uses ester groups to link maleimide or alkene groups to polymers. However, the ester group is susceptible to hydrolysis, potentially losing the benefits gained through bioconjugation. Here, we present a simple conjugation strategy that utilizes the amide bond stability of traditional 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide coupling while introducing site specificity. Hydrolytically stable maleimide-end-functionalized polymers for site-specific conjugation to free cysteines of proteins were synthesized using reversible addition-fragmentation chain-transfer (RAFT) polymerization. The alpha terminus of the polymers was amidated with a furan-protected aminoethyl maleimide using carbodiimide-based chemistry. Finally, the maleimide was exposed by a retro Diels-Alder reaction to yield the maleimide group, allowing for thiol-maleimide click chemistry for bioconjugation. A thermophilic cellulase from Fervidobacterium nodosum (FnCel5a) was conjugated using various strategies, including random 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling, site-specific hydroxyethyl maleimide (HEMI) end-functionalized coupling, hydroxyethyl acrylate (HEA) end-functionalized coupling, and amidoethyl maleimide (AEMI) end-functionalized coupling. Only the polymers conjugated by EDC and AEMI remained conjugated a week after attachment. This indicates that hydrolytically stable amide-based maleimides are an important bioconjugation strategy for conjugates that require long-term stability, while esters are better suited for systems that require debonding of polymers over time.