Polymer Conjugation to Enhance Cellulase Activity and Preserve Thermal and Functional Stability

Abstract

A thermophilic cellulase, FnCel5a, from Fervidobacterium nodosum was conjugated with various functional polymers including cationic, anionic, and strongly and weakly hydrogen bonding polymers. The activity of FnCel5a toward a high-molecular-weight carboxymethyl cellulose substrate was enhanced by polymer conjugation. Activity enhancements of 50% or greater observed for acrylamide and mixed N,N-dimethyl acrylamide-2-(N,N-dimethylamino)ethyl methacrylate polymers, suggesting that the greatest enhancements were caused by polymers capable of noncovalent interactions with the substrate. The conjugates were found to have nearly identical thermodynamic stability to the native enzyme, as assessed by free energy (ΔG), enthalpy (ΔH), and entropy (TΔS) parameters extracted from differential scanning fluorimetry. Polymers tended to confer comparable tolerance to high concentrations of dimethylformamide, with longer polymers typically enabling higher activity relative to shorter polymers. The new FnCel5a conjugates represent an advance in the production of cellulases that maintain activity at high temperatures or in the presence of denaturing organic solvents.

Polymer conjugation of proteins as a synthetic post-translational modification to impact their stability and activity

“…64 In addition to conjugation confirmation, the purity of the bioconjugate should be considered. Researchers have used techniques such as filtration 65 , dialysis 66,67 , and chromatography 23,68 to remove unreacted polymers.…”

“…141,142 In the presence of Page and coworkers also attempted to improve the activity of cellulase through the covalent attachment of acrylamide-and dimethyl acrylamide-based polymers to the FnCel5a enzyme. 65 FnCel5a is a thermophilic cellulase that efficiently degrades cellulose at an optimum temperature of 80 °C and pH 5. 143 The group tested activity of FnCel5a modified with nonionic poly(acrylamide) (Am) and poly(N,N-dimethyl acrylamide) (DMAm) chains and ionic poly(N,N-dimethyl acrylamide -acrylic acid) (DMAm/AA) and poly(dimethyl acrylamide -2-(N,N-dimethylamino) ethyl methacrylate) (DMAm/DMAEMA) using carboxymethylcelluose (CMC).…”

“…As mentioned before, Page and coworkers found that functional groups inherent to polymer chains can be effective in increasing activity if they are complementary to the target substrate. 65 The group also studied the effect of polymer conjugation on thermal, chemical, and thermodynamic stability using differential scanning fluorimetry (DSF) and studying activity following denaturation by DMF. DSF was used to determine thermal and thermodynamic stability of the conjugates when compared to the native enzyme, showing similar T m values for all samples.…”

“…Reprinted with permission. 65 Copyright 2017 American Chemical Society. Activity of native and polymer-conjugated lipase.…”

Photochemistry for Well‐Defined Polymers in Aqueous Media: From Fundamentals to Polymer Nanoparticles to Bioconjugates

“…In addition, the development of efficient metal‐free and other photocatalytic RAFT systems will likely lead to efficient aqueous polymerization under biologically applicable conditions. These research topics are anticipated to be a focus area in the future as the bioconjugate field moves toward industrially relevant enzymatic proteins and bioconjugates with biomedical applications …”