Protein-polymer bioconjugation, immobilization, and encapsulation: a comparative review towards applicability, functionality, activity, and stability.

Polymer-based biomaterials have received a lot of attention due to their biomedical, agricultural, and industrial potential. Soluble protein-polymer bioconjugates, immobilized proteins, and encapsulated proteins have been shown to tune enzymatic activity, improved pharmacokinetic ability, increased chemical and thermal stability, stimuli responsiveness, and introduced protein recovery. Controlled polymerization techniques, increased protein-polymer attachment techniques, improved polymer surface grafting techniques, controlled polymersome self-assembly, and sophisticated characterization methods have been utilized for the development of well-defined polymer-based biomaterials. In this review we aim to provide a brief account of the field, compare these methods for engineering biomaterials, provide future directions for the field, and highlight impacts of these forms of bioconjugation.

Tuning Polymer Composition Leads to Activity-Stability Tradeoff in Enzyme-Polymer Conjugates.

Protein-polymer conjugation provides an opportune means to adjust the local environment of proteins and enhance protein stability, performance, and solubility. Although much attention has been focused on tuning protein-polymer interactions, the properties of polymer-modified proteins may also be altered by polymer-polymer interactions. Herein, we sought to better understand the influence of polymer-polymer interactions on Candida rugosa lipase, which was modified with random co-polymers composed of sulfobetaine methacrylate (SBMA) and poly(ethylene glycol) methacrylate (PEGMA). Our findings suggest that there is an apparent activity-stability tradeoff as a function of polymer composition. Specifically, as the ratio of SBMA to PEGMA increased, lipase stability was enhanced, whereas activity decreased. By tuning the monomer ratio, we showed that lipase productivity could be optimized. These findings are discussed in the context of complex enzyme-polymer and polymer-polymer interactions and ultimately may enable more informed conjugate designs and improved enzyme productivity in industrial biotransformations under harsh or extreme conditions.

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.

Toward Next-Generation Biohybrid Catalyst Design: Influence of Degree of Polymerization on Enzyme Activity.

Due to their capacity to conduct complex organic transformations, enzymes find extensive use in medical and industrial settings. Unfortunately, enzymes are limited by their poor stability when exposed to harsh non-native conditions. While a host of methods have been developed to stabilize enzymes in non-native conditions, recent research into the synthesis of polymer-enzyme biohybrids using reversible deactivation radical polymerization approaches has demonstrated the potential of increased enzymatic activity in both native and non-native environments. In this manuscript, we utilize the enzyme lipase, as a model system, to explore the impact that modulation of grafted polymer molecular weight has on enzyme activity in both aqueous and organic media. We studied the properties of these hybrids using both solution-phase enzyme activity methods and coarse-grain modeling to assess the impact of polymer grafting density and grafted polymer molecular weight on enzyme activity to gain a deeper insight into this understudied property of the biohybrid system.

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

For more than 40 years, protein-polymer conjugates have been widely used for many applications, industrially and biomedically. These bioconjugates have been shown to modulate the activity and stability of various proteins while introducing reusability and new activities that can be used for drug delivery, improve pharmacokinetic ability, and stimuli-responsiveness. Techniques such as RDRP, ROMP and "click" have routinely been utilized for development of well-defined bioconjugate and polymeric materials. Synthesis of bioconjugate materials often take advantage of natural amino acids present within protein and peptide structures for a host of coupling chemistries. Polymer modification may elicit increased or decreased activity, activity retention under harsh conditions, prolonged activity in vivo and in vitro, and introduce stimuli responsiveness. Bioconjugation has resulted to modulated thermal stability, chemical stability, storage stability, half-life and reusability. In this review we aim to provide a brief state of the field, highlight a wide range of behaviors caused by polymer conjugation, and provide areas of future work.

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

This review article highlights recent developments in the field of photochemistry and photochemical reversible deactivation radical polymerization applied to aqueous polymerizations. Photochemistry is a topic of significant interest in the fields of organic, polymer, and materials chemistry because it allows challenging reactions to be performed under mild conditions. Aqueous polymerization is of significant interest because water is an environmentally benign solvent, and the use of water enables complex polymer self-assembly and bioconjugation processes to occur. This review focuses on powerful new developments in photochemical aqueous polymerization reactions and their applications to the synthesis of well-defined polymer nano-objects and bioconjugates. It is anticipated that these aqueous photopolymerizations will enable the next generation of self-assembled structures and biohybrid materials to be developed under mild and environmentally friendly conditions.

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

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.

Extraction of Thermodynamic Parameters of Protein Unfolding Using Parallelized Differential Scanning Fluorimetry.

Thermodynamic properties of protein unfolding have been extensively studied; however, the methods used have typically required significant preparation time and high protein concentrations. Here we present a facile, simple, and parallelized differential scanning fluorimetry (DSF) method that enables thermodynamic parameters of protein unfolding to be extracted. This method assumes a two-state, reversible protein unfolding mechanism and provides the capacity to quickly analyze the biophysical mechanisms of changes in protein stability and to more thoroughly characterize the effect of mutations, additives, inhibitors, or pH. We show the utility of the DSF method by analyzing the thermal denaturation of lysozyme, carbonic anhydrase, chymotrypsin, horseradish peroxidase, and cellulase enzymes. Compared with similar biophysical analyses by circular dichroism, DSF allows for determination of thermodynamic parameters of unfolding while providing greater than 24-fold reduction in experimental time. This study opens the door to rapid characterization of protein stability on low concentration protein samples.