Jason A. Berberich

Use of Salt Hydrate Pairs to Control Water Activity for Enzyme Catalysis in Ionic Liquids

Salt hydrate pairs were used to control water activity in the ionic liquid 1‐butyl‐3‐methylimidazolium hexafluorophosphate. It was shown that salt hydrate pairs behave essentially the same in ionic liquids as they do in organic solvents as long as they do not dissolve. Initial rate‐water activity profiles were prepared for the immobilized Candida antarctica lipase catalyzed synthesis of 2‐ethylhexyl methacrylate. The ability to use salt hydrate pairs for the control of water activity in ionic liquids should allow for improved comparison of enzyme activity and specificity in ionic liquids and conventional solvents.

Investigating the Impact of Polymer Functional Groups on the Stability and Activity of Lysozyme-Polymer Conjugates.

Polymers are often conjugated to proteins to improve stability; however, the impact of polymer chain length and functional groups on protein structure and function is not well understood. Here we use RAFT polymerization to grow polymers of different lengths and functionality from a short acrylamide oligomer with a RAFT end group conjugated to lysozyme. We show by X-ray crystallography that enzyme structure is minimally impacted by modification with the RAFT end group. Significant activity toward the negatively charged Micrococcus lysodeicticus cell wall was maintained when lysozyme was modified with cationic polymers. Thermal and chemical stability of the conjugates was characterized using differential scanning fluorimetry and tryptophan fluorescence. All conjugates had a lower melting temperature; however, conjugates containing ionic or substrate mimicking polymers were more resistant to denaturation by guanidine hydrochloride. Our results demonstrate that tailoring polymer functionality can improve conjugate activity and minimize enzymatic inactivation by denaturants.

Tunable stress relaxation behavior of an alginate-polyacrylamide hydrogel: comparison with muscle tissue.

Factors controlling the time-dependent mechanical properties of interpenetrating network (IPN) hydrogel materials are not well understood. In this study, alginate-polyacrylamide IPN were synthesized to mimic the stress relaxation behavior and elastic modulus of porcine muscle tissue. Hydrogel samples were created with single-parameter chemical concentration variations from a baseline formula to establish trends. The concentration of total monomer material had the largest effect on the elastic modulus, while concentration of the acrylamide cross-linker, N,N-methylenebis(acrylamide) (MBAA), changed the stress relaxation behavior most effectively. The IPN material was then tuned to mimic the mechanical response of muscle tissue using these trends. Swelling the hydrogel samples to equilibrium resulted in a dramatic decrease in both elastic modulus and stress relaxation behavior. Collectively, the results demonstrate that alginate-polyacrylamide IPN hydrogels can be tuned to closely mimic both the elastic and the viscoelastic behaviors of muscle tissue, although swelling detrimentally affects these desired properties.

Well-Defined Macromolecules Using Horseradish Peroxidase as a RAFT Initiase.

Enzymatic catalysis and control over macromolecular architectures from reversible addition-fragmentation chain transfer polymerization (RAFT) are combined to give a new method of making polymers. Horseradish peroxidase (HRP) is used to catalytically generate radicals using hydrogen peroxide and acetylacetone as a mediator. RAFT is used to control the polymer structure. HRP catalyzed RAFT polymerization gives acrylate and acrylamide polymers with relatively narrow molecular weight distributions. The polymerization is rapid, typically exceeding 90% monomer conversion in 30 min. Complex macromolecular architectures including a block copolymer and a protein-polymer conjugate are synthesized using HRP to catalytically initiate RAFT polymerization.

Toxicity effects of compressed and supercritical solvents on thermophilic microbial metabolism.

Selection of biocompatible solvents is critical when designing bioprocessing applications for the in situ biphasic extraction of metabolic end-products. The prediction of the biocompatibility of supercritical and compressed solvents is more complicated than for liquid solvents, because their properties can change significantly with pressure and temperature. The activity of the anaerobic thermophilic bacterium, Clostridium thermocellum, was studied when the organism was incubated in the presence of compressed nitrogen, ethane, and propane at 333 K and multiple pressures. The metabolic activity of the organisms in contact with compressed solvents was analyzed using traditional indicators of solvent biocompatibility, such as log P, interfacial tension, and solvent density. The toxicity of the compressed solvents was compared with the phase and molecular toxicity effects measured in liquid alkanes at atmospheric pressure. Inactivation increased with time in the presence of the compressed solvents, but was constant in the presence of atmospheric liquid solvents. Knowledge of molecular and phase toxicity provides a framework for the interpretation of C. thermocellum metabolism in contact with atmospheric and compressed solvents.

3D printing of an interpenetrating network hydrogel material with tunable viscoelastic properties

Interpenetrating network (IPN) hydrogel materials are recognized for their unique mechanical properties. While IPN elasticity and toughness properties have been explored in previous studies, the factors that impact the time-dependent stress relaxation behavior of IPN materials are not well understood. Time-dependent (i.e. viscoelastic) mechanical behavior is a critical design parameter in the development of materials for a variety of applications, such as medical simulation devices, flexible substrate materials, cellular mechanobiology substrates, or regenerative medicine applications. This study reports a novel technique for 3D printing alginate-polyacrylamide IPN gels with tunable elastic and viscoelastic properties. The viscoelastic stress relaxation behavior of the 3D printed alginate-polyacrylamide IPN hydrogels was influenced most strongly by varying the concentration of the acrylamide cross-linker (MBAA), while the elastic modulus was affected most by varying the concentration of total monomer material. The material properties of our 3D printed IPN constructs were consistent with those reported in the biomechanics literature for soft tissues such as skeletal muscle, cardiac muscle, skin and subcutaneous tissue.

Multisite Clickable Modification of Proteins Using Lipoic Acid Ligase

Approaches that allow bioorthogonal and, in turn, site-specific chemical modification of proteins present considerable opportunities for modulating protein activity and stability. However, the development of such approaches that enable site-selective modification of proteins at multiple positions, including internal sites within a protein, has remained elusive. To overcome this void, we have developed an enzymatic approach for multisite clickable modification based on the incorporation of azide moieties in proteins using lipoic acid ligase (LplA). The ligation of azide moieties to the model protein, green fluorescent protein (GFP), at the N-terminus and two internal sites using lipoic acid ligase was shown to proceed efficiently with near-complete conversion. Modification of the ligated azide groups with poly(ethylene glycol) (PEG), α-d-mannopyranoside, and palmitic acid resulted in highly homogeneous populations of protein-polymer, protein-sugar, and protein-fatty acid conjugates. The homogeneity of the conjugates was confirmed by mass spectrometry (MALDI-TOF) and SDS-PAGE electrophoresis. In the case of PEG attachment, which involved the use of strain-promoted azide-alkyne click chemistry, the conjugation reaction resulted in highly homogeneous PEG-GFP conjugates in less than 30 min. As further demonstration of the utility of this approach, ligated GFP was also covalently immobilized on alkyne-terminated self-assembled monolayers. These results underscore the potential of this approach for, among other applications, site-specific multipoint protein PEGylation, glycosylation, fatty acid modification, and protein immobilization.

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.

Chapter Five – Strategies for Biophysical Characterization of Protein–Polymer Conjugates

Protein-polymer conjugates are increasingly viewed as promising avenues to producing industrial enzymes with high activity capable of withstanding potentially harsh reaction conditions, or to designing novel therapeutics with triggered release, controlled masking, or increased resistance to proteolytic degradation. Common among these applications are the desire to improve the stability of protein-polymer conjugates to unfolding by exposure to chemicals or thermal stress. Thus, assays that allow researchers to robustly and easily characterize protein-polymer conjugates by obtaining thermodynamic parameters for folding stand to play an important role in the development of improved protein-polymer conjugates. Herein, we describe two techniques, differential scanning fluorimetry and intrinsic tryptophan fluorescence, used in our laboratories to obtain thermodynamic parameters of unfolding that allow for direct comparison of protein-polymer conjugates and the myriad effects of variations in attachment site, polymer identity, and polymer length. These two experiments, which are easily amenable to parallelization, are presented as high-throughput replacements for more traditionally employed circular dichroism experiments and as complements to functional chemical stability or functional thermal stability experiments. Each assay is presented in a parallelized format that allows for rapid scaling and high-throughput analysis of protein-polymer conjugate libraries. Descriptions of the assays include a discussion of advantages and disadvantages alongside protocol details and approaches to data analysis.

Investigating the Mechanism of Horseradish Peroxidase as a RAFT-Initiase

A detailed mechanistic and kinetic study of enzymatically initiated RAFT polymerization is performed by combining enzymatic assays and polymerization kinetics analysis. Horseradish peroxidase (HRP) initiated RAFT polymerization of dimethylacrylamide (DMAm) was studied. This polymerization was controlled by 2-(propionic acid)ylethyl trithiocarbonate (PAETC) in the presence of H2O2 as a substrate and acetylacetone (ACAC) as a mediator. In general, well controlled polymers with narrow molecular weight distributions and good agreement between theoretical and measured molecular weights are consistently obtained by this method. Kinetic and enzymatic assay analyses show that HRP loading accelerates the reaction, with a critical concentration of ACAC needed to effectively generate polymerization initiating radicals. The PAETC RAFT agent is required to control the reaction, although the RAFT agent also has an inhibitory effect on enzymatic performance and polymerization. Interestingly, although H2O2 is the substrate for HRP there is an optimal concentration near 1 mM, under the conditions studies, with higher or lower concentrations leading to lower polymerization rates and poorer enzymatic activity. This is explained through a competition between the H2O2 acting as a substrate, but also an inhibitor of HRP at high concentrations.