Increased cytoplasmic expression of PETase enzymes in E. coli.

Background: Depolymerizing polyethylene terephthalate (PET) plastics using enzymes, such as PETase, offers a sustainable chemical recycling route. To enhance degradation, many groups have sought to engineer PETase for faster catalysis on PET and elevated stability. Considerably less effort has been focused toward expressing large quantities of the enzyme, which is necessary for large-scale application and widespread use.

Rationalizing Diverse Binding Mechanisms to the Same Protein Fold: Insights for Ligand Recognition and Biosensor Design.

The engineering of novel protein-ligand binding interactions, particularly for complex drug-like molecules, is an unsolved problem, which could enable many practical applications of protein biosensors. In this work, we analyzed two engineered biosensors, derived from the plant hormone sensor PYR1, to recognize either the agrochemical mandipropamid or the synthetic cannabinoid WIN55,212-2. Using a combination of quantitative deep mutational scanning experiments and molecular dynamics simulations, we demonstrated that mutations at common positions can promote protein-ligand shape complementarity and revealed prominent differences in the electrostatic networks needed to complement diverse ligands.

Stabilization of an Infectious Enveloped Virus by Spray-Drying and Lyophilization.

Enveloped viruses are attractive candidates for use as gene- and immunotherapeutic agents due to their efficacy at infecting host cells and delivering genetic information. They have also been used in vaccines as potent antigens to generate strong immune responses, often requiring fewer doses than other vaccine platforms as well as eliminating the need for adjuvants. However, virus instability in liquid formulations may limit their shelf life and require that these products be transported and stored under stringently controlled temperature conditions, contributing to high cost and limiting patient access.

Rationalizing diverse binding mechanisms to the same protein fold: insights for ligand recognition and biosensor design.

The engineering of novel protein-ligand binding interactions, particularly for complex drug-like molecules, is an unsolved problem which could enable many practical applications of protein biosensors. In this work, we analyzed two engineer ed biosensors, derived from the plant hormone sensor PYR1, to recognize either the agrochemical mandipropamid or the synthetic cannabinoid WIN55,212-2. Using a combination of quantitative deep mutational scanning experiments and molecular dynamics simulations, we demonstrated that mutations at common positions can promote protein-ligand shape complementarity and revealed prominent differences in the electrostatic networks needed to complement diverse ligands.

Combinatorial High-Throughput Screening of Complex Polymeric Enzyme Immobilization Supports.

Recent advances have demonstrated the promise of complex multicomponent polymeric supports to enable supra-biological enzyme performance. However, the discovery of such supports has been limited by time-consuming, low-throughput synthesis and screening. Here, we describe a novel combinatorial and high-throughput platform that enables rapid screening of complex and heterogeneous copolymer brushes as enzyme immobilization supports, named combinatorial high-throughput enzyme support screening (CHESS).

Supra-biological performance of immobilized enzymes enabled by chaperone-like specific non-covalent interactions.

Designing complex synthetic materials for enzyme immobilization could unlock the utility of biocatalysis in extreme environments. Inspired by biology, we investigate the use of random copolymer brushes as dynamic immobilization supports that enable supra-biological catalytic performance of immobilized enzymes. This is demonstrated by immobilizing Bacillus subtilis Lipase A on brushes doped with aromatic moieties, which can interact with the lipase through multiple non-covalent interactions.

Effect of mechanical stresses on viral capsid disruption during droplet formation and drying.

Identification of the mechanisms by which viruses lose activity during droplet formation and drying is of great importance to understanding the spread of infectious diseases by virus-containing respiratory droplets and to developing thermally stable spray dried live or inactivated viral vaccines. In this study, we exposed suspensions of baculovirus, an enveloped virus, to isolated mechanical stresses similar to those experienced during respiratory droplet formation and spray drying: fluid shear forces, osmotic pressure forces, and surface tension forces at interfaces. DNA released from mechanically stressed virions was measured by SYBR Gold staining to quantify viral capsid disruption.

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 lipase, which was modified with random co-polymers composed of sulfobetaine methacrylate (SBMA) and poly(ethylene glycol) methacrylate (PEGMA).

Tuning the surface charge of phospholipid bilayers inhibits insulin fibrilization.

The interactions between proteins and materials, in particular lipid bilayers, have been studied extensively for their relevance in diseases and for the formulation of protein-based therapeutics and vaccines. However, the precise rules by which material properties induce favorable or unfavorable structural states in biomolecules are incompletely understood, and as a result, the rational design of materials remains challenging. Here, we investigated the influence of lipid bilayers (in the form of small unilamellar vesicles) on the formation of insulin amyloid fibrils using a fibril-specific assay (thioflavin T), polyacrylamide gel electrophoresis, and circular dichroism spectroscopy.

Biocatalytic 3D Actuation in Liquid Crystal Elastomers via Enzyme Patterning.

Liquid crystal elastomers (LCEs) are stimuli-responsive materials that undergo large shape transformations after undergoing an order-disorder transition. While shape reconfigurations in LCEs are predominantly triggered by heat, there is a considerable interest in developing highly specific triggers that work at room temperature. Herein, we report the fabrication of biocatalytic LCEs that respond to the presence of urea by covalently immobilizing urease within chemically responsive LCE networks.

Quantification of Metabolic Products from Microbial Hosts in Complex Media Using Optically Diffracting Hydrogels.

We herein describe a highly versatile platform approach for the in situ and real-time screening of microbial biocatalysts for enhanced production of bioproducts using photonic crystal hydrogels. This approach was demonstrated by preparing optically diffracting films based on polymerized -isopropylacrylamide that contracted in the presence of alcohols and organic acids. The hydrogel films were prepared in a microwell plate format, which allows for high-throughput screening, and characterized optically using a microwell plate reader.

Substitution of distal and active site residues reduces product inhibition of E1 from Acidothermus Cellulolyticus.

Cellulases are largely afflicted by inhibition from their reaction products, especially at high-substrate loading, which represents a major challenge for biomass processing. This challenge was overcome for endoglucanase 1 (E1) from Acidothermus cellulolyticus by identifying a large conformational change involving distal residues upon binding cellobiose. Having introduced alanine substitutions at each of these residues, we identified several mutations that reduced cellobiose inhibition of E1, including W212A, W213A, Q247A, W249A and F250A.

Chemically Triggered Changes in Mechanical Properties of Responsive Liquid Crystal Polymer Networks with Immobilized Urease.

Liquid crystal polymer networks (LCNs) are stimuli-responsive materials that can be programmed to realize spatial variation in mechanical response and undergo shape transformation. Herein, we report a process to introduce chemical specificity to the stimuli response of LCNs by integrating enzymes as molecular triggers. Specifically, the enzyme urease was immobilized in LCN films via acyl fluoride conjugation chemistry.

Understanding Design Rules for Optimizing the Interface between Immobilized Enzymes and Random Copolymer Brushes.

A long-standing goal in the field of biotechnology is to develop and understand design rules for the stabilization of enzymes upon immobilization to materials. While immobilization has sometimes been successful as a strategy to stabilize enzymes, the design of synthetic materials that stabilize enzymes remains largely empirical. We sought to overcome this challenge by investigating the mechanistic basis for the stabilization of immobilized lipases on random copolymer brush surfaces comprised of poly(ethylene glycol) methacrylate (PEGMA) and sulfobetaine methacrylate (SBMA), which represent novel heterogeneous supports for immobilized enzymes.

Faster Surface Ligation Reactions Improve Immobilized Enzyme Structure and Activity.

During integration into materials, the inactivation of enzymes as a result of their interaction with nanometer size denaturing "hotspots" on surfaces represents a critical challenge. This challenge, which has received far less attention than improving the long-term stability of enzymes, may be overcome by limiting the exploration of surfaces by enzymes. One way this may be accomplished is through increasing the rate constant of the surface ligation reaction and thus the probability of immobilization with reactive surface sites (i.

Rosetta-Enabled Structural Prediction of Permissive Loop Insertion Sites in Proteins.

While loop motifs frequently play a major role in protein function, our understanding of how to rationally engineer proteins with novel loop domains remains limited. In the absence of rational approaches, the incorporation of loop domains often destabilizes proteins, thereby requiring massive screening and selection to identify sites that can accommodate loop insertion. We developed a computational strategy for rapidly scanning the entire structure of a scaffold protein to determine the impact of loop insertion at all possible amino acid positions.

Mixed Phospholipid Vesicles Catalytically Inhibit and Reverse Amyloid Fibril Formation.

While many approaches to reduce fibrillation of amyloid-β (Aβ) have been aimed at slowing fibril formation, the degradation of fibrils remains challenging. We provide insight into fibril degradation as well as the inhibition of fiber formation by lipid vesicles composed of 1,2-dioleoyl--glycero-3-phosphocholine and 1,2-dioleoyl--glycero-3-phospho-(1'-rac-glycerol). In the presence of vesicles with the optimal lipid composition, fibril formation was inhibited up to 76%.

Polyelectrolyte Multilayers Enhance the Dry Storage and pH Stability of Physically Entrapped Enzymes.

Polyelectrolyte multilayers (PEMs) are attractive materials for immobilizing enzymes due to their unique ionic environment, which can prevent unfolding. Here, we demonstrated that the stability to dry storage and elevated pH were significantly enhanced when negatively charged nitroreductase (NfsB) was embedded in a PEM by depositing alternating layers of the enzyme and polycation (PC) onto porous silica particles. The PC strength (i.

Determinants for Efficient Editing with Cas9-Mediated Recombineering in .

In , editing efficiency with Cas9-mediated recombineering varies across targets due to differences in the level of Cas9:gRNA-mediated DNA double-strand break (DSB)-induced cell death. We found that editing efficiency with the same gRNA and repair template can also change with target position, promoter strength, and growth conditions. Incomplete editing, off-target activity, nontargeted mutations, and failure to cleave target DNA even if Cas9 is bound also compromise editing efficiency.

CRISPR/Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli.

Deep mutational scanning can provide significant insights into the function of essential genes in bacteria. Here, we developed a high-throughput method for mutating essential genes of Escherichia coli in their native genetic context. We used Cas9-mediated recombineering to introduce a library of mutations, created by error-prone PCR, within a gene fragment on the genome using a single gRNA pre-validated for high efficiency.

Reduced Enzyme Dynamics upon Multipoint Covalent Immobilization Leads to Stability-Activity Trade-off.

The successful incorporation of enzymes into materials through multipoint covalent immobilization (MPCI) has served as the foundation for numerous advances in diverse fields, including biocatalysis, biosensing, and chemical weapons defense. Despite this success, a mechanistic understanding of the impact of this approach on enzyme stability has remained elusive, which is critical for realizing the full potential of MPCI. Here, we showed that the stabilization of lipase upon MPCI to polymer brush surfaces resulted from the rigidification of the enzyme with an increase in the number of enzyme-brush attachments.

Stabilization of Fibronectin by Random Copolymer Brushes Inhibits Macrophage Activation.

We show that protein unfolding on biomaterials may be dramatically reduced via tuning the chemical heterogeneity of the protein-material interface. Specifically, using dynamic single-molecule methods, we confirmed that the transient structure and dynamics of fibronectin (FN) may be mediated through varying the composition of random copolymer brushes. The brushes, which themselves represent an intriguing biomaterial, were composed of oligoethylene glycol and sulfobetaine methacrylate and presumably stabilized FN through partitioning and/or segregation of the copolymers.

Protein adsorption measurements on low fouling and ultralow fouling surfaces: A critical comparison of surface characterization techniques.

Ultralow protein fouling behavior is a common target for new high-performance materials. Ultralow fouling is often defined based on the amount of irreversibly adsorbed protein (< 5 ng cm) measured by a surface ensemble averaging method. However, protein adsorption at solid interfaces is a dynamic process involving multiple steps, which may include adsorption, desorption, and irreversible protein denaturation.

Lipoic Acid Ligase-Promoted Bioorthogonal Protein Modification and Immobilization.

Protein bioconjugation benefits from precise regional and temporal control. One notable way of achieving this control is through the enzymatic attachment of bioorthogonal reactive handles to peptide recognition sequences that are genetically fused to target proteins of interest. The lipoic acid ligase variant, LplA, functionalizes proteins by covalently attaching an azide-bearing lipoic acid derivative to a 13-amino acid recognition sequence known as the lipoic acid ligase acceptor peptide (LAP).

Surface-Templated Nanobubbles Protect Proteins from Surface-Mediated Denaturation.

In this Letter, we report that surface-bound nanobubbles reduce protein denaturation on methylated glass by irreversible protein shell formation. Single-molecule total internal reflection fluorescence (SM-TIRF) microscopy was combined with intramolecular Förster resonance energy transfer (FRET) to study the conformational dynamics of nitroreductase (NfsB) on nanobubble-laden methylated glass surfaces, using reflection brightfield microscopy to register nanobubble locations with NfsB adsorption. First, NfsB adsorbed irreversibly to nanobubbles with no apparent desorption after 5 h.