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). Using a 384-well plate format, we synthesized arrays of three-component polymer brushes in the microwells using photoactivated surface-initiated polymerization and immobilized enzymes in situ. The utility of CHESS to identify optimal immobilization supports under thermally and chemically denaturing conditions was demonstrated usingBacillus subtilisLipase A (LipA). The identification of supports with optimal compositions was validated by immobilizing LipA on polymer-brush-modified biocatalyst particles. We further demonstrated that CHESS could be used to predict the optimal composition of polymer brushes a priori for the previously unexplored enzyme, alkaline phosphatase (AlkP). Our findings demonstrate that CHESS represents a predictable and reliable platform for dramatically accelerating the search of chemical compositions for immobilization supports and further facilitates the discovery of biocompatible and stabilizing materials.

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.

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.e., ligation efficiency). Here, the connection between ligation reaction efficiency and the retention of enzyme structure and activity was investigated by leveraging the extremely fast reaction of strained trans-cyclooctene (sTCOs) and tetrazines (Tet). Remarkably, upon immobilization via Tet-sTCO chemistry, carbonic anhydrase (CA) retained 77% of its solution-phase activity, while immobilization via less efficient reaction chemistries, such as thiol-maleimide and azide-dibenzocyclooctyne, led to activity retention of only 46% and 27%, respectively. Dynamic single-molecule fluorescence tracking methods further revealed that longer surface search distances prior to immobilization (>0.5 µm) dramatically increased the probability of CA unfolding. Notably, the CA distance to immobilization was significantly reduced through the use of Tet-sTCO chemistry, which correlated with the increased retention of structure and activity of immobilized CA compared to the use of slower ligation chemistries. These findings provide an unprecedented insight into the role of ligation reaction efficiency in mediating the exploration of denaturing hotspots on surfaces by enzymes, which, in turn, may have major ramifications in the creation of functional biohybrid materials.

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. Activity assays and confocal fluorescence imaging confirmed retention of urease activity after immobilization as well as widespread distribution of enzyme on the film. The addition of urea triggered a response in the LCN whereby newly generated ammonia reacted with free acyl fluorides to form benzamide moieties. These moieties were capable of dimerizing through the formation of supramolecular hydrogen bonds, which was reflected in a 4-fold increase in Young's modulus. Through dynamic mechanical analysis and calorimetry, we further confirmed that the degree of hydrogen bonding in the LCNs could be judiciously designed to fine-tune the mechanical properties and glass transition temperature. These findings demonstrate the untapped potential of biochemical mechanisms as molecular triggers in LCNs and open the door to the use of nucleophilic chemistries in modulating the mechanical properties of LCNs.

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. Tracking mutation frequency through deep sequencing revealed biases in the position and the number of the introduced mutations. We overcame these biases by increasing the homology arm length and blocking mismatch repair to achieve a mutation efficiency of 85% for non-essential genes and 55% for essential genes. These experiments also improved our understanding of poorly characterized recombineering process using dsDNA donors with single nucleotide changes. Finally, we applied our technology to target rpoB, the beta subunit of RNA polymerase, to study resistance against rifampicin. In a single experiment, we validate multiple biochemical and clinical observations made in the previous decades and provide insights into resistance compensation with the study of double mutants.

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. This approach is based on the Rosetta kinematic loop modeling protocol and was demonstrated by identifying sites in lipase that were permissive to insertion of the LAP peptide. Interestingly, the identification of permissive sites was dependent on the contribution of the residues in the near-loop environment on the Rosetta score and did not correlate with conventional structural features (e.g., B-factors). As evidence of this, several insertion sites (e.g., following residues 17, 47-49, and 108), which were predicted and confirmed to be permissive, interrupted helices, while others (e.g., following residues 43, 67, 116, 119, and 121), which are situated in loop regions, were nonpermissive. This approach was further shown to be predictive for ß-glucosidase and human phosphatase and tensin homologue (PTEN), and to facilitate the engineering of insertion sites through in silico mutagenesis. By enabling the design of loop-containing protein libraries with high probabilities of soluble expression, this approach has broad implications in many areas of protein engineering, including antibody design, improving enzyme activity, and protein modification.

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. This was evident by a 10-fold decrease in the rates of enzyme unfolding and refolding as well as a reduction of the intrinsic fluctuations of the folded and unfolded states, which was measured by single-molecule (SM) Förster Resonance Energy Transfer imaging. Moreover, our results illuminate an important trade-off between stability and activity as a function of this decrease in structural dynamics of the immobilized lipase. Notably, as the thermal stability of lipase increased, as indicated by the temperature optimum for activity of the enzyme, the specific activity of lipase decreased. This decrease in activity was attributed to a reduction in the essential motions of the folded state that are required for catalytic turnover of substrate. These results provide direct evidence of this effect, which has long been a matter of speculation. Furthermore, our findings suggest that the retention of activity and stabilization of an enzyme may be balanced by tuning the extent of enzyme attachment.

Modification of Lipase with Poly(4-acryloylmorpholine) Enhances Solubility and Transesterification Activity in Anhydrous Ionic Liquids.

Tuning the molecular interaction between enzymes and their solvent environment through polymer modification can greatly improve activity and thus utility in biocatalytic reactions. In this work, this approach was exploited to enhance the activity of lipase A (LipA) from Bacillus subtilis in anhydrous ionic liquids (ILs), which are highly attractive solvents for biocatalysis. Specifically, we showed that the transesterification activity of LipA in anhydrous 1-butyl-3-methyl imidazolium hexafluorophosphate ([BMIM][PF6]) was improved up to 19-fold via covalently conjugating the enzyme with the IL-soluble polymer poly(4-acryloylmorpholine) (PAcMO). The increase in transesterification activity correlated with an increase in LipA solubility in [BMIM][PF6] as well as, notably, the number of conjugated PAcMO repeat units. Light scattering results further showed that the attachment of PAcMO disrupted the aggregation of LipA in aqueous buffer, which was used as a proxy to understand the mechanism of activation of LipA in the IL, where aggregation was more pronounced. Additionally, using static light scattering, the Flory-Huggins interaction parameter (χ) for the polymer-IL interactions was determined (0.457). The favorable PAcMO-IL interactions presumably compensated for the unfavorable interactions between the enzyme and IL, which resulted in the improvement in dissolution and, in turn, activity due to reduced diffusional limitations. Through rationally considering χ, a similar approach may be used to tune the molecular interaction between other enzymes and ILs with other polymers, which has widespread implications for the enhancement of biocatalysis in ILs.

Connecting Protein Conformation and Dynamics with Ligand-Receptor Binding Using Three-Color Förster Resonance Energy Transfer Tracking.

Specific binding between biomolecules, i.e., molecular recognition, controls virtually all biological processes including the interactions between cells and biointerfaces, both natural and synthetic. Such binding often relies on the conformation of biomacromolecules, which can be highly heterogeneous and sensitive to environmental perturbations, and therefore difficult to characterize and control. An approach is demonstrated here that directly connects the binding kinetics and stability of the protein receptor integrin αvß3 to the conformation of the ligand fibronectin (FN), which are believed to control cellular mechanosensing. Specifically, we investigated the influence of surface-adsorbed FN structure and dynamics on αvß3 binding using high-throughput single-molecule three-color Förster resonance energy transfer (FRET) tracking methods. By controlling FN structure and dynamics through tuning surface chemistry, we found that as the conformational and translational dynamics of FN increased, the rate of binding, particularly to folded FN, and stability of the bound FN-αvß3 complex decreased significantly. These findings highlight the importance of the conformational plasticity and accessibility of the arginine-glycine-aspartic acid (RGD) binding site in FN, which, in turn, mediates cell signaling in physiological and synthetic environments.

Acylase-containing polyurethane coatings with anti-biofilm activity.

Due to the prevalence of biofilm-related infections, which are mediated by bacterial quorum sensing, there is a critical need for materials and coatings that resist biofilm formation. We have developed novel anti-biofilm coatings that disrupt quorum sensing in surface-associated bacteria via the immobilization of acylase in polyurethane films. Specifically, acylase from Aspergillus melleus was covalently immobilized in biomedical grade polyurethane coatings via multipoint covalent immobilization. Coatings containing acylase were enzymatically active and catalyzed the hydrolysis of the quorum sensing (QS) molecules N-butyryl-L-homoserine lactone (C4-LHL), N-hexanoyl-L-homoserine lactone (C6-LHL), and N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-LHL). In biofilm inhibition assays, immobilization of acylase led to an approximately 60% reduction in biofilm formation by Pseudomonas aeruginosa ATCC 10145 and PAO1. Inhibition of biofilm formation was consistent with a reduction in the secretion of pyocyanin, indicating the disruption of quorum sensing as the mechanism of the coating activity. Scanning electron microscopy further showed that acylase-containing coatings contained far fewer bacterial cells than control coatings that lacked acylase. Moreover, acylase-containing coatings retained 90% activity when stored dry at 37°C for 7 days and were more stable than the free enzyme in physiological conditions, including artificial urine. Ultimately, such coatings hold considerable promise for the clinical management of catheter-related infections as well as the prevention of infections in orthopedic applications (i.e., on hip and knee prostheses) and on contact lenses. Biotechnol. Bioeng. 2016;113: 2535-2543. © 2016 Wiley Periodicals, Inc.

Dense Poly(ethylene glycol) Brushes Reduce Adsorption and Stabilize the Unfolded Conformation of Fibronectin.

Polymer brushes, in which polymers are end-tethered densely to a grafting surface, are commonly proposed for use as stealth coatings for various biomaterials. However, although their use has received considerable attention, a mechanistic understanding of the impact of brush properties on protein adsorption and unfolding remains elusive. We investigated the effect of the grafting density of poly(ethylene glycol) (PEG) brushes on the interactions of the brush with fibronectin (FN) using high-throughput single-molecule tracking methods, which directly measure protein adsorption and unfolding within the brush. We observed that, as grafting density increased, the rate of FN adsorption decreased; however, surface-adsorbed FN unfolded more readily, and unfolded molecules were retained on the surface for longer residence times relative to those of folded molecules. These results, which are critical for the rational design of PEG brushes, suggest that there is a critical balance between protein adsorption and conformation that underlies the utility of such brushes in physiological environments.

Single-molecule resolution of protein structure and interfacial dynamics on biomaterial surfaces.

A method was developed to monitor dynamic changes in protein structure and interfacial behavior on surfaces by single-molecule Förster resonance energy transfer. This method entails the incorporation of unnatural amino acids to site-specifically label proteins with single-molecule Förster resonance energy transfer probes for high-throughput dynamic fluorescence tracking microscopy on surfaces. Structural changes in the enzyme organophosphorus hydrolase (OPH) were monitored upon adsorption to fused silica (FS) surfaces in the presence of BSA on a molecule-by-molecule basis. Analysis of >30,000 individual trajectories enabled the observation of heterogeneities in the kinetics of surface-induced OPH unfolding with unprecedented resolution. In particular, two distinct pathways were observed: a majority population (∼ 85%) unfolded with a characteristic time scale of 0.10 s, and the remainder unfolded more slowly with a time scale of 0.7 s. Importantly, even after unfolding, OPH readily desorbed from FS surfaces, challenging the common notion that surface-induced unfolding leads to irreversible protein binding. This suggests that protein fouling of surfaces is a highly dynamic process because of subtle differences in the adsorption/desorption rates of folded and unfolded species. Moreover, such observations imply that surfaces may act as a source of unfolded (i.e., aggregation-prone) protein back into solution. Continuing study of other proteins and surfaces will examine whether these conclusions are general or specific to OPH in contact with FS. Ultimately, this method, which is widely applicable to virtually any protein, provides the framework to develop surfaces and surface modifications with improved biocompatibility.

Molecular dynamics investigation of the ionic liquid/enzyme interface: application to engineering enzyme surface charge.

Molecular simulations of the enzymes Candida rugosa lipase and Bos taurus α-chymotrypsin in aqueous ionic liquids 1-butyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium ethyl sulfate were used to study the change in enzyme-solvent interactions induced by modification of the enzyme surface charge. The enzymes were altered by randomly mutating lysine surface residues to glutamate, effectively decreasing the net surface charge by two for each mutation. These mutations resemble succinylation of the enzyme by chemical modification, which has been shown to enhance the stability of both enzymes in ILs. After establishing that the enzymes were stable on the simulated time scales, we focused the analysis on the organization of the ionic liquid substituents about the enzyme surface. Calculated solvent charge densities show that for both enzymes and in both solvents that changing positively charged residues to negative charge does indeed increase the charge density of the solvent near the enzyme surface. The radial distribution of IL constituents with respect to the enzyme reveals decreased interactions with the anion are prevalent in the modified systems when compared to the wild type, which is largely accompanied by an increase in cation contact. Additionally, the radial dependence of the charge density and ion distribution indicates that the effect of altering enzyme charge is confined to short range (≤1 nm) ordering of the IL. Ultimately, these results, which are consistent with that from prior experiments, provide molecular insight into the effect of enzyme surface charge on enzyme stability in ILs.

Optically diffracting hydrogels for screening kinase activity in vitro and in cell lysate: impact of material and solution properties.

Optically diffracting films based on hydrogel-encapsulated crystalline colloidal arrays have considerable utility as sensors for detecting enzymaticphosphorylation and, thus, in screening small molecule modulators of kinases. In this work, we have investigated the impact of hydrogel properties, as well as the role of the ionic character of the surrounding environment, on the optical sensitivity of kinase responsive crystalline colloidal array-containing hydrogels. In agreement with a model of hydrogel swelling, the optical sensitivity of such materials increased as the shear modulus and the Flory-Huggins interaction parameter between polymer and solvent decreased. Additionally, elimination of extraneous charges in the polymer backbone by exploiting azide-alkyne click chemistry to functionalize the hydrogels with a peptide substrate for protein kinase A further enhanced the sensitivity of the optically diffracting films. Increasing peptide concentration and, in turn, immobilized charge within the hydrogel network was shown to increase the optical response over a range of ionic strength conditions. Ultimately, we showed that, by tuning the hydrogel and solution properties, as little as 0.1 U/µL protein kinase A could be detected in short reaction times (i.e., 2 h), which is comparable to conventional biochemical kinase assays. We further showed that this approach can be used to detect protein kinase A activity in lysate from HEK293 cells. The sensitivity of the resulting films, coupled with the advantages of photonic crystal based sensors (e.g., label free detection), makes this approach highly attractive for screening enzymatic phosphorylation.

Crystallographic Investigation of Imidazolium Ionic Liquid Effects on Enzyme Structure.

We present the first crystallographic insight into the interactions of an ionic liquid (IL) with an enzyme, which has widespread implications for stabilizing enzymes in IL media for biocatalysis. Structures of Bacillus subtilis lipase A (lipA) and an IL-stable variant (QM-lipA) were obtained in the presence of increasing concentrations of 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). These studies revealed that the [BMIM] cation interacts with surface residues through hydrophobic and cation-π interactions. Of specific interest was the disruption of internal stacking interactions of aromatic side chains by [BMIM], which provides structural evidence for the mechanism of enzyme denaturation by ILs. The interaction of [BMIM] and Cl ions with lipA was reduced by the stabilizing mutations Y49E and G158E in QM-lipA. Ultimately, these findings present the molecular basis for stabilizing enzymes from IL-induced inactivation, as well as the selection of ILs that are less denaturing.

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.

Label-free detection of missense mutations and methylation differences in the p53 gene using optically diffracting hydrogels.

We have developed a novel approach for DNA detection as well as genetic screening of mutations by uniquely combining DNA-responsive and optically diffracting materials. This approach entails the polymerization of a photonic crystal within a hydrogel network that alters the diffraction of light in response to a target DNA strand. The utility of this approach, which permits label-free sensing, was demonstrated via the detection of a target sequence from the DNA binding domain of the major tumor suppressor protein p53. Using a complementary capture probe strand, we were able to detect down to picomole concentrations of the target p53 sequence. Moreover, we demonstrated that this approach could readily detect a single base pair mutation in the target strand, which corresponds to the hotspot cancer mutation R175H in p53. The sensitivity of detection was increased by lowering the rate of annealing of the target strand and adjusting the solution ionic strength during optical characterization. Changes in ionic strength during characterization impact the melting temperature of the bound target DNA and the Donnan potential between the hydrogel and solution, which influence detection. We further showed that this approach is sensitive to epigenetic changes via the detection of a fully methylated form of the target p53 sequence. Ultimately, this approach represents a new paradigm for DNA detection and specifically genetic screening of p53 as well as other disease markers and nucleotide modifications that alter the properties of DNA (e.g., epigenetic alterations and adducts with chemical carcinogens).

Photonic crystal kinase biosensor.

We have developed a novel biosensor for kinases that is based on a kinase-responsive polymer hydrogel, which enables label-free screening of kinase activity via changes in optical properties. The hydrogel is specifically designed to swell reversibly upon phosphorylation of a target peptide, triggering a change in optical diffraction from a crystalline colloidal array of particles impregnated into the hydrogel. Diffraction measurements, and charge staining, confirmed the responsive nature of the hydrogel. Moreover, the change in diffraction of the hydrogel upon treatment with kinase exhibited a time- and dose-dependent response. A theoretical model for ionic polymer networks describes the observed optical response well and can be used to quantify the extent of phosphorylation.

Charge engineering of cellulases improves ionic liquid tolerance and reduces lignin inhibition.

We report a novel approach to concurrently improve the tolerance to ionic liquids (ILs) as well as reduce lignin inhibition of Trichoderma reesei cellulase via engineering enzyme charge. Succinylation of the cellulase enzymes led to a nearly twofold enhancement in cellulose conversion in 15% (v/v) 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). The improvement in activity upon succinylation correlated with the apparent preferential exclusion of the [Cl] anion in fluorescence quenching assays. Additionally, modeling analysis of progress curves of Avicel hydrolysis in buffer indicated that succinylation had a negligible impact on the apparent KM of cellulase. As evidence of reducing lignin inhibition of T. reesei cellulase, succinylation resulted in a greater than twofold increase in Avicel conversion after 170 h in buffer with 1 wt% lignin. The impact of succinylation on lignin inhibition of cellulase further led to the reduction in apparent KM of the enzyme cocktail for Avicel by 2.7-fold. These results provide evidence that naturally evolved cellulases with highly negative surface charge densities may similarly repel lignin, resulting in improved cellulase activity. Ultimately, these results underscore the potential of rational charge engineering as a means of enhancing cellulase function and thus conversion of whole biomass in ILs.

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. Incorporation of aromatic groups leads to a 50 °C increase in the optimal temperature of lipase, as well as a 50-fold enhancement in enzyme activity. Single-molecule FRET studies reveal that these supports act as biomimetic chaperones by promoting enzyme refolding and stabilizing the enzyme's folded and catalytically active state. This effect is diminished when aromatic residues are mutated out, suggesting the importance of π-stacking and π-cation interactions for stabilization. Our results underscore how unexplored enzyme-support interactions may enable uncharted opportunities for using enzymes in industrial biotransformations.