Scott Banta

Substrate channelling as an approach to cascade reactions

Millions of years of evolution have produced biological systems capable of efficient one-pot multi-step catalysis. The underlying mechanisms that facilitate these reaction processes are increasingly providing inspiration in synthetic chemistry. Substrate channelling, where intermediates between enzymatic steps are not in equilibrium with the bulk solution, enables increased efficiencies and yields in reaction and diffusion processes. Here, we review different mechanisms of substrate channelling found in nature and provide an overview of the analytical methods used to quantify these effects. The incorporation of substrate channelling into synthetic cascades is a rapidly developing concept, and recent examples of the fabrication of cascades with controlled diffusion and flux of intermediates are presented.

Protein Engineering in the Development of Functional Hydrogels

Proteins, which are natural heteropolymers, have evolved to exhibit a staggering array of functions and capabilities. As scientists and engineers strive to tackle important challenges in medicine, novel biomaterials continue to be devised, designed, and implemented to help to address critical needs. This review aims to cover the present advances in the use of protein engineering to create new protein and peptide domains that enable the formation of advanced functional hydrogels. Three types of domains are covered in this review: (a) the leucine zipper coiled-coil domains, (b) the EF-hand domains, and (c) the elastin-like polypeptides. In each case, the functionality of these domains is discussed as well as recent advancements in the use of these domains to create novel hydrogel-based biomaterials. As protein engineering is used to both create and improve protein domains, these advances will lead to exciting new biomaterials for use in a variety of applications.

Complete oxidation of methanol in biobattery devices using a hydrogel created from three modified dehydrogenases

Protein engineering involves the manipulation of amino acids to improve the properties of proteins. Breakthroughs are still being reported in the design and improvement of enzymes as well as efforts to improve structural proteins for biomaterials applications. Various protein and peptide domains have been engineered to create new functional materials for a variety of applications. Here we report an advancement of this approach where we create a new catalytic biomaterial by engineering of three dehydrogenase enzymes for self-assembly. When combined, the resulting new catalytic biomaterial is able to fully oxidize methanol to carbon dioxide and we demonstrate the application of this material as an anode modification in two types of enzymatic biobattery devices. Hydrogels can be created from proteins and peptides by outfitting them with cross-linking domains. Pioneering work by Tirrell and co-workers demonstrated that alpha-helical leucine zipper domains could be used to create peptides that self-assemble into hydrogels through coiled-coil interactions, and we have expanded on this line of research by demonstrating that these domains can be appended to globular proteins. These hydrogel constructs are crosslinked through both the coiled-coil motifs formed by the appended leucine zipper domains and through additional protein/protein interactions because of the quaternary structure of the proteins. So far, we have described the addition of helical appendages to fluorescent proteins, a thermostable alcohol dehydrogenase, an organophosphate hydrolase enzyme, and a small laccase enzyme. When the latter enzyme was combined with osmium-modified peptides, a bioelectrocatalytic hydrogel was formed that could reduce oxygen to water and could function as a cathode modification for a biobattery or enzymatic biofuel cell. In almost every case, the addition of the helical appendages has had a minimal impact on the catalytic activity of the enzymes, and robust hydrogels have been demonstrated. Here we extend this approach to create an enzymatic hydrogel that supports a functional synthetic metabolic pathway. Three NAD(H)-dependent dehydrogenase enzymes from different sources were modified for self-assembly. The first enzyme was a tetrameric alcohol dehydrogenase (ADH) from Bacillus stearothermophilus which oxidizes methanol to formaldehyde. The second enzyme was a tetrameric human aldehyde dehydrogenase (ALDH2) which oxidizes formaldehyde to formate. The final enzyme, a dimeric formate dehydrogenase (FDH1) from Saccharomyces cerevisiae, oxidizes formate to CO2. [7] When combined these enzymes produce a synthetic metabolic pathway capable of the complete oxidation of methanol. A schematic diagram of this reaction is as shown in Figure 1a. An alpha-helical leucine zipper domain (H) and randomly structured soluble peptide domain (S) were genetically appended to the N-termini of each of the three dehydrogenase genes. The three new bifunctional enzyme constructs (HSADH, HSALDH2, and HSFDH1) were overexpressed in E. coli and purified as described in the Supporting Information. HSADH and HSFDH1 were readily expressed and purified, while the HSALDH2 enzyme required the addition of the maltose binding protein (MBP) to enable functional expression. An intein domain was added between the MBP and HSALDH2 such that it spontaneously cleaved after expression within the cells and thus the HSALDH2 protein could be purified as though no fusion protein had been included. The kinetics of the purified bifunctionalized enzymes, all of which follow the ordered bi-bi kinetic mechanism, were measured in dilute solution to determine the impact of modifications on the kinetic parameters (Table 1 and Figure S3 in the Supporting Information). The kinetic parameters of the HSALDH2 enzyme were similar to those reported in the literature, while unexpectedly the kinetic parameters of both the HSADH and HSFDH1 enzymes were both found to be improved by the addition of the helical appendages. Both modified enzymes showed significant increases in catalytic efficiency (kcat/Km) as compared to the published values for the wild-type enzymes. The Michaelis constant (Km) for the substrate of HSADH was three orders of magnitude smaller than reported for the unmodified enzyme while the kcat value was found to increase by 120-fold. As a result, the catalytic efficiency (kcat/Km) of HSADH was increased six orders of magnitude. The catalytic efficiency of HSFDH1 was found to be increased by two orders of magnitude compared to literature values. The change in Km for the substrate was not significantly different, but the kcat value was two orders of magnitude higher than for the unmodified enzyme. We have previously observed that the addition of the helical appen[*] Dr. Y. H. Kim, Dr. E. Campbell, Prof. S. Banta Department of Chemical Engineering, Columbia University 500 West 120th Street, New York, NY 10027 (USA) E-mail: sbanta@columbia.edu Homepage: http://www.columbia.edu/~ sb2373

Bioelectrocatalytic hydrogels from electron-conducting metallopolypeptides coassembled with bifunctional enzymatic building blocks

Here, we present two bifunctional protein building blocks that coassemble to form a bioelectrocatalytic hydrogel that catalyzes the reduction of dioxygen to water. One building block, a metallopolypeptide based on a previously designed triblock polypeptide, is electron-conducting. A second building block is a chimera of artificial α-helical leucine zipper and random coil domains fused to a polyphenol oxidase, small laccase (SLAC). The metallopolypeptide has a helix–random-helix secondary structure and forms a hydrogel via tetrameric coiled coils. The helical and random domains are identical to those fused to the polyphenol oxidase. Electron-conducting functionality is derived from the divalent attachment of an osmium bis-bipyrdine complex to histidine residues within the peptide. Attachment of the osmium moiety is demonstrated by mass spectroscopy (MS-MALDI-TOF) and cyclic voltammetry. The structure and function of the α-helical domains are confirmed by circular dichroism spectroscopy and by rheological measurements. The metallopolypeptide shows the ability to make electrical contact to a solid-state electrode and to the redox centers of modified SLAC. Neat samples of the modified SLAC form hydrogels, indicating that the fused α-helical domain functions as a physical cross-linker. The fusion does not disrupt dimer formation, a necessity for catalytic activity. Mixtures of the two building blocks coassemble to form a continuous supramolecular hydrogel that, when polarized, generates a catalytic current in the presence of oxygen. The specific application of the system is a biofuel cell cathode, but this protein-engineering approach to advanced functional hydrogel design is general and broadly applicable to biocatalytic, biosensing, and tissue-engineering applications.

Functional assembly of a multi-enzyme methanol oxidation cascade on a surface-displayed trifunctional scaffold for enhanced NADH production

We report a simple and low-cost strategy that allows the sequential and site-specific assembly of a dehydrogenase-based multi-enzyme cascade for methanol oxidation on the yeast surface using the high-affinity interactions between three orthogonal cohesin-dockerin pairs. The multi-enzyme cascade showed 5 times higher NADH production rate than the non-complexed enzyme mixture, a result of efficient substrate channeling.

Detection of the Superoxide Radical Anion Using Various Alkanethiol Monolayers and Immobilized Cytochrome c

The superoxide radical anion (SO) is a critical biomarker for monitoring cellular stress responses. Electrochemical SO biosensors are frequently constructed through the covalent immobilization of cytochrome c (Cyt c) onto self-assembled monolayers (SAMs); however, a detailed comparison of these systems as well as configuration influence on SO detection is needed to enable robust applications. Two reaction pathways, oxidation of SO by the SAM-modified gold electrode or electron transfer through a protein and monolayer relay, may be involved during the electrochemical detection of SO with Cyt c, depending on the SAM that is used. Although electrodes with SAMs alone can exhibit a high sensitivity and low limit of detection (LOD) for the SO, they can suffer from a strong response to the presence of interferents such as hydrogen peroxide and ascorbic acid. Electrodes with immobilized Cyt c show decreased sensitivity, but exhibit better selectivity and resistance to fouling in complex media. Considering the trade-offs between sensitivity, selectivity, and LOD for SO detection, a bioelectrode made with Cyt c immobilized on dithiobis(succinimidyl)propionate (DTSP) appears to be the most suitable configuration. In phosphate buffer, the DTSP/Cyt c electrode has a sensitivity of 410 nA microM(-1) cm(-2) and an LOD for SO of 73 nM. Results are also presented for the detection of SO in a complex tissue culture media (MEM) with and without serum, and the sensitivity of the DTSP/Cyt c in MEM in the absence of serum increased to 640 nA microM(-1) cm(-2). By measuring SO with a DTSP/Cyt c electrode before and after the addition of a bolus of the superoxide dismutase (SOD) enzyme, the specificity of the SOD enzyme can be combined with the sensitivity of Cyt c system.

Development of a troponin I biosensor using a peptide obtained through phage display.

A small synthetic peptide with nanomolar affinity for cardiac troponin I (TnI), previously identified from a polyvalent phage displayed library, has been immobilized on a gold surface for TnI detection. The binding affinity of gold-immobilized peptides for TnI was studied and compared with that of phage-immobilized peptides. Quartz crystal microbalance (QCM), cyclic voltammetry, and electrochemical impedance spectroscopy (EIS) were used to monitor both the immobilization and target binding processes. All three techniques show that the binding is specific for TnI as compared to a streptavidin (SA) control. The response curves obtained at TnI concentrations ranging from 0 to 10 μg/mL, using both QCM and EIS, were also compared. For the EIS measurements, the sensitivity was 0.30 ± 0.030 normalized impedance/(μg/mL) and the limit of detection (LOD) was 0.34 μg/mL. Using the QCM, a sensitivity of 18 ± 1 Hz/(μg/mL) was obtained, corresponding to an LOD of 0.11 μg/mL. Although the QCM demonstrated a lower LOD as compared to EIS, the latter technique exhibited a larger linear dynamic range than QCM. In a relevant tissue culture milieu, Minimum Essential Media (MEM), the sensitivity of the EIS measurement was greater than that obtained in a phosphate buffer system (PBS). The kinetics of target binding using QCM were analyzed by two independent methods, and the dissociation constants (K(D) = 66 ± 4 nM and 17 ± 8 nM) were an order of magnitude higher than that calculated for the polyvalent phage particles (K(D) = 2.5 ± 0.1 nM). Even though the affinity of the immobilized peptides for TnI was somewhat reduced, overall, these results demonstrate that peptides obtained from the biopanning of phage display libraries can be readily used as sensing probes in biosensor development.

Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior

Cofactor specificity in the aldo‐keto reductase (AKR) superfamily has been well studied, and several groups have reported the rational alteration of cofactor specificity in these enzymes. Although most efforts have focused on mesostable AKRs, several putative AKRs have recently been identified from hyperthermophiles. The few that have been characterized exhibit a strong preference for NAD(H) as a cofactor, in contrast to the NADP(H) preference of the mesophilic AKRs. Using the design rules elucidated from mesostable AKRs, we introduced two site‐directed mutations in the cofactor binding pocket to investigate cofactor specificity in a thermostable AKR, AdhD, which is an alcohol dehydrogenase from Pyrococcus furiosus. The resulting double mutant exhibited significantly improved activity and broadened cofactor specificity as compared to the wild‐type. Results of previous pre‐steady‐state kinetic experiments suggest that the high affinity of the mesostable AKRs for NADP(H) stems from a conformational change upon cofactor binding which is mediated by interactions between a canonical arginine and the 2′‐phosphate of the cofactor. Pre‐steady‐state kinetics with AdhD and the new mutants show a rich conformational behavior that is independent of the canonical arginine or the 2′‐phosphate. Additionally, experiments with the highly active double mutant using NADPH as a cofactor demonstrate an unprecedented transient behavior where the binding mechanism appears to be dependent on cofactor concentration. These results suggest that the structural features involved in cofactor specificity in the AKRs are conserved within the superfamily, but the dynamic interactions of the enzyme with cofactors are unexpectedly complex. Biotechnol. Bioeng. 2010;107: 763–774.

Enzymatic biofuel cells utilizing a biomimetic cofactor

The performance of immobilized enzyme systems is often limited by cofactor diffusion and regeneration. Here, we demonstrate an engineered enzyme capable of utilizing the minimal cofactor nicotinamide mononucleotide (NMN(+)) to address these limitations. Significant gains in performance are observed with NMN(+) in immobilized systems, despite a decreased turnover rate with the minimal cofactor.

Replacing Antibodies: Engineering New Binding Proteins

Natures reliance on proteins to carry out nearly all biological processes has led to the evolution of biomolecules that exhibit a seemingly endless range of functions. Much research has been devoted toward advancing this process in the laboratory in order to create new proteins with improved or unique capabilities. The protein-engineering field has rapidly evolved from pioneering studies in engineering protein stability and activity to an application-driven powerhouse on the forefront of emerging technologies in biomedical engineering and biotechnology. A classic protein-engineering technique in the medical field has focused on manipulating antibodies and antibody fragments for various applications. New classes of alternative scaffolds have recently challenged this paradigm, and these structures have been successfully engineered for applications including targeted cancer therapy, regulated drug delivery, in vivo imaging, and a host of others. This review aims to capture recent advances in the engineering of nonimmunoglobulin scaffolds as well as some of the applications for these molecular recognition elements in the biomedical field.