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Jonathan S Dordick

Publications and source records attributed to Jonathan S Dordick.

At least 19 recordsLinked to original sources
PubMed2004

Structure and function of enzymes adsorbed onto single-walled carbon nanotubes.

We have examined the structure and function of two enzymes, alpha-chymotrypsin (CT) and soybean peroxidase (SBP), adsorbed onto single-walled carbon nanotubes (SWNTs). SBP retained up to 30% of its native activity upon adsorption, while the adsorbed CT retained only 1% of its native activity. Analysis of the secondary structure of the proteins via FT-IR spectroscopy revealed that both enzymes undergo structural changes upon adsorption, with substantial secondary structural perturbation observed for CT. Consistent with these results, AFM images of the adsorbed enzymes indicated that SBP retains its native three-dimensional shape while CT appears to unfold on the SWNT surface. This study represents the first in depth investigation of protein structure and function on carbon nanotubes, which is critical in designing optimal carbon nanotube-protein conjugates.

Animals↗
PubMed2004

Lipid-based nanotubes as functional architectures with embedded fluorescence and recognition capabilities.

A limited combinatorial strategy was used to synthesize a small library of soft lipid-based materials ranging from structurally unordered fibers to highly uniform nanotubes. The latter nanotubes are comprised of a bilayer structure with interdigitated alkyl chains associated through hydrophobic interactions. These tubes contain accessible 2,6-diaminopyridine linkers that can interact with thymidine and related nucleosides through multipoint hydrogen bonding, thereby quenching the intrinsic fluorescence of the aromatic linker. These results are the first example of a systematic strategy to design functional lipid nanotubes with precise structural and functional features.

Aminopyridines↗
PubMed2004

Bacterial P450-catalyzed polyketide hydroxylation on a microfluidic platform.

The incorporation of a multicomponent, cofactor-dependant P450 into a microfluidic biochip is demonstrated. The PikC hydroxylase Streptomyces venezuelae was incorporated into a PDMS-based microfluidic channel. The enzyme was immobilized to Ni-NTA agarose beads via in situ attachment following the addition of the beads to the microchannel. The enzyme loading was approximately 6 microg per mg of beads resulting in a microchannel loading of 10.7 mg/mL. This high enzyme loading enabled the rapid hydroxylation of the macrolide YC-17 to methymycin and neomethymycin in about equal amounts with a conversion of >90% at a flow rate of 70 nL/min. This high reactivity allowed rapid hydroxylation reactions to be performed with short residence times, which is critical for complex enzymes with limited inherent stability.

Bioreactors↗
PubMed2004

Chemoenzymatic synthesis and high-throughput screening of an aminoglycoside-polyamine library: identification of high-affinity displacers and DNA-binding ligands.

Chemoenzymatic parallel synthesis and high-throughput screening were employed to develop a multivalent aminoglycoside-polyamine library for use as high-affinity cation-exchange displacers and DNA-binding ligands. Regioselective lipase-catalyzed acylation, followed by chemical aminolysis, was used to generate vinyl carbonate and vinyl carbamate linkers, respectively, of the aminoglycosidic cores. These were further derivatized with polyamines, leading to library generation. A parallel batch-displacement assay was employed to identify the efficacy of the library candidates as potential displacers for protein purification. Using this approach, low-molecular-mass displacers with affinities higher than those previously observed have been identified. The aminoglycoside-polyamine library was also screened for DNA binding efficacy using an ethidium bromide displacement assay. These highly cationic molecules exhibited strong DNA-binding properties and may have potential for enhanced gene delivery.

Aminoglycosides↗
PubMed2004

Solid-phase chemoenzymatic synthesis of C-sialosides.

The chemoenzymatic regioselective acylation of Neu5Ac followed by SmI2-mediated C-glycosylation on a solid support is described for five C-glycosides. This method should facilitate the construction of combinatorial libraries of inhibitors of neuraminidase activity and hemagglutinin interaction as potential antiviral agents.

Acylation↗
PubMed2004

Silica nanoparticle size influences the structure and enzymatic activity of adsorbed lysozyme.

Adsorption of chicken egg lysozyme on silica nanoparticles of various diameters has been studied. Special attention has been paid to the effect of nanoparticle size on the structure and function of the adsorbed protein molecules. Both adsorption patterns and protein structure and function are strongly dependent on the size of the nanoparticles. Formation of molecular complexes is observed for adsorption onto 4-nm silica. True adsorptive behavior is evident on 20- and 100-nm particles, with the former resulting in monolayer adsorption and the latter yielding multilayer adsorption. A decrease in the solution pH results in a decrease in lysozyme adsorption. A change of protein structure upon adsorption is observed, as characterized by a loss in alpha-helix content, and this is strongly dependent on the size of the nanoparticle and the solution pH. Generally, greater loss of alpha helicity was observed for the lysozyme adsorbed onto larger nanoparticles under otherwise similar conditions. The activity of lysozyme adsorbed onto silica nanoparticles is lower than that of the free protein, and the fraction of activity lost correlates well with the decrease in alpha-helix content. These results indicate that the size of the nanoparticle, perhaps because of the contributions of surface curvature, influences adsorbed protein structure and function.

Adsorption↗
PubMed2004

Structural diversity of peroxidase-catalyzed oxidation products of o-methoxyphenols.

[reaction: see text] The biocatalytic oxidation of o-methoxyphenolic compounds led to a variety of oligophenols (dimers to pentamers) and some of their oxidation products. The reaction was carried out in an aqueous medium at room temperature with hydrogen peroxide as the terminal oxidant in a facile and green route to potentially bioactive compounds. Detailed structural information on the products of peroxidase-catalyzed oxidation of o-methoxyphenols is presented for the first time.

Catalysis↗
PubMed2004

Combinatorial formulation of biocatalyst preparations for increased activity in organic solvents: salt activation of penicillin amidase.

A combinatorial experimental technique was used to identify salts and salt mixtures capable of activating penicillin amidase in organic solvents for the transesterification of phenoxyacetate methyl ester with 1-propanol. Penicillin amidase was lyophilized in the presence of various chloride and acetate salts within 96-deep-well plates and catalytic rates measured to determine lead candidates for highly salt-activated preparations. The kinetics of the most active formulations were then further evaluated. These studies revealed that a formulation consisting of 98% (w/w) of a 1:1 KAc:CsCl salt mixture, 1% (w/w) enzyme, and 1% (w/w) potassium phosphate buffer was approximately 35,000-fold more active than the salt-free formulation in hexane, as reflected in values of V(max)/K(m). This extraordinary activation could be extended to more polar solvents, including tert-amyl alcohol, and to formulations with lower total salt contents. A correlation was found between the kosmotropic/chaotropic behavior of the salts (as measured by the Jones-Dole B coefficients) and the observed activation. Strongly chaotropic cations combined with strongly kosmotropic anions yielded the greatest activation, and this is likely due to the influence of the ions on protein-water and protein-salt interactions.

1-Propanol↗
PubMed2004

Biocompatibility of chemoenzymatically derived dextran-acrylate hydrogels.

The biocompatibility of chemoenzymatically generated dextran-acrylate hydrogels has been evaluated in vitro, using human foreskin fibroblasts, and in vivo, by subcutaneous and intramuscular implantation in Wistar rats for up to 40 days. In vitro tests show that hydrogel extracts only minimally reduced (<10%) the mitochondrial metabolic activity of fibroblasts. Direct contact of the hydrogels with cells induced a cellular proliferation inhibition index (CPII) of 50-80%, compared with a control, whereas through indirect contact, the CPII values were <16%, suggesting that the high CPII values achieved in the direct assay test were likely due to mechanical stress or limitations in oxygen diffusion. Hence, the hydrogels were noncytotoxic. Moreover, cell-material interaction studies show that these hydrogels were nonadhesive. Finally, histologic evaluation of tissue response to subcutaneous and intramuscular implants showed acceptable levels of biocompatibility, as characterized by a normal cellular response and the absence of necrosis of the surrounding tissues of the implant. In the first 10 days, the foreign-body reaction in the intramuscular implantation was more severe than in subcutaneous implantation, becoming identical after 30 days. In both cases, dextran hydrogels did not show signs of degradation 6 weeks postimplantation and were surrounded by a thin fibrous capsule and some macrophages and giant cells. This response is typical with a number of nondegradable biocompatible materials. These results indicate that dextran hydrogels are biocompatible, and may have suitable applications as implantable long-term peptide/protein delivery systems or scaffolds for tissue engineering.

Acrylates↗
PubMed2004

Hydration of enzyme in nonaqueous media is consistent with solvent dependence of its activity.

Water plays an important role in enzyme structure and function in aqueous media. That role becomes even more important when one focuses on enzymes in low water media. Here we present results from molecular dynamics simulations of surfactant-solubilized subtilisin BPN' in three organic solvents (octane, tetrahydrofuran, and acetonitrile) and in pure water. Trajectories from simulations are analyzed with a focus on enzyme structure, flexibility, and the details of enzyme hydration. The overall enzyme and backbone structures, as well as individual residue flexibility, do not show significant differences between water and the three organic solvents over a timescale of several nanoseconds currently accessible to large-scale molecular dynamics simulations. The key factor that distinguishes molecular-level details in different media is the partitioning of hydration water between the enzyme and the bulk solvent. The enzyme surface and the active site region are well hydrated in aqueous medium, whereas with increasing polarity of the organic solvent (octane --> tetrahydrofuran --> acetonitrile) the hydration water is stripped from the enzyme surface. Water stripping is accompanied by the penetration of tetrahydrofuran and acetonitrile molecules into crevices on the enzyme surface and especially into the active site. More polar organic solvents (tetrahydrofuran and acetonitrile) replace mobile and weakly bound water molecules in the active site and leave primarily the tightly bound water in that region. In contrast, the lack of water stripping in octane allows efficient hydration of the active site uniformly by mobile and weakly bound water and some structural water similar to that in aqueous solution. These differences in the active site hydration are consistent with the inverse dependence of enzymatic activity on organic solvent polarity and indicate that the behavior of hydration water on the enzyme surface and in the active site is an important determinant of biological function especially in low water media.

Acetonitriles↗
PubMed2003

Multienzyme catalysis in microfluidic biochips.

The attachment of enzymes to glass microfluidic channels has been achieved using a highly reactive poly(maleic anhydride-alt-alpha-olefin) (PMA)-based coating that is supplied to the microchannel in a toluene solution. The PMA reacts with 3-aminopropyltriethoxysilane groups linked to the glass surface to form a matrix that enables additional maleic anhydride groups to react with free amino groups on enzymes to give a mixed covalent-noncovalent immobilization support. Using a simple T-channel microfluidic design, with reaction channel dimensions of 200 microm wide (at the center), 15 microm deep, and 30 mm long giving a reaction volume of 90 nL, soybean peroxidase (SBP) was attached at an amount up to 0.6 microg/channel. SBP-catalyzed oxidation of p-cresol was performed in aqueous buffer (with 20% [v/v], dimethylformamide) containing H(2)O(2), with microfluidic transport enabled by electroosmotic flow (EOF). Michaelis-Menten kinetics were obtained with K(m) and V(max) values of 0.98 mM and 0.21 micromol H(2)O(2) converted/mg SBP per minute, respectively. These values are nearly identical to nonimmobilized SBP kinetics in aqueous-DMF solutions in 20-microL volumes in 384-well plates and 5-mL reaction volumes in 20-mL scintillation vials. These results indicate that SBP displays intrinsically native activity even in the immobilized form at the microscale, and further attests to the mild immobilization conditions afforded by PMA. Bienzymic and trienzymic reactions were also performed in the microfluidic biochip. Specifically, a combined Candida antarctica lipase B-SBP bienzymic system was used to convert tolyl acetate into poly(p-cresol), and an invertase-glucose oxidase SBP trienzymic system was used to take sucrose and generate H(2)O(2) for SBP-catalyzed synthesis of poly(p-cresol).

Catalysis↗
PubMed2003

Chemoenzymatic synthesis of neuraminic acid containing C-glycoside polymers.

[structure: see text] Two neuraminic acid-based, C-glycoside polymers were synthesized. Preliminary studies on one of these polymers showed potent neuraminidase inhibitory activity, suggesting potential utility as an antipathogenic surface coating for the preparation of antimicrobial biomaterials.

Anti-Bacterial Agents↗
PubMed2003

Solid-phase peptide synthesis by ion-paired alpha-chymotrypsin in nonaqueous media.

Solid-phase synthesis of dipeptides in low-water media was achieved using AOT ion-paired alpha-chymotrypsin solubilized in organic solvents. Multiple solvents and systematic variation of water activity, a(w), were used to examine the rate of coupling between N-alpha-benzyloxycarbonyl-L-phenylalanine methyl ester (Z-Phe-OMe) and leucine as a function of the reaction medium for both solid-phase and solution-phase reactions. In solution, the observed maximum reaction rate in a given solvent generally correlated with measures of hydrophobicity such as the log of the 1-octanol/water partitioning coefficient (log P) and the Hildebrand solubility parameter. The maximum rate for solution-phase synthesis (13 mmol/h g-enzyme) was obtained in a 90/10 (v/v) isooctane/tetrahydrofuran solvent mixture at an a(w) of 0.30. For the synthesis of dipeptides from solid-phase leucine residues, the highest synthetic rates (0.14-1.3 mmol/h g-enzyme) were confined to solvent environments that fell inside abruptly defined regions of solvent parameter space (e.g., log P > 2.3 and normalized electron acceptance index <0.13). The maximum rate for solid-phase synthesis was obtained in a 90/10 (v/v) isooctane/tetrahydrofuran solvent mixture at an a(w) of 0.14. In 90/10 and 70/30 (v/v) isooctane/tetrahydrofuran environments with a(w) set to 0.14, seven different N-protected dipeptides were synthesized on commercially available Tentagel support with yields of 74-98% in 24 h.

Catalysis↗
PubMed2003

Microfluidic peroxidase biochip for polyphenol synthesis.

An enzyme-containing microfluidic biochip has been developed for the oxidative polymerization of phenols. The biochip consists of a simple T-junction with two feed reservoirs 20 mm apart and a microreaction channel 30 mm long. The channel is 15 microm deep and 200 microm wide at the center, giving a reaction volume of 90 nL. The biochip was fabricated using conventional photolithographic methods on a glass substrate etched using a HF-based solution. Fluid transport was enabled using electroosmotic flow. Soybean peroxidase was used as the phenol oxidizing catalyst, and in the presence of p-cresol and H(2)O(2), essentially complete conversion of the H(2)O(2) (the limiting substrate) occurred in the microchannel at a flow rate of ca. 290 nL/min. Thus, peroxidase was found to be intrinsically active even upon dramatic scale-down as achieved in microfluidic reactors. These results were extended to a series of phenols, thereby demonstrating that the microfluidic peroxidase reactor may have application in high-throughput screening of phenolic polymerization reactions for use in phenolic resin synthesis. Finally, rapid growth of poly(p-cresol) on the walls of the microreaction channel could be performed in the presence of higher H(2)O(2) concentrations. This finding suggests that solution-phase peroxidase catalysis can be used in the controlled deposition of polymers on the walls of microreactors.

Bioreactors↗
PubMed2003

A combinatorial biocatalysis approach to an array of cholic acid derivatives.

A 39-member library of bile acid derivatives was prepared starting from 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid methyl ester using a combinatorial biocatalytic approach. A regioselective oxidation step, catalyzed by hydroxysteroid dehydrogenases, followed by an acylation step with a series of different acyl donors catalyzed by Candida antarctica lipase B, led to the modification of the bile acid scaffold. Each member of the library was obtained in high purity and good yield.

Acylation↗
PubMed2003

Si nanocolumns as novel nanostructured supports for enzyme immobilization.

Silicon nanocolumns have been used as novel supports for the high-density immobilization of enzymes. Silicon nanocolumns with diameters of ca. 50-100 nm and a height of 1 micron were constructed using glancing angle deposition. The surfaces were successively treated with 3-aminopropyltriethoxysilane (APTES) and then with an amine reactive polymer, poly(ethylene-alt-maleic anhydride), to attach soybean peroxidase (SBP) to the support. Optimal coverage of APTES, polymer, and SBP was obtained for incorporation of enzyme onto the sidewalls of the nanocolumns. SBP immobilized on the silicon nanocolumns demonstrated an enhancement in biocatalytic activity of 160% over that of the enzyme immobilized on flat silicon wafers with the same projected area. The enzymatic activity decreased with progressive washes for both supports. This decrease in the activity of enzyme was found to be primarily due to the intrinsic deactivation of immobilized enzyme on the silicon surface. Designing nanocolumns with optimal dimensions, spacing, and surface chemistry may lead to the development of high-density arrays of proteins for applications in biotechnology.

Adsorption↗
PubMed2002

High-throughput screening and quantitative structure-efficacy relationship models of potential displacer molecules for ion-exchange systems.

A technique is presented for the high-throughput screening of ion-exchange displacers. Potential displacers were employed to displace proteins in parallel batch ion-exchange experiments. The percentage of protein displaced from a particular stationary phase was then used as a parameter to rank the displacers. By employing this technique, a large number of molecules possessing a range of affinities and properties could be rapidly evaluated. This data was then used together with traditional and electron density-based transferable atom equivalent (TAE) molecular descriptors computed for the displacer molecules to produce quantitative structure-efficacy relationship (QSER) models using a genetic algorithm/partial least squares (GA/PLS) regression approach. The QSER models were generated using a portion of the protein-displacement data, with the remainder serving as a test set. Descriptor selection and model building was accomplished using a genetic algorithm/partial least squares approach. The resulting models were found to have high-correlation coefficients and could be used to accurately predict the behavior of molecules not included in the training set. In addition, the models were employed to examine a virtual library of displacers based on modifications of neomycin to provide further insight into displacer design. The results presented here indicate that it may be possible to design displacers that can dramatically improve the effective selectivity of ion-exchange chromatographic materials.

Algorithms↗