Co-reporter:Dr. Sanil Sreekumar;Zachary C. Baer;Dr. Elad Gross;Dr. Sasisanker Padmanaban;Konstantinos Goulas;Gorkem Gunbas;Dr. Selim Alayoglu; Harvey W. Blanch; Douglas S. Clark; F. Dean Toste
ChemSusChem 2014 Volume 7( Issue 9) pp:2445-2448
Publication Date(Web):
DOI:10.1002/cssc.201402244
Abstract
Biological and chemocatalytic processes are tailored in order to maximize the production of sustainable biodiesel from lignocellulosic sugar. Thus, the combination of hydrotalcite-supported copper(II) and palladium(0) catalysts with a modification of the fermentation from acetone–butanol–ethanol to isopropanol–butanol–ethanol predictably produces higher concentrations of diesel-range components in the alkylation reaction.
Co-reporter:Elizabeth Schneider, Douglas S. Clark
Biosensors and Bioelectronics 2013 Volume 39(Issue 1) pp:1-13
Publication Date(Web):15 January 2013
DOI:10.1016/j.bios.2012.05.043
Cytochrome P450s (CYPs) are a large family of heme-containing monooxygenase enzymes involved in the first-pass metabolism of drugs and foreign chemicals in the body. CYP reactions, therefore, are of high interest to the pharmaceutical industry, where lead compounds in drug development are screened for CYP activity. CYP reactions in vivo require the cofactor NADPH as the source of electrons and an additional enzyme, cytochrome P450 reductase (CPR), as the electron transfer partner; consequently, any laboratory or industrial use of CYPs is limited by the need to supply NADPH and CPR. However, immobilizing CYPs on an electrode can eliminate the need for NADPH and CPR provided the enzyme can accept electrons directly from the electrode. The immobilized CYP can then act as a biosensor for the detection of CYP activity with potential substrates, albeit only if the immobilized enzyme is electroactive. The quest to create electroactive CYPs has led to many different immobilization strategies encompassing different electrode materials and surface modifications. This review focuses on different immobilization strategies that have been used to create CYP biosensors, with particular emphasis on mammalian drug-metabolizing CYPs and characterization of CYP electrodes. Traditional immobilization methods such as adsorption to thin films or encapsulation in polymers and gels remain robust strategies for creating CYP biosensors; however, the incorporation of novel materials such as gold nanoparticles or quantum dots and the use of microfabrication are proving advantageous for the creation of highly sensitive and portable CYP biosensors.Highlights► An overview of the significance of CYP enzymes and CYP biosensors. ► A brief history of the development of CYP biosensors. ► A review of electrode types and modifications used to create CYP biosensors. ► Highlights of recent and/or novel studies on CYP biosensors. ► Focus on chemical and physical characterization of CYP electrodes.
Co-reporter:Jerome M. Fox, Seth E. Levine, Douglas S. Clark, and Harvey W. Blanch
Biochemistry 2012 Volume 51(Issue 1) pp:
Publication Date(Web):November 21, 2011
DOI:10.1021/bi2011543
Efforts to improve the activity of cellulases, which catalyze the hydrolysis of insoluble cellulose, have been hindered by uncertainty surrounding the mechanistic origins of rate-limiting phenomena and by an incomplete understanding of complementary enzyme function. In particular, direct kinetic measurements of individual steps occurring after enzymes adsorb to the cellulose surface have proven to be experimentally elusive. This work describes an experimental and analytical approach, derived from a detailed mechanistic model of cellobiohydrolase action, for determining rates of initial- and processive-cut product generation by Trichoderma longibrachiatum cellobiohydrolase I (TlCel7A) as it catalyzes the hydrolysis of bacterial microcrystalline cellulose (BMCC) alone and in the presence of Talaromyces emersonii endoglucanase II (TemGH5). This analysis revealed that the rate of TlCel7A-catalyzed hydrolysis of crystalline cellulose is limited by the rate of enzyme complexation with glycan chains, which is shown to be equivalent to the rate of initial-cut product generation. This rate is enhanced in the presence of endoglucanase enzymes. The results confirm recent reports about the role of morphological obstacles in enzyme processivity and also provide the first direct evidence that processive length may be increased by the presence of companion enzymes, including small amounts of TemGH5. The findings of this work indicate that efforts to improve cellobiohydrolase activity should focus on enhancing the enzyme’s ability to complex with cellulose chains, and the analysis employed provides a new technique for investigating the mechanism by which companion enzymes influence cellobiohydrolase activity.
Co-reporter:Matthew J. Traylor ; Jessica D. Ryan ; Eric S. Arnon ; Jonathan S. Dordick
Journal of the American Chemical Society 2011 Volume 133(Issue 37) pp:14476-14479
Publication Date(Web):July 18, 2011
DOI:10.1021/ja203172c
Metabolic stability measurements are a critical component of preclinical drug development. Available measurement strategies rely on chromatography and mass spectrometry, which are expensive and labor intensive. We have developed a general method to determine the metabolic stability of virtually any compound by quantifying cofactors in the mechanism of cytochrome P450 enzymes using fluorescence intensity measurements. While many previous studies have shown that simple measurements of cofactor depletion do not correlate with substrate conversion (i.e., metabolic stability) in P450 systems, the present work employs a reaction engineering approach to simplify the overall rate equation, thus allowing the accurate and quantitative determination of substrate depletion from simultaneous measurements of NADPH and oxygen depletion. This method combines the accuracy and generality of chromatography with the ease, throughput, and real-time capabilities of fluorescence.
Co-reporter:Paul W. Wolski, Douglas S. Clark and Harvey W. Blanch
Green Chemistry 2011 vol. 13(Issue 11) pp:3107-3110
Publication Date(Web):05 Oct 2011
DOI:10.1039/C1GC15691H
A rapid screen was developed to test the stability of proteins in ionic liquid–aqueous mixtures using green fluorescent protein (GFP) as a reporter. In at least one ionic liquid (IL), GFP retained 50% or more of its fluorescence in IL volume fractions as high as 75%. ILs that best preserved GFP fluorescence also showed the best retention of cellulase activity. Using this screen, two potential candidates for in situ enzymatic hydrolysis of biomass, 1,3-dimethylimidazolium dimethylphosphate (Mmim DMP) and 1-ethyl-3-methylimidazolium (Emim) lactate, were identified. A commercial Trichoderma reeseicellulase mixture retained activity in both ILs up to 40% (w/w) IL, and β-glucosidase remained active after incubation in 60% (w/w) Mmim DMP for 8 h, indicating the possibility of in situ cellulose hydrolysis in IL–water mixtures.
Co-reporter:Dr. Tae-Wan Kim;Dr. Harshal A. Chokhawala;Dr. Matthias Hess;Craig M. Dana;Zachary Baer;Dr. Alexer Sczyrba; Edward M. Rubin; Harvey W. Blanch; Douglas S. Clark
Angewandte Chemie International Edition 2011 Volume 50( Issue 47) pp:11215-11218
Publication Date(Web):
DOI:10.1002/anie.201104685
Co-reporter:Dr. Tae-Wan Kim;Dr. Harshal A. Chokhawala;Dr. Matthias Hess;Craig M. Dana;Zachary Baer;Dr. Alexer Sczyrba; Edward M. Rubin; Harvey W. Blanch; Douglas S. Clark
Angewandte Chemie International Edition 2011 Volume 50( Issue 47) pp:
Publication Date(Web):
DOI:10.1002/anie.201106146
Co-reporter:Umar Akbar, Dong-Sik Shin, Elizabeth Schneider, Jonathan S. Dordick, Douglas S. Clark
Tetrahedron Letters 2010 Volume 51(Issue 8) pp:1220-1225
Publication Date(Web):24 February 2010
DOI:10.1016/j.tetlet.2009.12.119
The natural flavonoid bergenin was directly immobilized onto carboxylic acid-functionalized controlled pore glass (carboxy-CPG) at 95% yield. Immobilized bergenin was brominated via chloroperoxidase in aqueous solution and then transesterified with vinyl butyrate in diisopropyl ether by subtilisin carslberg (SC) extracted into the organic solvent via ion-pairing. Enzymatic cleavage of 7-bromo-4-butyrylbergenin from carboxy-CPG (9.6% final yield) was accomplished using lipase B (LipB) in an aqueous/organic mixture (90/10 v/v of water/acetonitrile), demonstrating the feasibility of solid-phase biocatalysis of a natural product in aqueous and non-aqueous media.A natural flavonoid molecule, bergenin is attached to a solid support without a linker and is modified and cleaved enzymatically in both aqueous and organic media as a demonstration of solid-phase biocatalysis.
Co-reporter:Timothy A. Whitehead;Elizabeth Je
Biopolymers 2009 Volume 91( Issue 6) pp:496-503
Publication Date(Web):
DOI:10.1002/bip.21157
Abstract
An enticing possibility in nanotechnology is to use proteins as templates for the positioning of molecules in regular patterns with nanometer precision over large surface areas. However, the ability to redesign protein quaternary structure to construct new shapes remains underdeveloped. In the present work, we have engineered the dimensions of a filamentous protein, the γ prefoldin (γ PFD) from the hyperthermophile Methanocaldococcus jannaschii, and have achieved controllable attachment of filaments in a specific orientation on a carbon surface. Four different constructs of γ PFD were generated in which the coiled coils extending from the association domain are progressively truncated. Three of the truncation constructs form well-defined filaments with predictable dimensions according to transmission electron microscopy. Two of these constructs had 2D persistence lengths similar to that of γ PFD at 300–740 nm. In contrast, the 2D persistence length of the shortest truncation mutant was 3500 nm, indicating that the filament adsorbs along a different axis than the other constructs with its two rows of coiled coils facing out from the surface. The elastic moduli of the filaments range from 0.7–2.1 GPa, similar to rigid plastics and within the lower limit for proteins whose primary intermolecular interaction is hydrogen bonding. These results demonstrate a versatile approach for controlling the overall dimensions and surface orientation of protein filaments, and expand the toolbox by which to tune two overall dimensions in protein space for the creation of templated materials over a wide variety of conditions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 496–503, 2009.
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
Co-reporter:Nico Bruns Dr.;Katarzyna Pustelny;LisaM. Bergeron Dr.;TimothyA. Whitehead Dr.;DouglasS. Clark
Angewandte Chemie International Edition 2009 Volume 48( Issue 31) pp:5666-5669
Publication Date(Web):
DOI:10.1002/anie.200900554
Co-reporter:Nico Bruns Dr.;Katarzyna Pustelny;LisaM. Bergeron Dr.;TimothyA. Whitehead Dr.;DouglasS. Clark
Angewandte Chemie International Edition 2009 Volume 48( Issue 31) pp:
Publication Date(Web):
DOI:10.1002/anie.200990160
Co-reporter:Nico Bruns Dr.;Katarzyna Pustelny;LisaM. Bergeron Dr.;TimothyA. Whitehead Dr.;DouglasS. Clark
Angewandte Chemie 2009 Volume 121( Issue 31) pp:5776-5779
Publication Date(Web):
DOI:10.1002/ange.200900554
Co-reporter:Nico Bruns Dr.;Katarzyna Pustelny;LisaM. Bergeron Dr.;TimothyA. Whitehead Dr.;DouglasS. Clark
Angewandte Chemie 2009 Volume 121( Issue 31) pp:
Publication Date(Web):
DOI:10.1002/ange.200990162
Co-reporter:Elton P. Hudson;Jeffrey A. Reimer;Shannon D. Chase;Jonathan S. Dordick;Ross K. Eppler
PNAS 2008 Volume 105 (Issue 41 ) pp:15672-15677
Publication Date(Web):2008-10-14
DOI:10.1073/pnas.0804566105
Recent studies exploring the relationship between enzymatic catalysis and protein dynamics in the aqueous phase have yielded
evidence that dynamics and enzyme activity are strongly correlated. Given that protein dynamics are significantly attenuated
in organic solvents and that proteins exhibit a wide range of motions depending on the specific solvent environment, the nonaqueous
milieu provides a unique opportunity to examine the role of protein dynamics in enzyme activity. Variable-temperature kinetic
measurements, X-band electron spin resonance spectroscopy, 1H NMR relaxation, and 19F NMR spectroscopy experiments were performed on subtilisin Carlsberg colyophilized with several inorganic salts and suspended
in organic solvents. The results indicate that salt activation induces a greater degree of transition-state flexibility, reflected
by a more positive ΔΔS†, for the more active biocatalyst preparations in organic solvents. In contrast, ΔΔH† was negligible regardless of salt type or salt content. Electron spin resonance spectroscopy and 1H NMR relaxation measurements, including spin-lattice relaxation, spin-lattice relaxation in the rotating frame, and longitudinal
magnetization exchange, revealed that the enzyme's turnover number (kcat) was strongly correlated with protein motions in the centisecond time regime, weakly correlated with protein motions in the
millisecond regime, and uncorrelated with protein motions on the piconanosecond timescale. In addition, 19F chemical shift measurements and hyperfine tensor measurements of biocatalyst formulations inhibited with 4-fluorobenzenesulfonyl
fluoride and 4-ethoxyfluorophosphinyl-oxy-TEMPO, respectively, suggest that enzyme activation was only weakly affected by
changes in active-site polarity.
Co-reporter:Lisa M. Bergeron, Cecilia Lee, Talar Tokatlian, Volker Höllrigl, Douglas S. Clark
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2008 Volume 1784(Issue 2) pp:368-378
Publication Date(Web):February 2008
DOI:10.1016/j.bbapap.2007.11.011
Molecular chaperones play a central role in maintaining protein structure within a cell. Previously, we determined that the gene encoding a molecular chaperone, a thermosome, from the hyperthermophilic archaeon Methanocaldococcus jannaschii is upregulated upon lethal heat shock. We have recombinantly expressed this thermosome (rTHS) and show here that it is both stable and fully functional in aqueous solutions containing water-miscible organic co-solvents. Based on circular dichroism the secondary structure of rTHS was not affected by one-hour exposures to a variety of co-solvents including 30% v/v acetonitrile (ACN) and 50% methanol (MeOH). By contrast, the secondary structure of a mesophilic homologue, GroEL/GroES (GroE), was substantially disrupted. rTHS reduced the aggregation of ovalbumin and citrate synthase in 30% ACN, assisted refolding of citrate synthase upon solvent-inactivation, and stabilized citrate synthase and glutamate dehydrogenase in the direct presence of co-solvents. Apparent total turnover numbers of these enzymes in denaturing solutions increased by up to 2.5-fold in the presence of rTHS. Mechanistic models are proposed to help ascertain specific conditions that could enhance or limit organic solvent-induced chaperone activity. These models suggest that thermodynamic stability and the reversibility of enzyme unfolding play key roles in the effectiveness of enzyme recovery by rTHS.
Co-reporter:Moo-Yeal Lee;R. Anand Kumar;Sumitra M. Sukumaran;Michael G. Hogg;Jonathan S. Dordick;
Proceedings of the National Academy of Sciences 2008 105(1) pp:59-63
Publication Date(Web):December 26, 2007
DOI:10.1073/pnas.0708756105
We have developed a miniaturized 3D cell-culture array (the Data Analysis Toxicology Assay Chip or DataChip) for high-throughput
toxicity screening of drug candidates and their cytochrome P450-generated metabolites. The DataChip consists of human cells
encapsulated in collagen or alginate gels (as small as 20 nl) arrayed on a functionalized glass slide for spatially addressable
screening against multiple compounds. A single DataChip containing 1,080 individual cell cultures, used in conjunction with
the complementary human P450-containing microarray (the Metabolizing Enzyme Toxicology Assay Chip or MetaChip), simultaneously
provided IC50 values for nine compounds and their metabolites from CYP1A2, CYP2D6, and CYP3A4 and a mixture of the three P450s designed
to emulate the human liver. Similar responses were obtained with the DataChip and conventional 96-well plate assays, demonstrating
that the near 2,000-fold miniaturization does not influence the cytotoxicity response. The DataChip may therefore enable toxicity
analyses of drug candidates and their metabolites at throughputs compatible with the availability of compounds at early-stage
drug discovery.
Co-reporter:Boonchai B. Boonyaratanakornkit;Li Yan Miao
Extremophiles 2007 Volume 11( Issue 3) pp:495-503
Publication Date(Web):2007 May
DOI:10.1007/s00792-007-0063-4
Growth and transcriptional profiles of the deep-sea methanarchaeon Methanocaldococcus jannaschii were studied under sudden up-shifts of temperature and pressure. Application of 500 atm of hyperbaric pressure shifted the optimal growth temperature upwards by about 5°C in a high temperature–pressure bioreactor, and increased the specific growth rate threefold at 88°C. In contrast, pressure-shock from 7.8 to 500 atm over 15 min, the first such pressure up-shift reported for a piezophile, did not accelerate growth. High-pressure heat-shock from 88 to 98°C, a condition relevant to the turbulent in situ surroundings of deep-sea hydrothermal vents, resulted in termination of growth. Transcriptional profiles for cells grown at 88°C and 500 atm, heat-shocked at 500 atm, and pressure-shocked to 500 atm, shared a subset of genes whose differential expression was attributed to elevated pressure. In the pressure-shock case, this transcriptional response was evident despite the absence of a piezophilic growth response. In all, despite the piezophilic capacity and high-pressure origins of M. jannaschii, the core pressure response was remarkably limited and consisted of differential expression of genes encoding three hypothetical proteins and a gene involved in DNA recombination.
Co-reporter:Matthew J. Traylor, Jack Chai, Douglas S. Clark
Archives of Biochemistry and Biophysics (15 January 2011) Volume 505(Issue 2) pp:186-193
Publication Date(Web):15 January 2011
DOI:10.1016/j.abb.2010.10.002