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D S Clark

Publications and source records attributed to D S Clark.

At least 19 recordsLinked to original sources
PubMed2001

Analysis of an engineered sulfate reduction pathway and cadmium precipitation on the cell surface.

We previously have genetically engineered an aerobic sulfate reduction pathway in Escherichia coli for the generation of hydrogen sulfide and demonstrated the pathway's utility in the precipitation of cadmium. To engineer the pathway, the assimilatory sulfate reduction pathway was modified so that cysteine was overproduced. Excess cysteine was then converted by cysteine desulfhydrase to an abundance of hydrogen sulfide, which then reacted with aqueous cadmium to form cadmium sulfide. In this study, observations of various E. coli clones were combined with an analysis of kinetic and transport phenomena. This analysis revealed that cysteine production is the rate-limiting step in the engineered pathway and provided an explanation for the phenomenon of cell surface precipitation. An analytical model showed that cadmium sulfide must form at the cell surface because the rate of cadmium sulfide formation is extremely fast and the rate of sulfide transport is relatively slow.

Cadmium↗
PubMed2001

Towards more active biocatalysts in organic media: increasing the activity of salt-activated enzymes.

The activation of freeze-dried subtilisin Carlsberg (SC) in hexane has been systematically studied and partially optimized with respect to the freezing method, the addition of inorganic salts and lyoprotectants, the initial concentration and final weight percent of additives, and the amount of water added to the organic solvent. Activity and water content were found to correlate directly with the kosmotropicity of the activating salt (kosmotropic salts bind water molecules strongly relative to the strength of water-water interactions in bulk solution). Combinations of kosmotropic salts with known lyoprotectants such as poly(ethylene glycol) (PEG) and sugars did not yield an appreciably more active catalyst. However, the combination of the kosmotropic sodium acetate with the strongly buffering sodium carbonate activated the enzyme more than the individual additives alone. Enzyme activity was enhanced further by the addition of small amounts of water to the organic solvent. Under optimal conditions, enzyme activity in hexane was improved over 27,000-fold relative to the salt-free enzyme, reaching a catalytic efficiency that was within one order of magnitude of k(cat)/K(m) for hydrolysis of the same substrate in aqueous buffer. Further activation to attain even higher catalytic efficiencies may be possible with additional optimization.

Binding Sites↗
PubMed2001

Using isotopomer path tracing to quantify metabolic fluxes in pathway models containing reversible reactions.

As a more complete picture of the genetic and enzymatic composition of cells becomes available, there is a growing need to describe how cellular regulatory elements interact with the cellular environment to affect cell physiology. One means for describing intracellular regulatory mechanisms is concurrent measurement of multiple metabolic pathways and their interactions by metabolic flux analysis. Flux of carbon through a metabolic pathway responds to all cellular regulatory systems, including changes in enzyme and substrate concentrations, enzyme activation or inhibition, and ultimately genetic control. The extent to which metabolic flux analysis can describe cellular physiology depends on the number of pathways in the model and the quality of the data. Intracellular information is obtainable from isotopic tracer experiments, the most extensive being the determination of the isotopomer distribution, or specific labeling pattern, of intracellular metabolites. We present a rapid and novel solution method that determines the flux of carbon through complex pathway models using isotopomer data. This time-consuming problem was solved with the introduction of isotopomer path tracing, which drastically reduces the number of isotopomer variables to the number of isotopomers observed experimentally. We propose a partitioned solution method that takes advantage of the nearly linear relationship between fluxes and isotopomers. Whereas the stoichiometric matrix and the isotopomer matrix are invertible, simulated annealing and the Newton-Raphson method are used for the nonlinear components. Reversible reactions are described by a new parameter, the association factor, which scales hyperbolically with the rate of metabolite exchange. Automating the solution method permits a variety of models to be compared, thus enhancing the accuracy of results. A simplified example that contains all of the complexities of a comprehensive pathway model is presented.

Algorithms↗
PubMed2001

Stable sol-gel microstructured and microfluidic networks for protein patterning.

We demonstrate the formation of micropatterned sol-gel structures containing active proteins by patterning with polydimethylsiloxane (PDMS) microchannels. To transport sol solution efficiently into the hydrophobic PDMS microchannels, a hydrophilic-hydrophobic block copolymer was used to impart hydrophilicity to the PDMS microchannels. Poor adhesion of the micropatterned gel structure onto glass slides was improved by treating the glass surface with a polymeric substrate. To minimize cracks in the gel microstructure, hybrid matrices of interpenetrating organic and inorganic networks were prepared containing the reactive organic moieties polyvinylalcohol or polyvinylpyrrolidone. Retention of biochemical activity within the micropatterned gel was demonstrated by performing immunobinding assays with immobilized immunoglobulin G (IgG) antibody. The potential application of microfluidics technology to immobilized-enzyme biocatalysis was demonstrated using PDMS-patterned microchannels filled with trypsin-containing sol-gels. This work provides a foundation for the microfabrication of functional protein chips using sol-gel processes.

Catalysis↗
PubMed2001

Variations in the enantioselectivity of salt-activated subtilisin induced by lyophilization.

Including excess salt during lyophilization has been shown to increase the activity of freeze-dried subtilisin Carlsberg (SC) in anhydrous media by over 20,000-fold [Ru et al. (1999) Biotechnol Bioeng 63:233-241]. In the present study, salt-activated SC (KCl-SC) showed a 30% enhancement in enantioselectivity compared to the salt-free enzyme in a variety of organic solvents. Activity toward both enantiomers of N-acetyl-phenylalanine methyl ester (APME) increased in tandem by 2-3 orders of magnitude in all solvents, indicating that the mechanism of salt activation is inherent to the enzyme and does not strongly favor one enantiomer over the other. However, activity and enantioselectivity of salt-activated SC could be manipulated through changes in the lyophilization conditions. Variations in lyophilization time, initial KCl concentration, and initial lyophilization volume altered enantioselectivity over 2-fold. The changes in enantioselectivity reflected the activity for the L enantiomer, while the activity toward the D enantiomer was mostly unaffected. The results indicate that the lyophilization time and final water content of the KCl-SC are important determinants of enzyme activity for the L enantiomer, suggesting that the favored reaction is more sensitive to the structural integrity of the salt-activated enzyme.

Freeze Drying↗
PubMed2001

Activation of enzymes for nonaqueous biocatalysis by denaturing concentrations of urea.

Urea is one of the most commonly used denaturants of proteins. However, herein we report that enzymes lyophilized from denaturing concentrations of aqueous urea exhibited much higher activity in organic solvents than their native counterparts. Thus, instead of causing deactivation, urea effected unexpected activation of enzymes suspended in organic media. Activation of subtilisin Carlsberg (SC) in the organic solvents (hexane, tetrahydrofuran, and acetone) increased with increasing urea concentrations up to 8 M. Active-site titration results and activity assays indicated the presence of partially unfolded but catalytically active SC in 8 M urea; however, the urea-modified enzyme retained high enantioselectivity and was ca. 80 times more active than the native enzyme in anhydrous hexane. Likewise, the activity of horseradish peroxidase (HRP) lyophilized from 8 M urea was ca. 56 times and 350 times higher in 97% acetone and water-saturated hexane, respectively, than the activity of HRP lyophilized from aqueous buffer. Compared with the native enzyme, the partially unfolded enzyme may have a more pliant and less rigid conformation in organic solvents, thus enabling it to retain higher catalytic activity. However, no substantial activation was observed for alpha-chymotrypsin lyophilized from urea solutions in which the enzyme retained some activity, illustrating that the activation effect is not completely general.

Acetone↗
PubMed2001

Aerobic sulfide production and cadmium precipitation by Escherichia coli expressing the Treponema denticola cysteine desulfhydrase gene.

The cysteine desulfhydrase gene of Treponema denticola was over-expressed in Escherichia coli to produce sulfide under aerobic conditions and to precipitate metal sulfide complexes on the cell wall. When grown in a defined salts medium supplemented with cadmium and cysteine, E. coli producing cysteine desulfhydrase secreted sulfide and removed nearly all of the cadmium from solution after 48 h. A control strain produced significantly less sulfide and removed significantly less cadmium. Measurement of acid-labile sulfide and energy dispersive X-ray spectroscopy indicated that cadmium was precipitated as cadmium sulfide. Without supplemental cysteine, both the E. coli producing cysteine desulfhydrase and the control E. coli demonstrated minimal cadmium removal.

Aerobiosis↗
PubMed2001

Probing stability-activity relationships in the thermophilic proteasome from Thermoplasma acidophilum by random mutagenesis.

Structural perturbations (L65H, V12L/M27T, F35V) generated by random mutation of the beta-subunit were used to probe the relationship between stability and activity in the thermophilic proteasome from Thermoplasma acidophilum. The optimum temperature for activity of each mutant (approximately 95 degrees C) remained unchanged; however, each mutant was significantly less stable than the wild type. Stability, therefore, is not the factor limiting high-temperature activity. Interestingly, mutation L65H drastically reduced stability without affecting specific activity over a wide temperature range, providing evidence that activity and stability can be decoupled. To investigate the nature of the flexibility introduced by mutation, stability of the proteasome was examined under pressure. The application of 10,000 psi stabilized the wild-type proteasome 3.4 fold at 97 degrees C. When inactivation temperatures were chosen such that the rate of inactivation of the mutants was similar to that of the wild type, mutants with changes at the intersubunit interfaces (L65H and V12L/M27T) were similarly stabilized. Pressure was less effective in stabilizing mutant F35V, however, in which the substitution may have introduced a new pathway for inactivation.

Cysteine Endopeptidases↗
PubMed2001

Mechanism of pressure-induced thermostabilization of proteins: studies of glutamate dehydrogenases from the hyperthermophile Thermococcus litoralis.

In this study, we investigated the effect of pressure on protein structure and stability at high temperature. Thermoinactivation experiments at 5 and 500 atm were performed using the wild-type (WT) enzyme and two single mutants (D167T and T138E) of the glutamate dehydrogenase (GDH) from the hyperthermophile Thermococcus litoralis. All three GDHs were stabilized, although to different degrees, by the application of 500 atm. Interestingly, the degree of pressure stabilization correlated with GDH stability as well as the magnitude of electrostatic repulsion created by residues at positions 138 and 167. Thermoinactivation experiments also were performed in the presence of trehalose. Addition of the sugar stabilized all three GDHs; the degree of sugar-induced thermostabilization followed the same order as pressure stabilization. Previous studies suggested a mechanism whereby the enzyme adopts a more compact and rigid structure and volume fluctuations away from the native state are diminished under pressure. The present results on the three GDHs allowed us to further confirm and refine the proposed mechanism for pressure-induced thermostabilization. In particular, we propose that pressure stabilizes against thermoinactivation by shifting the equilibrium between conformational substates of the GDH hexamer, thus inhibiting irreversible aggregation.

Enzyme Stability↗
PubMed2001

Chemical denaturation and elevated folding temperatures are required for wild-type activity and stability of recombinant Methanococcus jannaschii 20S proteasome.

The 20S proteasome from the extreme thermophile Methanococcus jannaschii (Mj) was purified and sequenced to facilitate production of the recombinant proteasome in E. coli. The recombinant proteasome remained in solution at a purity level of 80-85% (according to SDS PAGE) following incubation of cell lysates at 70 degrees C. Temperature-activity profiles indicated that the temperature optima of the wild-type and recombinant enzymes differed substantially, with optimal activities occurring at 119 degrees C and 95 degrees C, respectively. To ameliorate this discrepancy, two recombinant enzyme preparations were produced, each of which included denaturation of the proteasome by 4 M urea followed by high-temperature (85 degrees C) dialysis. The wild-type temperature optimum was restored, but only if proteasome subunits were denatured and refolded prior to assembly (a preparation designated as alpha & beta). In contrast, when proteasome assembly preceded denaturation (designated alpha + beta) the optimum temperature was raised to a lesser degree. Moreover, the alpha & beta and alpha + beta preparations had apparent thermal half-lives at 114 degrees C of 54.2 and 26.2 min, respectively, and the thermostability of the less stable enzyme was more sensitive to a reduction in pH. Attainment of wild-type activity and stability thus required the proper folding of both the alpha- and beta-subunits prior to proteasome assembly. Consistent with this behavior, dual-scanning calorimetry (DSC) measurements revealed differences in the reassembly efficiency of the two proteasome preparations. The ability to produce structural conformers with dramatically different thermal optima and thermostabilities may facilitate the determination of molecular forces and structural motifs responsible for enzyme thermostablity and high-temperature activity.

Calorimetry, Differential Scanning↗
PubMed2000

Chemoenzymatic construction of a four-component Ugi combinatorial library.

The chemoenzymatic preparation of a nine-member Ugi condensation library is described. The carboxylic acid and amine precursors are based on 3-hydroxybutyrate and 4-amino-1-butanol, respectively, and have been acylated selectively using a variety of acyl donors catalyzed by porcine pancreatic lipase. The enzyme is selective for the hydroxyl functionalities on both precursors, thereby yielding 3-acyl-butyric acid and 4-amino-1-acyl compounds. These enzymatically generated derivatives were then subjected to a four-component Ugi condensation reaction in the presence of acetaldehyde and methyl isocyanoacetate. Isolated yields of the alpha-(acylamino)amide Ugi products ranged from 72-95%. The inherent chemoselectivity of enzymatic catalysis may play an increasingly important role in expanding the structural diversity that can be achieved by chemical multicomponent condensation reactions.

3-Hydroxybutyric Acid↗
PubMed2000

Intrinsic effects of solvent polarity on enzymic activation energies.

The effect of organic solvents on subtilisin Carlsberg catalysis has been investigated with the aid of a thermodynamic analysis. Saturation solubility experiments were performed to provide a quantitative measure of substrate desolvation from the reaction medium. This enabled calculation of the intrinsic enzymic activation energy and resulted in a linear free energy relationship with respect to solvent polarity. The results indicate that the intrinsic activation energy of subtilisin catalysis is lowest in polar organic solvents, which may be due to transition state stabilization of the enzyme's polar transition state for transesterification.

Bacillus↗
PubMed2000

Posterior capsule opacification.

Posterior capsule opacification (PCO) is the most common complication following primary cataract surgery. Advances in intraocular lens (IOL) designs that have reduced the amount of PCO following surgery have been made. The understanding of how the IOL design effects PCO has also advanced. Lenses that provide a mechanical barrier between it and the posterior lens capsule seem to inhibit PCO to a greater degree. Intracapsular rings are now being explored to test and enhance this barrier effect. Major advances in the elimination of lens epithelial cells at the time of surgery especially by pharmacologic means have also been made. An immunotoxin specific for human lens epithelial cells shows promise and is under latter phase clinical development.

Cataract↗
PubMed2000

Metabolic engineering of an aerobic sulfate reduction pathway and its application to precipitation of cadmium on the cell surface.

The conversion of sulfate to an excess of free sulfide requires stringent reductive conditions. Dissimilatory sulfate reduction is used in nature by sulfate-reducing bacteria for respiration and results in the conversion of sulfate to sulfide. However, this dissimilatory sulfate reduction pathway is inhibited by oxygen and is thus limited to anaerobic environments. As an alternative, we have metabolically engineered a novel aerobic sulfate reduction pathway for the secretion of sulfides. The assimilatory sulfate reduction pathway was redirected to overproduce cysteine, and excess cysteine was converted to sulfide by cysteine desulfhydrase. As a potential application for this pathway, a bacterium was engineered with this pathway and was used to aerobically precipitate cadmium as cadmium sulfide, which was deposited on the cell surface. To maximize sulfide production and cadmium precipitation, the production of cysteine desulfhydrase was modulated to achieve an optimal balance between the production and degradation of cysteine.

Aerobiosis↗
PubMed2000

Engineering hydrogen sulfide production and cadmium removal by expression of the thiosulfate reductase gene (phsABC) from Salmonella enterica serovar typhimurium in Escherichia coli.

The thiosulfate reductase gene (phsABC) from Salmonella enterica serovar Typhimurium was expressed in Escherichia coli to overproduce hydrogen sulfide from thiosulfate for heavy metal removal (or precipitation). A 5.1-kb DNA fragment containing phsABC was inserted into the pMB1-based, high-copy, isopropyl-beta-D-thiogalactopyranoside-inducible expression vector pTrc99A and the RK2-based, medium-copy, m-toluate-inducible expression vector pJB866, resulting in plasmids pSB74 and pSB77. A 3. 7-kb DNA fragment, excluding putative promoter and regulatory regions, was inserted into the same vectors, making plasmids pSB103 and pSB107. E. coli DH5alpha strains harboring the phsABC constructs showed higher thiosulfate reductase activity and produced significantly more sulfide than the control strains under both aerobic and anaerobic conditions. Among the four phsABC constructs, E. coli DH5alpha (pSB74) produced thiosulfate reductase at the highest level and removed the most cadmium from solution under anaerobic conditions: 98% of all concentrations up to 150 microM and 91% of 200 microM. In contrast, a negative control did not produce any measurable sulfide and removed very little cadmium from solution. Energy-dispersive X-ray spectroscopy revealed that the metal removed from solution precipitated as a complex of cadmium and sulfur, most likely cadmium sulfide.

Cadmium↗
PubMed1999

Optimizing the salt-induced activation of enzymes in organic solvents: effects of lyophilization time and water content.

The addition of simple inorganic salts to aqueous enzyme solutions prior to lyophilization results in a dramatic activation of the dried powder in organic media relative to enzyme with no added salt. Activation of both the serine protease subtilisin Carlsberg and lipase from Mucor javanicus resulting from lyophilization in the presence of KCl was highly sensitive to the lyophilization time and water content of the sample. Specifically, for a preparation containing 98% (w/w) KCl, 1% (w/w) phosphate buffer, and 1% (w/w) enzyme, varying the lyophilization time showed a direct correlation between water content and activity up to an optimum, beyond which the activity decreased with increasing lyophilization time. The catalytic efficiency in hexane varied as much as 13-fold for subtilisin Carlsberg and 11-fold for lipase depending on the lyophilization time. This dependence was apparently a consequence of including the salt, as a similar result was not observed for the enzyme freeze-dried without KCl. In the case of subtilisin Carlsberg, the salt-induced optimum value of kcat/Km for transesterification in hexane was over 20,000-fold higher than that for salt-free enzyme, a substantial improvement over the previously reported enhancement of 3750-fold (Khmelnitsky, 1994). As was found previously for pure enzyme, the salt-activated enzyme exhibited greatest activity when lyophilized from a solution of pH equal to the pH for optimal activity in water. The active-site content of the lyophilized enzyme samples also depended upon lyophilization time and inclusion of salt, with opposite trends in this dependence observed for the solvents hexane and tetrahydrofuran. Finally, substrate selectivity experiments suggested that mechanism(s) other than selective partitioning of substrate into the enzyme-salt matrix are responsible for salt-induced activation of enzymes in organic solvents.

Bacillus↗
PubMed1999

Combinatorial biocatalysis: taking the lead from nature.

Combinatorial biocatalysis is an emerging technology in the field of drug discovery. The biocatalytic approach to combinatorial chemistry uses enzymatic, chemoenzymatic, and microbial transformations to generate libraries from lead compounds. Important recent advances in combinatorial biocatalysis include iterative derivatization of small molecules and complex natural products, regioselectively controlled libraries, novel one-pot library syntheses, process automation, and biocatalyst enhancements.

Automation↗