Control Of Redox Potential Research Articles

Developing cell-free biology for industrial applications

Although cell-free protein synthesis has been practiced for decades as a research tool, only recently have advances suggested its feasibility for commercial protein production. This focused review, based on the 2005 Amgen Award lecture, summarizes the relevant progress from the Swartz laboratory. When our program began, projected costs were much too high, proteins with disulfide bonds could not be folded effectively, and no economical scale-up technologies were available. By focusing on basic biochemical reactions and by controlling cell-free metabolism, these limitations have been methodically addressed. Amino acid supply has been stabilized and central metabolism activated to dramatically reduce substrate costs. Control of the sulfhydral redox potential has been gained and a robust disulfide isomerase added to facilitate oxidative protein folding. Finally, simple scale-up technologies have been developed. These advances not only suggest production feasibility for pharmaceutical proteins, they also provide enabling technology for producing patient-specific vaccines, for evolving new enzymes to enable biological hydrogen production from sunlight, and for developing new and highly effective water filters. Although many challenges remain, this newly expanded ability to activate and control protein production holds much promise for both research and commercial applications.

Impact on the remobilization of trace metals in lake sediments by micro-organisms

蓄积在湖泊沉积物中污染物质某些情况下可以成为威胁上覆水体水质安全的二次污染源．根据贵州省阿哈湖季节性缺氧的特性,通过控制氧化还原状况,设计了对该湖沉积物-水柱的原样/抑菌条件的培养实验．实验发现,微生物活动对界面附近氧化还原反应具有控制作用;改变水体的含氧状况可以显著影响上覆水体水质,包括表观性状和水体中污染物含量．聚类分析结果表明,早期成岩过程中Mn,Ga,SO₄^2-,Cu,Cr,Pb和Co,Ni,Fe,Sc,V,Rb两大类分别具有相似的地球化学行为．根据实验结果,计算了厌氧培养过程中,微量元素的最大释放量,发现铁、锰在厌氧过程中可以大量向水体释放．;It is of great concerns on some pollutants cumulated in lake sediments to become the source of pollution, under certain circumstances, causing serious harm on overlying water quality. In this research, experiment was designed based on the fact of seasonal hypoxia near sediment-water interface (SWI) in Lake Aha,Guizhou Province. By means of the control of redox potential, together with microbiological activities, it was found that microbiological activities has significant influences on the redox reactions happened around SWI; and the overlying water quality can be changed remarkably, along with the variation of redox potential, including transparency, odor and poUutant contents. The cluster analysis of experimental data demonstrated that two main groups with similar geochemical behavior during diagenesis process, could be sorted as Mn,Ga, SO₄^2-,Cu,Cr,Pb and Co,Ni,Fe, Sc,V, Rb. Finally, the maximum release of some metals when lake water turned to hypoxia, were calculated, of which plenty of Fe and Mn found can be released into overlying water.

On-Line Measuring Method for the Concentration of Peroxodisulphate

Although the analytical determination of peroxodisulphate is state of the art, there is no well-established on-line measuring method for this substance. This paper presents an on-line monitoring device for peroxodisulphate concentration that uses a coulometric measuring method. The method is based on the redox potential control in a miniaturised electrochemical reactor. This paper presents the design of the measuring cell and describes the integration of this cell in an on-line measuring device which can be applied in a process environment. Experimental results illustrate its dynamic characteristics and accuracy.

Control of Redox Potential by Deprotonation of Coordinated 1H-Imidazole in Complexes of 2-(1H-Imidazol-2-yl)pyridine

AbstractComplexes [ML3]2+ of the bidentate ligand 2‐(1H‐imidazol‐2‐yl)pyridine were prepared with iron(II), cobalt(II), and ruthenium(II). The electronic spectra suggest the ligand to be a weaker σ‐donor and π‐acceptor than the closely related 2,2′‐bipyridine. The complexes are readily deprotonated by addition of base, and the effect of the deprotonation is to lower the MIII/MII redox potential by roughly 900 mV. This is roughly 75% of the drop observed for related complexes of 2,6‐di‐1H‐imidazol‐2‐ylpyridine, and suggests the effect to be largely coulombic in origin.

Cyclometalated N, N-dimethylbenzylamine ruthenium(II) complexes [Ru(C 6HR 1R 2R 3- o-CH 2NMe 2)(bpy)(RCN) 2]PF 6 for bioapplications: synthesis, characterization, crystal structures, redox properties, and reactivity toward PQQ-dependent glucose dehydrogenase

Cyclometalated derivatives of ring-substituted N, N-dimethylbenzylamines with controlled redox potentials as potent mediators of bioelectrochemical electron transport are reported. The cycloruthenation of R 1R 2R 3C 6H 2CH 2NMe 2 (R 1, R 2, R 3 = H, Me, t BuO, MeO, NMe 2, F, CF 3, CN, NO 2) by [(η 6-C 6H 6)RuCl(μ-Cl)] 2 in the presence of NaOH/KPF 6 in acetonitrile or pivalonitrile affords cyclometalated complexes [(η 6-C 6H 6)Ru(C 6HR 1R 2R 3- o-CH 2NMe 2)(RCN)]PF 6 [R = Me ( 1) and R = CMe 3 ( 2)] in good yields. Reactions of complexes 1 and 2 with 2,2′-bipyridine (bpy) in acetonitrile or pivalonitrile result in dissociation of η 6-bound benzene and the formation of [Ru(C 6HR 1R 2R 3- o-CH 2NMe 2)(bpy)(RCN) 2]PF 6 [R = Me ( 3) and R = CMe 3 ( 4)]. All new compounds have been fully characterized by mass spectrometry, 1H/ 13C NMR, and IR spectroscopy. An X-ray crystal structural investigation of complex 1 (R 1/R 2/R 3 = H/H/H) and two complexes of type 3 (R 1/R 2/R 3 = MeO/H/H, MeO/MeO/H) has been performed. Acetonitrile ligands of 3 are mutually cis and the σ-bound carbon is trans to one of the bpy nitrogens. Measured by the cyclic voltammetry in MeOH as solvent, the redox potentials of complexes 3 for the Ru II/III feature cover the range 320–720 mV (versus Ag/AgCl) and correlate linearly with the Hammett ( σ p + + σ m ) constants. Complexes 3 mediate efficiently the electron transport between the active site of PQQ-dependent glucose dehydrogenase (PQQ = pyrroloquinoline quinone) and a glassy carbon electrode. Determined by cyclic voltammetry the second order rate constant for the oxidation of the reduced (by d-glucose) enzyme active site by Ru III derivative of 3 (R 1/R 2/R 3 = H) (generated electrochemically) is as high as 4.8 × 10 7 M −1 s −1 at 25 °C and pH 7.

A method for cultivating plants under controlled redox intensities in hydroponics

An automated, low-cost hydroponic system was developed for growing single plants at controlled redox potentials (Eh) for extended periods. The system features a millivoltmeter with high and low set-point relays and offers the investigator full control of Eh within the entire redox potential range encountered in natural soils (−300 to +700 mV) with an accuracy of ±50 mV around the set-point value. Eh is lowered in the nutrient solution by adding the reducing agent titanium citrate. Conversely, Eh is elevated by bubbling the nutrient solution with air. The system’s ability to control Eh is demonstrated using data from a test experiment involving Phragmites australis plants grown at three redox levels (−150, +150 and +550 mV). Some frequently encountered factors that were potentially responsible for erroneous control are discussed. The buffer capacity and the relationship between pH and Eh were determined for the nutrient solution. These parameters, along with plant-mediated changes in pH, not only determine the time interval between pH adjustments, but also affect the Eh fluctuations in the continuously aerated experimental units. The variability and drift in readings from the platinum (Pt) electrodes, used to measure Eh, was investigated in order to relate electrode reliability to system performance. The variation in Eh readings for clean Pt electrodes was lower (S.E.=4.3 mV, n=23) than that of the calomel reference electrodes (S.E.=6.8 mV, n=23), the difference fixed across solutions of different poise. The response time (drift) of Pt electrodes in hydroponics (low-poised nutrient solution) was compared to pH-buffered quinhydrone (medium-poised) and flooded alluvial silt (well-poised). Drift increased from the low-poised nutrient solution, where Eh readings stabilized within minutes, to high-poised flooded soil, where Eh readings showed a downward drift for ca. 50 h. Overall, it is concluded that both the Pt electrodes and the calomel reference electrodes perform well in hydroponics. The extent to which the high NaCl salinity of titanium citrate (ca. 84‰) may limit the growth of plants is also discussed relative to the proportion of titanium citrate in the nutrient solution.

Flavin reduction potential tuning by substitution and bending

Computations on a series of flavin derivatives with the B3LYP hybrid density functional and 6-311G(2d,2p) basis set indicate that the ionization potentials (IPs) of the anionic semiquinone and hydroquinone states serve as accurate predictors of the flavin one- and two-electron reduction potentials. The relation between the semiquinone IPs and the two-electron reduction potentials, E m, is ΔIP sq − /Δ E m=3.69±0.18 meV/mV, with a very similar relation obtained for the flavin derivatives' Kohn–Sham highest occupied molecular orbital (KS-HOMO) energies, E KS-HOMO vs. E m. Interestingly, these good correlations between vertical IPs and E ms are observed even though the second reduction step, Fl sq⇌Fl red − involves significant conformational changes for a number of the derivatives. In fact, the flavin derivatives can be divided roughly into two categories. Those derivatives with high E ms are either planar in the anionic reduced state, or else a negligible amount of energy (<kT) is required for planarizing them. Flavin derivatives of low potential however possess significant conformational energy (>kT), and tend to have larger butterfly bends. The flavin parent compound, lumiflavin, represents the dividing point between these two categories, a result with possibly interesting biological implications for the conformational control of flavin redox potentials by enzymes. B3LYP/6-311G(2d,2p) calculations on lumiflavin constrained to various butterfly bend angles show that the oxidized and semiquinone states (both anionic and protonated at N 5) resist bending, with the oxidized state being by far the stiffest. On the other hand, the optimum geometry of the fully reduced state is bent by 15.9° in the anionic state and 24.4° in the neutral state. Full MP2 geometry optimizations confirm the reduced flavin butterfly bend, however the bend angles are larger than the DFT results: 28.7° and 32.6° for the anionic and neutral states, respectively. The relation of the N 5 and N 10 pyramidalization to the flavin butterfly bend is discussed. The results indicate that a protein-enforced flavin conformation should have significant and differing effect on each of the one electron reduction steps.

Probing electrostatic interactions in cytochrome c using site-directed chemical modification.

This communication reports the generation of an electrostatic probe using chemical modification of methionine side chains. The alkylation of methionine by iodoacetamide was achieved in a set of Saccharomyces cerevisiae iso-1-cytochrome c mutants, introducing the nontitratable, nondelocalized positive charge of a carboxyamidomethylmethionine sulfonium (CAMMS) ion at five surface and one buried site in the protein. Changes in redox potential and its variation with temperature were used to calculate microscopic effective dielectric constants operating between the probe and the heme iron. Dielectric constants (epsilon) derived from deltadeltaG values were not useful due to entropic effects, but epsilon(deltadeltaH) gave results that supported the theory. The effect on biological activity of surface derivatization was interpreted in terms of protein-protein interactions. The introduction of an electrostatic probe in cytochrome c often resulted in marked effects on activity with one of two physiological partners: cytochrome c reductase, especially if introduced at position 65, and cytochrome c oxidase, if at position 28.

Reciprocating constructed wetlands for treating industrial, municipal and agricultural wastewater

Scientists at the Tennessee Valley Authority (TVA), and in collaboration with the U.S. Environmental Protection Agency (EPA), are continuing to develop and refine an innovative wastewater treatment system referred to as reciprocating subsurface-flow constructed wetlands. Reciprocation relates to patented improvements in the design and operation of paired subsurface-flow constructed wetlands, such that contiguous cells are filled and drained on a frequent and recurrent basis. This operating technique turns the entire wetland system into a fixed-film biological reactor, in which it is possible to control redox potential in alternating aerobic and anaerobic zones. Reciprocating systems enable manipulation of wastewater treatment functions by controlling such parameters as hydraulic retention time, frequency of reciprocation, reciprocation cycle time, depth of reciprocation, and size and composition of substrate. These improved wetland technologies have been used for treating municipal/domestic wastewater, high strength animal wastewater, and mixed wastewater streams containing acids, recalcitrant compounds, solvents, antifreeze compounds, heavy metals, explosives, and fertilizer nutrients. Results from selected treatability studies and field demonstrations will be summarized with respect to conceptual design and treatment efficacy.

Electrochemical Vat Dyeing Combination of an Electrolyzer with a Dyeing Apparatus

A standard dyeing apparatus for yarn on X-cones has been coupled to a multicathode electrolyzer for indirect reduction of dispersed oxidized vat dyes. An alkaline solution containing 0.01 M iron-triethanolamine complex was used as mediator solution. In test runs current efficiencies better than 80% were obtained at cell currents up to 10 A. The cathode was built up from 10 insulated sheets of steel fabric with a total area of 0.96 m2. In batch experiments cones of cotton yarn with an average mass of 900 g yarn were dyed. Different dyes were applied in light to dark shades at a dyeing temperature of 60°C. During the electrochemical dyeing process the cell must carry out different functions: reduction of dissolved oxygen, indirect reduction of dispersed oxidized vat dye, buildup of a certain reduction capacity against air oxygen, and control of redox potential in the dyeing apparatus by adjustment of cell current. A description of the concentrations of dissolved oxygen and reduced form of the mediator during electrolysis can be achieved by a simple mathematical model, which permits efficient optimization of important technical parameters for process design and scale-up. © 2001 The Electrochemical Society. All rights reserved.

Heme redox potential control in de novo designed four-alpha-helix bundle proteins.

The effects of various mechanisms of metalloporphyrin reduction potential modulation were investigated experimentally using a robust, well-characterized heme protein maquette, synthetic protein scaffold H10A24 [¿CH(3)()CONH-CGGGELWKL.HEELLKK.FEELLKL.AEERLKK. L-CONH(2)()¿(2)](2). Removal of the iron porphyrin macrocycle from the high dielectric aqueous environment and sequestration within the hydrophobic core of the H10A24 maquette raises the equilibrium reduction midpoint potential by 36-138 mV depending on the hydrophobicity of the metalloporphyrin structure. By incorporating various natural and synthetic metalloporphyrins into a single protein scaffold, we demonstrate a 300-mV range in reduction potential modulation due to the electron-donating/withdrawing character of the peripheral macrocycle substituents. Solution pH is used to modulate the metalloporphyrin reduction potential by 160 mV, regardless of the macrocycle architecture, by controlling the protonation state of the glutamate involved in partial charge compensation of the ferric heme. Attempts to control the reduction potential by inserting charged amino acids into the hydrophobic core at close proximity to the metalloporphyrin lead to varied success, with H10A24-L13E lowering the E(m8.5) by 40 mV, H10A24-E11Q raising it by 50 mV, and H10A24-L13R remaining surprisingly unaltered. Modifying the charge of the adjacent metalloporphyrin, +1 for iron(III) protoporphyrin IX or neutral for zinc(II) protoporphyrin IX resulted in a loss of 70 mV [Fe(III)PPIX](+) - [Fe(III)PPIX](+) interaction observed in maquettes. Using these factors in combination, we illustrate a 435-mV variation of the metalloporphyrin reduction midpoint potential in a simple heme maquette relative to the about 800-mV range observed for natural cytochromes. Comparison between the reduction potentials of the heme maquettes and other de novo designed heme proteins reveals global trends in the E(m) values of synthetic cytochromes.

Scale-up of citric acid fermentation by redox potential control

To obtain high citric acid productivity in Aspergillus niger fermentation on beet molasses substrate, a certain redox potential profile with two maxima (260 and 280 mV) and two minima (180 and 80 mV) must be maintained. The most effective regulation of redox potential is by regulation of aeration and agitation. It has been shown that control of redox potential by aeration and agitation is a most successful method for scale-up from 10-L laboratory scale to the 100- and 1000-L pilot-plant scale, even in geometrically dissimilar stirred-tank reactors. Copyright 1999 John Wiley & Sons, Inc.

Bioleaching of copper sulphide concentrate using extreme thermophilic bacteria

An extreme thermophilic, iron-sulphur oxidising bacterial culture was isolated and adapted to tolerate high metal and solids concentrations at 70°C Following isolation and adaptation, the culture was used in a continuous multi-stage pilot plant employing standard mechanically agitated and aerated tanks, for the bioleaching of a mixed secondary copper sulphide-chalcopyrite concentrate. The culture exhibited stable leach performance over the period of pilot plant operation and overall copper extractions of higher than 97% were maintained. It was shown, however, that solids concentration had a significant effect on the copper bioleach kinetics. Increased solids concentrations resulted in a drop in redox potential and copper extractions in the first stage reactor. Batch chemical ferric leaches, performed at controlled redox potentials, indicated that reduced residence times may be possible in bioleach processes, using thermophiles, if high redox potential levels can be maintained.

The effect of redox potential changes on reductive dechlorination of pentachlorophenol and the degradation of acetate by a mixed, methanogenic culture

The effect of changes in redox potential on methanogenesis from acetate, and on the reductive dechlorination of pentachlorophenol (PCP), was evaluated using a computer-monitored and feedback-controlled bioreactor. PCP was transformed via 2,3,4, 5-tetrachlorophenol (2,3,4,5-TeCP) to 3,4,5-trichlorophenol (3,4, 5-TCP). In 6- to 12-d experiments, pH, acetate concentration, and temperature were held constant; the redox potential, defined here as the potential measured at a platinum electrode (EPt), was maintained at different set points, while transformation of multiple PCP additions was monitored. Without redox potential control, the value of EPt for the culture was approximately -0.26 V (vs. SHE). The value of EPt was elevated from -0.26 V for periods up to 10 h by computer-controlled addition of H2O2 or K3Fe(CN)6. Methanogenesis continued during a relatively mild shift of EPt to -0.2 V with H2O2, but was halted when EPt was raised to -0.1 V with either H2O2 or K3Fe(CN)6. Methanogenesis resumed when EPt returned to -0.26 V. During periods in which EPt was elevated significantly and methanogenesis stopped, transformation of PCP and 2,3,4,5-TeCP continued at progressively slower rates, but the rate of 2,3,4, 5-TeCP transformation was diminished to a greater extent. When a small volume of pure H2 was added to the reactor headspace, while EPt was maintained at -0.1 V, reductive dechlorination rates increased dramatically. Lower H2 concentrations during periods of oxidant addition, perhaps due to the effect of the oxidant on H2-producing bacteria, may contribute to decreased reductive dechlorination rates. Copyright 1999 John Wiley & Sons, Inc.

Influence of Redox Potential on Product Distribution in Clostridium thermosuccinogenes

Clostridium thermosuccinogenes are the only known anaerobic thermophilic bacteria that ferment inulin to succinate and acetate as major products and formate, lactate, and ethanol as minor products. In this study, organic acid production in 2-L fermentations having an initially low (−300 to −330 mV) or high (−220 to −250 mV) redox potential was compared for two strains of C. thermosuccinogenes (DSM 5808 and DSM 5809). Although DSM 5809 consistently provided higher succinate yield, high variability in results was attributed to the absence of redox control during the fermentations, and, therefore, fermentations at three controlled redox potentials (−240, −275, and −310 mV) were conducted. At an intermediate redox potential (−275 mV), the succinate yield was the greatest (0.36 g of succinate/g of hexose unit), whereas ethanol yield was the least (0.02 g/g). Redox potential did not significantly affect acetate or lactate formation. At controlled redox potential of −275 mV, the growth of DSM 5809 on three substrates was also compared: inulin, fructose, and glucose. DSM 5809 had similar growth rates when inulin (0.20/h) or glucose (0.21/h) was the carbon source but grew more slowly when fructose (0.16/h) was the carbon source. Also, the specific rate of utilization of fructose by DSM 5809 was higher (0.89 g of fructose/[g of biomass·h]) compared to glucose (0.53 g/[g·h]) or inulin (0.55 g/[g·h]). Succinate was the major product formed by DSM 5809 fermenting inulin (0.50 g/[g·h]) or glucose (0.36 g/[g·h]), and ethanol was the principal product when DSM 5809 fermented fructose (0.54 g/[g·h]).

THE FUNCTIONAL SIGNIFICANCE OF THE HYPORHEIC ZONE IN STREAMS AND RIVERS

▪ Abstract The hyporheic zone is an active ecotone between the surface stream and groundwater. Exchanges of water, nutrients, and organic matter occur in response to variations in discharge and bed topography and porosity. Upwelling subsurface water supplies stream organisms with nutrients while downwelling stream water provides dissolved oxygen and organic matter to microbes and invertebrates in the hyporheic zone. Dynamic gradients exist at all scales and vary temporally. At the microscale, gradients in redox potential control chemical and microbially mediated nutrient transformations occurring on particle surfaces. At the stream-reach scale, hydrological exchange and water residence time are reflected in gradients in hyporheic faunal composition, uptake of dissolved organic carbon, and nitrification. The hyporheic corridor concept describes gradients at the catchment scale, extending to alluvial aquifers kilometers from the main channel. Across all scales, the functional significance of the hyporheic zone relates to its activity and connection with the surface stream.

A Critical Review of Stabilization/Solidification Technology

The processes and techniques of stabilization/solidification (S/S) have matured into an accepted, and important, part of environmental technology. As a result, a great many S/S methods have been promoted recently and offered for the treatment of hazardous and other wastes from industry, municipalities, and government sources. The purpose of this article is review these methods critically in light of the current regulatory atmosphere that controls and often mandates their use.An overview of the technology is provided. The generic and proprietary S/S processes (chemical processes, physical processes, and thermal processes) are described. Of these, six generic chemical processes dominate the field and encompass nearly all of the treatment work and proprietary products to date: Portland cement, cement/fly ash, cement/soluble silicate, lime/flyash, kiln dust, and phosphate.Current and anticipated major waste streams using S/S technology today are summarized. The S/S approach for each of these are reviewed. For metals, the primary factors affecting immobilization are pH control, chemical speciation, and redox potential control. For organics, immobilization of constituents can be broken down into two primary classifications: reactions that destroy or alter organic compounds, and physical processes such as adsorption and encapsulation.

Redox Potential Controls the Structure and DNA Binding Activity of the Paired Domain

Pax proteins are transcriptional regulators controlling a variety of cell fates during animal development. This role depends on the intact function of the paired (Prd) domain that is able to recognize specific DNA sequences. The Prd domain is composed of two distinct helix-turn-helix subdomains, PAI and RED. Molecular functions of Pax proteins are subjected to different levels of regulation involving both pre-translational and post-translational mechanisms. By using Pax-5 and Pax-8 recombinant proteins, we demonstrate that the binding activity of the Prd domain is regulated through the oxidation/reduction of conserved cysteine residues. Mass spectrometry analysis and mutagenesis experiments demonstrate that the redox regulation is accomplished through the reversible formation of an intramolecular disulfide bridge involving the cysteines present in the PAI subdomain, whereas the RED subdomain appears quite insensitive to redox potential. Circular dichroism experiments indicate that only the reduced form of the Prd domain is able to undergo the proper conformational change necessary for sequence-specific DNA binding. Nuclear extracts from different cell lines contain an activity that is able to reduce the Paired domain and, therefore, to control the DNA binding activity of this protein. Immunodepletion of nuclear extracts demonstrate that the protein Ref-1 contributes to the redox regulation of the Prd DNA binding activity. Given the modular nature of the Prd domain and the independent DNA binding specificity of the PAI and RED subdomains, we propose that this control mechanism should be involved in "switching" among different DNA sequences and therefore different target genes.

Generating controlled reducing environments in aerobic recombinant Escherichia coli fermentations: Effects on cell growth, oxygen uptake, heat shock protein expression, and in vivo CAT activity

The independent control of culture redox potential (CRP) by the regulated addition of a reducing agent, dithiothreitol (DTT) was demonstrated in aerated recombinant Escherichia coli fermentations. Moderate levels of DTT addition resulted in minimal changes to specific oxygen uptake, growth rate, and dissolved oxygen. Excessive levels of DTT addition were toxic to the cells resulting in cessation of growth. Chloramphenicol acetyltransferase (CAT) activity (nmoles/μg total protein min.) decreased in batch fermentation experiments with respect to increasing levels of DTT addition. To further investigate the mechanisms affecting CAT activity, experiments were performed to assay heat shock protein expression and specific CAT activity (nmoles/μg CAT min.). Expression of such molecular chaperones as GroEL and DnaK were found to increase after addition of DTT. Additionally, sigma factor 32 (σ32) and several proteases were seen to increase dramatically during addition of DTT. Specific CAT activity (nmoles/μg CAT min.) varied greatly as DTT was added, however, a minimum in activity was found at the highest level of DTT addition in E. coli strains RR1 [pBR329] and JM105 [pROEX-CAT]. In conjunction, cellular stress was found to reach a maximum at the same levels of DTT. Although DTT addition has the potential for directly affecting intracellular protein folding, the effects felt from the increased stress within the cell are likely the dominant effector. That the effects of DTT were measured within the cytoplasm of the cell suggests that the periplasmic redox potential was also altered. The changes in specific CAT activity, molecular chaperones, and other heat shock proteins, in the presence of minimal growth rate and oxygen uptake alterations, suggest that the ex vivo control of redox potential provides a new process for affecting the yield and conformation of heterologous proteins in aerated E. coli fermentations. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 248–259, 1998.

Generating controlled reducing environments in aerobic recombinantEscherichia coli fermentations: Effects on cell growth, oxygen uptake, heat shock protein expression, and in vivo CAT activity

The independent control of culture redox potential (CRP) by the regulated addition of a reducing agent, dithiothreitol (DTT) was demonstrated in aerated recombinant Escherichia coli fermentations. Moderate levels of DTT addition resulted in minimal changes to specific oxygen uptake, growth rate, and dissolved oxygen. Excessive levels of DTT addition were toxic to the cells resulting in cessation of growth. Chloramphenicol acetyltransferase (CAT) activity (nmoles/microgram total protein min.) decreased in batch fermentation experiments with respect to increasing levels of DTT addition. To further investigate the mechanisms affecting CAT activity, experiments were performed to assay heat shock protein expression and specific CAT activity (nmoles/microgram CAT min.). Expression of such molecular chaperones as GroEL and DnaK were found to increase after addition of DTT. Additionally, sigma factor 32 (sigma32) and several proteases were seen to increase dramatically during addition of DTT. Specific CAT activity (nmoles/microgram CAT min. ) varied greatly as DTT was added, however, a minimum in activity was found at the highest level of DTT addition in E. coli strains RR1 [pBR329] and JM105 [pROEX-CAT]. In conjunction, cellular stress was found to reach a maximum at the same levels of DTT. Although DTT addition has the potential for directly affecting intracellular protein folding, the effects felt from the increased stress within the cell are likely the dominant effector. That the effects of DTT were measured within the cytoplasm of the cell suggests that the periplasmic redox potential was also altered. The changes in specific CAT activity, molecular chaperones, and other heat shock proteins, in the presence of minimal growth rate and oxygen uptake alterations, suggest that the ex vivo control of redox potential provides a new process for affecting the yield and conformation of heterologous proteins in aerated E. coli fermentations.