Stability and sulfur-reduction activity in non-aqueous phase liquids of the hydrogenase from the hyperthermophile Pyrococcus furiosus.

Hydrogenase from the hyperthermophilic archaeon, Pyrococcus furiosus, catalyzes the reversible activation of H(2) gas and the reduction of elemental sulfur (S degrees ) at 90 degrees C and above. The pure enzyme, modified with polyethylene glycol (PEG), was soluble (> 5 mg/mL) in toluene and benzene with t(1/2) values of more than 6 h at 25 degrees C. At 100 degrees C the PEG-modified enzyme was less stable in aqueous solution (t(1/2) approximately 10 min) than the native (unmodified) enzyme (t(1/2) approximately 1 h), but they exhibited comparable H(2) evolution, H(2) oxidation, and S degrees reduction activities at 80 degrees C. The H(2) evolution activity of the modified enzyme was twice that of the unmodified enzyme at 25 degrees C. The PEG-modified enzyme did not catalyze S degrees reduction (at 80 degrees C) in pure toluene unless H(2)O was added. The mechanism by which hydrogenase produces H(2)S appears to involve H(2)O as the proton source and H(2) as the electron source. The inability of the modified hydrogenase to catalyze S degrees reduction in a homogeneous non-aqueous phase complicates potential applications of this enzyme.

A biological process for the reclamation of flue gas desulfurization gypsum using mixed sulfate-reducing bacteria with inexpensive carbon sources.

A combined chemical and biological process for the recycling of flue gas desulfurization (FGD) gypsum into calcium carbonate and elemental sulfur is demonstrated. In this process, a mixed culture of sulfate-reducing bacteria (SRB) utilizes inexpensive carbon sources, such as sewage digest or synthesis gas, to reduce FGD gypsum to hydrogen sulfide. The sulfide is then oxidized to elemental sulfur via reaction with ferric sulfate, and accumulating calcium ions are precipitated as calcium carbonate using carbon dioxide. Employing anaerobically digested municipal sewage sludge (AD-MSS) medium as a carbon source, SRBs in serum bottles demonstrated an FGD gypsum reduction rate of 8 mg/L/h (10(9) cells)(-1). A chemostat with continuous addition of both AD-MSS media and gypsum exhibited sulfate reduction rates as high as 1.3 kg FGD gypsum/m(3)d. The increased biocatalyst density afforded by cell immobilization in a columnar reactor allowed a productivity of 152 mg SO(4) (-2)/Lh or 6.6 kg FGD gypsum/m(3)d. Both reactors demonstrated 100% conversion of sulfate, with 75-100% recovery of elemental sulfur and chemical oxygen demand utilization as high as 70%. Calcium carbonate was recovered from the reactor effluent on precipitation using carbon dioxide. It was demonstrated that SRBs may also use synthesis gas (CO, H(2), and CO(2) in the reduction of gypsum, further decreasing process costs. The formation of two marketable products-elemental sulfur and calcium carbonate-from FGD gypsum sludge, combined with the use of a low-cost carbon source and further improvements in reactor design, promises to offer an attractive alternative to the landfilling of FGD gypsum.

Biodesulfurization of flue gases and other sulfate/sulfite waste streams using immobilized mixed sulfate-reducing bacteria.

Sulfur dioxide (SO2) is one of the major pollutants in the atmosphere that cause acid rain. Microbial processes for reducing SO2 to hydrogen sulfide (H2S) have previously been demonstrated by utilizing mixed cultures of sulfate-reducing bacteria (SRB) with municipal sewage digest as the carbon and energy source. To maximize the productivity of the bioreactor for SO2 reduction in this study, various immobilized cell bioreactors were investigated: a stirred tank with SRB flocs and columnar reactors with cells immobilized in either potassium-carrageenan gel matrix or polymeric porous BIO-SEP beads. The maximum volumetric productivity for SO2 reduction in the continuous stirred-tank reactor (CSTR) with SRB flocs was 2.1 mmol of SO2/(h.L). The potassium-carrageenan gell matrix used for cell immobilization was not durable at feed sulfite concentrations greater than 2000 mg/L (1.7 mmol/(h.L)). A columnar reactor with mixed SRB cells that had been allowed to grow into highly stable BIO-SEP polymeric beads exhibited the highest sulfite conversion rates, in the range 16.5 mmol/(h.L) (with 100% conversion) to 20 mmol/(h.L) (with 95% conversion). The average specific activity for sulfite reduction in the column, in terms of dry weight of SRB biomass, was 9.5 mmol of sulfite/(h.g). In addition to flue gas desulfurization, potential applications of this microbial process include the treatment of sulfate/sulfite-laden wastewater from the pulp and paper, petroleum, mining, and chemical industries.

Enzymatic catalysis in organic solvents: Polyethylene glycol modified hydrogenase retains sulfhydrogenase activity in toluene.

Naturally occurring enzymes may be modified by covalently attaching hydrophobic groups that render the enzyme soluble and active in organic solvents, and have the potential to greatly expand applications of enzymatic catalysis. The reduction of elemental sulfur to hydrogen sulfide by a hydrogenase isolated from Pyrococcus furiosus has been investigated as a model system for organic biocatalysis. While the native hydrogenase catalyzed the reduction of sulfur to H(2)S in aqueous solution, no activity was observed when the aqueous solvent was replaced with anhydrous toluene. Hydrogenase modified with PEG p-nitrophenyl carbonate demonstrated its native biocatalytic ability in toluene when the reducing dye, benzyl viologen, was also present. Neither benzyl viologen nor PEG p-nitrophenyl carbonate alone demonstrated reducing capability. PEG modified cellulase and benzyl viologen were also incapable of reducing sulfur to H(2)S, indicating that the enzyme itself, and not the modification procedure, is responsible for the conversion in the nonpolar organic solvent. Sulfide production in toluene was tenfold higher than that produced in an aqueous system with equal enzyme activity, demonstrating the advantages of organic biocatalysis. Applications of bio-processing in nonaqueous media are expected to provide significant advances in the areas of fossil fuels, renewable feedstocks, organic synthesis, and environmental control technology. (c) 1996 John Wiley & Sons, Inc.

In vitro measurement and screening of monoclonal antibody affinity using fluorescence photobleaching.

Antibody screening is a routine in vitro assay in monoclonal antibody development and production. We have recently adapted the fluorescence photobleaching method to quantify antibody mass transport and binding parameters in bulk solution (Kaufman and Jain, 1990, 1991). The present study uses this in vitro method to screen a series of monoclonal antibodies (IgG) developed against the rabbit VX2 carcinoma tumor line. These experiments indicate that the three antibodies recognize distinct epitopes on the tumor, with equilibrium binding constants of 1.3 +/- 0.5, 5.1 +/- 3.6 and 2.0 +/- 1.1 x 10(7) M-1 for the antibodies RVC-184, RVC-626 and RVC-779, respectively. The antibody diffusion coefficient revealed no dependent upon protein concentration or antigen bead volume fraction within the ranges investigated. It was demonstrated experimentally that the interactions conformed to a reaction limited binding model of fluorescence recovery, that the system was at equilibrium, and that non-specific binding due to the fluorescein probe was not significant. Once the non-reactive fraction of antibody is determined, this photobleaching technique does not require perturbation or physical separation of the unbound species. As such, it has many potential applications including in vivo investigation of binding parameters.

Effect of bivalent interaction upon apparent antibody affinity: experimental confirmation of theory using fluorescence photobleaching and implications for antibody binding assays.

The affinity of a monoclonal antibody for its tumor-associated antigen is one of several parameters governing in vivo monoclonal antibody distribution. However, there is a lack of apparent correlation between the affinity of a bivalent monoclonal antibody measured using equilibrium binding experiments and its in vivo delivery. Furthermore, differences in the reported affinity for identical antibody/antigen pairs are quite common in the literature. In this paper, both of these discrepancies are addressed in terms of variation in avidity due to bivalent interaction. The enhancement of avidity afforded by bivalent attachment is addressed theoretically by extending the model of Crothers and Metzger (Immunochemistry, 9: 341-357, 1972). Theoretical assessment of Lineweaver-Burk, Scatchard, Steward-Petty, Langmuir, fluorescence recovery after photobleaching, and Sips models demonstrates quantitatively that the measured affinity using equilibrium binding experiments may vary over four orders of magnitude with similar variation in experimental cellular antigen density. Further, the measured affinity is a function of the experimental protocol. Predictions of avidity enhancement were confirmed experimentally using fluorescence recovery after photobleaching. These experiments measured the equilibrium binding constant and concentration of binding sites for an immunoglobulin G monoclonal antibody and its F(ab) fragment directed against the rabbit VX2 carcinoma cell line. Bivalent binding data agree quantitatively with those predicted by the bivalent model with no adjustable parameters. It is concluded that bivalent equilibrium binding constants are useful only in antibody screening, where experimental conditions are identical for all series. They must be used with caution in predicting in vivo antibody distribution, and it is recommended that the intrinsic, monovalent affinity be measured in tandem with any bivalent antibody study as a standard reference.

Measurement of mass transport and reaction parameters in bulk solution using photobleaching. Reaction limited binding regime.

Fluorescence recovery after photobleaching (FRAP) has been used previously to investigate the kinetics of binding to biological surfaces. The present study adapts and further develops this technique for the quantification of mass transport and reaction parameters in bulk media. The technique's ability to obtain the bulk diffusion coefficient, concentration of binding sites, and equilibrium binding constant for ligand/receptor interactions in the reaction limited binding regime is assessed using the B72.3/TAG-72 monoclonal antibody/tumor associated antigen interaction as a model in vitro system. Measurements were independently verified using fluorometry. The bulk diffusion coefficient, concentration of binding sites and equilibrium binding constant for the system investigated were 6.1 +/- 1.1 x 10(-7) cm2/s, 4.4 +/- 0.6 x 10(-7) M, and 2.5 +/- 1.6 x 10(7) M-1, respectively. Model robustness and the applicability of the technique for in vivo quantification of mass transport and reaction parameters are addressed. With a suitable animal model, it is believed that this technique is capable of quantifying mass transport and reaction parameters in vivo.

Quantification of transport and binding parameters using fluorescence recovery after photobleaching. Potential for in vivo applications.

Fluorescence Recovery After Photobleaching (FRAP) has been used extensively in the study of transport and binding in biological media in vitro. The present study adapts and further develops FRAP so that it may be utilized for the in vivo quantification of binding parameters. The technique is validated in vitro by measuring mass transport and binding parameters for the Concanavalin A/Mannose binding system (a diffusion-limited system). The pseudo-equilibrium constant (the product of the equilibrium constant and the total concentration of binding sites) for this system was determined to be 26 +/- 15 which compares favorably with literature values ranging between 16 and 32. The applicability of this technique to measure parameters for monoclonal antibody/antigen interactions in a thin tissue preparation such as the rabbit ear chamber tissue preparation is also examined. Unlike other methods for measuring binding parameters, this is the only technique which has the potential to measure parameters relevant to antibody delivery in vivo. The proposed technique is noninvasive and does not require a priori knowledge of, independent measurement of, or variation in the concentration of binding sites or total concentration of binding species.

Recombinant interleukin-1 alpha and recombinant tumor necrosis factor alpha synergize in vivo to induce early endotoxin tolerance and associated hematopoietic changes.

Endotoxin, the lipopolysaccharide (LPS) derived from gram-negative bacteria, invokes a wide range of responses in susceptible hosts. It is known that virtually all responses to LPS are mediated by the action of macrophage-derived cytokines (such as interleukin-1 [IL-1], tumor necrosis factor [TNF], and others) which are produced principally by macrophages and maximally within several hours of LPS administration. One manifestation of LPS administration which is not well understood is the phenomenon of "early endotoxin tolerance." In response to a single sublethal injection of LPS, experimental animals become refractory to challenge with a homologous or heterologous LPS preparation 3 to 4 days later. Animals rendered tolerant exhibit mitigated toxicity and a reduced capacity to produce circulating cytokines (i.e., colony-stimulating factor or interferon) in response to the challenge LPS injection. Previous studies have also shown that this state of transient, acquired hyporesponsiveness to LPS is accompanied by a marked increase in the size of cells in the bone marrow which are enriched in numbers of macrophage progenitors. In this study, we examined the capacity of recombinant IL-1 or recombinant TNF or both to induce early endotoxin tolerance and its associated hematopoietic changes. Neither cytokine alone was able to mimic LPS for induction of tolerance. Combined administration of recombinant IL-1 and recombinant TNF doses which were not toxic when administered individually led to synergistic toxicity (as assessed by death or weight loss). However, within a nontoxic range, the two cytokines synergized to induce a significant reduction in the capacity to produce colony-stimulating factor in response to LPS, as well as the characteristic increase in bone marrow cell size and macrophage progenitors shown previously to be associated with LPS-induced tolerance.

Induction of colony stimulating factor in vivo by recombinant interleukin 1 alpha and recombinant tumor necrosis factor alpha 1.

In response to a potent inflammatory challenge, such as Gram-negative endotoxin, a number of cytokines are induced that, in turn, mediate many of the pathophysiologic alterations associated with endotoxicity. In this study, we have observed two endotoxin-associated monokines, recombinant interleukin-1 alpha (rIL 1 alpha) and recombinant tumor necrosis factor alpha (rTNF alpha), to induce colony stimulating factor (CSF) in vivo. The CSF activities produced in response to rIL 1 alpha or rTNF alpha gave rise to a mixture of granulocyte-macrophage colonies and were induced in a dose- and time-dependent fashion, peaking within 3 hr of cytokine injection (preceding peak CSF induction by endotoxin by several hours). Combined injection of suboptimal concentrations of rIL 1 alpha and rTNF alpha were additive, and simultaneous injection of optimal concentrations of each failed to increase CSF levels over that observed with either cytokine alone. Unlike endotoxin, neither cytokine induced interferon in vivo. These findings extend our understanding of the cytokine cascade that is operative in an inflammatory response and may account for many of the observed hematopoietic alterations that accompany inflammation.