Harvey W. Blanch

Protein adsorption at the oil/water interface: characterization of adsorption kinetics by dynamic interfacial tension measurements.

The dynamics of protein adsorption at an oil/water interface are examined over time scales ranging from seconds to several hours. The pendant drop technique is used to determine the dynamic interfacial tension of several proteins at the heptane/aqueous buffer interface. The kinetics of adsorption of these proteins are interpreted from tension/log time plots, which often display three distinct regimes. (I) Diffusion and protein interfacial affinity determine the duration of an initial induction period of minimal tension reduction. A comparison of surface pressure profiles at the oil/water and air/water interface reveals the role of interfacial conformational changes in the early stages of adsorption. (II) Continued rearrangement defines the second regime, where the resulting number of interfacial contacts per protein molecule causes a steep tension decline. (III) The final regime occurs upon monolayer coverage, and is attributed to continued relaxation of the adsorbed layer and possible build-up of multilayers. Denaturation of proteins by urea in the bulk phase is shown to affect early regimes.

Enzymatic catalysis in a supercritical fluid

The enzyme alkaline phosphatase, EC 3. 1. 3. 1, was found to be active in a supercritical carbo dioxide solvent system. A batch reaction of disodium p-nitrophenyl phosphate with a 0.1 vol. % water solution in supercritical CO2 at 100 atm and 35°C produced p-nitrophenol when catalyzed by alkaline phosphatase.

Protein‐protein and protein‐salt interactions in aqueous protein solutions containing concentrated electrolytes

Protein-protein and protein-salt interactions have been obtained for ovalbumin in solutions of ammonium sulfate and for lysozyme in solutions of ammonium sulfate, sodium chloride, potassium isothiocyanate, and potassium chloride. The two-body interactions between ovalbumin molecules in concentrated ammonium-sulfate solutions can be described by the DLVO potentials plus a potential that accounts for the decrease in free volume available to the protein due to the presence of the salt ions. The interaction between ovalbumin and ammonium sulfate is unfavorable, reflecting the kosmotropic nature of sulfate anions. Lysozyme-lysozyme interactions cannot be described by the above potentials because anion binding to lysozyme alters these interactions. Lysozyme-isothiocyanate complexes are strongly attractive due to electrostatic interactions resulting from bridging by the isothiocyanate ion. Lysozyme-lysozyme interactions in sulfate solutions are more repulsive than expected, possibly resulting from a larger excluded volume of a lysozyme-sulfate bound complex or perhaps, hydration forces between the lysozyme-sulfate complexes.

The transient responses of hybridoma cells to nutrient additions in continuous culture: II. Glutamine pulse and step changes

The transient and steady‐state responses of hybridoma growth and metabolism to glutamine pulse and step changes have been examined. Metabolic quotients are reported for oxygen, glucose, lactate, ammonia, glutamine, alanine, and other amino acids. The specific glutamine consumption rate increased rapidly after all glutamine additions, but the responses of the glucose and oxygen consumption rates and the cell concentration were found to depend on the intial feed glutamine concentration. The glucose consumption rate was 1.4‐10.9 times that of glutamine, and serine and branched‐chain amino acids were consumed in larger amounts at the higher glucose: glutamine uptake ratios. It was estimated that maintenance accounted for ca. 60% of the cellular ATP requirements at specific growth rates ranging from 0.57 to 0.68 day−1.

BUBBLE COALESCENCE IN STAGNANT LIQUIDS

Abstract The coalescence behavior of bubbles in aqueous solutions is investigated, A sharp transition on coalescence frequency is observed with increasing solute concentration. The mechanism of coalescence appears to be very different for volatile and non-volatile solutes.

Enzymatic Oxidation of Cholesterol Aggregates in Supercritical Carbon Dioxide

Fundamental studies of enzyme-solvent interactions can be conducted with supercritical fluids because small changes in pressure or temperature may bring about great changes in the properties of a single solvent near its critical point. Cholesterol oxidase is active in supercritical carbon dioxide and supercritical carbon dioxide-cosolvent mixtures. Variations in solvent power caused by pressure changes or by the addition of dopants affected the rate of enzymatic oxidation of cholesterol by altering the structure of cholesterol aggregates.

Transient responses of hybridoma cells to lactate and ammonia pulse and step changes in continuous culture

Ammonia and lactate are the major byproducts from mammalian cells grown in medium containing glutamine and glucose. Both can be toxic to cells, and may limit the productivity of commercial bioreactors. The transient and steady-state responses of hybridoma growth and metabolism to lactate and ammonia pulse and step changes in continuous suspension culture have been examined. No inhibition was observed at 40 mM lactate. Cell growth was inhibited by 5 mM ammonia, but the cells were able to adapt to ammonia concentrations as high as 8.2 mM. Ammonia production decreased and alanine production increased in response to higher ammonia concentrations. Increased ammonia concentrations also inhibited glutamine and oxygen consumption. The specific oxygen consumption rate decreased by an order of magnitude after an ammonia pulse to 18 mM. Under these conditions, over 90% of the estimated ATP production was due to glycolysis and a large fraction of glutamine was converted to lactate.

Analysis of affinity separations: I: Predicting the performance of affinity adsorbers

Abstract The analysis of affinity separations has lagged behind the rapid increase in new applications. The purpose of this work is to outline an approach to analysis that will aid the design and optimization of large-scale affinity separations. This paper is an introduction to the theory of fixed bed adsorption as it applies to affinity chromatography. This theory provides analytical tools for evaluating and predicting industrial-scale affinity column performance, based on data from laboratory-scale experiments. Design equations are included for continuous separations on a rotating annular chromatograph and for batch operation of the adsorption and wash steps. The design equations are derived from a general model by introducing appropriate simplifying assumptions for rate-limiting mass transfer and sorption steps.

Swelling properties of acrylamide-based ampholytic hydrogels: comparison of experiment with theory

Abstract Ampholytic hydrogels were synthesized by copolymerizing acrylamide with specialty monomers. Two monomers, [(methacrylamido)propyl]trimethylammonium chloride (MAPTAC) and sodium styrene sulfonate (SSS), were copolymerized with acrylamide to form one ampholytic hydrogel. Also, the zwitterionic monomers N-(3-sulfopropyl)-N-methacrylamidopropyl-N-dimethylammonium betaine (SB1) and N-(3-sulfopropyl)-N-methacroyloxyethyl-N,N-dimethylammonium betaine (SB2) were both, in turn, copolymerized with acrylamide to form ampholytic hydrogels. Swelling equilibria were measured in water and in aqueous sodium chloride solutions ranging in concentration from 10−5 to 5 M. Antipolyelectrolyte behaviour was observed for the ampholytic hydrogels prepared; hydrogel swelling increases the sodium chloride concentration rises. To demonstrate theoretically antipolyelectrolyte behaviour for ampholytic hydrogels, we incorporate the Debye-Huckel theory of electrolyte solutions into a Flory-type swelling model to account for Coulombic interactions between fixed and mobile ions. Calculated swelling equilibria are in qualitative agreement with experiment.

In-situ recovery of butanol during fermentation

End product inhibition can be reduced by the in situ removal of inhibitory fermentation products as they form. Extractive fermentation, in which an immiscible organic solvent is added to the fermentor in order to extract inhibitory products, was applied to the acetone-butanol fermentation. Six solvents or solvent mixtures were tested in batch extractive fermentations: kerosene, 30 wt% tetradecanol in kerosene, 50 wt% dodecanol in kerosene, oleyl alcohol, 50 wt% oleyl alcohol in a decane fraction and 50 wt% oleyl alcohol in benzyl benzoate. The best results were obtained with oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate. In normal batch fermentation of Clostridium acetobutylicum, glucose consumption is limited to about 80 kg/m3 due to the accumulation of butanol in the broth. In extractive fermentation using oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate, over 100 kg/m3 of glucose can be fermented. Removal of butanol from the broth as it formed also increased the rate of butanol production. Maximum volumetric butanol productivity was increased by as much as 60% in extractive fermentation compared to batch fermentation. Butanol productivities obtained in extractive fermentation compare favorably with other in situ product removal fermentations.