bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Engineering of Polysaccharide Structure: Production of Variants of Xanthan Gum in Xanthomonas campestris
Authors: Hassler Randal A. , Doherty Daniel H.
Published: March 2005

Xanthan gum is an extracellular heteropolysaccharide produced by the bacterium Xanthomonas campestris. Xanthan has wide commercial application as a viscosifier of aqueous solutions. Previously, through genetic engineering, a set of mutants defective in the xanthan biosynthetic pathway has been obtained. Certain mutants were shown to synthesize and polymerize structural variants of the xanthan repeating unit and thus produce “variant xanthans”. Initial studies of solution viscosities of these polymers, presented here, indicate that the variants have rheological properties similar to, but not identical with, xanthan. These results indicate that acetylation and pyruvylation can affect the viscometric properties of xanthan. Specifically, the presence of pyruvate increases viscosity, whereas acetate decreases viscosity. In addition, the elimination of sugar residues from xanthan side chains also has a major effect on viscosity. Compared to wild‐type xanthan, polymer lacking the terminal mannose (polytetramer) is a poor viscosifier. In contrast, polymer lacking both the terminal mannose and glucuronic acid (polytrimer) is a superior viscosifier, on a weight basis. There is a negative effect of acetylation on the viscosity of polytetramer xanthan, but there is seemingly no effect of acetylation on polytrimer xanthan viscosity. The further study of these materials should provide insight into the relationship between xanthan structure and rheological behavior.
Experimental and Theoretical Evidence for Convective Nutrient Transport in an Immobilized Cell Support
Authors: Bringi V. , Dale B. E.
Published: March 2005

Even though immobilized‐cell reactors possess several engineering advantages over free‐cell reactors, their full potential has not been realized because mass transfer often limits the rate of nutrient supply and product removal from immobilized cell supports. We studied the interaction between mass transfer and reaction kinetics in the anaerobic conversion of glucose to CO2 and ethanol by yeast immobilized in a porous rotating disk on the agitator shaft of a conventional CSTR. A Sherwood number correlation was used to show that external mass‐transfer resistances were negligible under typical operating conditions. The modulus of Weisz based on observable reaction parameters was used to gauge the importance of pore diffusion limitations. Under conditions for which significant pore diffusion effects and hence low effectiveness factors (η =ca. 0.1) would be predicted, the observed reaction rates were much higher than expected (η=ca. 1), suggesting that pore diffusion limitations were at least partially relieved by convective transport of glucose into the support. Two possible mechanisms of convective transport are discussed. We hypothesize that gas evolution was responsible for the convective enhancement of glucose supply.
Recovery of Clostridium thermosulfurogenes Produced β‐Amylase by (Hydroxypropyl)methylcellulose Partition
Authors: Miranda Everson A. , Berglund Kris A.
Published: March 2005

A procedure for recovering Clostridium thermosulfurogenes produced β‐amylase from fermentation broth by partition was developed. The partition was achieved by addition of ammonium sulfate to an aqueous solution of the enzyme with (hydroxypropyl)methylcellulose. The β‐amylase‐containing pellet formed upon centrifugation could be redissolved and the polymer recovered by a second salt addition. The process was not dependent on polymer/enzyme solution pH, but it was affected by temperature, polymer nominal molecular weight and loading, and fermentation carbon source. Unlike more traditional aqueous‐phase partitions, such as poly(ethylene glycol)/dextran, the current approach appeared to be biospecific.
Representation of Phase Equilibrium Behavior of Antibiotics
Authors: Zhu Yizu , Evans Lawrence B. , Chen Chau‐Chyun
Published: March 2005

The phase equilibrium behavior of biomolecules is important both in understanding the partition mechanisms and in the design and optimization of downstream recovery processes. Chen et al. (1989) proposed a molecular thermodynamic framework that successfully represents the liquid‐solid equilibrium behavior of amino acids and small peptides as functions of temperature, ionic strength, solvent compositions, and pH. Based on this theoretical framework, this paper presents recent results in representing the liquid‐solid equilibrium behavior (solubilities) and the liquid‐liquid equilibrium behavior (phase partitioning) of β‐lactam antibiotics, which are amino acid derivatives and important chemotherapeutic agents.
Defining an Optimal Carbon Source/Methionine Feed Strategy for Growth and Cephalosporin C Formation by Cephalosporium acremonium
Authors: Vicik Steven M. , Fedor Anthony J. , Swartz Randall W.
Published: March 2005

The effect of the method of methionine addition, growth‐limiting carbon source (glucose vs sucrose), and culture growth rate on cephalosporin C production was investigated in a Cephalosporium acremonium defined medium fed batch fermentation. Batch addition of methionine, at a concentration of 3 g/L, prior to the start of a fed sucrose fermentation was found to interfere with the ability of the culture to utilize this sugar, thus limiting growth and decreasing cephalosporin C production. Batch methionine addition had no effect on glucose‐limited cultures. Concurrent exponential feeding of methionine with sucrose improved both culture growth and productivity. Under the control of identical carbon source limiting feed profiles, sucrose was observed to support greater cephalosporin C production than glucose. Optimal cephalosporin C production in a C. acremonium defined medium fed batch fermentation was obtained through controlling culture growth during the rapid growth phase at a relatively low level with respect to μmax (μ∼0.036 h−1) until achieving a desired cell mass with a concurrent sucrose and methionine feed, followed by maintaining relatively vigorous growth (μ∼0.01 h−1) with sucrose for the duration of the fermentation.
Intrinsic Growth and Fermentation Rates of Alginate‐Entrapped Saccharomyces cerevisiae
Authors: Hannoun Betty J. M. , Stephanopoulos Gregory
Published: March 2005

Experiments were conducted under anaerobic conditions to determine the intrinsic specific rates of growth, glucose uptake, and ethanol production for Saccharomyces cerevisiae immobilized in calcium alginate. The simultaneous processes of diffusion and reaction in an alginate membrane were analyzed to determine the intrinsic specific growth and reaction rates from glucose and ethanol concentration measurements made outside the alginate phase. Under anaerobic conditions, the specific growth rate of immobilized S. cerevisiae decreased by 20% compared to the growth rate for suspended cells. The specific glucose uptake rate and specific ethanol production rate increased by a factor of 4 compared to those for suspended cells. The ethanol yield remained the same, and the biomass yield decreased to one‐fifth the yield for suspended cells. Further experiments were conducted under aerobic conditions to investigate the effects of dissolved oxygen concentration on the specific rates of growth, glucose uptake, and ethanol production of immobilized S. cerevisiae. Although the obtained results cannot be interpreted unambiguously, oxygen appears to affect immobilized cells in a way similar to the way it affects suspended cells, while the observed differences between immobilized and suspended cells under anaerobic conditions remain the same in the presence of oxygen. Due to rate variability with oxygen, the obtained results are representative of average rate values. Experimental data and simulations indicate that glucose mitigates oxygen effects, yielding relatively small rate variations with aeration rates and relatively constant ethanol yields.
Fluidized Bed Adsorption for Whole Broth Extraction
Authors: Gailliot F. P. , Gleason C. , Wilson J. J. , Zwarick J.
Published: March 2005

Fluidized bed adsorption using a high‐density synthetic resin has proven to be an invaluable technique for separating novel compounds from unfiltered fermentation broths during the very early stages of fermentation development, where product concentrations are typically in the parts per million range. Previous initial downstream processing strategies consisted of cell separation from whole broth or direct extraction with water‐immiscible solvents, both of which resulted in lengthy time cycles, conflicts with existing operations requiring the use of high‐cost centrifugal separators, and environmental/solvent recovery concerns. Laboratory and subsequent pilot plant process development work along with concomitant improvements in yield, quality, and time cycles are presented for one of several fluidized bed processes piloted in Merck's Natural Product Isolation facility.
Practical Considerations in the Measurement of Culture Fluorescence
Authors: Coppella Steven J. , Rao Govind
Published: March 2005

We have examined practical considerations associated with the use of a commercially available fluorescence probe for in situ measurements in bioreactors. The optical path length of the measurement was first determined and a flow cell subsequently designed. The environment (agitation/aeration rates) of the probe was found to have a significant influence on the measurement. These effects were eliminated by placing the probe in a recycle loop using the flow cell. Fluorescence measurement in the recycle loop was verified to be representative of the cell sample and to not affect cell metabolism.
Microstructure of Mixed Gelatin‐Egg White Gels: Impact on Rheology and Application to Microparticulation
Authors: Ziegler G. R.
Published: March 2005

The microstructure of mixed gelatin‐egg white gels was examined by using both light and electron microscopy. Interpenetrating polymer network morphology was observed in mixed gels that were macroscopically single‐phase systems. For two‐phase gels, phase volumes were determined by optical image analysis and used to predict the composite shear modulus. Rheological properties were related to observable changes in the gel microstructure. For systems containing 5.6% egg white protein and greater than 5% gelatin, the dispersed egg white phase formed capsules of roughly 1–2 μm in diameter, with a dense outer coat and a less dense interior containing voids. From this, a method for the microparticulation of proteins based on aqueous phase partitioning was developed.
Growth and Anthocyanin Accumulation in Carrot Cell Suspension Cultures Growing on Fructose, Glucose, or Their Mixtures
Authors: Zwayyed S. K. , Frazier G. C. , Dougall D. K.
Published: March 2005

Carrot suspension cultures have been grown on glucose, fructose, and mixtures of these two sugars and the effects of these sugars on growth and anthocyanin accumulation measured. Several parameters of growth decrease with increasing glucose in the medium. The anthocyanin accumulated remains relatively low and constant until the glucose exceeds 50% of the sugar, when the anthocyanin accumulation increases with increased glucose. The data suggest that glucose and fructose are metabolized differently in these carrot cultures despite the interconversion of their 6‐phosphates in glycolysis. Ways in which the metabolism of these sugars might differ are discussed, as is the possibility of increasing the accumulation of anthocyanin by alterations in the way these sugars are made available to the cultures.
Manipulation of Heterogeneous Hybridoma Cultures for Overproduction of Monoclonal Antibodies
Authors: Mckinney Katherine L , Dilwith Robert , Belfort Georges
Published: March 2005

In searching for ways to manipulate heterogeneous hybridoma cell cultures (ATCC HB124) to obtain increased production of monoclonal antibodies (IgG2a), we have selected for a higher secreting but slower growing subpopulation using the level of fluorescent surface‐associated antibodies and a fluorescence‐activated cell sorter. Cell surface fluorescence was found to be correlated with specific antibody secretion rate over the short term but not with intracellular antibody content. Also, the specific secretion rate of a heterogeneous population of hybridoma cells grown in batch culture has been shown to be inversely correlated with an increase in either the initial cell concentration or the medium antibody concentration. Several experiments suggest that an upper limit exists for medium antibody concentration, above which antibody is degraded at the same rate at which it is produced. Should other cell lines behave similarly, strategies for overproduction of monoclonal antibodies suggested herein could be profitably used in industry.
Application of Immobilized Yeast Cells to Sparkling Wine Fermentation
Authors: Gòdia Francesc , Casas Carles , Solàg Carles
Published: March 2005

The production of sparkling wine with yeast immobilized in natural gels was investigated. Cell release from the beads to the wine was studied by using two different techniques: simple gel beads and beads with an external layer free of cells around them. In the first technique, 2% calcium alginate beads with 109 cells/g of bead and a bead dosage of 12 g/bottle were the optimal conditions to completely avoid cell leakage. When externally coated beads were employed, the dosage could be reduced to 6 g/bottle and the wine remained absolutely free of cells at the end of the fermentation. In both cases, the organoleptical properties of the sparkling wine produced with immobilized cells were found to be improved with respect to the conventional product.
Intracellular Ice Formation during the Freezing of Hepatocytes Cultured in a Double Collagen Gel
Authors: Hubel A , Toner M , Cravalho E. G , Yarmush M. L , Tompkins R. G
Published: March 2005

During freezing, intracellular ice formation (IIF) has been correlated with loss in viability for a wide variety of biological systems. Hence, determination of IIF characteristics is essential in the development of an efficient methodology for cryopreservation. In this study, IIF characteristics of hepatocytes cultured in a collagen matrix were determined using cryomicroscopy. Four factors influenced the IIF behavior of the hepatocytes in the matrix: cooling rate, final cooling temperature, concentration of Me2SO, and time in culture prior to freezing. The maximum cumulative fraction of cells with IIF increased with increasing cooling rate. For cultured cells frozen in Dulbecco's modified Eagle's medium (DMEM), the cooling rate for which 50% of the cells formed ice (B50) was 70°C/min for cells frozen after 1 day in culture and decreased to 15°C/min for cells frozen after 7 days in culture. When cells were frozen in a 0.5 M Me2SO + DMEM solution, the value of B50 decreased from 70 to 50°C/min for cells in culture for 1 day and from 15 to 10°C/min for cells in culture for 7 days. The value of the average temperature for IIF (Tuf) for cultured cells was only slightly depressed by the addition of Me2SO when compared to the IIF behavior of other cell types. The results of this study indicate that the presence of the collagen matrix alters significantly the IIF characteristics of hepatocytes. Thus freezing studies using hepatocytes in suspension are not useful in predicting the freezing behavior of hepatocytes cultured in a collagen matrix. Furthermore, the weak effect of Me2SO on IIF characteristics implies that lower concentrations of Me2SO (0.5 M) may be just as effective in preserving viability. Finally, the value of B50 measured in this study indicates that cooling rates nearly an order of magnitude faster than those previously investigated could be used for cryopreservation of the hepatocytes in a collagen gel.
Use of Various Measurements for Biomass Estimation
Authors: Chattaway T. , Demain A. L. , Stephanopoulos Gregory
Published: March 2005

Electron, carbon, and nitrogen balances can be thought of as relationships among the time rates of change of the various compounds participating in a fermentation process. As such, they define the minimum number of necessary measurements from which the remaining rates can be determined through the use of the balances. All possibilities, however, are not equivalent, and some of them lead to singularities and solutions of high sensitivity. These possibilities are reviewed in this paper, and suggestions are offered in regard to the combination of rate measurements from which robust biomass estimates can be produced.
A Novel Extractive Fermentation Process for Propionic Acid Production from Whey Lactose
Authors: Lewis Vivian P. , Yang Shang‐Tian
Published: March 2005

An extractive fermentation process was developed to produce propionate from lactose. The bacterium Propionibacterium acidipropionici was immobilized in a spiral wound, fibrous matrix packed in the reactor. Propionic acid is the major product from lactose fermentation, with acetic acid and carbon dioxide as byproducts. Propionic acid is a strong inhibitor to this fermentation. A tertiary amine was used to selectively extract propionic acid from the bioreactor, hence enhancing reactor productivity by over 100 %. We also speculate that by selectively extracting propionic acid, lactose metabolism can be directed to yield more propionate and less byproducts. Other advantages of extractive fermentation include better pH control (by removing acid products) and a purer product. The propionic acid present in the extractant can be easily stripped with small amounts of an alkaline solution, resulting in a concentrated propionate salt. The extractant was also regenerated in this stripping step. Thus, the process is energy‐efficient and economically attractive.
Glass Transition in Poly(vinylpyrrolidone): Effect of Molecular Weight and Diluents
Authors: Buera Maria Del Pilar , Levi Guy , Karel Marcus
Published: March 2005

Amorphous poly(vinylpyrrolidone) (PVP) varying in molecular weight (MW) (10 000, 24 000, and 40 000) was used to study the effect of molecular weight on the glass transition temperature (Tg). Tg was measured by differential scanning calorimetry (DSC). Blends with several diluents (water, butylamine, glycine, hexanal, xylose, glucose, and lysine) were also analyzed and the effect of blend composition on Tg was evaluated. The effect of average molecular weight and of composition in the blends of PVP‐plasticizer on Tg was well described by previously reported relationships developed for synthetic polymers. The quantitative results obtained can be used for formulation of mixtures with known Tg and for prediction of their behavior in storage.
Prediction of pH in Model Systems Pressurized with Carbon Dioxide
Authors: Meyssami Behrouz , Balaban Murat O. , Teixeira Arthur A.
Published: March 2005

Mathematical models were developed to predict the pH of model liquid food systems (pure water, ascorbic acid‐water, citric acid‐water, and ascorbic acid‐citric acid‐water) pressurized with carbon dioxide, as functions of pressure (0–34.50 MPa), temperature (32,37, and 42° C), and component concentrations [(0–1.107) X10−3 M for ascorbic acid, (0 to 6.080) × 10−4 M for citric acid]. For all simulation systems, predicted pH values closely followed experimentally measured pH values. In the pure water‐CO2 systems, pH was a strong function of pressure but a weak function of temperature. In ascorbic acid‐water‐CO2, citric acid‐water‐CO2, and ascorbic acid‐citric acid‐water‐CO2 systems, pH was relatively insensitive to CO2 pressure and was a strong function of acid concentrations. This method could be a first step toward predicting the pH of orange juice and other liquid food systems under pressurized CO2.
Designing Enzymes for Use in Organic Solvents
Authors: Dordick Jonathan S.
Published: March 2005

Enzymes are routinely used in organic solvents where numerous reactions of interest to synthetic and polymer chemists can be performed with high selectivity. Recently, it has become apparent that the catalytic properties of an enzyme can be tailored to a specific catalytic requirement by the use of solvent and protein engineering. The former involves altering the polarity, hydrophobicity, water content, etc., of the organic milieu, while the later applies site‐directed mutagenesis to alter the physicochemical properties of the biocatalyst. The dominant effects of organic solvents on enzyme structure and function, and the potential of solvent and protein engineering to design enzymes to function optimally in organic media, are the major foci of this review.
Bioprocess Development To Improve Foreign Protein Production from Recombinant Streptomyces
Authors: Delacruz Neslihan , Payne Gregory F. , Smith Jeffrey M. , Coppella Steven J.
Published: March 2005

Bioprocessing strategies to improve production of the heterologous protein parathion hydrolase from recombinant Streptomyces lividans were investigated. Initial limitations to increased production were overcome by using large amounts of nutrients and feeding these nutrients throughout the fermentation. Batch addition of such large amounts of nutrients resulted in byproduct acid accumulation. Our data suggest that byproducts resulted from incomplete utilization of peptide medium ingredients and not from an overflow of glucose catabolism. Over extended fed‐batch operation, oxygen transfer became limiting and these limitations were overcome by sparging oxygen‐enriched gas. When cultivation was continued past about 90 h, we observed that despite nutrient feeding and oxygen enrichment enzyme activities no longer increased. Our results show that during such late cultivation periods the rates of enzyme synthesis and deactivation became balanced. If synthesis is prevented, either by a nutritional limitation or by the addition of the protein synthesis inhibitor chloramphenicol, enzyme activities were observed to decrease. Since deactivation rate constants in these experiments were similar to those observed in cell‐free studies, and because extracellular protease activities were not detected in our fermentation, it appears that deactivation results from the inherent instability of the parathion hydrolase enzyme.
Bioreactor Scale‐Up for the Oxygen‐Sensitive Culture Bacillus subtilis: The Influence of Stirrer Shaft Geometry
Authors: Bourne John R. , Zurita Eddy P. , Heinzle Elmar
Published: March 2005

The relative amounts of the metabolites acetoin and 2,3‐butanediol, formed in a Bacillus subtilis culture, depend strongly on the dissolved oxygen concentration (DO). Hence any factor determining DO can influence the product ratio Ac/Bu. Experiments in a 0.045 m3 stirred bioreactor, fitted with three Rushton turbines, have shown different product ratios when the diameter of the drive shaft was changed and all other conditions were kept constant. This unexpected result was attributed to changes in the power consumption and the pumping and gas handling capacities of the turbines. B. subtilis was cultivated in three geometrically similar vessels (0.045, 0.45, and 4.5 m3) using a constant superficial gas velocity (0.0075 m s−1). The product ratio and the volumetric mass transfer coefficient were essentially independent of scale when the gassed power input per unit volume was constant. This scale‐up result reflects the importance of adequate oxygenation of this culture, which was violated when the shaft diameter was not scaled with the vessel diameter. Geometrical similarity was an important factor in the scale‐up of this fermentation.
On the Theoretical Determination of Optimal Times for Biomass Production in Batch Cultures of Aggregate‐Forming Cells
Authors: Silva Teresa R. , Malcata F. Xavier
Published: March 2005

A model based on transient substrate and population balances coupled with Monod kinetics for growth has been formulated using several simplifying assumptions for batch cultures of cells which form biomass aggregates. The functional form of the analytical solution of this system of equations in its most generic aspect has been obtained and applied in the calculation of the time period corresponding to the maximum average rate of biomass increase.
Optimization of Growth Methods and Recombinant Protein Production in BTI‐Tn‐5B1‐4 Insect Cells Using the Baculovirus Expression System
Authors: Wickham T. J. , Nemerow G. R.
Published: March 2005

A novel insect cell line from Trichoplusia ni, BTI‐Tn 5B1‐4 (Tn 5), was compared to Spodoptera frugiperda, Sf 9, cells for production of two recombinant secreted proteins: truncated Epstein‐Barr viral attachment protein (EBV gp105) and truncated, soluble tissue factor (sTF). Under optimum conditions for both cell lines, Tn 5 cells produced 28‐fold more secreted sTF than Sf 9 cells, respectively, on a per cell basis. The total production of gp105 was similar for the two cell lines. However, Tn5 cells secreted gp105 much more efficiently, resulting in 5‐fold higher levels in the extracellular medium. Despite these increases, Tn 5 cells are attachment‐dependent, and protein production is sensitive to the cell density (cells/cm2), unlike the Sf9 cell line which can be easily grown and scaled up in cell suspension cultures without significantly affecting its per cell production. Thus, protein production from Tn 5 cells above 0.1L scales was optimized with respect to cell density using standard techniques for the growth of attachment‐dependent cells. Roller bottles precoated with DEAE‐based microcarriers and suspension cultures employing collagen‐coated microcarriers were found to be effective ways of culturing Tn 5 cells. Predetermined optimal cell densities were used to produce EBV gp105 in microcarrier‐coated roller bottles or in suspension cultures using collagen‐coated microcarriers at concentrations close to those observed in tissue culture flasks.
The Change of Pressure Drop during Large‐Scale Chromatography of Viscous Samples
Authors: Felinger Attila , Guiochon Georges
Published: March 2005

The variation of the inlet pressure during the injection and the elution of a band of a viscous compound, or pressure‐drop profile, was calculated in overloaded elution chromatography, assuming a significant difference between the viscosities of the solvent and the pure solute. The viscosity of the mixture was calculated using the Grunberg—Nissan equation. By means of the numerical integration of the mass balance equation, the concentration profile along the length of the column was calculated. Then, using the instantaneous concentration profile to derive the local viscosity, hence the local pressure gradient, the pressure drop was determined by integrating the local pressure gradient along the column. The change of pressure drop calculated is significant only when the concentration of the injected sample is very high. If a dilute sample is injected, even large sample volumes will not really change the pressure drop. The maximum pressure drop observed in each case is practically independent of the absorption isotherm. The only determining factors are the solute concentration in the sample, the amount of sample injected, and the solute diffusivity.
A Study of Process Interactions between Cell Disruption and Debris Clarification Stages in the Recovery of Yeast Intracellular Products
Authors: Clarkson Andrew I. , Lefevre Patrick , Titchener‐Hooker Nigel J.
Published: March 2005

The effects of varying the levels of cell disruption achieved by high‐pressure homogenization upon subsequent centrifugal separation of cell debris have been defined by measuring sedimentation velocity distributions of the cell debris. Using this data together with particle size distributions, a computer‐based model of a disk stack centrifuge has been used to predict the efficiency of centrifugal separation. These predictions have been compared with both laboratory scale and pilot plant scale experimental results. A similar sedimentation velocity technique has been used to characterize the properties of the floes formed by addition of poly (ethylene imine) to a borax‐clarified homogenate supernatant. The variations of floe settling properties with respect to polymer concentration are defined.
Crossflow Microfiltration of Yeast Suspensions in Tubular Filters
Authors: Redkar Sanjeev G. , Davis Robert H.
Published: March 2005

Crossflow microfiltration experiments were performed on yeast suspensions through 0.2‐μm pore size ceramic and polypropylene tubes at various operating conditions. The initial transient flux decline follows dead‐end filtration theory, with the membrane resistance determined from the initial flux and the specific cake resistance determined from the rate of flux decline due to cake buildup. For long times, the observed fluxes reach steady or nearly steady values, presumably as a result of the cake growth being arrested by the shear exerted at its surface. The steady‐state fluxes increase with increasing shear rate and decreasing feed concentration, and they are nearly independent of transmembrane pressure. The steady‐state fluxes for unwashed yeast in deionized water or fermentation media are typically 2–4 times lower than those predicted by a model based on the properties of nonadhesive, rigid spheres undergoing shear‐induced back‐diffusion. In contrast, the steady‐state fluxes observed for washed yeast cells in deionized water are only 10–30% below the predicted values. The washed yeast cells also exhibited specific cake resistances that are an order of magnitude lower than those for the unwashed yeast. The differences are due to the presence of extracellular proteins and other macromolecules in the unwashed yeast suspensions. These biopolymers cause higher cell adhesion and resistance in the cake layer, so that the cells at the top edge are not free to diffuse away. This is manifested as a concentration jump from the edge of the cake layer to the sheared suspension adjacent to it. The shear‐induced diffusion model includes a dimensionless parameter (referred to as the crossflow integral) that accounts for the concentration jump as well as the viscosity and diffusivity properties of a given suspension, and this parameter was determined for the unwashed yeast suspensions by fitting the experimental flux data to the model. The resulting values of this parameter are significantly lower than those for rigid spheres but show relatively little variation between experiments at different operating conditions for a given filter, indicating that the parameter is an intrinsic property of the suspension/membrane system. However, a significant difference in the parameter values was obtained between the ceramic and polypropylene membranes, indicating that the adhesive and fouling properties of the cake depend on the membrane material with which it is in contact. The resulting fluxes obtained from the model using single values for the membrane resistance, specific cake resistance, and crossflow integral for each data set (ceramic and polypropylene filters) are in good agreement with the measured fluxes. Inertial lift theory with no adjustable parameters was also compared to the data. It was found to overpredict the steady‐state fluxes at high shear rates and underpredict the fluxes at low shear rates.
Liquid‐Liquid Extraction of a Recombinant Protein with a Reverse Micelle Phase
Authors: Pires M. J. , Cabral J. M. S.
Published: March 2005

Recombinant cytochrome bs was extracted to a reversed micelle phase of a cationic surfactant (hexadecyltrimethylammonium bromide, CTAB) in cyclohexane/decanol and back‐extracted to a fresh aqueous phase. Depending on the experimental conditions, i.e., temperature, pH, and ionic strength, the extraction is controlled by either hydrophobic (pH close to the pI and high ionic strength) or electrostatic (pH far from the pI and low ionic strength) interactions. The protein interacts strongly with the surfactant aggregate once it is extracted to the organic phase, and it is very difficult to back‐extract it to a new aqueous phase.
Competitive Immunosorbent Assays for Biotin Using Bifunctional Unilamellar Vesicles
Authors: Jones Matthew A. , Kilpatrick Peter K. , Carbonell Ruben G.
Published: March 2005

Competitive immunosorbent assays for the model antigen biotin were performed using both unilamellar vesicles with covalently attached biotin and horseradish peroxidase (HBVs) and commercially available biotin‐labeled horseradish peroxidase (B‐HRP) as the enzyme‐labeled antigen. The assays were performed using anti‐biotin antibody (ABA) surface densities ranging from one‐tenth to full monolayer coverage. It was found that assays using HBVs strongly depended on the antibody surface density, while assays using B‐HRP were relatively insensitive to the antibody surface density. The HBV assay dependence on the ABA surface density was most likely due to multiple point attachment of vesicles to the surface. The lowest detectable antigen concentration (least detectable dose) foT vesicles (∼ 10−9 M) was an order of magnitude lower than the value found for B‐HRP (∼ 10−8 M). The sensitivity (slope of the response vs biotin concentration curve) of assays with B‐HRP was comparable to the sensitivity of assays with HBVs at low antibody surface density, probably due to less extensive multipoint attachment. It was also found that assays could be performed with vesicles at antibody surface densities that were at least 5 times lower, in terms of the bulk antibody concentrations used to coat the wells, than antibody surface densities at which B‐HRP gave comparable signals (∼1 ΔA/min).
Solvent Selection and Productivity in Multiphase Biotransformation Systems
Authors: Santhanam Harish K. , Shreve Gina S.
Published: March 2005

The 11‐β hydroxylation of the steroid precursor Reichstein's substance S (cortexolone) to hydrocortisone by Curvularia lunata cells is used as a model system for investigating the basis of the effect of solvent choice on multiphase biotransformation reaction kinetics. The effect of solvent choice and phase ratio on cellular toxicity and productivity is examined in batch suspension cultures. In general, the greater the miscibility of the solvent with the aqueous phase, the more cellular toxicity it exhibits. However, the presence of solvent at concentrations above the solubility limit showed no increased toxic effect over that achieved with saturation concentrations. Toxicity experiments indicate that of the solvents examined octane is the least toxic to the Curvularia lunata substance S biotransformation system under consideration. The production of hydrocortisone is determined to be approximately 20 times greater during the growth phase than during the stationary phase. The phase ratio of the organic solvent added has a significant effect on the productivity of the system. It was found that there is an increase in productivity with an increase in the phase ratio of the organic solvent added. Reaction kinetics are examined, and the increase in productivity with increasing amounts of organic solvent is attributed to the increase in concentration of the steroid reactant in the system.
Enzymatic Esterification of Diols in Reverse Micellar Media
Authors: Yang Ching‐Lun , Gulari Erdogan
Published: March 2005

With the help of highly dynamic reverse micellar media, lipase‐catalyzed esterification reactions can be carried out at ambient conditions without any agitation. Having low water contents in reverse micellar media, these reversible reactions favor the esterification direction instead of the normal hydrolysis catalyzed by lipases. A proposed enzyme kinetic model has successfully predicted the reaction mechanism of lauric acid with 1,3‐propanediol in reverse micellar media. However, experiments conducted showed a likelihood of substrate inhibition at higher lauric acid concentrations (>70 mM) and a strong bonding effect among 1,3‐propanediol, water, and AOT head groups. The effect of water concentration in the reaction media was shown to be important. The optimal Wo's for achieving the greatest initial reaction rate and final conversion were 9 and 4 for Candida rugosa and Rhizopus delemar, respectively. For both lipases, reverse micellar media experiments showed a lipase localization possibility.
On‐Line Exhaust Gas Analysis of Volatiles in Fermentation Using Mass Spectrometry
Authors: Oeggerli A. , Heinzle E.
Published: March 2005

Liquid‐phase concentrations of volatile compounds were determined on‐line via gasphase analysis using mass spectrometry. It has been shown theoretically as well as experimentally that almost every volatile compound commonly produced by microorganisms can be measured by the method presented, if its partial pressure in the fermentor off‐gas exceeds 1 μbar. This has been shown in a baker's yeast as well as a Bacillus subtilis culture. The experimental results also indicated that these measurements are not controlled by mass‐transfer kinetics. A dimensionless parameter, E, allows us to check whether the analysis is determined by thermodynamics (Henry coefficient) or by mass‐transfer rates.
Fractal Analysis of Antigen‐Antibody Binding Kinetics: Biosensor Applications
Authors: Sadana Ajit , Ram Aruna Beela
Published: March 2005

The diffusion‐limited binding kinetics of antigen (or antibody) in solution to antibody (or antigen) immobilized on a biosensor surface is analyzed within a fractal framework. The estimated fractal dimension increases linearly from 2.28 to 2.75 for the antirabbit IgG (antibody) (Nygren, H.; Stenberg, M. J. Colloid Interface Sci. 1985, 107, 560–566) as the R1S1 antibody concentration in solution increases 28‐fold from 6 to 170 μg/mL‐Similarly, for the binding of ferritin (antigen) in solution by a “sandwich” immunoassay to antiferritin (antibody) immobilized on a surface (Bluestein, B. I.; Craig, M.; Slovacek, R., Stundter, L.; Urciuoli, C; Walizak, I.; Luderer, A. In Biosensors with Fiberoptics; Wise, D., Wingard, L. B., Jr., Eds.; The Humana Press, Inc.: Clifton, NJ, 1991; pp 181–223), the estimated fractal dimension decreases from 1.33 to 1.02 as the initial concentration of ferritin increases from 20 to 1000 μg/mL. The analysis attempts to provide novel physical insights into the reactions occurring on the surface and the structure or nature of the surface. Diffusional limitations inherent in such systems need to be uncoupled from reaction kinetics and complexities, such as heterogeneity, that exist on the surface.
Energetic Effect of Vitreoscilla Hemoglobin Expression in Escherichia coli: An On‐Line 31P NMR and Saturation Transfer Study
Authors: Chen Ruizhen , Bailey James E.
Published: March 2005

A newly developed on‐line 31P NMR system has been used to investigate the effect of Vitreoscilla hemoglobin (VHb) expression on the steady‐state level of ATP in Escherichia coli under oxygen‐limited conditions. The VHb‐expressing strain GR021 exhibited a higher growth rate on a minimal fed‐batch medium after the onset of oxygen limitation and maintained the same level of ATP pool and transmembrane ΔpH as the slower growing wild‐type strain MG1655, indicating that the net ATP accumulation rate (the rate at which ATP increases per unit volume of culture per unit time) was enhanced in the VHb‐expressing strain. Further direct evidence of the enhanced flux of Pi‐ATP (catalyzed by ATPase) in GR021 was obtained in a saturation transfer study on respiring cells. VHb‐expressing cells respiring on succinate exhibited a Pi‐ATP flux 30 ± 5% higher than that of an otherwise isogenic strain that did not express VHb.
Modification of the Enzyme Enantioselectivity by Product Inhibition
Authors: Rakels J. L. L. , Caillat P. , Straathof A. J. J. , Heijnen J. J.
Published: March 2005

A kinetic explanation of solvent effects on the enantioselective hydrolysis of racemic methyl and ethyl 2‐chloropropionate by carboxylesterase NP has been studied. After a general ping‐pong model for the kinetic resolution of chiral esters was defined, the progress curves of the reactions were analyzed, and a simplified uni‐uni four‐parameter model was selected. First‐order rate constants and alcohol inhibition constants were determined for both enantiomers. Enantiomeric excess values were correctly predicted from the model. According to the kinetic description, the enantioselectivity could be enhanced or decreased by adding the product alcohol as a cosolvent. It appeared that these changes could be predicted quantitatively by enantioselective product inhibition for alcohol concentrations <10%. At higher alcohol concentrations additional changes in enantioselectivity occurred, which may be due to well‐known changes in enzyme conformation, alteration of the solvent properties, or enzyme‐solvent interactions. Similar results were found for hydrolyses catalyzed by α‐chymotrypsin and Candida cylindracea lipase.
Turbidimetric Titration Study of the Interaction of Proteins with Acrylic Polyampholytes
Authors: Nath Sunil , Patrickios Costas S. , Hatton T. Alan
Published: March 2005

A study of the interaction of proteins [soybean trypsin inhibitor (0.3–1 mg/mL)] with dilute solutions of synthetic polyampholytes (0.05–0.4 mg/mL) composed of acrylic (or methacrylic) acid, (dimethylamino)ethyl methacrylate, and methyl methacrylate was undertaken experimentally using turbidimetric titration. The polyampholytes had a number‐average molecular weight of 4000 (block polyampholytes) or 70000 (random polyampholytes) and a polydispersity index of only 1.3. Alone, the polyampholytes showed a tendency to self‐aggregate around their isoelectric point. Therefore, polyampholyte‐polyampholyte interactions (self‐aggregation) and polyampholyte‐protein complexation behavior were studied as a function of pH (3–9), ionic strength (0–0.75 M) and polymer dosage (50–400 mg of polymer/g of protein). Large increases in turbidity (>500%) were observed for protein‐polyampholyte mixtures (compared to polyampholyte alone) in the case of both block and random copolymers. The observed turbidity increases could be due to protein‐polymer interactions or to enhanced precipitation of polymer in the presence of protein. To differentiate between these alternatives, turbidimetry experiments were carried out at different dosages by (a) keeping the protein concentration constant and varying the polymer concentration and (b) keeping the polymer concentration constant and varying the protein concentration in small steps; they demonstrated that the turbidity increases were indeed due to protein‐polymer interactions. These experiments offer a general methodology to distinguish between intermolecular protein‐polymer and intermolecular polymer‐polymer interactions.
Constitutive Overexpression of Secreted Heterologous Proteins Decreases Extractable BiP and Protein Disulfide Isomerase Levels in Saccharomyces cerevisiae
Authors: Robinson Anne Skaja , Wittrup K. Dane
Published: March 2005

High‐level gene expression does not always lead to corresponding high‐level secretion of heterologous proteins in yeast. The rate‐limiting step in many cases has been shown to exit from the endoplasmic reticulum (ER). Within the ER, the correct folding of secreted proteins is required for export competence; hence, the cellular proteins involved in these events are likely to be important for efficient secretion. We have found that the extractable levels of two ER‐resident proteins involved in folding—heavy chain binding protein (BiP) and protein disulfide isomerase (PDI)‐‐are significantly reduced by prolonged constitutive overexpression of human granulocyte colony stimulating factor (GCSF), human erythropoietin, or Schizosaccharomyces pombe acid phosphatase. However, the rate of BiP synthesis measured in pulse‐chase radiolabeling experiments is not reduced by GCSF overexpression, and galactose‐directed transcription of the BiP gene does not restore normal BiP protein levels once they have been depleted. The observed loss of lumenal resident proteins, either by proteolysis or irreversible aggregation, is expected to contribute significantly to the inefficiency of foreign protein secretion in yeast.
Potential for Bioremediation of Xenobiotic Compounds by the White‐Rot Fungus Phanerochaete chrysosporium
Authors: Paszczynski Andrzej , Crawford Ronald L.
Published: March 2005

The white‐rot fungi produce an unusual enzyme system, characterized by a specialized group of peroxidases, that catalyzes the degradation of the complex plant polymer lignin. This ligninolytic system shows a high degree of nonspecificity and oxidizes a very large variety of compounds in addition to lignin. Among these compounds are numerous environmental pollutants. Thus, the white‐rot fungi show considerable promise as bioremediation agents for use in the restoration of environments contaminated by xenobiotic molecules. One white‐rot fungus, Phanerochaete chrysosporium, has been studied in great detail with regard to ligninolytic enzymes and the degradation of anthropogenic chemicals. It has been widely promoted as a bioremediation agent. This article examines literature concerning the degradation of xenobiotic compounds by Phanerochaete chrysosporium and attempts to critically assess this organism's real potential as a bioremediation tool.
Suppressed Acid Formation By Cofeeding of Glucose and Citrate in Bacillus Cultures: Emergence of Pyruvate Kinase as a Potential Metabolic Engineering Site
Authors: Goel Akshay , Lee Jinwon , Domach Michael M. , Ataai Mohammad M.
Published: March 2005

Microbial cultures typically produce acids when metabolizing the common carbon source, glucose. Acid production not only represents a waste of carbon, but its accumulation can limit cell concentration and culture stability, thereby reducing productivity. On the basis of prior work, acid production was attributed to be due to a mismatch between glycolytic and tricarboxylic acid (TCA) cycle capacities. To suppress acid production, a strategy entailing adding citrate to glucose minimal medium proved extremely effective. The effect of citrate on in‐vivo flux distribution was quantified using a detailed flux‐model. When the molar glucose—citrate ratio was varied between 3 and 6, a significant reduction in glycolytic flux and essentially complete suppression of acid formation was found as compared to chemostat cultures grown solely on glucose. Adding other biosynthetic precursors such as glutamine did not invoke the same suppression, thus indicating that citrate's effect is at the regulatory level. We hypothesized that the reduction of glycolytic flux in the presence of citrate results from its transport being coupled with the uptake of divalent metal ions. Citrate transport alters the intracellular balance of metal ions which in turn could trigger a sophisticated series of metabolic events leading to reduction of the activities of the pyruvate kinase and phosphofructokinase (PFK), the regulatory enzymes of glycolysis. On the basis of this scenario and other regulatory information, pyruvate kinase has emerged as a potential metabolic engineering site. It's deactivation in Bacillus subtilis or Escherichia coli strains is expected to yield constructs with a much lower tendency for making acid byproducts.