bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding isotherms for soluble immobilized affinity ligands from spectral titration
Authors: Mayes A. G. , Eisenthal R. , Hubble J.
Published: December 2004

The method of spectral titration has been applied to binding equilibria between proteins and soluble immobilized ligands and evaluated using the interaction between Cibacron blue–dextran conjugates and lysozyme. The method is both simple and rapid and provides a convenient screening technique for characterization of soluble adsorbents designed for use in aqueous two‐phase affinity extraction or as liquid‐phase models for affinity chromatography systems. The results indicate that regardless of ligand density a constant 28% of the total coupled dye is available for high‐affinity protein binding at saturation. The dissociation constant for the dye–protein interaction, however, decreases with dye loading. The potential for kinetic investigations has been demonstrated using a stopped‐flow apparatus. The results indicate that a simple rate equation is inadequate to describe the data for lysozyme binding to dye–dextran conjugates. A modified model, which better describes the data, was developed by including a second rate limiting process, the transition from stacked to unstacked dye ligands on the dextran backbone. This effect could have practical significance for protein binding kinetics in affinity chromatography, especially in high‐performance liquid affinity chromatography applications where mass transfer is rapid. © 1992 John Wiley & Sons, Inc.
On‐line monitoring of hybridoma cell growth using a laser turbidity sensor
Authors: Konstantinov Konstantin B. , Pambayun Rindit , Matanguihan Ricaredo , Yoshida Toshiomi , Perusicn Cheryl M. , Hu Wei‐Shou
Published: December 2004

A high‐sensitivity turbidity probe was used for on‐line monitoring of the cell concentration in batch hybridoma cultivation. Good correlation between off‐line cell counts and the linearized sensor signal was found. The quality of the signal was sufficiently high to provide for on‐line estimation of the specific growth rate using an efficient filtering procedure. These positive results suggest that such laser turbidity sensors will facilitate development of systems for on‐line monitoring and control of animal cell cultivations. © 1992 John Wiley & Sons, Inc.
Biodegradation of chlorophenol mixtures by Pseudomonas putida
Authors: Dapaah Samuel Y. , Hill G. A.
Published: December 2004

The dynamic growth behavior of Pseudomonas putida has been studied when resting calls were inoculated into a growth medium containing inhibitory concentrations of mixtures of phenol and monochlorophenols. Resting cells inoculated into single carbon substrate media did not demonstrate enhanced cell lysis by any of the phenol substrates. The apprarent death rate was reduced as the concentrations of phenol or chlorophenols were increased. This behavior was modeled by employing a constant specific death rate (kd = 0.0075 h−1) and assuming all organic species result in a lag‐phase, specific growth rate which may be larger or smaller than kd.

Logarithmic biomass growth on pure monochlorophenols did not occur within 2 weeks after inoculation. Logarithmic growth phases were only observed when the monochlorophenols were cometabolized with phenol. The delay time over which the lag phase exists increased exponentially with phenol concentration and linearly with monochlorophenol concentration. The log growth yield coefficient decreased linearly with monochlorophenol concentration.

The lag‐phase, specific growth rate was found to decrease exponentially with the concentration of monochlorophenols. This resulted in a 50% lag growth rate inhibition for both 3‐ and 4‐chlorophenol of 9 ppm and for 2‐chlorophenol of only 2 ppm. The new, empirical correlations are shown to closely model the complete lag and log growth behavior ot P. putida on phenol and chlorophenol mixtures. © 1992 John Wiley & Sons, Inc.
Cultivator for NMR studies of suspended cell cultures
Authors: Meehan A. J. , Eskey C. J. , Domach M. M. , Koretsky A. P.
Published: December 2004

When nuclear magnetic resonance (NMR) spectroscopy is employed for physiological experiments with suspended cells, providing for adequate nutrient and oxygen delivery is particularly important, because the inherent insensitivity of NMR requires that concentrated cell suspensions be used. In addition, it is desirable to be able to manipulate the growth rate of cells during a NMR experiment. To address these concerns, a continuous cell cultivator that provides convective oxygen and nutrient transport has been constructed for NMR experiments. The NMR detector coil is located within the cultivator volume. The location is advantageous because the rapid exchange of cells in and out of the coil leads to a small apparent spin lattice relaxation time, thus allowing for rapid pulsing and fast signal averaging. In this article we present the physical principles on which the cultivator's design is based. 31P spectra showing the response of continuously cultivated Saccharomyces cerevisiae cultures to a phosphate bolus and growth rate shift are then given. © 1992 John Wiley & Sons, Inc.
Kinetics of sterilization ofLactobacillus breviscells by the application of high voltage pulses
Authors: Jayaram Sheshakamal , Castle G. S. P. , Margaritis Argyrios
Published: December 2004

The technique of irreversible electroporation has been successfully applied to cause a lethal effect onLactobacillus breviscells suspended in phosphate buffer solution, Na2HPO4/NaH2PO4· H2O (0.845/0.186 mM) between parallel plane electrodes. Tests were carried out at different temperatures (24,45,60, and 80°C) to determine if there was a synergistic effect of temperature and electric pulse treatment on the destruction ofL. brevis. Experimental results indicate that the viability (logN/N0; whereN0andNare the number of cells survived per milliliter before and after pulse voltage application, respectively) ofL. brevisdecreased with electric field strengthEand temperatureTand treatment timett. The relations between log(N/N0) andttand log(N/N0) andEindicate that higher field strengths are more effective than higher treatment times in causing destruction ofL. breviscells. It was also found that as the temperature of the liquid medium containingL. breviscells increased from 24 to 60°C, the death rate ofL. breviscells increased with a decrease in the total treatment timett(pulse width × number of pulses applied). The application of an electric field strengthE= 25 kV/cm at 60°C and treatment timett= 10 ms resulted in very high destruction levels ofL. breviscells (N/N0= 10−9). In comparison with existing steam sterilization technology, this new method of sterilization using relatively low temperature and short treatment time could prove to be an excellent method to minimize thermal denaturation of important nutrient components in liquid media. © John Wiley & Sons, Inc.
Effect of subunit dissociation, denaturation, aggregation, coagulation, and decomposition on enzyme inactivation kinetics: I. First‐order behaviour
Authors: Lencki Robert W. , Arul Joseph , Neufeld Ronald J.
Published: December 2004

Enzyme inactivation kinetics typically follows what would appear to be simple first‐order behavior. However, the inactivation process is known to involve a number of reversible (decomposition, denaturation) as well as irreversible (decomposition, aggregation, and coagulation) reactions. These reactions can combine to form a wide variety of reaction pathways which can potentially demonstrate complex inactivation kinetics. However, it was shown that with appropriate assumptions with regard to the relative magnitudes of the various reaction rates, many complex inactivation pathways can demonstrate apparent first‐order behavior. Thus, with this analysis, a more accurate interpretation of the slope of an activity versus time semi‐log plot can be obtained. © 1992 John Wiley & Sons, Inc.
Comparison of maximum production rates and optimum operating/design parameters in overloaded elution and displacement chromatography
Authors: Felinger Attila , Guiochon Georges
Published: December 2004

The results of a study of the optimization of the experimental conditions for maximum production rate in overloaded elution and displacement chromatography are discussed. This study is based on the use of the equilibrium‐dispersive model of chromatography and the competitive Langmuir isotherms to calculate individual band profiles in the elution and displacement modes, and of a simplex algorithm to optimize the production rate. The operating parameters (sample size, mobile phase velocity, and the displacer concentration in the displacement model) and the column design parameters (column length and average particle diameter) are optimized simultaneously. Binary mixtures having relative concentrations 3:1 and 1:3, and separation factors of 1.2 to 1.8 are investigated. One of our major results is that, in both modes of chromatography, the maximum production rate is achieved at very low values of the retention factors, k′, much lower than those used in current practice. In all cases, unless k′ exceeds greatly that optimum value, the production rate is higher in overloaded elution than in displacement chromatography. This is particularly true for the extraction of a minor component, which is eluted second. © 1993 John Wiley & Sons, Inc.
Rheological characterization of media containing Penicillium chrysogenum
Authors: Pedersen Annemarie Gade , Bundgaard‐Nielsen Mikael , Nielsen Jens , Villadsen John , Hassager Ole
Published: December 2004

Samples from fed‐batch fermentations of Penicillium chrysogenum on complex medium are rheologically characterized. The behavior is well described by a power law model for which the parameters are estimates. Furthermore, two types of model media are characterized and compared with the real fermentation samples. Xanthan solutions are found to mimic the rheological properties of the filamentous fungi much better than carboxymethyl cellulose (CMC) solutions. © 1993 John Wiley & Sons, Inc.
Scaleup of ajmalicine production by plant cell cultures of Catharanthus roseus
Authors: Schlatmann J. E. , Nuutila A. M. , Van Gulik W. M. , ten Hoopen H. J. G. , Verpoorte R. , J Heijnen J.
Published: December 2004

The effect of scaleup on he production of ajmalicine by a Catharanthus roseus cell suspension culture in a selected induction medium were studied. In preliminary experiments it was observed that the culture turned brown and the production was inhibited upon transfer from a shake flask to a stirred bioreactor with forced aeration. Two factors were recognized as the potential origin of the differences between shake flask and bioreactor cultures: gas composition and mechanical shear forces. These factors were studied separately.

By recirculating a large part of the exhaust gas, a comparable gas regime was obtained in a bioreactor as occurred in a shake flask cultures. This resulted in the absence of browning and a similar pattern of ajmalicine production as observed in shake flasks. The effect of shear forces could not be demonstrated. However, the experiments showed that the culture may be very sensitive to liquid phase concentrations of gaseous compounds. The effects of kLa, aeration rate, CO2 production rate, and influent gas phase CO2 concentration on the liquid phase CO2 concentration are discussed. © 1993 John Wiley & Sons, Inc.
Continuous bioconversion of n‐octane to octanoic acid by recombinant Escherichia coli (alk+) growing in a two‐liquid‐phase Chemostat
Authors: Favre‐Bulle Oliver , Weenink Erik , Vos Tera , Preusting Hans , Witholt Bernard
Published: December 2004

Escherichia coli is able to grow on sugars in the presence of a bulk n‐alkane phase. When E. coli is equipped with the alk genes from Pseudomonas oleovorans, the resulting recombinant strain converts n‐alkanes into the corresponding alkanoic acids. To study the effects of growth rate and exposure to a bulk apolar phase on the physiology and the productivity of E. coli, we have grown this microorganism in two‐liquid‐phase continuous cultures containing 5% (v/v) n‐octane.

In contrast to batch cultures of wild‐tape E. coli grown in the presence of n‐octane, cells remained viable during the entire continuous culture, which lasted 200 h. Bioconversion of n‐octane to n‐octanoic acid by a recombinant E. coli (alk+) in a two‐liquid‐phase continuous culture was made possible by optimizing both the recombinant host strain and the conditions of culturing the organism. Continuous production in such two‐phase systems has been maintained for the least 125 h without any changes in the product concentration in the fermentation medium. The volumetric productivity was determined as a function of growth rate and showed a maximum at a dilution rate D = 0.32 h−1, reaching a continuous production rate of 0.5 g octanoate/L · h (4 tons/m3 · year). © 1993 John Wiley & Sons, Inc.
Relieving effects of glycine and methionine from acetic acid inhibition in Escherichia coli fermentation
Authors: Han Keehyun , Hong Juan , Lim Henry C.
Published: December 2004

Among amino acids screened for their potential to relieve wild and recombinant Escherichia coli from the negative effects of acetic acid, glycine, and methionine showed a sparing effect. In the presence of 2 g/L of acetic acid, addition of 0.5 g/L of glycine or methionine resulted in either a complete recovery or a further enhancement in the specific growth rate, while the enhancement was significant but not fully complete in the presence of 4 g/L of acetic acid. The addition of 0.5 g/L of methionine alleviated the negative effect of acetic acid on recombinant E. Coli growth to produce more β‐lactamase, which was encoded by plasmid pUC18. In continuous fermentation the methionine effect on recombinant. E. coli metabolism depended on dilution rate; at high dilution rates, above 0.4 h−1, the methionine addition enhanced β‐lactamase production and reduced acetic acid formation, while at low dilution rates, below 0.3 h −1, the effect was reversed. In def‐batch fermentation with wild‐type E. Coli, cell growth rate and cell yield from glucose were enhanced with methionine addition, while the acetic acid concentration reached over 4 g/L. © 1993 John Wiley & Sons, Inc.
Are water‐immiscibility and apolarity of the solvent relevant to enzyme efficiency?
Authors: Narayan V. Satya , Klibanov Alexander M.
Published: December 2004

The question of whether the solvent's water‐immiscibility is relevant to enzymatic activity was addressed by assaying four different hydrolases (three lipases and one protease) in nine anhydrous solvents of similar hydrophobicities of which four were infinitely miscible with water and five were not. For no enzyme was a jump in activity observed upon a transition from water‐miscible to water‐immiscible solvent. The relevance of solvent apolarity to enzymatic efficiency was also examined. To this end, three groups of isomeric anhydrous solvents were selected where within each group of isomeric anhydrous solvents were selected where within each group one solvent was apolar (i.e., lacked a permanent dipole moment). For none of the four enzymes studied was activity significantly higher in apolar solvents than in their polar counterparts. Thus we conclude that often‐cited solvent's immiscibility with water and apolarity by themselves are irrelevant to enzymatic activity. © 1993 John Wiley & Sons, Inc.
Calculation of entropy change accompanying growth of Escherichia coli K‐12 on succinic acid
Authors: Battley Edwin H.
Published: December 2004

The ΔS of one unit carbon formula weight of Escherichia coli K‐12 cells, when grown on succinic acid, was calculated to be −80.13 J/deg. This value could then be used to calculate the entropy change accompanying the anabolism and metabolism of succinic acid to be 30.82 J/deg and 32.40 J/mol deg, respectively. The entropy of one unit carbon formula weight of dried E. Coli K‐12 cells is calculated to be 94.40 J/deg, which when divided by the mass of these cells becomes 3.90 J/g deg. The corresponding entropy of succinic acid is 2.77 J/g deg, making it apparent that the entropy per unit mass of the cells is greater than that of the substrate. It might be thought that because the cells appear to be so much more complex than the substrate, the cells should have a lesser entropy per unit mass than the substrate. That this does not appear to be true leads to the conclusion that the macromolecular organization (informational content?) of the cells contributes only in a very minor way to the total physical entropy of cells. © 1993 John Wiley & Sons, Inc.
Analysis of substrate protection of an immobilized glucose isomerase reactor
Authors: Houng Jer‐Yiing , Yu Hong‐Yang , Chen Kuo‐Cheng , Tiu Carlos
Published: December 2004

The effect of substrate protection on enzyme deactivation was studied in a differential bed and a packed bed reactor using a commercial immobilized glucose isomerase (Swetase, Nagase Co.). Experimental data obtained from differential bed reactor were analyzed based on Briggs‐Haldane kinetics in which enzyme deactivation accompanying the protection of substrate was considered. The deactivation constant of the enzyme‐substrate complex was found to be about half of that of the free enzyme. The mathematical analysis describing the performance of a packed bed reactor under the considerations of the effects of substrate protection, diffusion resistance, and enzyme deactivation was studied. The system equations for the packed bed reactor were solved using an orthogonal collocation method. The presence of substrate protection and the diffusion effect within the enzyme particles resulted in an axial variation of effectiveness factor, ηD, along the length of the packed bed. The axial distribution profile of ηD was found to be dependent on the operation temperature, Based on the effect of substrate protection, a better substrate feed policy could be theoretically found for promoting productivity in long‐term operation. © 1993 John Wiley & Sons, Inc.
Mathematical modeling of growth and alkaloid production in Claviceps purpurea batch fermentation
Authors: Miličić S. , Velušček J. , Kremser M. , Sočič H.
Published: December 2004

An new systematic approach for describing Claviceps purpurea growth and ergot alkaloid production during batch fermentation is presented. The model is based on microbial life, as the main characteristic for microbial development during fermentation process. The aging process of the microorganism is represented by life function, defined in microbial life space. The life space is defined as a measure in which the observer follows the development of a biosystem through physiological and morphological changes of a microorganism. As a consequence of such approach the relativistic theory is recognized. To validate the model developed, a test on growth and alkaloid synthesis data from an industrial batch fermentation was performed. © 1993 John Wiley & Sons, Inc.
Production of indole alkaloids by selected hairy root lines of Catharanthus roseus
Authors: Bhadra Rajiv , Vani Sundeep , Shanks Jacqueline V.
Published: December 2004

Hairy root cultures of Catharanthus roseus were established by infection of seedlings with Agrobacterium rhizogenes 15834. Approximately 150 transformants from four different. C. roseus cultivars were screened for desirable traits in growth and indole alkaloid production. Five hairy root clones grew well in liquid culture with doubling times similar to those reported for cell suspensions. Fast growing clones had similar morphologies, characterized by thin, straight, and regular branches with thin tips. The levels of key alkaloids, ajmalicine, serpentine, and catharanthine, in these five clones, also compared well with literature data from cell suspensions, yet HPLC and GC‐MS data indicate the presence of vindoline in two clones at levels over three orders of magnitude greater than the minute amounts reported in cell culture. These results suggest that further optimization may result in hairy roots as a potential source of vindoline and catharanthine, the two monomers necessary to synthesize that antineoplastic drug, vinblastine. © 1993 John Wiley & Sons, Inc.
Dissolution of sulphur particles by Thiobacillus ferrooxidans: Substrate for unattached cells
Authors: Shrihari  , Bhavaraju S. R. , Modak Jayant M. , Kumar R. , Gandhi K. S.
Published: December 2004

The growth of Thiobacillus ferrooxidans on sulphur is known to proceed through the attachment of cells to the sulphur particles. Experiments, However, show that the cells in the liquid phase, which are not attached to the sulphur particles, also grow. It has been shown through the use of a two‐compartment membrane reactor that this increase is partially due to the release of ions, corresponding to partially oxidized of sulphur, into the solution by the attached cells. The main soluble ion has been found to the thiosulphate, but traces of sulphite have also been detected. © 1993 John Wiley & Sons, Inc.
Mechanism of microbial flotation using Thiobacillus ferrooxidans for pyrite suppression
Authors: Ohmura Naoya , Kitamura Keiko , Saiki Hiroshi
Published: December 2004

Microbial desulfurization might be developed as a new process for the removal of pyrite sulfur from coal sluries such as coal–water mixture (CWM). An application of iron‐oxidizing bacterium Thiobacillus ferrooxidans to flotation would shorten the periods of the microbial removal of pyrite from some weeks by leaching methods to a few minutes. The floatability of pyrite in flotation was mainly reduced by T. ferrooxidans itself rather than by other microbial substances in bacterial culture as additive of flotation liquor. Floatability was suppressed within a few seconds by bacterial contact. The suppression was proportional to increasing the number of cells observed between bacterial adhesion and the suppression of floatability. If 25% of the total pyrite surface area covered with the bacteria, pyrite floatability would be completely depressed. Bacteria that lost their iron‐oxidizing activities by sodium cyanide treatment were also able to adhere to pyrite and reduced pyrite floatability as much as normal bacteria did. Thiobacillus ferrooxidans ATCC 23270, T‐1, 9, and 11, which had different iron‐oxidizing abilities, suppressed floatability to similar‐levels. The oxidizing ability of bacteria did not influence the suppressing effect. These results showed the mechanism of the suppression of pyrite floatability by bacteria. Quick bacterial adhesion to pyrite induced floatability suppression by changing the surface property from hydrophobic. The quick adhesion of the bacterium was the novel function which worked to change the surface property of pyrite to remove it from coal. © 1993 John Wiley & Sons, Inc.
Instability caused by high strength of cheese whey in a UASB reactor
Authors: Yan J. Q. , Lo K. V. , Pinder K. L.
Published: December 2004

The anaerobic digestion of cheese whey was studied in a UASB reactor. The profiles of the reactor, i.e., the distributions of the substrate concentration and pH under different operating conditions were developed. From the concentrations of substrates measured at various levels above the bottom of the reactor, two reaction stages, namely acidogenesis and methanogenesis, were distinguished. The instability caused by high influent concentration was interpreted as the accumulation of VFAs in the acidogenic stage beyond the assimilative capacity of the methanogenic stage. A range of stable operating conditions was predicted from the results of the profile measurements. The optimal influent concentration was found to be between 25 and 30 g COD/L at an HRT of 5 days for system stability. Other options fro stability control were discussed. © 1993 John Wiley & Sons, Inc.
A statistical analysis of the effect of substrate utilization and shear stress on the kinetics of biofilm detachment
Authors: Peyton Brent M. , Characklis W. G.
Published: December 2004

One of the least understood processes affecting biofilm accumulation is detachment. Detachment is the removal of cells and cell products from an established biofilm and subsequent entrainment in the bulk liquid. The goal of this research was to determine the effects of shear stress and substrate loading rate on the rate of biofilm detachment.

Monopopulation Pseudomonas aeruginosa and undefined mixed population biofilms were grown on glucose in a RotoTorque biofilm reactor. Three levels of shear stress and substrate loading rate were used to determine their effects on the rate of detachment. Suspended cell concentrations were monitored to determine detachment rates, while other variables were measured to determine their influence on the detachment rate. Results indicate that detachment rate is directly related to biofilm growth rate and that factors which limit growth rate will also limit detachment rate. No significant influence of shear on detachment rate was observed.

A new kinetic expression that incorporates substrate utilization rate, yield, and biofilm thickness was compared to published detachment expressions and gives a better correlation of data obtained both in this research and from previous research projects, for both mono‐ and mixed‐population biofilms. © John Wiley & Sons, Inc.
Rheological properties of mammalian cell culture suspensions: Hybridoma and HeLa cell lines
Authors: Shi Yaun , Ryu Dewey D. Y. , Ballica Rabia
Published: December 2004

Data on viscous (η′) and elastic (η″) components of the complex viscosity versus oscillatory angular frequency (0.01 to 4.0 rad/s) with increasing strains were obtained for hybridoma cell (62′D3) and HeLa cell (S3) suspensions in PBS at 0.9 (mL/mL) cell volume fraction using a Weissenberg rheogoniometer equipped with two parallel plate geometry at ambient temperature. Both cell suspensions exhibited shear thinning behavior. From the measured viscoelastic properties, the yield stress was calculated. Hybridoma cell suspension (15 μm as the mean diameter of cells) showed the yield stress at 550 dyne/cm2 that was 1.8 times higher than the value of HeLa cell suspension (22 μm mean diameter) as measured at the oscillatory angular frequency, 4.0 rad/s. The apparent viscosities of HeLa cell suspension at four concentrations and varying steady shear rate were also determined using the Brookfield rotational viscometer. The yield stress to steady shear test was about 130 dyne/cm2 for HeLa cell suspension at 0.9 (mL/mL) cell volume fraction. The apparent viscosity was in the range about 1 ∼ 1000 Poise depending on the cell concentration and shear rate applied. A modified semiempirical Mooney equation, \documentclass{article}\pagestyle{empty}\begin{document}$ \eta = \eta _0 \exp [K\dot \gamma ^{ - \beta } \phi /(1 - K''\sigma \phi _c /D)] $\end{document} was derived based on the cell concentration, the cell morphology, and the steady shear rate. The β, shear rate index, was estimated as 0.159 in the range of shear rate, 0.16 to 22.1 s−1, for the cell volume fractions from 0.6 to 0.9 (mL/mL). In this study, the methods of determining the shear sensitivity and the viscous and the elastic components of mammalian cell suspensions are described under the steady shear field. © 1993 John Wiley & Sons, Inc.
The adsorptive immobilization of phospholipids D mediated by calcium ions
Authors: Lambrecht Romy , Ulbrich‐Hofmann Renate
Published: December 2004

Immobilization of phospholipase D from cabbage was studied with the aim of stabilizing the enzyme for its use in synthesis of phospholipids. It was shown that phospholipase D can be immobilized by adsorption to polymeric carriers containing long chain anchor groups such as octadecyl, octyl, or other alkyl residues. Starting from the crude enzyme, phospholipase D activity is preferentially bound (up to 100%) in competition with contaminating proteins. A prerequisite of high binding rates is the presence of calcium ions, which play a mediating role in the adsorption process. The maximum activity of the carrier‐enzyme complexes depends upon the calcium concentration in the immobilization process and the carrier material (≥10mM CaCl2 with octadecyl‐Si40, ≥40 mM CaCl2 with octyl‐sepharose and butyl‐fractogel). Immobilization of phospholipase D to octyl‐sepharose was shown to result in a distinctly increased storage stability and an enlarged pH‐optimum range for the catalytic activity. Operational stability of different phospholipase D‐carrier complexes was compared. © 1993 Wiley & Sons, Inc.
Mathematical model for the luminol chemiluminescence reaction catalyzed by peroxidase
Authors: Li Lin , Arnold Mark A. , Dordick Jonathan S.
Published: December 2004

A kinetic model that accurately describes intensity vs. time reaction profiles for the chemiluminescence reaction between luminol and hydrogen peroxide, as catalyzed by horseradish perioxdase, is derived and evaluated. A set of three differential equations is derived and solved to provide intensity time information for the first 200 seconds of the reaction. The model accurately predicts intensity‐time profiles when literature values are used for all but one of the reaction rate constants. Furthermore, the model predicts a nonlinear curve for plots of light intensity versus the initial hydrogen peroxide concentration. Experimental data confirm that such plots are nonlinear. Finally, a linear double‐reciprocal plot is predicted by the model and the experimental data verify this relationship. © 1993 Wiley & Sons, Inc.
Viability testing and characterization of germination of fungal spores by automatic image analysis
Authors: Paul G. C. , Kent C. A. , Thomas C. R.
Published: December 2004

Fungal spores are used in the laboratory for culture maintenance and at laboratory and other scales as inocula for fermentations. The spore swelling and germination processes constitute a major part of the lag phase, and the subsequent culture morphology and productivity can be greatly influenced by the initial concentration and condition of the spores. An image analysis method has been developed for assessing the viability and the germination characteristics of fungal spores in submerged cultures. Structural variations during germination, i.e., swelling, germ tube formation, and germ tube elongation, are measured in terms of distributions of spore volumes and of germ tube lengths and volumes. These measurements are fully automatic and give a very rapid assessment of spore viability. This image analysis method might be used as a tool in culture maintenance and for determining the quality of inocula for fungal fermentations. © 1993 John Wiley & Sons, Inc.
Solid‐State nuclear magnetic resonance investigation of solvent dependence of tyrosyl ring motion in an enzyme
Authors: Burke Paul A. , Griffin Robert G. , Klibanov Alexander M.
Published: December 2004

Tyrosyl ring motions in α‐lytic protease were investigated by solid‐state deuterium nuclear magnetic resonance (NMR) spectroscopy in lyophilized enzyme powder, in powder suspended in organic solvents, and in aqueous crystals. Ring flipping rates were determined by examining deuterium quadrupole echo line shapes. Of the four Tyr residues in the enzyme, one was flipping at the slow (≤103 s−1) and one at the fast (≥107 s−1) exchange limit of the line shape experiment in all the environments tested. Flipping rates of the remaining two Tyr residues depended markedly on the solvent, with the lowest flipping rates (≤103 s−1 for both residues) observed in the enzyme powder, whether dry or suspended in hydrophobic tert‐butyl methyl ether. In hydrophilic dioxane and acetonitrile, the mobility of these residues increased to 104 and 105 s−1. The latter rate rose further to 106 s−1 in the hydrated hydrophilic solvents and to ≥107 s−1 in aqueous crystals. The deuterium spectrum of native α‐lytic protease was compared with that of the enzyme whose active center was covalently modified with an inhibitor, which binds next to Tyr‐123, constraining its ring. This experiment revealed that water addition to acetonitrile specifically increased the flipping rate of this active center residue. Librational motions (“wobbling”), estimated by their effect on spin–lattice relaxation times, were slowest in the anhydrous solvents, intermediate in the hydrated solvents, and fastest in the aqueous crystals. Thus, α‐lytic protease is more rigid in organic solvents than in water, as judged by mobility of its tyrosyl residues. Water stripping by hydrophilic solvents did not increase enzyme rigidity, nor were there clear correlations between mobility and either enzymatic activity or solvent dielectric constant. © 1993 John Wiley & Sons, Inc.
Micelle‐supported electroenzymology: Succinate dehydrogenation by escherichia coli fumarate reductase in decylubiquinone and octyl glucoside micelles
Authors: Kinnear Kathleen T. , Monbouquette Harold G.
Published: December 2004

The concept of micelle‐supported electroenzymology is demonstrated using a system consisting of the membrane enzyme Escherichia coli fumarate reductase (FRD), the amphiphilic coenzyme analogue decylubiquinone (DU), the micelle‐forming surfactant n‐octyl glucoside (OG), and a gold electrode. The OG micelles provide a hydrophobic, membrane mimetic medium for FRD and DU to exchange electrons while the gold electrode serves to regenerate DU. When succinate is presented to the FRD/DU/OG micelle system, electroenzymatic oxidation of succinate to fumarate occurs as evidenced using cyclic voltammetry. DU is shown to be the only electroactive species in the system; and as increasing amounts of succinate are added, the expected increase in the peak anodic (oxidative) current and decrease in the peak cathodic (reductive) current are observed. The peak anodic current approaches a limiting value with succinate concentration in qualitative agreement with simple Michaelis–Menten enzyme kinetics. When the strong competitive inhibitor oxaloacetate is added, enzymatic oxidation of succinate is inhibited as indicated by no change in the peak anodic and cathodic currents with increasing succinate concentration. © 1993 John Wiley & Sons, Inc.
Intracellular pH‐based controlled cultivation of yeast cells: II. Cultivation methodology
Authors: Sureshkumar G. K. , Mutharasan R.
Published: December 2004

Intracellular pH (pHi) was measured on‐line in a bioreactor using a fluorescent pHi indicator, 9‐aminoacridine, and controlled fed‐batch cultivations of yeast cells based on pHi (FB‐pHi) were performed. In FB‐pHi cultivations, automated glucose additions were made to the culture in response to culture pHi. The average ethanol (an‐aerobic product) yield was significantly lower [0.12 g g−1 glucose in fed‐batch pHi cultivations with 100 ppm glucose additions (FB‐pHi‐100 cultivation) vs. 0.48 g g−1 glucose in batch] and cell yield was higher (0.54 g g−1 glucose in FB‐pHi‐100 cultivation vs. 0.3 g g−1 glucose in batch) compared to batch cultivation. An expression has been derived to calculate changes in pHi from measured fluorescence values when the cell concentration increases during growth. Cultivations based on pHi, performed with different magnitudes of glucose addition (100, 50, and 10 ppm additions), showed that lower magnitudes of glucose addition resulted in lower ethanol yields while cell yield remained unaffected. The ratio of specific oxygen uptake rate to specific glucose uptake rate (OUR/GUR) increased with decreased in magnitude of glucose additions in FB‐pHi cultivations, suggesting that the culture aerobic state was higher when the magnitude of glucose addition was lower. The average cell productivity in FB‐pHi cultivations was 29% higher than in batch cultivation. Cells were also cultivated at high OUR conditions, and the results are compared with other cultivations. © 1993 John Wiley & Sons, Inc.
Alginate–chitosan coacervation in production of artificial seeds
Authors: Tay Ling‐Fong , Khoh Leng‐Khim , Loh Chiang‐Shiong , Khor Eugene
Published: December 2004

Survival of secondary embryoids of winter oilseed rape (Brassica napus ssp. oleifera cv. Primor) has been used as an assay for the development of artificial seeds involving complex coacervation of alginate (polyanion) with chitosan (polycation). Germination frequency of 100% was achieved for encapsulated embryoids when alginate formed the inner matrix and chitosan the outer layer. When the matrix makeup was reversed, there was no germination of embryoids. The artificial seeds produced were hardened in dilute alkaline solutions of NaOH and Ca(OH)2. An optimum setting time could be selected based on a quantitative measurement of resistance of hardened capsules to compression and the germination frequency of the encapsulated embryoids. © 1993 John Wiley & Sons, Inc.
Controlled release process to recover heterologous glycosylphosphatidylinositol membrane anchored proteins from CHO cells
Authors: Kennard M. L. , Food M. R. , Jefferies W. A. , Piret J. M.
Published: December 2004

A semicontinuous process has been developed to recover heterologous proteins at increased concentrations and purities. Proteins attached to mammalian cell membranes by glycosylphosphatidylinositol (GPI) anchors can be selectively released into the supernatant by the enzyme phosphatidylinositol–phospholipase C (PI–PLC). Chinese hamster ovary (CHO) cells, genetically engineered to express the GPI anchored, human melanoma antigen (p97), were used as a model system. These cells were grown in protein containing growth medium. During a brief harvesting phase the medium was replaced by phosphate buffered saline (PBS) containing 10 mU/mL of PI–PLC and the GPI anchored protein was cleaved from the cell surface and recovered in soluble form at up to 30% purity. After harvesting, the cells were returned to growth medium where the protein was re‐expressed within 40 h. The growth rate, viability, and protein production of cells, repeatedly harvested over a 44‐day period, were not adversely affected. This continuous cyclic harvesting process allowed recovery of a heterologous protein at high purity and concentrations and could be applied to the recovery of other GPI anchored proteins and genetically engineered GPI anchored fusion proteins. © 1993 John Wiley & Sons, Inc.
Cultivation of virus antigen in fibroblast cells using a glass fiber bed reactor
Authors: Junker B. H. , Chiou T. , Wang D. I. C. , Buckland B. C.
Published: December 2004

The anchorage‐dependent cell line, MRC‐5, was cultivated successfully on glass fibers with diameters ranging from 24 to 120 μm, despite vast differences in substrate curvature. Multilayer cell growth was observed, particularly for fiber diameters 30 μm and below, which differed from the typical monolayer growth observed in T‐flask cultivations. Cells were maintainable at a reduced incubation temperature and were demonstrated to support virus replication for the 21‐day antigen production period. Direct microscopic observation, along with indirect calculations, indicated that only a small fraction (about 10%) of the total available fiber surface area was occupied by cells. Thus, productivity per unit surface area was replaced by productivity per unit medium volume when evaluating fiber bed performance. Antigen and protein yields, as well as nutrient uptakes, were 1.5‐ to 2.5‐fold greater than parallel T‐flask cultures when compared on this basis. Corresponding available surface area–based values were 10‐ to 15‐fold lower for the fiber bed reactor. The multilayer cell morphology obtained in the fiber bed was attractive for antigen production when immobilized in a column reactor system. © 1993 John Wiley & Sons, Inc.
Optimal operating policy of the ultrafiltration membrane bioreactor for enzymatic hydrolysis of cellulose
Authors: Lee Seung‐Goo , Kim Hak‐Sung
Published: December 2004

The dilution rate of an ultrafiltration membrane bioreactor in the enzymatic hydrolysis of cellulose was optimized using the kinetic model developed by Fan and Lee.4 The sequence of optimal dilution rates was found to generally consist of an initial period of a minimal value (batch period), a subsequent period of maximum dilution rate, a period of a second batch, and a final period of a singular dilution rate. The effects of operating conditions, such as β‐glucosidase activity, operating time, maximum dilution rate, substrate feeding rate, and enzyme‐to‐substrate ratio on both the conversion yield and the sequence of optimal dilution rates were investigated. To evaluate the validity of kinetic model employed in this work, enzymatic hydrolysis was carried out using α‐cellulose as a substrate in the ultrafiltration membrane bioreactor. The experimental data were well consistent with the simulation results. © 1993 John Wiley & Sons, Inc.
Modeling lipolysis in a reversed micellar system: Part I. Conventional batch reactor
Authors: Prazeres D. M. F. , Lemos F. , Garcia F. A. P. , Cabral J. M. S.
Published: December 2004

Triglyceride hydrolysis using Chromobacterium viscosum lipase B in a reversed micellar media was investigated in a batch‐type reactor. The dynamic evolution of the product concentration was analyzed using several mechanistic models, both from the literature and developed in this work. A kinetic model with nonlinear product inhibition was found to be the most adequate for the description of batch hydrolysis data over an extensive range of time and substrate concentration. The obtained rate equation described the time course of not only the reactions performed in this work, at different water contents (W0 = 7, 24) and pH values, but also the experimental results obtained in the literature with a Candida rugosa lipase. © 1993 John Wiley & Sons, Inc.
Peroxidase and laccase as catalysts for removal of the phenylhydrazide protecting group under mild conditions
Authors: Semenov Anatoly N. , Lomonsova Irina V. , Berezin Vasilly I. , Titov Mikhail I.
Published: December 2004

A new method is proposed for the removal of the phenylhydrazide protecting group by the action of peroxidase or laccase, the enzymes attributed to the class of oxidoreductases. The deblocking procedure is performed under mild oxidative conditions, i.e., aqueous solution and neutral or close to neutral pH. Such mild oxidizing agents as 1 mM H2O2 and air oxygen are used for unmasking. The method is available for the deblocking of both α‐ and γ‐carboxyl groups. The enzyme‐catalyzed removal of the phenylhydrazide protecting group causes no oxidative modification nor destruction of methionine or tryptophan side chains. © 1993 John Wiley & Sons, Inc.
Membrane formation by interfacial cross‐linking of chitosan for microencapsulation of Lactococcus lactis
Authors: Groboillot A. F. , Champagne C. P. , Darling G. D. , Poncelet D. , Neufeld R. J.
Published: December 2004

Lactic acid bacteria were microencapsulated within cross‐linked chitosan membranes formed by emulsification/interfacial polymerization. The technique was modified and optimized to provide biocompatible conditions during encapsulation involving the use of mineral oils as the continuous phase and chitosan as the membrane material. Chitosan cross‐linked with hexamethylene diisocyanate or glutaraldehyde resulted in strong membranes, with a narrow size distribution about a mean diameter of 150 μm. Cell viability and activity was demonstrated by the acidification of milk. Loss of acidification activity during microencapsulation was recovered in subsequent fermentations to levels similar to that of free cell fermentations. © 1993 John Wiley & Sons, Inc.
Effects of rheological properties and mass transfer on plant cell bioreactor performance: Production of tropane alkaloids
Authors: Ballica Rabia , Ryu Dewey D. Y.
Published: December 2004

Volumetric mass transfer coefficients, KLa were measured over an aeration rate range from 0.1 to 1.0 vvm in a 1.2‐L draft‐tube‐type airlift bioreactor for different Datura stramonium cell concentrations and correlated with superficial air velocity and rheological properties of the cell suspension. The measured KLa values (17–40 h−1) for a cell volume fraction of 0.2 (v/v) were approximately 2 times higher than those for the highest cell concentrations tested (cell volume fraction 0.7–0.8 v/v). Cell suspensions exhibited yield stress and pseudoplastic behavior. This behavior was described by the Casson model. The estimated yield stress values depended upon cell concentration with an exponent of 4.0. An empirical correlation based on the data for plant cell suspensions exhibiting yield stress was developed in order to determine aeration strategy for the plant cell cultivation in draft‐tube‐type airlift bioreactors:

Aeration rates above 1.0 vvm caused a significant drop in cell yield and product content. Maximum growth and production were obtained at 0.6 vvm aeration. The cell and product yields obtained at 1.7 vvm were 2.8 times lower than the maximum values (25 g cell DW/L and 73.8 mg tropane alkaloid/L). The effects of the increased aeration rates on cell yield were also evaluated in terms of Reynolds stress. It was found that there was a relation between cell damage and the estimated Reynolds stress. The Reynolds stress estimated for the same aeration rate decreased with increasing cell concentration, suggesting that cells in the cultures at low cell concentrations are subjected to hydrodynamic damage. In the experiments with the cell cultures having a cell concentration of 0.3 (v/v), approximately 70% reduction in cell concentration was observed when the Reynolds stress was increased from 10 to 50 dyn/cm2. © 1993 John Wiley & Sons, Inc.
On‐line monitoring of monoclonal antibodies in animal cell culture using a grating coupler
Authors: Polzius R. , Bier F. F. , Bilitewski U. , Jäger V. , Schmid R. D.
Published: December 2004

A grating coupler was used for the on‐line determination of monoclonal antibodies produced in perfused animal cell bioreactor. The device was connected with the culture vessel via a flow‐injection analysis (FIA) system, which was controlled automatically. Specific antimouse lgG antibodies were immobilized on the surface of the sensor‐chip. After injection of the sample, the binding of mouse lgG was observed in real time. The regeneration of the binding sites of the immobilized antibodies using an acidic solution allowed the on‐line detection of produced monoclonal antibodies in the range of 10 to 150 μg/mL. In contrast to other techniques coupled to bioprocesses, the developed method represents a regenerable direct immunosensor. Results were compared with standard ELISA techniques (off‐line) and a competitive immunochemical assay using the grating coupler (off‐line). © 1993 John Wiley & Sons, Inc.
Transfer vectors for maximal expression of passenger genes in the Bombyx mori nuclear polyhedrosis virus expression system
Authors: Johnson Russell R. , Schmiel Deborah , Iatrou Kostas , Gedamu Lashitew
Published: December 2004

A series of Bombyx mori nuclear polyhedrosis virus (Bm‐NPV) transfer vectors has been developed containing various lengths of the polyhedrin promoter, including sequences 3′ of the initiation codon. The ATG initiation codon was mutated in some of these vectors to allow for the production of authentic nonfusion proteins. The ability of the various polyhedrin promoter constructs to direct expression of foreign gene sequences was assessed using two test genes, chloramphenicol acetyl transferase (cat), and human metallothionein II. Accumulation of cat mRNA and nonfused protein was low when only polyhedrin promoter sequences to –8 (relative to the translational start site of polyhedrin mRNA) were included in the transfer vector, but cat expression was comparable with that of the wild‐type polyhedrin gene when promoter sequences to +5 were present. Further addition of polyhedrin gene sequences to +26 or +94 resulted in no further increase in expression. Similar results were obtained for expression of human metallothionein II, where constructs encoding polyhedrin–metallothionein fusion proteins containing polyhedrin sequences to at least +5 resulted in high levels of mRNA and protein accumulation. The expression vectors containing the +5, +26, or +94 BmNPV polyhedrin promoter can thus be used to direct maximal levels of production of nonfused proteins (when the polyedrin ATG has been mutated) or of fusion proteins, depending on which is more suitable for a particular application. These new vectors are a useful addition to those presently available and should increase the utility of the BmNPV expression system for large‐scale protein production. © 1993 John Wiley & Sons, Inc.