bluuebunny/crossref_metadata_2025 huggingface dataset:
Application of quantitative image analysis to a mammalian cell line grown on microcarriers
Authors: Pons Marie‐Noëlle , Wagner Anne , Vivier Harvé , Marc Annie
Published: December 2004

Quantitative image analysis has been applied to the monitoring of cultures of a mammalian cell line on microcarriers. Procedures have been developed to investigate microcarrier colonization and cluster formation and to determine the eventual modification of cell size during cultivation using scanning electron microscopy (SEM) microphotography. The human kidney tumor cells (TCL 598) on which the procedures were tested underwent a slight size decrease during the development of the first cell layer on the microcarriers. The cluster size and the cell size remained constant during the culture stationary phase.
Oxygen transfer properties of bubbles in animal cell culture media
Authors: Zhang S. , Handa‐Corrigan A. , Spier R. E.
Published: December 2004

Oxygen transfer rates were determined in a bubble aerated animal cell bioreactor. It was found that the oxygen transfer rates increased in the following order: large bubbles (≈5 mm diameter) < intermediate bubbles (≈1 mm diameter) < micron‐sized bubbles (≈100 μm diameter). Under certain conditions, the micron‐sized bubbles were capable of achieving oxygen transfer rate up to 100 h−1, a 10–20‐fold higher transfer rate than the large bubbles. The effects of medium composition on oxygen transfer rates were different for the three ranges of bubbles studied. For the large bubbles, oxygen transfer rates decreased with increasing medium complexity. The lowest oxygen transfer rate was found in new‐born calf serum (NBCS) and/or Pluronic F‐68 supplemented media. For the intermediate and micron‐sized bubbles, supplementation with NBCS into the culture media resulted in decreased oxygen transfer rate. However, further supplementation with Pluronic F‐68 enhanced oxygen transfer rate greatly for both types of bubbles. The highest oxygen transfer rate was found for micron‐sized bubbles in Pluronic F‐68 supplemented media containing antifoam agent and NBCS.
Effects of high product and substrate inhibitions on the kinetics and biomass and product yields during ethanol batch fermentation
Authors: Thatipamala R. , Rohani S. , Hill G. A.
Published: December 2004

In ethanol fermentation, instantaneous biomass yield of the yeast Saccharmoyces cerevisiae was found to decrease (from 0.156 to 0.026) with increase in ethanol concentration (from 0 to 107 g/L), indicating a definite relationship between biomass yield and product inhibition. A suitable model was proposed to describe this decrease which incorporates the kinetic parameters of product inhibition rather than pure empirical constants. Substrate inhibition was found to occur when substrate concentration is above 150 g/L. A similar definite relationship was observed between substrate inhibition and instantaneous biomass yield. A simple empirical model is proposed to describe the declines in specfic growth rate and biomass yield due to substrate inhibition. It is observed that product inhibition does not have any effect on product yield whereas substrate inhibition significantly affects the product yield, reflecting a drop in overall product yield from 0.45 to 0.30 as the initial substrate concentration increases from 150 to 280 g/L. These results are expected to have a significant influence in formulating optimum fermentor design variables and in developing an effective control strategy for optimizing ethanol producitivity.
A rapid, simple immunofluorometric assay: Development and characterization
Authors: Park Hongwoo , Wang Daniel I. C. , Yarmush Martin L.
Published: December 2004

A novel two‐site immunofluorometric assay, which includes only one incubation step and one separation step, is described. The assay is based on using small‐sized beads (0.5 μm diameter) as a soild support and measuring the unbound fraction of labeled antibody in the liquid. The use of a mixture of the solid‐phase and labeled antibodies at different concentrations makes it possible to determine antigen concentration over a wide range, without an initial sample dilution. Two assays were developed: one using anti‐IgG polyclonal antibodies and the other using antibovine serum albumin monoclonal antibodies. The detection range of the polyclonal antibody assay using 30‐minute incubation was 0.2 to 40 μg/mL for human IgG standard. The detection range of the monoclonal antibody assay was 0.2 to 14 μg/mL for bovine serum albumin (BSA) standard with 2 minutes required for the incubation. The interassay variability for the BSA measurement was 1.9% at 4.0 μg/mL of BSA and intra‐assay variability was 2.3% at 3.2 μg/mL of BSA. The principles of this assay can be applied in the measurement of any protein in a rapid and reproducible way. © 1992 John Wiley & Sons, Inc.
Reaction and diffusion in a gel membrane reactor containing immobilized cells
Authors: De Backer L. , Devleminck S. , Willaert R. , Baron G.
Published: December 2004

To investigate the effect of diffusional limitations and heterogeneous cell distribution in a gel‐immobilized cell system, a gel membrane reactor has been constructed. The reactor consists essentially of a gel layer with immobilized cells, flanked by two well‐mixed chambers. Through one chamber substrate is pumped, and this chamber is the equivalent of the outside of a spherical gel bead. The second closed measuring chamber contains a small quantity of liquid that can equilibrate with the inside surface of the membrane, eventually after a long transient. Analysis of the liquid in this chamber can give direct information on substrate and product concentrations at the gel surface, and is and indication of the situation in the center of a gel bead. The gel membrane reactor appears to be an excellent tool to study diffusion and reaction in a gel‐containing immobilized cells. A mathematical model with time‐ and position‐dependent cell concentration and diffusion coefficient is described. Experimental data show the effective diffusion coefficient of glucose in an alginate gel to decrease with yeast cell concentration. Moreover, kinetic parameters could be determined, using the mathematical model. Microscopic analysis confirmed the proliferation of the gel‐entrapped microorganisms in the outer layer of the matrix, as predicted by the model. Potentially, this type of reactor has a clear potential to study the physiology of gel‐immobilized cells. © 1992 John Wiley & Sons, Inc.
Cell cycle model for growth rate and death rate in continuous suspension hybridoma cultures
Authors: Linardos Theodor I. , Kalogerakis Nicolas , Behie Leo A.
Published: December 2004

As a result of recent advances in flow cytometry, renewed interest is shown in modeling the kinetic behavior of cells in culture on the basis of cell cycle parameters. An important but often overlooked kinetic variable in hybridoma cultures is the cell death rate. Not only the overall cell growth but also the kinetics of nutrient metabolism and monoclonal antibody production have been shown to depend on the cell death rate in continuous suspension hybridoma cultures. The present study shows that the death rate in hybridoma cultures is proportional to the fraction of cells arrested in the G1 phase of the cell cycle. The steady‐state cell age distributions in the various phases of the division cycle have been calculated analytically. A simple mathematical model has been used to produce the profiles of the cycling and arrested cell fractions with respect to the dilution rate. The calculated steady‐state growth rate, death rate, and viability profiles are shown to be in agreement with recently published experimental data from continuous suspension hybridoma cultures. © 1992 John Wiley & Sons, Inc.
Physiological effects of TGFα‐PE40 expression in recombinant Escherichia coli JM109
Authors: George H. A. , Powell A. L. , Dahlgren M. E. , Herber W. K. , Maigetter R. Z. , Burgess B. W. , Stirdivant S. M. , Greasham R. L.
Published: December 2004

Physiological effects of isopropyl‐thiogalactopyranoside (IPTG) induction were examined in Escherichia coli strain JM109 expressing a fusion protein composed of transforming growth factor alpha and a 40‐kD portion of Pseudomonas aeruginosa exotoxin A (TGFα‐PE40) under control of the tac promoter. Fermentations at the 15‐L scale in complex medium compared growth and metabolite profiles of the untransformed JM109 host strain, the strain transformed with the vector lacking the TGFα‐PE40 open reading frame (JM109[pKK2.7]), and the strain with the complete plasmid for TGFα‐PE40 expression (JM109[pTAC‐TGF57‐PE40]). Metabolite and growth profiles of JM109 (pTAC‐TGF57‐PE40) cultures changed significantly in IPTG‐induced versus uninduced cultures. Prior to induction, glucose was metabolized to acetate or completely oxidized to CO2. Following induction, pyruvate was also excreted in addition to acetate. In the absence of inducer, pyruvate was excreted by JM109 (pTAC‐TGF57‐PE40) only when dissolved oxygen levels fell to less than 10% of saturation (microaerobic rather than anaerobic conditions). The untransformed JM109 host strain or JM109 (pKK2.7) did not excrete pyruvate in the presence or absence of inducer, although JM109 (pKK2.7) exhibited a pattern of growth following addition of IPTG that closely resembled JM109 (pTAC‐TFG57‐PE40). Fermentations of JM109 (pTAC‐TFG57‐PE40) in a synthetic medium supported lower expression levels, but resulted in similar alterations in metabolite profiles. Induction in synthetic medium resulted in pyruvate excretion without further acetate accumulation. Taken together, these data suggest that one consequence of TGFα‐PE40 expression in JM109 is altered patterns of pyruvate oxidation. © 1992 John Wiley & Sons, Inc.
Separation of fructose and glucose mixture by zeolite Y
Authors: Cheng Y. L. , Lee T. Y.
Published: December 2004

Na−, K−, Ba−, and Ca−Y were employed for the separation of fructose and glucose in an adsorption column. Effects of temperature, solvent flow rate, amount of mixture injection, and exchangeable cations on the separation were investigated. Efficiency of separation was used as a criterion to characterize the effectiveness of the separation. The transport and kinetic parameters for the column separation were also presented. From simple pulse experiments and moment analysis, the obtained process information of equilibrium and dynamic parameters might be used to design, operate, and control the separation column. © 1992 John Wiley & Sons, Inc.
Analysis of productivity in lysis‐deficient lambda expression systems
Authors: Padukone N. , Peretti S. W. , Ollis D. F.
Published: December 2004

The integrated state of λ in the host chromosome in lysogeny can be combined with its extrachromosomal replication in the lytic state to achieve high cloned gene productivities. Our previous studies on λ expression systems21,22 have shown 100% segregational stability of the cloned gene in lysogeny and cloned gene product levels up to 15% of total cell protein in a mutant lytic state. However, the expression phase of systems based on Escherichia coli JM109 and JM105 showed partial lysis of the productive culture despite a mutation in the lysis gene S of the lambda vector resulting in extracellular release of the cloned gene product. In the current study, we have eliminated partial lysis in the expression phase of λ systems and conducted a detailed comparative analysis of these systems in relation to maximization of cloned gene productivity. The elimination of partial cell lysis by using a nonpermissive strain Y1089 did not enhance product yields vs. earlier systems that exhibited partial lysis. The elimination of nonessential λ protein production by construction of a new vector NP326 did not yield higher product yields presumably because of the small fraction of these proteins in the lytic state. Temperature induction of the lysogen Y1089(NM1070) resulted in higher product levels than direct infection of Y1089 by the phage vector at a high multiplicity. Using infection experiments, we found the promoter lacUV5 in the vector λZEQS to yield threefold higher product levels than lac in NM1070, suggesting possible further enhancement of productivity with stronger promoters. The occurrence or absence of partial lysis in λ systems could be used beneficially to achieve extracellular or intracellular product as desired. The large capacity of λ vectors for insert DNA suggests potential applications in obtaining highly amplified levels of operons and multienzyme systems. © 1992 John Wiley & Sons, Inc.
Ethanol production from eucalyptus wood hemicellulose hydrolysate by Pichia stipitis
Authors: Ferrari M. D. , Neirotti E. , Albornoz C. , Saucedo E.
Published: December 2004

Ethanol production was evaluated from eucalyptus wood hemicellulose acid hydrolysate using Pichia stipitis NRRL Y‐7124. An initial lag phase characterized by flocculation and viability loss of the yeast inoculated was observed. Subsequently, cell regrowth occurred with sequential consumption of sugars and production of ethanol. Polyol formation was detected. Acetic acid present in the hydrolysate was an important inhibitor of the fermentation, reducing the rate and the yield. Its toxic effect was due essentially to its undissociated form. The fermentation was more effective at an oxygen transfer rate between 1.2 and 2.4 mmol/L h and an initial pH of 6.5. The hydrolysate used in the experiences had the following composition (expressed in grams per liter): xylose 30, arabinose 2.8, glucose 1.5, galactose 3.7, mannose 1.0, cellobiose 0.5, acetic acid 10, glucuronic acid 1.5, and galacturonic acid 1.0. The best values obtained were maximum ethanol concentration 12.6 g/L, fermentation time 75 h, fermentable sugar consumption 99% ethanol yield 0.35 g/g sugars consumed, and volumetric ethanol productivity 4 g/L day. © 1992 John Wiley & Sons, Inc.
Cellulase activity in commercial lipase preparations
Authors: Derksen Johannes T. P. , Boswinkel Gerard , Cuperus F. Petrus
Published: December 2004

Polymeric membranes are increasingly used as supports for the immobilization of enzymes in bioreactors. One of the more common reactor types employed in lipase‐catalyzed hydrolysis of oils, contains modified cellulose as a membrane material. We found that this type of material is readily attacked by cellulase present in several commercially available lipase preparations. This leads to membrane damage, reactor instability, and leakage. We conclude that cellulose membranes are not suitable as supports in bioreactors for the immobilizartion of these lipases. The development of alternative membranes is currently in progress. © 1992 John Wiley & Sons, Inc.
Intracellular ribonucleotide pools as a tool for monitoring the physiological state of in vitro cultivated mammalian cells during production processes
Authors: Ryll Thomas , Wagner Roland
Published: December 2004

Intracellular analysis has been shown to be useful as a tool for control production processes based on in vitro cultivated hybridoma and recombinant animal cells. Nucleotides were found to present the best target as they reflect the exact physiological state of a culture. Following the progress of batch, perfused, and chemostat cultures cell specific regularities were found in various biochemical correlations which allowed the generation of three characteristic parameters based on the interaction of particular nucleotides: the nucleotide triphosphate (NTP) ratio ([ATP + GTP]/[UTP + CTP]), the uridine (U) ratio (UTP/UDP–GNAc), and the combined ratio NTP/U ([UDP–GNAc (ATP + GTP)/ UTP (UTP + CTP)]). These allowed a direct description of the growth cycle by means of specific values or behavior for every phase of the culture. In particular, the critical phase of entrance into the phase of reduced growth was predicted up to 24 h earlier than was possible with the classical method of microscopic cell control. A specific function for the application of in vitro cultivated processes is proposed with an NTP‐to‐U plot which combines the results obtained by the cell analysis and which offers a tool for the control and regulaiton of cell growth–derived procedures. © 1992 John Wiley & Sons, Inc.
The equilibrium and kinetics of N‐acetyl‐tryptophan phenylethyl ester synthesis by agarose–chymotrypsin in organic media
Authors: Blanco Rosa M. , Guisán José M. , Halling Peter J.
Published: December 2004

The synthesis of N‐acetyl tryptophan phenylethyl ester in organic media is catalyzed by suspended agarose beads with multipoint covalently attached chymotrypsin. A dilute aqueous phase is trapped within the gel beads and may be manipulated separately from the organic phase. The equilibrium position becomes more favorable as the solvent polarity decreases, with Keq increasing 38 times between 2‐butanone and 1,1,1‐trichloroethane. The more apolar solvents also give faster kinetics. Addition of cosolvents (up to 10% dimethylformamide or 20% acetonitrile) does not affect the rate but does substantially reduce the equilibrium yield, presumably also by making the organic phase more polar. With trichloroethane as solvent the enzyme appears to be kinetically saturated with 1M phenylethanol. Doubling this concentration also does not cause the expected increase in equilibrium conversion, probably again because Keq is reduced in the more polar organic phase. Increased temperature raises the reaction rate as expected but has little effect on the equilibrium. © 1992 John Wiley & Sons, Inc.
Influence of oxygen adsorption on the dynamic KLa measurement in three‐phase slurry reactors
Authors: Boon M. , Meeder T. A. , Heijnen J. J. , Luyben K. Ch. A. M.
Published: December 2004

To check for possible mass transfer limitations of oxygen and/or carbon dioxide in kinetic experiments on microbial desulphurization of coal, it is important to properly measure the volumetric mass transfer coefficient (kLa) especially at high slurry densities. Volumetric mass transfer coefficients of oxygen, at different solid hold‐up values (εs = 0 to 0.28) of coal slurries (dpar < 100 * 10−6 m), were measured in a lab scale fermentor and in a lab scale pachuca tank, using the dynamic gas–liquid absorption method. It was shown that serious errors could occur due to oxygen adsorption at the coal surface. Using the data of an independently measured adsorption isotherm, the real kLa could be calculated from the measured apparent kLa. The results show a kLa decrease of 40% to 50% at a volumetric solid hold‐up of 28%. Estimation of the oxygen and carbon dioxide transfer rates, from the measured mass transfer coefficients, indicates that the stirred fermentor is suitable for kinetic experiments at high slurry densities, whereas the pachuca tank and shake flask are not. © 1992 John Wiley & Sons, Inc.
Poly(ethylenimine)‐reinforced liquid‐core capsules for the cultivation of hybridoma cells
Authors: Hsu Yuan‐Lung , Chu I‐Ming
Published: December 2004

The mechanical stability of liquid‐core alginate‐poly‐L‐lysine (PLL) capsules used for encapsulation of hybridoma cells can be greatly enhanced by the inclusion of poly(ethylenimine) (PEI) in the hardening solution containing calcium chloride. The PEI can also reinforce the PLL‐coated carboxymethyl‐celluose liquid‐core capsules. The cultivation of murein hybridoma CT04 in these two capsules was carried out. Cell concentrations higher than 108 cells/mL per capsule were obtained with ca. 80% of the specific antibody productivity as the freely suspended cells. These capsules could withstand severe agitation and aeration in an air‐lift reactor over a period of 3 weeks with minimal damage. © 1992 John Wiley & Sons, Inc.
Effect of subunit dissociation, denaturation, aggregation, coagulation, and decomposition on enzyme inactivation kinetics: II. Biphasic and grace period behavior
Authors: Lencki Robert W. , Arul Joseph , Neufeld Ronald J.
Published: December 2004

A model previously developed to characterize enzymatic in activation behavior was used to explain the non‐first‐order biphasic and grace period phenomena that are often observed with oligomeric enzymes. Luciferase and urease were used as model enzyme such as luciferase, the oligomer initially dissociates reversibly into two native monomer species. These native monomers can then reversibly denature and irreversibly aggregate and coagulate. With the hexamer, urease, two trimers are formed that can subsequently aggregate to form an inactive hexamer. The dissociated monomer species of luciferase do not possess catalytic activity, so the inactivation mechanism, is biphasic; the first slope of a first‐order kinetic plot is influenced by the reversible oligomer/monomer/denatured‐monomer transition. Whereas the second slope is associated with either irreversible aggregation or coagulation. In contrast, the trimer of urease has the same activity as the hexamer; therefore, during the intitial hexamer–trimer transition, little activity loss occurs. However, as the trimer concentration increases, activity decreases as a result of trimer aggregation. As a result, grace period inactivation behavior is observed. © 1992 John Wiley & Sons, Inc.
Effects of the kinetics of osmotic pressure variation on yeast viability
Authors: Gervais P. , Marechal P. A. , Molin P.
Published: December 2004

The variation rate of the osmotic pressure increase was found to have a great effect on the viability of yeasts subjected to hyperosmotic stress. A low intensity of the increase rate of osmotic pressure could maintain an important viability of the cells (about 90 to 100%) even for very high levels of osmotic pressure (about 108Pa). The viability level was found to be highly dependent on the physiological state of the cells: Variations in the properties of the cell membrane were supposed to be involved in such a dependence. © 1992 John Wiley & Sons, Inc.
Some observations on protease production in continuous suspension cultures of Bacillus firmus
Authors: Moon Seung‐Hyeon , Parulekar Satish J.
Published: December 2004

Invariance of culture conditions in steady state continuous cultures make these a very valuable tool to study the influence of various culture parameters on cell growth and synthesis of primary and secondary metabolites. The result of a parametric study on production of protease in continuous suspension cultures of Bacillus firmus NRS 783 are reported in this article. This strain is a superior producer of an alkaline protease with major application in the detergent industry. The parameters investigated include dilution rate and concentrations of yeast extract, ammonium, and inorganic phosphate in the bioreactor feed, glucose being the principal carbon source in all experiments. The regulatory effects of the key culture parameters on cell growth, synthesis and secretion of protease, and production of acetic acid are investigated. The relations among the specific cell growth rate, specific utilization rates of the principal carbon, nitrogen, and phosphorous sources, and specific production rates of two nonbiomass products, viz., acetic acid and protease, are examined, and the effects of the manipulated culture parameters on these relations, specific protease activity, and yields of cell mass, protease, and acetic acid on the basis of the principal carbon, nitrogen, and phosphorous sources are studied. An increase in dilution rate led to increases in specific utilization rates of the principal carbon, nitrogen, and phosphorous sources and specific production rates of acetic acid and protease and decreases in bulk activities/concentrations of the three products (acetic acid, cell mass, and protease). As a result, the productivities of the three species were maximized at an intermediate dilution rate. Increased supply of yeast extract (a rich source of amino acids, proteins, and vitamins, besides being an additional source of carbon, nitrogen, and phosphorus) promoted cell mass formation but reduced protease production per unit cell mass. Increased supply of nitrogen and phosphorous sources stimulated protease synthesis up to certain threshold levels and repressed the enzyme synthesis beyond the threshold levels. With increased supply of the nitrogen source, the phosphorous source was more efficiently utilized for cell growth and protease synthesis. Stable maintenance of continuous cultures of B. firmus over prolonged period is demonstrated in this study. © 1993 John Wiley & Sons, Inc.
Effects of oxygen and ethanol on recombinant yeast fermentation for hepatitis B virus surface antigen production: Modeling and simulation studies
Authors: Shi Yuan , Ryu Dewey D. Y. , Yuan Wei‐Kang
Published: December 2004

A model was formulated to examine the competitive growth of two phenotypes (Leu+ and Leu−) and the product formation with recombinant Saccharomyces cerevisiae strain DBY‐745, which contains the shuttle vector pYGH3‐16‐s with the foreign gene HBsAg (hepatitis B virus surface antigen) as well as experimental fedbatch fermentation data. The important state variables and the process parameters evaluated include (1) the ratio of the plasmid‐free cell concentration to the plasmid‐containing cell concentration (ρ = X−X+), (2) the expression of human hepatitis B surface antigen g (CH), (3) the glucose consumption (S), (4) the ethanol production (/), (5) the change of working volume (V) in the fermentor, (6) the different specific growth rates of two phenotype cells, and (7) the plasmid loss frequency coefficient (α ). These variables and other parameters were carefully defined, their correlations were studied, and a mathematical model using a set of nonlinear ordinary differential equations (ODEs) for fed‐batch fermentation was then obtained based on the theoretical considerations and the experimental results. The extended Kalman filter (EKF) methods was applied for the best estimate of these variables based on the experimentally observable variables: ρV, and g (CH). Each of these variable was affected by random measuring errors under the different operating conditions. Simulation results presented for verification of the model agreed with our observations and provided useful information relevant to the operation and the control of the fedbatch recombinant yeast fermentation. The method of predicting an optimal profile of the cell growth was also demonstrated under the different dissolved oxygen concentrations. © 1993 John Wiley & Sons, Inc.
Development of bacterial cytochrome P‐450cam (Cytochrome m) production
Authors: Horowitz Jeffrey B. , Vilker Vincent L.
Published: December 2004

Cytochrome P‐450cam monooxygenase is an important bacterial redox enzyme system with potential commercial value for detoxifying trace hydrocarbon contaminants, catalyzing regiospecific hydroxylations, and amperometric biosensing. The present study was undertaken to increase productivity of this enzyme, which is induced in its host, pseudomonas putida PpG 786, by D(+)‐camphor. Culture processes were studied in batch, fed‐batch, and continuous modes to evaluate metabolic behavior and develop constitutive equations for specific rate of growth (μ), camphor utilization (qp). Fed‐batch culture was characterized by an extended linear growth phase which is often encountered in hydrocarbon fermentations. Inhibition by the camphor solvent, dimethylformamide, was assessed. Production of the terminal protein of the p‐450cam enzyme system, cytochrome m, was shown to depend on growth medium iron content in fed‐batch culture and was increased by 130% over previously protocols by eliminating iron deficiency. A continuous process that enables greater production rates was developed by using oxygen enrichment while simultaneously reducing gas throughput. Camphor and oxygen requirements were determined for fedbatch and continuous growth. © 1993 John Wiley & Sons, Inc.
Complete design analysis of a continuous sterilizer for fermentation media containing suspended solids
Authors: Armenante Piero M. , Shen Li Yuan
Published: December 2004

An improved mathematical model was developed to design a continuous sterilizer for liquid fermentation media containing suspended solids. Unsteady‐state energy balances were used to determine the temperature distribution in the liquid medium and in the solid particles as a function of time and position within the particle, as the medium flows through the sterilizer. Such temperature profiles were used to determine the level of microbial reduction achieved by each component of the sterilizer and by the entire sterilization process. A considerable difference exists between the temperature in the particle core and in the surrounding liquid. This has a significant impact on the degree of sterility achieved by the process. The level of microbial reduction in the particles was found to be tens and or even hundreds of order of magnitude lower than the corresponding level achieved in the liquid. The role of the particle material on the degree of sterilization was also investigated. Solid materials typically found in fermentation media, such as wood or flour clumps were found to offer considerable resistance to the sterilization of the organisms lodged inside them. © 1993 Wiley & Sons, Inc.
Simultaneous enzymatic/electrochemical determination of glucose and L‐glutamine in hybridoma media by flow‐injection analysis.
Authors: Meyerhoff Mark E. , Trojanowicz Marek , Palsson Bernhard O.
Published: December 2004

A split‐stream flow‐injection analysis system is described for simultaneous determination of glucose and L‐glutamine in serum‐free hybridoma bioprocesses media. Amperometric measurement of glucose is based on anodic oxidation of hydrogen peroxide produced by immobilized glucose oxidase within a triple layer membrane of an integrated flow‐through glucose‐selective biosensor. Determination of L‐glutamine is based on quantitating ammonium ions produced in a flow‐through enzymes reactor containing immobilized glutaminase enzyme, and subsequent downstream potentiometric detection of these ions by a nonacting‐based ion‐selective polymer membrane electrode. Endogenous potassium and ammonium ion interference in the L‐glutamine determination are eliminated by using a novel in‐line tubular cation‐exchange membrane unit to exchange these interferent species for cations undetectable by the membrane electrode. The first generation split‐steam flow‐injections system can assay 12 samples/h using direct injections of 50 μL of media samples, with linear responses to glucose in the range of 0.03 to 30mM, and log‐linear response to L‐glutamine from 0.1 to 10 mM. © 1993 Wiley & Sons, Inc.
Kinetic resolution of racemic glycidyl butyrate using a multiphase membrane enzyme reactor: Experiments and model verification
Authors: Wu Dauh‐Rurng , Cramer Steven M. , Belfort Georges
Published: December 2004

A laboratory‐scale multiphase hollow fiber membrane reactor was employed to investigate the lipase‐catalyzed enzymatic resolution of racemic glycidyl butyrate. A mathematical formulation was feveloped to simulate the performance of this system. Model parameters were determined independently (except the effective rate constant, ks) and incorporated in the model simulations. In this study, two modes of operation are considered: subtractive resolution, in which the unreacted substrate is recovered in the organic stream; and product recovery, where the optically pure product of the enzymatic reaction is recovered in the aqueous stream. Good agreement was obtained between theoretical predictions and experimental results under a variety of conditions. The effect of mass transport limitations on the performance of this system was investigated. An increase in enzyme loading resulted in a higher Thiele modulus due to an elevated rate constant as well as a concomitant decrease in the effective diffusivity. Optical purity decreased in both subtractive resolution and product recovery at higher Thiele modulus with the effect being more pronounced in the product recovery mode. Finally, normalized plots were established to describe the effect of enzyme immobilization on both the effective enzymes activity and effective diffusivity. © 1993 Wiley & Sons, Inc.
Sterilization of various diameter dead‐ended tubes
Authors: Young Jack H.
Published: December 2004

Effect of tube diameter on steam‐in‐place sterilization of dead‐ended tubes was studied by examining temperature profiles and rates of kill of Bacillus stearothermophilus spores. Time required for sterilization was determined for 9.4‐cm‐long tubes with various inside diameters from 0.4 to 1.7 cm. Sterilization time increased with decreasing tube diameter. Experimentally measured kill kinetics in 1.7‐cm tubes were in agreement with those predicted if measured temperatures represented saturated steam. A 12‐log spore reduction was achieved in 1.7‐cm diameter vertical and horizontal tubes in less than 63 minutes. For smaller diameter tubes, entrapped air remained after 2 hours and rates of kill were very dependent on position within the tube, tube diameter, and tube orientation with respect to the gravitational vector. Times to achieve a 1‐log drop in spore population in the smaller tubes were as much as 10 times greater than those expected if measured temperatures represented saturated steam. Sterilization was not achieved throughout the 0.4–cm tubes. Recommendations are made for including steam bleeders or using prevaccum cycles for these smaller diameter tubes. © 1993 John Wiley & Sons, Inc.
Cell retention–chemostat studies of hybridoma cells—analysis of hybridoma growth and metabolism in continuous suspension culture in serum‐free medium
Authors: Hiller G. W. , Clark D. S. , Blanch H. W.
Published: December 2004

The steady‐state metabolic parameters for a hybridoma cell line have been determined in continuous suspension–perfusion culture over a wide range of perfusion rates and cell bleed rates. Significant increases in viable cell concentrations and volumetric productivities were achieved at high perfusion rates and low cell bleed rates. At the low growth rates examined in this study, cellular metabolism shifted to become more oxidative, and as a result, the fraction of consumed substrate converted to inhibitory metabolic by‐products was reduced. Specific antibody productivity was found to be non–growth associated. © 1993 John Wiley & Sons, Inc.
Lipase activity in vesicular systems: Characterization of candida cylindracea lipase and its activity in polymerizable dialkylammonium surfactant vesicles
Authors: Mosmuller E. W. J. , Franssen M. C. R. , Engbersen J. F. J.
Published: December 2004

Lipase from Candida cylindracea (CCL) was incorporated into polymerizable positively charged dialkylammonium bromide surfactant vesicles. The enzyme was incorporated by the use of the dehydration–rehydration method or by incubation. In the latter case, trapping efficiencies of up to 100% could be obtained. Activities of free and vesicle‐incorporated CCL were tested for three triglycerides: triacetin, tributyrin, and tricaprylin. Enzyme activity was lowest in homogeneous mixtures (triacetin and small concentrations of tributyrin) and highest in heterogeneous mixtures (tricaprylin and high concentrations of tributyrin). Entrapment in vesicular systems is advantageous, especially in homogeneous reaction mixtures and in the case of the production of insoluble fatty acid (caproate), because inhibition by the acid can be suppressed. The influence of several surface‐active additives, including vesicles, on the activity of lipase in triglyceride assays was tested. Vesicles have a positive influence on the activity, whereas other positively charged additives act as inhibitors. In the case of tricaprylin assays, the positively charged additives increase the activity. Finally, tryptic digestion for free and incorporated CCL were compared. Free CCL is readily inactivated, whereas incorporated enzyme is protected from proteolytic degradation. © 1993 John Wiley & Sons, Inc.
Fed‐batch cultivation of recombinant escherichia coli JM103 and production of the fusion protein SPA::EcoRI in a 60‐L working volume airlift tower loop reactor
Authors: Brandes Lutz , Wu Xiaoan , Bode Jens , Rhee Jong II , Schügerl Karl
Published: December 2004

SPA::EcoRI fusion protein was produced by Escherichia coli JM103 carrying the multicopy expression plasmid pMTC48, the multicopy repressor plasmid pRK248, and the multicopy protection plasmid pEcoR4 in a 60‐L working volume airlift tower loop reactor on M9 minimal medium with glucose. Cell mass concentration, total cell count, number of colony‐forming units, specific growth rate, yield coefficient, and metabolite (acetate, pyruvate, succinate, lactate, ethanol) concentrations were monitored during the growth phase and gene expression. Gene expression was induced by temperature shift or chemically by isopropyl‐thiogalactosidase in the airlift tower loop reactor (ALTR) at constant cultivation time and in a small stirred tank reactor at different cultivation times. During induction, the cultivation medium was supplemented with concentrated Luria–Bertani (LB) medium. The intracellular enzyme activity was evaluated as a function of the time after the start of the induction. It was found that the reduction of the glucose concentration and increase of the dissolved oxygen concentration reduced the acetate produced and increased the intracellular enzyme activity. © 1993 John Wiley & Sons, Inc.
Expression of epoxide hydrolase in insect cells: A focus on the infected cell
Authors: Wang Min‐Ying , Vakharia Vikram , Bentley William E.
Published: December 2004

Insect cell culture and the baculovirus vector expression system have emerged to be a promising production technique for heterologous proteins. In this article, expression characteristics for membrane‐bound epoxide hydrolase are examined. A generic process is presented whereby cells are grown in serum‐free media supplemented with serum and then resuspended in serum‐free media to simplify purification after infection. The infected cells retain significant metabolic activity during the postinfection stage. Thus, maintaining nutrient supply during the postinfection period is critical, and a low stirring rate will result in oxygen depletion and shift the metabolism of the infected cells toward lactate production which then lowers product yield. This is the first report indicating that glucose is supplied from sucrose decomposition and then metabolized for viral DNA and recombinant protein production in recombinant baculovirus insect expression system. © 1993 John Wiley & Sons, Inc.
Subzero temperature operating biosensor utilizing an organic solvent and quinoprotein glucose dehydrogenase
Authors: Sode Koji , Nakasono Satoshi , Tanaka Mitsuharu , Matsunaga Tadashi
Published: December 2004

A subzero temperature operating biosensor was constructed using immobilized quinoprotein glucose dehydrogenase (PQQGDH), glassy carbon electrode, soluble electron mediator (ferrocene monocarboxylic acid), and an organic solvent, ethylene glycol, as an antifreezing reagent. Using this biosensor, glucose concentration can be determined even at −7°C. At this temperature, the response was 20% of that obtained at 20°C. This is the first study describing a subzero temperature operating biosensor. © 1993 John Wiley & Sons, Inc.
Esterification of N‐benzyloxycarbonyldipeptides in ethanol–water with immobilized papain
Authors: Kawashiro Katsuhiro , Ishizaki Hideyuki , Sugiyama Shigeru , Hayashi Hiromu
Published: December 2004

The esterification of some N‐benzyloxycarbonyl (Z)‐dipeptides in ethanol‐containing water was investigated using papain as a catalyst. The esterification took place in ethanol containing a samll amount of water (2% v/v, pH 9) with free papain at room temperature. The yield (after 24 h) of the ethyl ester was in the range of 25% to 50%. Any peptide bond cleavage of the substrates was not observed during esterification, indicating that the unfavorable amidase activity of papain was well depressed under these conditions. However, dipeptides having a D‐amino amino acid (Z‐valyl‐D‐alanine) or a bulky amino acid (Z‐valylvaline) at the C‐terminal position could not be esterified. It was found that the immobilization of papain on Amberlite XAD‐8 increased the yield of the ester significantly as compared with free papain. In the esterification of Z‐valylalanine using immobilized papain, the optimum water content, pH of an added buffer, and temperature were found to be 2% (v/v), 9, and 40°C, respectively. The water content affected the yield of the product ester significantly.© 1993 John Wiley & Sons, Inc.
Kinetic models for synthesis by a thermophilic alcohol dehydrogenase
Authors: Ford Jonathan B. , Askins K. Janine , Taylor Kenneth B.
Published: December 2004

Alcohol dehydrogenase (E. C. 1.1.1.1) from Thermoanaerobium brockii at 25°C and at 65°C is more active with secondary than primary alcohols. The enzyme utilizes NADP and NADPH as cosubstrates better than NAD and NADH. The maximum velocities (Vm) for secondary alcohols at 65°C are 10 to 100 times higher than those at 25°C, whereas the Km values are more comparable.

At both 25°C and 65°C the substrate analogue 1,1,1,3,3,3‐hexafluoro‐2‐propanol inhibited the oxidation of alcohol competitively with respect to cyclopentanol, and uncompetitively with respect to NADP. Dimethylsulfoxide inhibited the reduction of cyclopentanone competitively with respect to cyclopentanone, and uncompetitively with respect to NADPH. As a product inhibitor, NADP was competitive with respect to NADPH. These results demonstrate that the enzyme binds the nucleotide and then the alcohol or ketone to form a ternary complex which is converted to a product ternary complex that releases product and nucleotide in that order.

At 25°C, all aldehydes and ketones examined inhibited the enzyme at concentrations above their Michaelis constants. The substrate inhibition by cyclopentanone was incomplete, and it was uncompetitive with respect to NADPH. Furthermore, cyclopentanone as a product inhibitor showed intercept‐linear, slope‐parabolic inhibition with respect to cyclopentanol. These results indicate that cyclopentanone binds to the enzyme–NADP complex at high concentrations. The resulting ternary complex slowly dissociates NADP and cyclopentanone.

At 65°C, all of the secondary alcohols, with the exception of cyclohexanol, show substrate activation at high concentration. Experiments in which NADP was the variable substrate and cyclopentanol as the constant–variable substrate over a wide range of concentrations gave double reciprocal plots in which the intercepts showed substrate activation and the slopes showed substrate inhibition. These results indicate that the secondary alcohols bind to the enzyme–NADPH complex at high concentrations and that the resulting ternary complex dissociates NADPH faster than the enzyme–NADPH complex. © 1993 John Wiley & Sons, Inc.
Bioconversion of lactose/whey to fructose diphosphate with recombinant saccharomyces cerevisiae cells
Authors: Compagno C. , Tura A. , Ranzi B. M. , Martegani E.
Published: December 2004

Genetically engineered Saccharomyces cerevisiae strains that express Escherichia coli β‐galactosidase gene are able to bioconvert lactose or whey into fructose‐1,6‐diphosphate (FDP). High FDP yields from whey were obtained with an appropriate ratio between cell concentration and inorganic phosphate. The biomass of transformed cells can be obtained from different carbon sources, according to the expression vector bearing the lacZ gene. We showed that whey can be used as the carbon source for S. cerevisiae growth and as the substrate for bioconversion to fructose diphosphate. © 1993 John Wiley & Sons, Inc.
Immobilized Coffea arabica cell culture using a bubble‐column reactor with controlled light intensity
Authors: Kurata Hiroyuki , Furusaki Shintaro
Published: December 2004

Coffea arabica cells immobilized by calcium alginate gel were photocultured using a bubble‐column reactor under controlled light intensity. This process was carried out after their alkaloid productivity was improved by increasing the cell density in the initial gel matrix and preculturing the immobilized cells in the dark prior to light irradiation. The cells were grown in the form of a biofilm on gel beads, producing 100 mg/L of purine alkaloids in a 24‐day batch culture. Alkaloid production was relatively constant with respect to light intensity changes, and also cell growth was not suppressed much at high light intensity, with these behaviors being different from those obtained using suspended cells. These phenomena are explained by estimating the light intensity gradient within the cell‐immobilizing particles and by measuring the viable cell distribution within them. It subsequently suggests that the subsurface cells affect both the production and growth behaviors. © 1993 John Wiley & Sons, Inc.
Extractive separation of penicillin G by facilitated transport via carrier supported liquid membranes
Authors: Lee Chau‐Jen , Yeh Huey‐Jiuan , Yang Wu‐Jung , Kan Chin‐Rang
Published: December 2004

The facilitated transport of penicillin G (Pen G), through a supported liquid membrane with Amberlite LA‐2 dissolved in 1‐decanol, supported on a microporous polypropylene membrane, were studied. The distribution coefficient was obtained from a batch extraction experiment. The effects of flow rate, carrier concentration, initial concentration of Pen G, and the pH of feed and stripping phases on the transport rate of Pen G through the supported liquid membrane were also investigated. The results are in agreement with theoretical predictions, and it is demonstrated that the transport of Pen G through the supported liquid membrane is controlled simultaneously by mass transfer across both aqueous and liquid membranes. © 1993 John Wiley & Sons, Inc.
Substantial overproduction of antibodies by applying osmotic pressure and sodium butyrate
Authors: Oh Steve K. W. , Vig Pooja , Chua Florence , Teo W. K. , Yap Miranda G. S.
Published: December 2004

Much of the current cell technology has enabled increased antibody production levels due to judicious nutrient feeding to raise cell densities and design better bioreactors. This study demonstrates that hybridomas can be hyperstimulated to produce higher immunoglobulin (lg) levels by suppressing cell growth and increasing culture longevity through adaptation to higher osmolarity media and addition of sodium butyrate. Prior to adaptation, cells placed in higher osmotic pressures (350 and 400 mOsm) were severely suppressed in growth down to 25% of the control (300 mOsm), although total lg titers achieved were similar to the control, approximately 140 mg/L. After a week of adaptation to 350 and 400 mOsm media, cell growth was not as dramatically suppressed, but considerably higher lg levels were attained at these elevated osmolarities. The highest yield of 265 mg/L was obtained at 350 mOsm compared to 140 mg/L at 300 mOsm, while maximum viable cell numbers dropped from 35 × 105 cells/mL to 31 × 105 cells/mL and culture longevity was extended by 20 h more than the control. Sodium butyrate, known to enhance protein production in other cell types, was then supplemented at a range of concentrations between 0.01 and 0.4 mM to the 350 mOsm culture to further enhance the lg levels. Butyrate at a concentration of 0.1 mM, in combination with osmotic pressure at 350 mOsm, further elevated the lg levels to 350 mg/L. Concomitantly, maximum viable cell numbers were reduced to 22 × 105 cells/mL, but culture longevity was extended by 40 h in the 0.1 mM butyrate supplemented culture compared to the control condition. Specific antibody productivity, qMab, continued to stay high during the stationary phase and was further elevated during the decline phase: thus, overall lg levels can be increased by 2.3 times by combining osmotic pressure and butyrate treatment. © 1993 John Wiley & Sons, Inc.