bluuebunny/crossref_metadata_2025 huggingface dataset:
Monitoring and modeling density‐dependent growth of anchorage‐dependent cells
Authors: Ruaan R.‐C. , Tsai G.‐J. , Tsao G. T.
Published: December 2004

The density‐dependent growth of Chinese hamster ovary (CHO) cells was monitored on‐line by using an inverted microscope. A flow system was employed for cell cultivation so that nutrient concentration could be maintained and metabolic wastes were removed. With the help of video image analysis, local cells density could be accurately calculated and cell motility and exposed cell surface area could be estimated. A computer program which accounted for change of sell size and translocation of cells was developed to stimulate dell growth. The stimulated results of the population dynamics and the variations in cell size showed good agreement with our experimental observations, Cell motility and initial cell distribution on the substratum were found to have strong effect on cell growth. © 1993 John Wiley & Sons, Inc.
Effect of carbon dioxide concentration on the bioleaching of a pyrite–arsenopyrite ore concentrate
Authors: Nagpal Soumitro , Dahlstrom Donald , Oolman Timothy
Published: December 2004

The effect of carbon dioxide concentration on the bacterial leaching of a pyrite‐arsenopyrite ore concentrate was studied in continuous‐flow reactors. Steady‐state operation with two feed slurry densities, 6 wt% and 16 wt% solids, were tested for the effect of carbon dioxide concentration. Bacterial growth rates were estimated via the measurement of carbon dioxide consumption rates. Aqueous‐phase carbon dioxide concentrations in excess of 10 mg/L were found to be inhibitory to bacterial growth. © 1993 John Wiley & Sons, Inc.
Air slugs entrapped cross‐flow filtration of bacterial suspensions
Authors: Lee Cheng‐Kan , Chang We‐Gwo , Ju Yi‐Hsu
Published: December 2004

A novel cross‐flow technique for membrane filtration of bacterial cell suspensions was established. This is an air slugs entrapped cross‐flow method in which air slugs were generated by introducing air into the cross‐flow stream. As air slugs moved along with cross‐flow, the disturbance of cell sublayer formation on membrane surface was enhanced. As a consequence, filtration flux was improved and stabilized. The effect of air slugs on improving filtration flux was more pronounced in filtering gram‐negative Escherichia coli cell than grampositive Brevibacterium flavum cell. Moreover, air slug was about 50% more effective on reducing filtration resistance using ultrafiltration (UF) membrane of 300,000 molecular weight cutoff (MWCO) than microfiltration (MF) membrane of 0.2 μm. ©1993 John Wiley & Sons, Inc.
Dramatic enhancement of enzymatic activity in organic solvents by lyoprotectants
Authors: Debulis Katherine , Klibanov Alexander M.
Published: December 2004

When seven different hydrolytic enzymes (four proteases and three lipases) were lyophilized from aqueous solution containing a ligand, N‐Ac‐L‐Phe‐NH2, their catalytic activity in anhydrous solvents was far greater (one to two orders of magnitude) than that of the enzymes lyophilized without the ligand. This ligand‐induced activation was expressed regardless of whether the substrate employed in organic solvents structurally resembled the ligand. Furthermore, nonligand lyoprotectants [sorbitol, other sugars, and poly(ethylene glycol)] also dramaticaliy enhanced enzymatic activity in anhydrous solvents when present in enzyme aqueous solution prior to lyophilization. The effects of the ligand and of the lyoprotectants were nonadditive, suggesting the same mechanism of action. Excipient activated and nonactivated enzymes exhibited identical activities in water. Also, addition of the excipients directly to suspensions of nonactivated enzymes in organic solvents had no appreciable effect on catalytic activity. These observations indicate that the mechanism of the excipient‐induced activation is based on the ability of the excipients to alleviate reversible denaturation of enzymes upon lyophilization. Activity enhancement induced by the excipients is displayed even after their removal by washing enzymes with anhydrous solvents. Subtilisin Carlsberg, lyophilized with sorbitol, was found to be a much more efficient practical catalyst than its “regular” counterpart. © 1993 John Wiley & Sons, Inc.
Monitoring glutamine in animal cell cultures using a chemiluminescence fiber optic biosensor
Authors: Cattaneo M. V. , Luong J. H. T.
Published: December 2004

Together with flow injection analysis (FIA), a chemiluminescence (CL) fiber optic biosensor system has been developed for determining glutamine in animal cell cultures. Glutaminase (GAH) and glutamate oxidase (GLO) were onto separate porous aminopropyl glass beads via glutaraldehyde activation and packed to form an enzyme column. These two enzymes acted in sequence on glutamine to produce hydrogen peroxide, which was then reacted with luminol in the presence of ferricyanide to produce a light signal. An anion exchanger was introduced on‐line to eliminate interfering endogenous glutamate in view of its negative charge at pH above 3.22 (isoelectric pH). Among several resins tested, the acetate form was most effective, and this type of ion exchanger also effectively adsorbed uric acid, acetaminophen, and aspartic acid.

There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 1 to 100 μM. A complete analysis could be performed in 2 min, including sampling and washing with a good reproducibility (± 4.4%). Both the bi‐enzymic and ion exchange columns were useful for at least 500 analyses when the biosensor system was applied for the glutamine determination in murine hybridoma cell cultures and insect cell cultures. The values obtained compared well with those of HPLC, thus validating the applicability of the CL fiber optic system. © 1993 John Wiley & Sons, Inc.
A comparison of oxygenation methods fro high‐density perfusion culture of animal cells
Authors: Zhang S. , Handa‐Corrigan A. , Spier R. E.
Published: December 2004

A perfusion culture system was developed to investigate the oxygenation of high‐density hybridoma cell cultures. The culture system was composed of a stirred‐tank bioreactor and an external microfiltration hollow fiber cartridge for medium perfusion. Cell growth and antibody production were examined with large bubble (≈5 mm in diameter), micron‐sized bubble (≈ 80 μm in diameter), and silicone tubing oxygenation techniques. Comparable cell growth and monoclonal antibody (MAb) production were found for both the micron‐sized and large oxygenation methods, provided that large bubbles were enriched with pure oxygen. Relatively low cell growth and MAb production were attained with the bubble‐free silicone tubing oxygenation. It is concluded that direct bubble oxygenation can be applied successfully in high‐density animal cell cultures, provided that the culture medium is supplemented with Pluronic F‐68. The accumulation of ammonia in the culture medium rather than oxygen limitation was found to be one of the possible problems that eventually inhibited cell growth. This and the fouling of the filtration cartridge during long‐term cultivation were found to be more problematic than simple bubble oxygenation of high‐density cell culture. The micron‐sized bubble oxygenation method is highly recommended for high‐density animal cell cultures, provided that Pluronic F‐68 is supplemented into the culture medium. © 1993 John Wiley & Sons, Inc.
A structured model describing carbon and phosphate limited growth of Catharanthus roseus plant cell suspensions in batch and chemostat culture
Authors: van Gulik W. M. , ten Hoopen H. J. G. , Heijnen J. J.
Published: December 2004

The growth of plant cell suspension cultures of Catharanthus roseus in batch fermentors was studied at different initial phosphate levels of the medium. On the basis of the observations and existing knowledge with respect to phosphate metabolism in cultured C. roseus cells, a structured mathematical model was developed for the description of the kinetics of growth and intracellular accumulation of glucose and phosphate, as a function of glucose and phosphate supply. It was shown that the model offers not only good description of the growth of the cells in batch culture at different initial phosphate levels, but also provided a satisfactory description of the growth in glucose limited chemostats. © 1993 Wiley & Sons, Inc.
Purification and characterization of a highly thermostable glucose isomerase produced by the extremely thermophilic eubacterium, Thermotoga maritima
Authors: Brown Stephen H. , Sjøholm Carsten , Kelly Robert M.
Published: December 2004

Thermotoga maritima, among the most thermophilic eubacteria currently known, produces glucose isomerase when grow in the presence of xylose. The purified enzyme is a homotetramer with submit molecular Wight of about 45,000. It has a number of features in common with previously described glucose isomerases‐pH optimum of 6.5 to 7.5, presence of activesite histidine, requirement for metal cations such as Co2+ and Mg2+, and preference for xylose as substrate. In addition, it has significant sequence/structural homology with other glucose isomerases, as shown by both N‐terminal sequencing and immunological crossreactivity. The T. maritima enzyme is distinguished by its extreme thermostability–a temperature optimum of 105 to 110°C, and an estimated half‐life of 10 minutes at 120°C, pH 7.0. The high degree of thermostability, coupled with a neutral to slightly acid pH optimum, reveal this enzyme to be a promising candidate for improvement of the industrial glucose isomerization process © 1993 Wiley & Sons, Inc.
Kinetics of competitive inhibition and cometabolism in the biodegradation of benzene, toluene, and p‐xylene by two Pseudomonas isolates.
Authors: Chang Myung‐Keun , Voice Thomas C. , Criddle Craig S.
Published: December 2004

Two Pseudomonas species (designated strains B1 and X1) were isolated from an aerobic pilot‐scale fluidized bed reactor treating groundwater containing benzene, toluene, and p‐xylene (BTX). Strain B1 grew with benzene and toluene as the sole sources of carbon and energy, and it cometabolized p‐xylene in the presence of toluene. Strain X1 grew on toluene and p‐xylene, but not benzene. In single substrate experiments, the appearance of biomass lagged the consumption of growth substrates, suggesting that substrate uptake may not be growth‐rate limiting for these substrates. Batch tests using paired substrates (BT, TX, or BX) revealed competitive inhibition and cometabolic degradation patterns. Competitive inhibition was modeled by adding a competitive inhibition term to the Monod expression. Cometabolic transformation of nongrowth substrate (p‐xylene) by strain B1 was quantified by coupling xylene transformation to consumption of growth substrate (toluene) during growth and to loss of biomass during the decay phase. Coupling was achieved by defining two transformation capacity terms for the cometabolizing culture: one that relates consumption of growth substrate to the consumption of nongrowth substrate, and second that relates consumption of biomass to the consumption of nongrowth substrate. Cometabolism increased decay rates, and the observed yield for strain B1 decreased in the presence of p‐xylene. © 1993 Wiley & Sons, Inc.
Effect of fluorochrome location in protein A on sensing efficiency of IgG
Authors: Huang Ping Yu , Lee Cheng S.
Published: December 2004

Fragment B of protein A conjugated with fluorescein at various lysine residues is prepared and separated by using a DEAE column in anion‐exchange chromatography. The binding of IgG Fc to fragment B contributes to an additional positive electric potential around fragment B. The change in the local electrostatic environment and pH can then be specifically monitored by measuring the fluorescence intensity of fluorescein conjugated with fragment B before and after the introduction of IgG. The studies for the quantitative dependence of fluorescein location on the effectiveness of fluorescein for sensing the protein A–IgG reaction are presented and discussed. © 1993 John Wiley & Sons, Inc.
Regeneration of NADH in a bioreactor using yeast cells immobilized in alginate fiber: I. Method and effect of reactor variables
Authors: Stevenson E. , Ibbotson P. G. , Spedding P. L.
Published: December 2004

Saccharomyces cerevisiae cells immobilized in a calcium alginate fiber reactor were used as a source of alcohol dehydrogenase for the NAD+‐to‐NADH reaction. The reaction was catalyzed by enzyme in cells on the surface of the fiber. Internal diffusional effects were present. The enzyme cell concentration was optimized by harvesting cells finally grown under anaerobic conditions. The results were expressed as an apparent reaction rate constant that was independent of NAD+ and excess ethanol concentration, was slightly affected by flow rate above a minimum value, and increased with immobilized cell concentration in the fiber. The reaction was complete after 6 to 7 h under optimal conditions of 36°C and 9.5 pH. The latter was 0.5 pH units above the free enzyme optimum, indicating that microenvironmental effects were in evidence. © 1993 John Wiley & Sons, Inc.
Construction, expression, and analysis of recombinant HIV gp41 constructs containing a novel cellular binding domain
Authors: Kestler D. P. , Henderson L. A. , Noti J. D.
Published: December 2004

The gp41 polypeptide of human immunodeficiency virus (HIV) contains an immunosuppressive domain, an epitope which elicits specific cytolytic T cell responses to HIV, and a complement Clq interactive domain. In addition, a synthetic peptide called CS3, derived from gp41 (amino acids 576–593 of gp160) and contiguous with the major immunodominant domain, binds to cellular proteins and may be important in HIV entry/fusion. In order to further investigate the role of the CS3 region of gp41 in cellular binding and to investigate other properties of gp41, sufficient quantities of this polypeptide must be readily available. We have therefore cloned the region of the HIV genome between nucleotides 7891 and 8188 (corresponding to amino acids 541–639 of gp160) into a series of procaryotic expression vectors. The resulting clones express a recombinant polypeptide of gp41 (r41). Two of these recombinants, pMAL‐cRl/r41 and pGEMEX‐2/r41, expressed the highest and most consistent levels of r41 as judged by both sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blot analysis. With the pMAL‐cRl/r41 construct, r41 was expressed as a fusion to the maltose‐binding protein (MBP) and, following purification by affinity chromatography, was cleaved from MBP by factor Xa protease digestion. MBP/r41 may be useful for studies of a reported gp41 cellular binding domain and may facilitate studies involving other functions ascribed to this region of gp41. © 1993 John Wiley & Sons, Inc.
Esterification reactions catalyzed by lipases in microemulsions: The role of enzyme localization in relation to its selectivity
Authors: Stamatis H. , Xenakis A. , Provelegiou M. , Kolisis F. N.
Published: December 2004

The activity of lipases from Rhizopus delemar, Rhizopus arrhizus, and Penicillium simplicissimum entrapped in microemulsions formulated by bis‐(2‐ethylhexyl)sulfo‐succinate sodium salt (AOT) in isooctane has been studied in esterification reactions of various aliphatic alcohols with fatty acids. The effect of the nature of the fatty acids (chain length) and of the alcohols (primary, secondary, or tertiary; chain length; cyclic structures) on the lipase activities was investigated in relation to the reverse micellar structure. The lipases tested showed a selectivity regarding the structure of the substrates used when hosted in the AOT/isooctane microemulsion systems. Penicillium simplicissimum lipase showed higher reaction rates in the esterification of long chain alcohols as well as secondary alcohols. Primary alcohols had a low reaction rate and tertiary a very slow rate of esterification. Long chain fatty acids were better catalyzed as compared to the shorter ones. Rhizopus delemar and R. arrhizus lipases showed a preference for the esterification of short chain primary alcohols, while the secondary alcohols had a low rate of esterification and the tertiary ones could not be converted. The reaction of medium chain length fatty acids was also better catalyzed than in the case of the long ones. The observed lipase selectivity appeared to be related to the localization of the enzyme molecule within the micellar microstructure due to the hydrophobic/hydrophilic character of the protein. The reverse micellar structural characteristics, as well as the localization of the enzyme, were examined by fluorescence quenching measurements and spectroscopical studies. © 1993 John Wiley & Sons, Inc.
Transport of 1‐μm latex particles in pseudomonas aeruginosa biofilms
Authors: Drury William J. , Stewart Philip S. , Characklis William G.
Published: December 2004

Fluorescent latex microbeads added to a Pseudomonas aeruginosa biofilm as tracers of particle movement penetrated the biofilm and remained in it much longer than predicted by a model of advective displacement due to cell growth. Beads with a nominal diameter of 1 μm that were added in the bulk fluid became distributed throughout the biofilm depth. Some microbeads penetrated to the substratum within the 24‐h bead addition period. The biofilms had a mean thickness of approximately 34 μm but have been previously shown to be quite rough. Measured rates of bead release from the biofilm corresponded to first order time coefficients of 0.01–0.03 h−1. These bead release rates were approximately an order of magnitude less than the predicted time scale of advective transport, which is just the experimentally measured specific cellular growth rate of 0.15 h−1. Computer simulations of bead transport using the biofilm model BIOSIM were compared with bead release rate data and with bead position distributions within the biofilm as determined by microscopic examination of thin cross sections of embedded biofilm. The model predicted much faster release of beads from the biofilm than actually occurred. It is hypothesized that both the ability of beads to penetrate the biofilm and the unexpectedly low advective displacement velocity of particles in the biofilm were due to the rough nature of the biofilm. © 1993 John Wiley & Sons, Inc.
A three‐phase pattern in growth, monoclonal antibody production, and metabolite exchange in a hybridoma bioreactor culture
Authors: Bushell M. E. , Bell S. L. , Scott M. F. , Snell K. , Spier R. E. , Wardell J. N. , Sanders P. G.
Published: December 2004

The use of partial cubic spline data interpolation for the calculation of volumetric metabolite exchange rates suggested the existence of three distinct metabolic phases during bioreactor culture of a hybridoma cell line. During phase 1, a rapid amino acid uptake rate and ammonia release rate were observed. The growth rate was low and glutamine synthetase activity fell. In phase 2, maximum growth rate and minimum glutamine assimilation and ammonium production rates were observed. Attempts to corroborate the apparent ammonia assimilation in this phase using 15NH4Cl resulted in low incorporation rates into alanine and glutamine. Maximum glutamine synthetase activity took place during this period. Maximum antibody production rate was observed during phase 3 during which peaks in glutamine assimilation, ammonia release, and glutamine synthetase activity were observed. The apparent existence of the three phases prompted us to carry out Northern blot analysis of glutamine synthetase RNA at appropriate times during the process. This revealed a pattern of appearance and dis‐appearance of mRNA consistent with the three phases indicated by the fermentation parameters. © 1993 John Wiley & Sons, Inc.
A synergistic kinetics model for enzymatic cellulose hydrolysis compared to degree‐of‐synergism experimental results
Authors: Converse A. O. , Optekar J. D.
Published: December 2004

It is demonstrated that a two‐enzyme component synergistic model can account for the observation that the degree of synergism goes through a maximum as the total enzyme concentration is increased. The degree of synergism is low at low enzyme concentration because the extent of conversion is low and therefore the cellulose chain ends, present originally, are not exhausted; thus the action of the cellobiohydrolase (CBH) is not dependent on the chain ends generated by the endoglucanase (EG). The degree of synergism declines at high enzyme concentration due to saturation of adsorption sites with CBH, thus decreasing the generation of chain ends by EG. © 1993 John Wiley & Sons, Inc.
Production of recombinant proteins in high‐density insect cell cultures
Authors: Reuveny S. , Kim Y. J. , Kemp C. W. , Shiloach J.
Published: December 2004

The effect of the growth phase of Spodoptera frugiperda (Sf9) cells on the production of recombinant proteins (β‐galactosidase and glucocerebrosidase) was investigated. Cells infected with the recombinant Autographa californica nuclear polyhedrosis virus at the late exponential and stationary phases yielded low quantities of expressed protein. Highest enzyme yields were obtained using Sf9 cells from the early exponential phase (0.9 mg β‐galactosidase/106 cells and 1.7 μg glucocerebrosidase/106 cells). Infection of resuspension of cells collected from various phases of growth in fresh medium resulted in 75% restoration of maximal expression levels. This finding suggested either nutrient limitation or waste product accumulation as the cause of the decrease in productivity at the latter phases of growth. Further experiments revealed that the highest productivity levels could be obtained with cultures of Sf9 cells grown in a fermentor to a cell concentration of 4 × 106 mL−1. The medium needed to be replaced prior to infection with the recombinant virus and supplemented with a mixture of glucose, L‐glutamine, and yeastolate ultrafiltrate. © 1993 John Wiley & Sons, Inc.
A bioassay of thyrotrophin by photografted mammalian cells onto polymeric supports
Authors: D'Ambrosio Andrea , Bellobono Ignazio Renato , Marangoni Francesca , Rebulla Paolo , Sirchia Girolamo
Published: December 2004

Viable and functionally responsive human thyroid follicular cells, suspended in a commercial polyester acrylate diluted with tripropyleneglycol diacrylate, photoinitiated, and photocatalyzed with a proprietary photocatalytic system based on a synergic mixture of vanadium (V) t‐butoxide and i‐propoxide, have been immobilized as monolayers onto polystyrene plates. Bioassay of thyrotrophin in immobilized cell cultures yielded, by log‐log plot of the dose–response curve, a slope (0.92 ± 0.02) in close agreement with that (0.91) reported for cells immobilized by physical adsorption. The decisive role of photocatalyst in the photografting procedure has also been shown experimentally. A mechanism is suggested by which cells are anchored, rapidly and with stable chemical bonds, onto one of the two acrylate functions of the monomer–prepolymer mixture, the other one being simultaneously responsible for photochemical grafting onto the support. © 1993 John Wiley & Sons, Inc.
The cellulose‐binding domain (CBDCex) of an exoglucanase from Cellulomonas fimi: Production in Escherichia coli and characterization of the polypeptide
Authors: Ong Edgar , Gilkes Neil R. , Miller Robert C. , Warren R. Antony J. , Kilburn Douglas G.
Published: December 2004

The gene fragment encoding the cellulose‐binding domain (CBD) of an exoglucanase (Cex) from Cellulomonas fimi was subcloned and expressed in Escherichia coli. Transcription from the lac promoter coupled with translation from a consensus prokaryotic ribosome binding site led to the production of large quantities of CBDCex (up to 25% total soluble cell protein). The polypeptide leaked into the culture supernatant (up to 50 mg · L−1), facilitating one‐step purification by affinity chromatography on cellulose. The 11‐kDa polypeptide reacted with Cex antiserum. Absence of free thiols indicated that the two Cys residues of CBDCex form a disulfide bridge. It had the same N‐terminal amino acid sequence as CBDCex prepared from Cex by proteolysis, plus two additional N‐terminal amino acid residues (Ala and Ser) encoded by the Nhel site introduced during plasmid construction. CBDCex bound to a variety of β‐1, 4‐glycans with different affinities and saturation levels. Adsorption to bacterial microcrystalline cellulose was dependent on the temperature, but not on the pH. © 1993 John Wiley & Sons, Inc.
Stabilization of heterodimeric enzyme by multipoint covalent immobilization: Penicillin G acylase from Kluyvera citrophila
Authors: Guisán José M. , Alvaro Gregorio , Fernandez‐Lafuente Roberto , Rosell Cristina M. , Garcia Jose L. , Tagliani Auro
Published: December 2004

We have developed a strategy for immobilization–stabilization of penicillin G acylase (PGA) from Kluyvera citrophila by controlled multipoint covalent attachment to agarose–aldehyde gels. This enzyme is composed by two dissimilar subunits noncovalently bound. Thus, in this article we establish clear correlations between enzyme stabilization and the multipoint immobilization and/or between enzyme stabilization and the involvement of the two subunits in the attachment of them to the support. We have demonstrated that important thermal stabilizations of derivatives were only obtained through a very intense enzyme–support multipoint attachment involving the whole enzyme molecule. In this way, we have prepared derivatives preserving more than 90% of catalytic activity and being more than 1000‐fold more stable than soluble and one‐point attached enzyme. In addition, the involvement of the two subunits in the covalent attachment to the support has proved to be essential to develop interesting strategies for reactivation of inactivated enzyme molecules [e.g., by refolding of immobilized PGA after previous unfolding with urea and sodium dodecyl sulfate (SDS)]. © 1993 John Wiley & Sons, Inc.
Lipase catalyzed esterification of glycidol in organic solvents
Authors: Martins João F. , Da Ponte Manuel Nunes , Barreiros Susana
Published: December 2004

We studied the resolution of racemic glycidol through esterification with butyric acid catalyzed by porcine pancreatic lipase in organic media. A screening of seven solvents (log P values between 0.49 and 3.0, P being the n‐octanol–water partition coefficient of the solvent) showed that neither log P nor the logarithm of the molar solubility of water in the solvent provides good correlations between enantioselectivity and the properties of the organic media. Chloroform was one of the best solvents as regards the enantiomeric purity (e. p.) of the ester produced. In this solvent, the optimum temperature for the reaction was determined to be 35°C. The enzyme exhibited maximum activity at a water content of 13 ± 2% (w/w). The enantiomeric purity obtained was 83 ± 2% of (S)‐glycidyl butyrate and did not depend on the alcohol concentration or the enzyme water content for values of these parameters up to 200 mM and 25% (w/w), respectively. The reaction was found to follow a BiBi mechanism. © 1993 John Wiley & Sons, Inc.
A new approach for modeling cellulase–cellulose adsorption and the kinetics of the enzymatic hydrolysis of microcrystalline cellulose
Authors: Nidetzky Bernd , Steiner Walter
Published: December 2004

Two fractions of substrate in microcrystalline cellulose which differ in their adsorption capacities for the cellulases and their susceptibility to enzymatic attack have been identified. On the basis of a two‐substrate hypothesis, mathematical models to describe enzyme adsorption and the kinetics of hydrolysis have been derived. A new nonequilibrium approach was chosen to predict cellulase–cellulose adsorption. A maximum binding capacity of 76 mg protein per gram substrate and a half‐maximum saturation constant of 26 filter paper units (FPU) per gram substrate have been calculated, and a linear relationship of hydrolysis rate vs. adsorbed protein has been found. The fraction of substrate more easily hydrolyzed, as calculated from hydrolysis data, represents 19% of the total effective substrate concentration. This fraction is only slightly different from that of other celluloses and has been estimated to be 27% and 30% for NaOH‐ and H3PO4‐swollen cellulose, respectively. The effective substrate concentration is equal to the maximum amount of the substrate which can be converted during exhaustive hydrolysis. This in turn is determined by the overall degradability of the substrate by the cellulases (85–90% for microcrystalline cellulose) and by the cellobiose concentration during hydrolysis. The kinetic model is based on a summation of two integrated first‐order reactions with respect to the effective substrate concentration. Furthermore, it includes the principal factors influencing the reaction rates: the ratio of filter paper and β‐glucosidase units per gram substrate and the initial substrate concentration. © 1993 John Wiley & Sons, Inc.
Development of a recovery and recycle process for a pseudomonas lipase used for large‐scale enzymatic synthesis
Authors: Junker B. H. , Bhupathy M. , Buckland B. C.
Published: December 2004

Enzymes are potential catalysts for a wide range of large‐scale chemical synthesis steps, particularly when the creation of a specific chiral center is desired. The efficient recycling of the enzyme catalyst and the removal of carryover impurities were crucial factors in the improvement of a stereoselective ester hydrolysis step used in the synthesis of a selective leukotriene antagonist. In this enzymatic reaction step, the substrate and product were both largely insoluble, while the enzyme was soluble in the aqueous reaction mixture. Microfiltration and ultrafiltration of the slurry reaction mother liquor indicated near 100% enzyme protein recovery, while activity recovery was about 70% to 80%. These activity losses might be accounted for by enzyme degradation (1 to 2 mg/L · h) during the 40‐hour reaction period. Dissolved impurities, principally a diacid byproduct, in the enzyme recycling stream were reduced 60% to 70% by either lowering the solution pH to 4.0 or raising the solution ionic strength to 1 M. © 1993 John Wiley & Sons, Inc.
Plant cell suspension culture rheology
Authors: Curtis Wayne R. , Emery Alden H.
Published: December 2004

The results of rheological measurements on 10 different plant cell suspension cultures are presented. Nicotiana tabacum (tobacco) suspension cultures grown in serial batch subculture display high viscosity and power law rheology. This “undesirable” rheology is shown to be a result of elongated cell morphology. The rheology of Papaver somniferum (poppy) cell suspensions is quite different; poppy suspensions behave as Newtonian fluids and have relatively low viscosity (less than 15 cP) at fresh cell densities up to 250 g/L. This flow behavior can be attributed to a lack of elongation in batch‐grown poppy cells. A simple correlation for the viscosity as a function of cell density is developed for poppy suspensions up to 300 g fresh weight (FW)/L. It is shown that tobacco cells do not elongate when grown in semicontinuous culture (daily media replacement). These semicontinuously cultured cells have rheological behavior that is indistinguishable from that of poppy, further confirming the dependence of rheology on plant cell morphology. The rheology of a wide variety of other plant suspensions at 200 g FW/L is presented. Most cell suspensions, including soybean, cotton, bindweed, and potato, display low viscosities similar to poppy suspensions. Only carrot and atriplex exhibit slight pseudoplastic behavior which corresponded to a slight degree of cellular elongation for these cultures. This demonstrates that complex rheology associated with elongated cell morphology is much less common than low‐viscosity Newtonian behavior. High viscosity in plant cell culture is therefore not an intrinsic characteristic of plant cells but, instead, is a result of the ability to grow cultures to extremely high cell densities due to low biological oxygen demand. © 1993 John Wiley & Sons, Inc.
The yield equations in the modeling and control of bioprocesses
Authors: Andrews G. F.
Published: December 2004

Two basic questions in bioprocess modeling are how many rate equations must be specified and which processes (substrate uptake, product formation, etc.) they should describe. The number of rate equations is constrained by the yield equations, which represent the balances of reducing power, energy in the form of ATP, and the various elements involved in microbial metabolism. These balances are derived from a simplified picture that divides metabolism into catabolic, anabolic, respiratory, and product formation pathways. The linear growth equation for aerobic metabolism and the Ludeking–Piret equation for product formation by fermentation are derived from these balances, and the yield coefficients are related to the metabolic parameters, YATP (P/O), etc. The use of oxygen for purposes other than respiration is included in the analysis and extends the idea of a constant “yield on available electrons” to very reduced substrates. These balances specify the number of degrees of freedom, i.e., the number of pieces of information required to complete the description of the system. This information may be in the form of measurements, knowledge of the biochemical pathways, or rate equations. The number of rate measurements available (usually two, the consumption rates of O2 and CO2) versus the number needed defines the state estimation problem in bioprocess control. Rate equations usually specify the biomass growth rate, but it may be preferable to specify the specific consumption rate of the limiting nutrient. © 1993 John Wiley & Sons, Inc.
Microbial degradation of quinoline: Kinetic studies with Comamonas acidovorans DSM 6426
Authors: Miethling R. , Hecht V. , Deckwer W.‐D.
Published: December 2004

The microbial degradation of quinoline by Comamonas acidovorans was studied in a laboratory scale stirred tank reactor. In continuous culture experiments using quinoline as a sole source of carbon and nitrogen, it was shown by means of mass balances that quinoline was converted completely to biomass, carbon dioxide, and ammonia. Degradation rates up to 0.7 g/L h were obtained. Measured yield coefficients Yx/s for quinoline were about 0.7 g/g, which is in agreement with the theoretical value for complete mineralization. Kinetic constants based on Haldane substrate inhibition were evaluated. The values were μmax = 0.48 h−1, Ki = 69 mg/L, and Ks < 1.45 mg/L. © 1993 John Wiley & Sons, Inc.
Fatty acid production characteristics of fungi with particular emphasis on gamma linolenic acid production
Authors: Kennedy Max J. , Reader Sarah L. , Davies R. Julian
Published: December 2004

The fatty acid production characteristics of fungi are described. These characteristics are the relationship between the oil content of the cell and the fatty acid content of the oil. For example, for polyunsaturated fatty acid (PUFA) production by Mucor hiemalis IPD 51, the oil content of the cell and the GLA content of the oil are coupled. For fungal production of some PUFA, synthesized after the rate‐limiting step in the fatty acid anabolic chain, a maximum fatty acid production model was developed to link the fatty acid content of the oil and the oil content of the cell. Maximum volumetric productivity of gamma linolenic acid (GLA) by molds was found to occur at a specific GLA content of the oil. For example, for M. hiemalis IPD 51, a maximum volumetric of 4.7 mg GLA/L · h was produced at a GLA content of the oil of 8% to 10%. Similarly for Mucor circinelloides v. Tieghem IPD 155 a maximum volumetric productivity of 4.8 mg GLA/L · h was produced at a GLA content of the oil of 14% to 16%. These results imply that, when screening microorganisms for GLA or other fatty acid production, a number of medium compositions need to be evaluated to determine the tradeoff between oil content of the cell and fatty acid content of the oil. © 1993 John Wiley & Sons, Inc.
Growth rate effects on fundamental transport properties of bacterial populations
Authors: Mercer James R. , Ford Roseanne M. , Stitz Jill L. , Bradbeer Clive
Published: December 2004

In many natural environments, bacterial populations experience suboptimal growth due to the competition with other microorganisms for limited resources. The chemotactic response provides a mechanism by which bacterial populations can improve their situation by migrating toward more favorable growth conditions. For bacteria cultured under suboptimal growth conditions, evidence for an enhanced chemotactic response has been observed previously. In this article, for the first time, we have quantitatively characterized this behavior in terms of two macroscopic transport coefficients, the random motility and chemotactic sensitivity coefficients, measured in the stopped‐flow diffusion chamber assay. Escherichia coli cultured over a range of growth rates in a chemostat exhibits a dramatic increase in the chemotactic sensitivity coefficient for D‐fucose at low growth rates, while the random motility coefficient remains relatively constant by comparison. The change in the chemotactic sensitivity coefficient is accounted for by an independently measured increase in the number of galactose‐binding proteins which mediate the chemotactic signal. This result is consistent with the relationship between macroscopic and microscopic parameters for chemotaxis, which was proposed in the mathematical model of Rivero and co‐workers. © 1993 John Wiley & Sons, Inc.
Multigradient method for optimization of slow biotechnological processes
Authors: Tammisola Jussi , Ojamo Heikki , Kauppinen Veli
Published: December 2004

A new method (named a “jumping spider”) is introduced for the optimization of slow biotechnological processes. The more traditional sequential experimentation (i.e., gradient search, simplex, etc.) is not well suited for slow dynamic processes, e.g., plant cell culture and differentiation. Therefore, a more simultaneous approach is proposed. A large number of initial experiments are performed, on the basis of which several of the initial experiments are selected as starting points. A search is then performed simultaneously from several gradient directions and the optimum is estimated by a quadratic approximation. In simulations, the spider generally climbs up the slopes quickly and the final estimator yields good maximum point estimates even on a complex topography. The spider may even approach more than one local maximum point simultaneously. As a model application, the average xylitol conversion rate ofCandida guilliermondiiwas optimized in relation to cultivation volume (oxygen availability) and the concentration of nitrogen and phosphorus in the medium. A threefold increase in xylitol production was obtained with three experimental steps. © 1993 John Wiley & Sons, Inc.
Growth and production modeling in hybridoma continuous cultures
Authors: Cazzador Lorenzo , Mariani Luigi
Published: December 2004

Several experimental data on continuous cultures of hybridoma cells show that monoclonal antibody productivity is a decreasing function of dilution rate. It has been suggested that this unusual behavior may be due to the arrest of a fraction of cycling cells at a critical point of Phase G1. Although this hypothesis has been recently investigated by using population balance models, mathematical analysis has been performed without accounting for the dynamics of the arrested cells properly. In this article, a more general and accurate approach is presented and new specific assumptions are introduced to characterize the arrest and the later progress through the cycle. Two different models (stochastic and deterministic) and two different critical points for the arrest (at the beginning and at the end of G1) are considered. The cell cycle parameters are estimated so that data predicted by the model fit those reported in the literature. In particular, the fraction of arrested cells, the cell arrest probability, and the mean cell generation time are computed as functions of the dilution rate. Results so far obtained predict that there is an optimal value of dilution rate for maximizing specific production rate of monoclonal antibody. © 1993 John Wiley & Sons, Inc.
Screening of concanavalin A‐bead cellulose conjugates using an enzyme thermistor with immobilized invertase as the reporter catalyst
Authors: Dočolomanský Peter , Gemeiner Peter , Mislovičová Danica , Štefuca Vladimír , Danielsson Bengt
Published: December 2004

Screening and design of immobilized biocatalysts (IMBs) is a time‐consuming process. An ideal process should be universal, fast, convenient, precise, and reproducible. Many of these requirements are met by enzymic flow microcalorimeters, also known as enzyme thermistors (ETs) or thermal assay probes (TAPs). Adaptation of ETs to real measurements of reaction rates requires coupling of the mathematical description of the reaction‐diffusion phenomena in the ET column with heat balance and, subsequently, experimental verification of the mathematical model. This article presents such a process developed as an adaptation of ETs for the characterization of the microkinetic properties of IMBs and their further application for screening of IMBs. The IMBs characterized were the preparations of invertase, biospecificaly adsorbed on concanavalin A conjugated to activated bead cellulose. © 1994 John Wiley & Sons, Inc.
Surfactant pretreatment of a polysulfone ultrafilter for reduction of antifoam fouling
Authors: Yamagiwa Kazuaki , Kobayashi Hiroe , Onodera Masayuki , Ohkawa Akira , Kamiyama Yoshiyasu , Tasaka Kentaro
Published: December 2004

Effectiveness of surfactant precoat treatment of the polysulfone ultrafilter was first investigated for reduction of membrane fouling in ultrafiltration of antifoam. Fifteen different surfactants, including alcohols and synthetic nonionic surfactants, were tested. In general, pretreatment with nonionic surfactant gave a larger flux than that with alcohol did. The flux increase by pretreatment with nonionic surfactant depended on a hydrophile lipophile balance (HLB) value and type of hydrophobic tail. The most effective surfactant for reducing antifoam fouling among the 15 surfactants was Brij‐58 which has an HLB value of 16 and a straight alkyl hydrophobic chain. The ultrafiltration flux of the membrane treated with Brij‐58 was almost three times larger than that of untreated membrane. The precoat treatment with Brij‐58 was the most effective for reducing antifoam fouling in terms of rejection properties.

Furthermore, flux was also improved by the surfactant pretreatment in ultrafiltration of model process streams, such as fermentation media, broth, and yeast suspension with or without antifoam. The surfactant Brij‐58 was found to be more effective for reducing membrane fouling in ultrafiltration of model stream YG compared with ethanol or Brij‐35. The mean flux increase by the pretreatment with Brij‐58 was about 80% in ultrafiltration of the model stream without antifoam. When antifoam was added to the model stream, flux was almost doubled by the pretreatment with Brij‐58. The effectiveness of surfactant precoat treatment for reducing membrane fouling was also confirmed in terms of rejection properties. © 1994 John Wiley & Sons, Inc.
A mathematical model for the bacterial oxidation of a sulfide ore concentrate
Authors: Nagpal Soumitro , Dahlstrom Donald , Oolman Timothy
Published: December 2004

The effect of dilution rate and feed solids concentration on the bacterial leaching of a pyrite/arsenopyrite ore concentrate was studied. A mathematical model was developed for the process based on the steady‐state data collected over the range of dilution rates (20 to 110 h) and feed solids concentrations (6 to 18% w/v) studied. A modified Monod model with inhibition by arsenic was used to model bacterial ferrous ion oxidation rates. The model assumes that (i) pyrite and arsenopyrite leaching occurs solely by the action of ferric iron produced from the bacterial oxidation of ferrous iron and (ii) bacterial growth rates are proportional to ferrous ion oxidation rate. The equilibrium among the various ionic species present in the leach solution that are likely to have a significant effect on the bioleach process were included in the model. © 1994 John Wiley & Sons, Inc.
Multifactorial experimental design for optimizing transformation: Electroporation of Streptococcus thermophilus
Authors: Marciset Olivier , Mollet Beat
Published: December 2004

A multifactorial process was used to optimize transformation of Streptococcus thermophilus by electroporation. Simple experimental designs were applied to study three, four, or five factors in eight experiments. Four qualitative factors, growth and recovering media, and plasmid and bacterial strains, were studied empirically. Eight quantitative factors, including electrical, physiological, and chemical parameters, were studied by fractional factorial designs. Effects of individual parameters as well as interactions between them were investigated and optimized. Optimization was performed for one S. thermophilus strain, ST11, and proved to work for all other tested strains of the same species. Transformation efficiencies of 9 × 102 to 6 × 105 transformants per microgram DNA were achieved, depending on the strains and vectors used. © 1994 John Wiley & Sons, Inc.
A 3‐year experience of quality control and quality assurance in the multisite delivery of a lymphocyte‐based cellular therapy for renal cell carcinoma
Authors: du Moulin Gary C. , Stack Jessica , Pitkin Zorina , Chew‐Darke Judy , Cyr Carolyn , White Adrienne , Ho Letinh , Shen Yuan‐Jin , Hamilton Dylan , Davies Brian , Charles Carla , Conti Evelyn , Liu Victor
Published: December 2004

Autolymphocyte therapy (ALT) is outpatient‐based adoptive immunotherapy using ex vivo–activated memory T‐cells. To support the safe and reproducible delivery of ALT at three cell processing facilities (Boston, MA; Atlanta, GA; Orange, CA) we created a comprehensive quality assurance/quality control program compliant with recent FDA guidance relevant to activated lymphocytes and somatic cell therapies. Each facility performed extensive QC testing to ensure sterility, viability, and proper cell yield. Additonally, several QC tests were performed at Cellocr′s centralized reference laboratory to monitor cell potency and identity of the ex vivo‐processed lymphocytes. We report here the successful implementation of this QA/QC program for ALT which has resulted in the safe preparation and delivery of cell infusion products amounting to over 3600 treatments at seven clinical sites nationwide. We believe this program will serve as a model for other cellular therapies.