bluuebunny/crossref_metadata_2025 huggingface dataset:
Response dynamics of 26‐, 34‐, 39‐, 54‐, and 80‐kDa proteases in induced cultures of recombinant Escherichia coli
Authors: Harcum Sarah W. , Bentley William E.
Published: December 2004

Several researchers have demonstrated that the presence of a heterologous protein in recombinant Escherichia coli elicits a response similar to the heat‐shock response, which includes enhanced protease expression. The present work detects, quantifies, and characterizes intracellular protease activity in E. coli that are “shocked” by the induction of a recombinant protein, CAT, which is an endogenous protein in some E. coli strains. A novel, sodium dodecyl sulfate gelatin poly‐acrylamide gel electrophoresis (SDS‐GPAGE) method is used to detect, quantify, and characterize the presence of these proteases. A hypothesis is proposed which links the amplified protease activity to a temporary depletion of specific amino acid pools, and a stringent‐like stress response. © 1993 John Wiley & Sons, Inc.
Utilization of glucose and amino acids in insect cell cultures: Quantifying the metabolic flows within the primary pathways and medium development
Authors: Ferrance Jerome P. , Goel Akshay , Ataai Mohammad M.
Published: December 2004

The current understanding of insect cell metabolism is very limited. In order to gain some insight into the growth and metabolism of insect cells Spodoptera frugiperda (Sf9), a comprehensive characterization of culture conditions for cells grown in the IPL‐41 medium was made by measuring the amino acid composition of the growth medium and the cell extract, the macromolecular composition of the cells (DNA, RNA, and protein), medium concentrations of various metabolites and sugars, and the evolved CO2. Since in the IPL‐41‐based serum‐free medium all of the amino acids except cysteine are in great excess of what is needed by the cells for energy and protein production, a medium formulation with an osmolarity similar to the IPL‐41 but with a lower amino acid content than IPL‐41 was also developed. The new medium also lacks maltose and sucrose (contains only glucose), supported cell growth to a high cell density of 8 × 106 cells/mL. The cellular and energetic yields indicated that a tight coupling between the biosynthetic and energetic reactions was attained for cells grown in the new medium. Moreover, it was found that the intermittent feeding of glucose may not be required as the cell yield and growth rate were comparable whether the same total amount of glucose was provided intermittently or was included initially in the medium. The eventual cessation of growth in the new medium is believed to be due to the amino acid limitation because concentrations of both glutamine and glutamate were very low at the end of the growth phase. Thus, further optimization, which may include higher initial glutamine in the medium or its intermittent feeding, could lead to a further increase in the cell density. Finally, a stoichiometrically based analysis of metabolic reactions confirmed the operation of the key pathways and was used to quantify the distribution of metabolites among primary metabolic reactions. The quantitative flow values were used to highlight some key aspects of insect cell metabolism. © 1993 John Wiley & Sons, Inc.
Removal of phenols from wastewater by soluble and immobilized tyrosinase
Authors: Wada Shinji , Ichikawa Hiroyasu , Tatsumi Kenji
Published: December 2004

An enzymatic method for removal of phenols from industrial wastewater was investigated. Phenols in an aqueous solution were removed after treatment with mushroom tyrosinase. The reduction order of substituted phenols is catechol > p‐cresol > p‐chlorophenol > phenol > p‐methoxyphenol. In the treatment of tyrosinase alone, no precipitate was formed but a color change from colorless to dark‐brown was observed. The colored products were removed by chitin and chitosan which are available abundantly as shellfish waste. In addition, the reduction rate of phenols was observed to be accelerated in the presence of chitosan. Tyrosinase, immobilized by using amino groups in the enzyme on cation exchange resins, can be used repeatedly. By treatment with immobilized tyrosinase, 100% of phenol was removed after 2 h, and the activity was reduced very little even after 10 repeat treatments. © 1993 John Wiley & Sons, Inc.
Comments on “in search of a thermodynamic description of biomass yields for the chemotrophic growth of microorganisms”
Authors: Roels J. A.
Published: December 2004

In a recent publication, Heijnen and van Dijken (Biotechnol. Bioeng. 39: 833–858, 1992) reviewed the state of the art regarding the use of macroscopic methods in the correlation of biomass yields in growing microorganisms. In their article, reference is made to this author's work of some 10 years ago.

Heijnen and van Dijken introduce the Gibbs' energy dissipation as a concept with favorable characteristics compared with various other approaches, including thermodynamic efficiency, as introduced by this author.

In this communication, it will be shown that the “dissipation” and the “thermodynamic efficiency” description are completely equivalent and that there can be no preference for one of these in terms of rigor or characteristics. © 1993 John Wiley & Sons, Inc.
Response to comments on “in search of a thermodynamic description of biomass yields for the chemotropic growth of microorganisms”
Authors: Heijnen J. J. , van Dijken J. P.
Published: December 2004

It is shown that for the thermodynamic description of microbial growth there does not exist complete equivalency between the “efficiency” and “Gibbs energy dissipation” approach. The reasons for this absence of equivalency are discussed, and it is argued that the “dissipation approach” has much more favorable properties compared with the efficiency approach. © 1993 John Wiley & Sons, Inc.
A method for simultaneous determination of solubility and transfer coefficient of oxygen in aqueous media using off‐gas mass spectrometry
Authors: Dorresteijn R. C. , de Gooijer C. D. , Tramper J. , Beuvery E. C.
Published: December 2004

A static method was developed that simultaneously determined the solubility of oxygen and the oxygen‐transfer coefficient in a stirred bioreactor. It was based on the static method developed by van Sonsbeek et al. to determine the ka in a liquid‐impelled loop reactor. Only physical properties of the liquid were used to determine both parameters using a mass spectrometer. Data about the solubility of oxygen in water are available from the literature. Therefore, the solubility of oxygen in water was used to compare our data with published data. Furthermore, the solubility of oxygen in trypticase soy broth was compared to literature data. No significant deviations between our data and literature data could be observed. Our static method and the commonly applied dynamic method to determine the oxygen‐transfer coefficient yielded similar results. The effect of temperature on the oxygen‐transfer coefficient could be expressed as the activation energy needed for the transition of oxygen from the gas to the water phase. This was verified using the Arrhenius equation. © 1994 John Wiley & Sons, Inc.
High pressure EPR studies of protein mobility in reversed micelles
Authors: Affleck Rhett , Clark Douglas S. , Kamat Sanjay , Russell Alan J.
Published: December 2004

We have investigated the effect of pressure on structural properties of subtilisin solubilized in reversed micelles of Tween‐85/isopropanol in hexane. Electron paramagnetic resonance (EPR) spectra of spin‐labeled enzyme indicate a reduction in spin‐label mobility when the enzyme is transferred from aqueous solution to the microemulsion. One explanation for the spectral broadening is a change in the protein's active‐site conformation and/or dynamics. However, over a W0 range of 80 to 180, EPR spectroscopy could detect no change in the enzyme's environment, conformation, or molecular dynamics. The EPR spectra also contained a contribution from free spin label located in an environment with a polarity roughly between that of propanol and bulk water. No changes in the polarity surrounding the free spin label nor in the enzyme's structural properties were evident at pressures up to 10,000 psi. Previous work has demonstrated that pressure can be used to manipulate the size of some reversed micelles, and the EPR data indicated that for this system such pressure tuning of micellar properties will not adversely affect the structure of solubilized enzyme. © 1994 John Wiley & Sons, Inc.
Structured model of genetic control via the lac promoter in Escherichia coli
Authors: Laffend L. , Shuler M. L.
Published: December 2004

A model that describes induction of protein synthesis from lac‐based promoters has been developed and incorporated into the single‐cell model of Escherichia coli with transcriptional and translational modifications. Unlike previous models of lac‐based promoters, this model allows a priori prediction of the intracellular parameters controlling transcription from lac‐based promoters with only the extracellular levels of substrate and inducer as inputs. Because of the structural detail of the model, it is possible to simulate different genetic constructions for comparison, such as Laclq strains versus wild‐type cells, or including lacl on a multicopy plasmid. Expression from lac to tac promoters is predicted to yield 5% and 30% of the total cellular protein, respectively, with a pBR322‐type plasmid. The model predicts the experimental observation that the Laclq strain is not as fully induced as the wild‐type strains, even at higher inducer concentrations. Additionally, the model predicts the right order of magnitude of protein production from lac and tac promoters when mechanisms for attenuation of transcription at lower translational efficiency are considered. Finally, the model predicts that for high copy number systems ribosomes become limiting in the synthesis of plasmid‐encoded proteins. © 1994 John Wiley & Sons, Inc.
Continuous reaction‐separation process for enzymatic esterification in supercritical carbon dioxide
Authors: Marty Alain , Combes Didier , Condoret Jean‐Stéaphane
Published: December 2004

The study of enzymatic esterification by an immobilized lipase in supercritical carbon dioxide (SCCO2) and in n‐hexane, described in our previous works, was extended to continuous operation in a tubular fixed bed. The modeling of the reaction vessel operation was achieved through the use of the simple plug flow model coupled with the appropriate kinetic equation. Comparison with experiments proved to be satisfactory. The study of the postreactional separation, an important feature when using SCCO2, was undertaken experimentally and good selectivities and product recovery were obtained. © 1994 John Wiley & Sons, Inc.
Kinetics of leucrose formation from sucrose by dextransucrase
Authors: Böker Mathias , Jördening Hans‐Joachim , Buchholz Klaus
Published: December 2004

Leucrose formation from sucrose and fructose by dextransucrase is of practical interest. It has been investigated at different experimental conditions, including the influence of temperature on reaction rate and selectivity. Under appropriate conditions high product yield can be obtained. Furthermore, a model is presented that allows interpretation of the experimental data.
Extraction of rIL‐2 inclusion bodies from Escherichia coli using cross‐flow filtration
Authors: Meagher M. M. , Barlett R. T. , Rai V. R. , Khan F. R.
Published: December 2004

A cross‐flow membrane filtration process was developed for the recovery of rIL‐2 inclusion bodies from homogenized Escherichia coli. The membrane extraction process was comprised of a two‐step diafiltration followed by an extraction with 7 M GuHCl and a 40–fold dilution of the solubilized inclusion bodies into 0.01 M Tris–HCl, 0.035 M NaCl, pH 7.9. The first diafiltration was with a 0.03 M Tris–HCl, 5 mM ethylenediaminetetraacetic acid (EDTA), pH 8, followed by a diafiltration with 1.75 M GuHCl. All of the insoluble rIL‐2 was retained behind the membrane, whereas a GuHCl wash solubilized approximately 15% of the rIL‐2. The membrane process increased the yield of rIL‐2 in the diluted extract by threefold as compared to a similar centrifuge process with a significant increase in purity as determined by reverse‐phase high‐performance liquid chromatography (HPLC). © 1994 John Wiley & Sons, Inc.
Adsorption of BSA on strongly basic chitosan: Equilibria
Authors: Yoshida Hiroyuki , Nishihara Hideki , Kataoka Takeshi
Published: December 2004

Equilibrium isotherms for adsorption of bovine serum albumin (BSA) on a new adsorbent, a strongly basic crosslinked chitosan (Chitopearl 2503), which is hard and is not compressed by pressure in a column, have been presented and compared with diethylaminoethyl (DEAE) Sepharose Fast Flow (hard gel). In Chitopearl 2503, when only buffer existed in the BSA solution, the isotherm was not affected by the initial concentration of BSA but it was affected by pH considerably. The isotherm was favorable when pH ≥ pl (≅ 4.8). When NaCl existed in the BSA solution, the amount of BSA absorbed on the resin decreased with increasing concentration of NaCl. When the concentration of NaCl was 200 mol/m3, the resin did not adsorb BSA at all. The equilibrium data were correlated by the Langmuir equation reasonably well. The BSA may be adsorbed mainly by electrostatic attraction between negatively charged BSA and positively charged quanternary ammonium groups at pH > pl and by protonation reaction of the primary ammonium groups by weak acid groups of BSA at pH = pl. These are confirmed by measuring the amount of inorganic ion exchanged for BSA. In DEAE Sepharose Fast Flow, the isotherm was favorable when pH > pl but unfavorable ar pH = pl. The saturation capacity of BSA on Chitopearl 2503 is about 1.3 to 2.2 times larger than that on DEAE Sepharose Fast Flow. © 1994 John Wiley & Sons, Inc.
Synthesis of dipeptide precursors with an immobilized thermolysin in ethyl acetate
Authors: Nagayasu Takeshi , Miyanaga Masamitsu , Tanaka Takaaki , Sakiyama Takaharu , Nakanishi Kazuhiro
Published: December 2004

N‐(Benzyloxycarbonyl)‐L‐aspartyl‐L‐phenylalanine methyl ester (Z‐AspPheOMe), a precursor of the aspartame, and N‐(benzyloxycarbonyl)‐L‐phenylalanyl‐Lphenylalanine methyl ester (Z‐PhePheOMe) were synthesized from the respective amino acid derivatives with an immobilized thermolysin (EC 3.4.24.4) in ethyl acetate. Various factors affecting the synthesis of these dipeptide precursors were clarified. The initial synthetic rate was the highest at the water content of 3.5% for both reactions. The substrate concentration dependencies of the initial synthetic rate of Z‐AspkPheOMe and Z‐PhePheOMe with the immobilized enzyme in ethyl acetate were different from those in an aqueous buffer solution saturated with ethyl acetate but similar to those in the aqueous/organic biphasic system using the free enzyme. Particularly, the initial synthetic rate of Z‐AspPhOMe increased in order higher than first order with respect to the concentration of L‐phenylalanine methyl ester (PheOMe), whereas it decreased sharply with the concentration of N‐(benzyloxycarbonyl)‐L‐aspartic acid (Z‐Asp). Such kinetic behavior could be explained by regarding the inside of the immobilized enzyme as being a biphasic mode composed from the organic phase and aqueous phase where the enzymatic reaction takes place. The reaction in the aqueous/organic biphasic system using the free enzyme could be simulated by taking into consideration the partition of the substrate and the initial rate of synthesis in the aqueous buffer saturated with ethyl acetate. Based on this analysis, the rate of reaction with the immobilized enzyme in ethyl acetate could also be predicted. Z‐AsPheOMe and Z‐PhePheOMe were synthesized by the fed‐batch method where the acid component of the substrate was intermittently added during the course of reaction and by the batch method. In the synthesis of Z‐AspPheOMe, the synthetic rate and maximum yield of reaction as well as the stability of the immobilized enzyme were higher in the fed‐batch reaction than those in the batch reaction. In the synthesis of Z‐PhePheOMe, the results obtained by both methods were similar. © 1994 John Wiley & Sons, Inc.
Glycolipid membrane anchored recombinant protein production from CHO cells cultGred on porous microcarriers
Authors: Kennard M. L. , Piret J. M.
Published: December 2004

Recombinant proteins were harvested from Chinese hamster ovary (CHO) cells by a controlled release process, which increased the purity and concentration of the harvested protein. Recombinant human melano‐transferrin (p97) was expressed linked to the outer surface of CHO cells by a glycosyl‐phosphatidylinositol (GPI) membrane anchor. Cells were grown to confluence in T‐flask culture, and the p97 harvested by replacing the growth medium for 30 min with phosphate‐buffered saline (PBS) containing 10 mU/mL phosphatidylinositol‐phospholipase C (PI‐PLC). The GPI anchor was selectively cleaved by PI‐PLC. In fresh medium, the CHO cells regained over 95% of their p97 expression within 40 h. The process was repeated for eight harvests. Harvested protein concentrations varied from 1.5 to 3.8 μg/mL due to difficulties in maintaining stable confluent T‐flask cultures. Harvesting from cells growing on porous microcarriers was investigated to increase p97 product concentrations and to overcome culture stability problems. Semicontinuous cultures were maintained in spinners for up to 76 days with average bioreactor cell densities of over 107 cell/mL. The p97 was harvested at up to 100 μg/mL and 30% purity with protein production remaining stable for 4 harvest cycles. Production of high levels of p97 from CHO cells was maintained at 0.5% serum. © 1994 John Wiley & Sons, Inc.
A differential scanning calorimetric study of chymotrypsin in the presence of added polymers
Authors: Otamiri Marina , Adlercreutz Patrick , Mattiasson Bo
Published: December 2004

Scanning calorimetry measurements of different amounts of chymotrypsin in water alone gave a temperature of denaturation (Td) value of 54°C. However, when high‐molecular‐weight poly(ethylene glycol) was added to aqueous solutions of chymotrypsin, the thermostability of the enzyme was enhanced. For example, the addition of 20% (w/w) of poly(ethylene glycol) of molecular weight of 100,000 increased the Td/ value to 66°C. In toluene containing various amounts of added water, ethyl cellulose was used to improve the thermostability of chymotrypsin. For this system, a Td value of 82°C was obtained with a 20% (w/w/) concentration of ethyl cellulose and 2% (v/v) of added water. Polymers in these solvents interact with water, which could otherwise denature the enzyme; polymers also from complexes with enzyme molecules to produce a more stable structure. © 1994 John Wiley & Sons, Inc.
Fuzzy supervisory control of glutamic acid production
Authors: Kitsuta Yoichi , Kishimoto Michimasa
Published: December 2004

In glutamic acid fermentation, the molasses feeding policy and time of penicillin addition significantly affected glutamic acid production, and a fuzzy supervisory control system was developed for their quasi‐optimal regulation.

From the trend of the experimental data, production rules and membership functions of fuzzy inference were devised to determine the quasi‐optimum molasses feeding policy and penicillin addition time. A computer with multitasking operating system was used for the construction of the control system with fuzzy inferencing, which decided the control policy every minute, and the feed rate was controlled automatically. The pattern of residual sugar concentration was almost the same as that of maximum glutamic acid production under manual operation. Using the computer control system, stable production was maintained at the highest level of 71 to 75 g/L. © 1994 John Wiley & Sons, Inc.
Unified kinetic treatment for growth on dual nutrients
Authors: Haas Charles N.
Published: December 2004

This paper presents a new formulation for the analysis of growth kinetics on multiple nutrients. The baseline for the theory is the concept that a noninteractive growth process occurs among perfectly substitutable nutrients if the locus of points of the substrate concentrations producing equal growth rate is linear. A deviation function is then defined with respect to this base case, and several models for this function are suggested. The underlying theory is taken by analogy with mixture thermodynamics. The proposed formulation is tested against data in the literature on growth under substitutable and complementary substrate mixtures. © 1994 John Wiley & Sons, Inc.
Biochemistry of growth inhibition by ammonium ions in mammalian cells
Authors: Ryll Thomas , Valley Ulrich , Wagner Roland
Published: December 2004

The intracellular pool of UDP‐N‐acetylglucosamine and UDP‐N‐acetylgalactosamine has been shown to act as a central target during the inhibitory action of ammonium ions in vitro cultivated mammalian cell cultures. This pool has been demonstrated to be elevated at the end of a batch cultivation and very quickly as a response to exogenously applied ammonium chloride by using four different cell lines (hybridoma, BHK, CHO, and Ltk−929). The amount of enlarged UDP aminohexoses is correlated to the inhibitor concentration and additionally dependent on the cell line. The formation of the UDP sugars is associated with a transient reduction of the UTP pool. Moreover, the quick formation of UDP‐GNAc is strictly dependent on the presence of glucose and ammonium. Both metabolites act as biochemical precursors. Additionally, the formation of UDP‐GNAc after ammonium application has been shown to increase with an elevated cultivation pH and to be independent of the inhibition of transcription and translation processes. The intracellular amount of UDP‐GNAc correlates with the level of growth inhibition in mammalian cell lines. © 1994 John Wiley & Sons, Inc.
Improvement of the extraction of penicillin acylase from Escherichia coli cells by a combined use of chemical methods
Authors: Novella Isabel S. , Fargues Claire , GréVillot Georges
Published: December 2004

Several techniques for protein extraction were tested for recovering penicillin acylase from a recombinant strain of Escherichia coli. These techniques include chemical [guanidine hydrochloride, Triton X‐100, ethylenediaminetetraacetic acid (EDTA), ethanol/toluene], physical (sonication, freeze‐and‐thawing), and enzymatic (lysozyme) treatments. Best results were obtained with the combined use of guanidine and EDTA. This extraction procedure was optimized, and it was found that 95% of the enzyme was extracted after a 10 m/M EDTA plus 10 mM guanidine treatment at room temperature for 10 h. The purification factor was 25 when compared to disruption by sonication. This extraction method could avoid purification steps for particular applications. © 1994 John Wiley & Sons, Inc.
Hyphal vocuolation and fragmentation inpenicillium chrysogenum
Authors: Paul G. C. , Kent C. A. , Thomas C. R.
Published: December 2004

A link between vacuolation and fragmentation of Penicillium chrysogenum mycelia in stirred tank submerged fermentations is reported. Quantitative information on vocuolation and morphology was obtained by image analysis. In fed‐batch fermentations the coincidence of the events of rapid vacuolation and the fall of the mean total and main hyphal lengths suggests that hyphal fragmentation is not necessarily due to “shear” alone. The physiological state of the hyphae, characterized by the proportions of vaccuoles, was found to have a significant influence on the breakage of mycelial hyphae, It was found that the fragmentation was greater when the hyphae became heavily vacuolated following nutrient limitation in the culture, i.e., during the switch from the rapid growth to the production phase. © 1994 John Wiley & Sons, Inc.
Solid‐state enzyme deactivation in air and in organic solvents
Authors: Toscano Giuseppe , Pirozzi Domenico , Maremonti Michele , Greco Guido
Published: December 2004

Thermal deactivation of solid‐state acid phosphates (E.C. 3.1.3.2, from potato) is analyzed, both in the presence and in the absence of organic solvents. The thermal deactivation profile departs from first order kinetics and shows an unusual activity. The process is described by a phenomenological equation, whose theoretical implications are also discussed. The total amount of buffer salts in the enzyme powder dramatically affects enzyme stability in the range 70×C to 105×C. The higher salt/protein ratio increases the rate of thermal deactivation. The deactivation rate is virtually unaffected by the presence of organic solvents, independent of their hydrophilicity. © 1994 John Wiley & Sons, Inc.
Ion exchange immobilization of changed β‐galactosidase fusions for lactose hydrolysis
Authors: Heng Meng H. , Glatz Charles E.
Published: December 2004

The use of charged peptides fused to enzymes for immobilization onto ion‐exchange membranes was explored for the enzyme ×‐galactosidase. The additional charged peptides, containing 1, 5, 11, and 16 aspartates, fused to ×‐galactosidase, for the most part did not interfere with the kinetic behavior for lactose hydrolysis. There was a 2‐fold decline in Vm for the 16‐aspartate fusion, but the others were quite similar to the wild type enzyme (BGWT). BGWT and the fusions all retained approximately 50% of their activities when adsorbed onto ion‐exchange membranes. In contrast to BGWT, the enhanced binding strength of the 11 aspartate fusion provided the ability to hydrolyze whey permeate at 0.3 M ionic strength without enzyme leakage, and to immobilize the enzyme directly from diluted cell extract with 83% purity. © 1994 John Wiley & Sons, Inc.
Modified monoclonal antibody production kinetics kappa/gamma mRNA levels, and metabolic activities in a murine hybridoma selected by continuous Culture
Authors: Merten O.‐W. , Moeurs D. , Keller H. , Leno M. , Palfi G. E. , Cabanié L . , Couvé E.
Published: December 2004

During long‐term continuous culture of the hybridoma cell line 11317, a better‐producing subclone (I1317‐SF11), giving improved productivity, has been selected. The comparison of the original cell line (I1317‐DC) with this subclone revealed that although the growth patterns of both clones were similar, both in continuous and in batch cultures, considerable differences could be seen between the clones with respect to monoclonal antibody (MAB) accumulation, MAB production rate, the levels of mRNA coding for heavy and light chains of IgG, and some metabolic activities. In continuous culture as well as in batch culture, I1317‐SF11 showed increased levels of mRNA coding for kappa and gamma chains compared with I1317‐DC and/or a modified ratio of the mRNA species when compared to that in I1317‐DC. Using pulse experiments, it could be established that the biosynthesis of both chains was augmented in I1317‐SF11. Although the kappa and gamma mRNA levels were modified or inversed for I1317‐SF11, the cells always synthesized more kappa than gamma chains. The overall increase in the synthetic activity of I1317‐SF11 is suggested as one reason for the considerable increase of IgG productivity and product accumulation in continuous culture as well as in repeated batch cultures. Tests concerning metabolic activity revealed that I1317‐SF11 had a predominantly glycolytic metabolism independent of growth requirements, whereas for I1317‐DC the metabolism became increasingly glycolytic with increased growth. The antibody yield coefficient of I1317‐SF11 on glutamine was significantly higher than that of I1317‐DC for the continuous culture, whereas the antibody coefficients on glucose were almost similar for both clones under the different culture conditions used. Both antibody coefficients were considerablly influenced by the specific growth rate.

All these facts together lead to the conclusion that subclone I1317‐SF11 uses more of the energy available, or it was the energy and/or precursors available for the synthesis and production of MAB more efficiently than the thesis and production of MAB more efficiently than the original cell line. Although the levels of mRNA coding for heavy and light chains of IgG were modified, it could be confirmed that the overall regulation of MAB‐synthesis and ‐production occurs post‐translationally and that at higher growth rates, more biosynthetic activity is diverted to biomass production. © 1994 John Wiley & Sons, Inc.
Specific quantification of trichoderma reesei cellulases in reconstituted mixtures and its application to cellulase–cellulose binding studies
Authors: Nidetzky Bernd , Claeyssens Marc
Published: December 2004

Specific quantifications of the major cellulolytic components of the Trichoderma reesei enzyme complex, i.e., endoglucanases I and III and cellobiohydrolases I and II, are described and, employing a defined mixture of these four cellulases reconstituted according to the composition of the native Trichoderma cellulase complex, used to determine the binding of each individual component onto filter paper. During substrate degradation by this enzyme mixture, the specific adsorption of each individual cellulase gradually increases and no preferential binding of one enzyme component in any particular phase of cellulose hydrolysis is found. T. reesei cellobiohydrolases I and II admixed with endoglucanases I and III represent a “full‐value” cellulase system that is capable of degrading semicrystalline cellulose efficiently. In comparison with the crude Trichoderma enzyme complex, almost identical adsorption properties and similar hydrolytic efficiency are found for the reconstituted mixture. © 1994 John Wiley & Sons, Inc.
Enhancement of monoclonal antibody yield by hybridoma fed‐batch culture, resulting in extended maintenance of viable cell population
Authors: Bushell M. E. , Bell S. L. , Scott M. F. , Spier R. E. , Wardell J. N. , Sanders P. G.
Published: December 2004

Hybridoma batch cultures were extended using feed formulations based on nutrient consumption measured during different batch culture phases when (a) growth but negligible antibody production was taking place; (b) maximum antibody production rate and declining viable cell growth rate were observed. Strategy (a) was the more successful (2.8‐fold compared with 1.8‐fold antibody titer increase) and maintained cell viability for longer. Analysis of the effects of omitting individual amino acids yielded results which were consistent with those from the feeding experiment © 1994 John Wiley & Sons, Inc.
A mathematical model for the leukocyte filtration process
Authors: Bruil Anton , Beugeling Tom , Feijen Jan
Published: December 2004

Leukocyte filters are applied clinically to remove leukocytes from blood. In order to optimize leukocyte filters, a mathematical model to describe the leukocyte filtration process was developed by modification of a general theoretical model for depth filtration. The model presented here can be used to predict the time‐dependent leukocyte filtration as a function of cell–cell interaction in the filter, filter efficiency, filter capacity, filter dimensions, and leukocyte concentration in the suspension applied to the filter. The results of different leukocyte filtration experiments previously reported in the literature could be well described by the present model. © 1995 John Wiley & Sons, Inc.
Metal recognition by in‐vitro selection
Authors: Gupta Kanika , Yin John
Published: December 2004

We propose an in vitro selection strategy to identify bacteriophage variants that recognize metal ions in solution. In 6 M urea, phage T7 loses 99.9% of its activity in less than 5 min. Inactivation is accelerated by gold, but slowed by zinc and magnesium. Selection of phage over five generations in the presence of gold, zinc, and magnesium increases phage half‐lives 4‐, 10‐, and 70‐fold, respectively. As selections are repeated, phage become increasingly dependent on the specific metal employed in the selection, indicating the suitability of the strategy for optimization of metal‐ion recognition. © 1995 John Wiley & Sons, Inc.
Determination of the respiration quotient in mammalian cell culture in bicarbonate buffered media
Authors: Bonarius Hendrik P. J. , de Gooijer Cornelis D. , Tramper Johannes , Schmid Georg
Published: December 2004

The determination of the respiration quotient (RQ = CER/OUR) has not been used so far as a tool for understanding animal cell metabolism. This is due to problems in measuring the carbon dioxide evolution rate (CER) rather than the oxygen uptake rate (OUR). The determination of the CER is complicated by the use of bicarbonate in the medium. Using liquid and gas balances we have derived an equation for continuous culture to quantify the amount of CO2 that comes from the bicarbonate in the feed. Under cell‐free conditions, values predicted by this equation agree within 4% with the experimental results. In continuous culture using hybridoma cells, the CO2 from the feed, as determined by an IR‐gas analyzer, was found to represent a significant amount of the total measured CO2 in the off‐gas (50% in a suboptimal, and 30% in high‐growth medium). Furthermore, the problem of CO2 loss from the medium during medium preparation and storage was solved using both a theoretical and an experimental approach. RQ values in continuous culture were evaluated for two different growth media. Small but significant differences in RQ were measured, which were matched by differences in specific antibody rates and other metabolic quotients. In a medium with Primatone RL, an enzymatic hydrolysate of animal cell tissue that causes a more than twofold increase in cell density, the RQ was found to be 1.05, whereas in medium without Primatone RL (but containing amino acids equivalent in composition and concentration to Primatone RL) the RQ was found to be 0.97. We suggest the RQ to be a useful parameter for estimating the physiological state of cells. Its determination could be a suitable tool for both the on‐line control of animal cell cultivations and the understanding of cell metabolism. © 1995 John Wiley & Sons, Inc.
Effects of substrate branching characteristics on kinetics of enzymatic depolymerization of mixed linear and branched polysaccharides: II. Amylose/glycogen α‐amylolysis
Authors: Park Jun T. , Rollings James E.
Published: December 2004

Enzymatic depolymerization of polysaccharides with α‐amylase has been studied in mixed aqueous dimethylsulfoxide (DMSO)/water solvents. Polysaccharide substrate chemical compositions, configurational structures, and bonding pattersn are known to affect observed enzymatic reaction kinetics. The branching structures of polysaccharides and their effects on the kinetic mechanisms of depolymerization reactions via endo‐acting hydrolyzing enzyme was studied via size exclusion chromatography coupled to low angle laser light scattering (SEC/LALLS). The glycogen branching structure is a heterogeneously distributed “cluster” structure rather than a homogeneously distributed “treelike” structure. The action pattern of α‐amylase on glycogen, which is composed of highly branched clusters, as end‐products, has a “pseudo‐exo‐attack” in contrast to an expected “endoattack” as seen in the hydrolysis of amylose or amylopectin substrates. These effects of branched substrates for mixed amylose/glycogen α‐amylolysis have been predicted and demonstrated by both experimental and theoretical analysis using the kinetic model presented in this report. The “lumped” kinetic model employed, assumes that the enzyme simultaneously attacks both linear and branched substrates. In general, excellent agreement between the model predictions and the experimental observations, both qualitatively and quantitatively, was obtained. © 1995 John Wiley & Sons, Inc.
Protein extration from an aqueous phase into a reversed micellar phase: Effect of water content and reversed micellar composition
Authors: Hilhorst R. , Sergeeva M. , Heering D. , Rietveld P. , Fijneman P. , Wolbert R. B. G. , Dekker M. , Bijsterbosch B. H.
Published: December 2004

In the system composed of the cationic surfactant TOMAC (10 mM), the nonionic (co)surfactant Rewopal HV5 (2 mM), and octanol (0.1% v/v) in isooctane, reversed micelles are formed upon contact with an aqueous phase containing 50 mM ethylene diamine. α‐Amylase can be transferred from the aqueous phase into reversed micelles in the pH range 9.5 to 10.5 and re‐extracted into a second aqueous phase of different composition. The size of the reversed micelles (as reflected in the water content of the organic phase) can be varied by changes in percentage of octanol, type of counterion in the aqueous phase, or in the number of ethoxylate head groups of the nonionic surfactant. An increase in size results in transfer at lower pH values. Experiments in which the charge density in the reversed micellar interface was changed by incorporation of charged derivatives of the nonionic surfactant, without influencing the water content, revealed that an increased charge density facilitated transfer, resulting in a broader transfer profile. Replacement of TOMAC by other quaternary ammonium surfactants differing in number and length of tails revealed that, of the 14 surfactants tested, only 2 gave appreciable amounts of transfer. The amount of transfer is related to the dynamics of phase separation of the surfactants: those giving a poor phase separation inactivate the enzyme. This inactivation is caused by electrostatic interactions between the charged surfactant head groups and charged groups on the enzyme. Electrostatic interactions are the first step of transfer, and can result in either incorporation in a reversed micelle, or, if reversed micelle formation is slow, in enzyme inactivation. © 1995 John Wiley & Sons, Inc.
The viability of Scenedesmus bicellularis cells immobilized on alginate screens following nutrient starvation in air at 100% relative humidity
Authors: Kaya Valentino M. , Picard Gaston
Published: December 2004

The viability of algal cells immobilized on screens and starved in a water‐saturated air stream was studied at the laboratory scale. This new process for wastewater biotreatment has been developed using immobilized cells, which were starved in air, to obtain a high rate of nutrient removal. A unicellular green microalgae, Scenedesmus bicellularis, was isolated from secondary decantation tanks at an urban wastewater treatment plant and grown in a synthetic medium for 12 days. The cells were then concentrated by centrifugation and immobilized on alginate screens. The screens were then inserted in a photochamber saturated at 100% relative humidity and subjected to a photoperiod of 16 h in the light and 8 h in the dark, with an illumination of 150 μE m−2 s−1 provided by fluorescent lamps. After 48 h of nutrient starvation, the immobilized cells were used for the removal of ammonium and orthophosphate from a synthetic secondary wastewater effluent in a plexiglass reactor. During the sequential operation of starvation followed by incubation in the presence of nutrients, fast growth of viable cells in the gel matrix was obtained and there was no appreciable decay of chlorophyll a or cell activity. When these immobilized and starved cells were incubated in wastewater, ammonium (NH4+) and orthophosphate (PO43−) ions were quickly taken up from medium. After three successive 2‐h exposures to wastewater, immobilized algal cells were freed by dissolving the Ca‐alginate with phosphate as 0.2 M Na3PO4 and resuspended in fresh culture medium. Results indicate that free cells transferred to rich medium remained viable, but the growth rate revealed that the viable cells decreased their culturability. © 1995 John Wiley & Sons, Inc.
Effect of single nutrient limitation of poly‐β‐hydroxybutyrate molecular weight distribution in alcaligens europhus
Authors: Asenjo J. A. , Schmidt A. S. , Andersen P. R. , Andrews B. A.
Published: December 2004

The effect of magnesium and phosphate limitation on the molecular weight distribution of poly‐β‐hydroxybutyrate (PHB) in Alcaligens europhus in cotinuons culture has been stuied. Conditions of nitrogen limitation both with glucose excess (above ca. 20 g/L) and without excess were investigated Under N‐limitation and glucose excess, Mw decreases when the magnesium content is decreased below 50% (19.7 mg/L) of the basal medium content; this also results in a broadenng of molecular weight distribution (Mw/Mn) from 2 to 5 and a decrease in Mw fron 2 × 106 to 0.9 × 106. Below 20% of the basal content of magnesium (7.9 mg/L) these two trends were reversed. This behaviour was not observed in the absence of glucose excess, phshate had virtually no effect on PHB Mw or its distribution, whereas wih no (or little) glucose excess Mw of the PHB decreased with phosphate concentrations below 50% of the basal level (0.705 g/L). Hence, in continuous or fed‐batch cultures, in addition to nitrogen limitation to alklow for PHB accumulation, it is necesary to control both the addition of glucose (no excess) and also to maintain magnesium limitation (ca. 25% of basal medium level, 9.9 mg/L) and phosphate above 50% of he basal level (0.705 g/L). Thus, when broadening of molecular weight destribution (increase in Mw/Mn) is observed at the end of fed‐batch culture it is probably caused by phosphate limitation and/or glucose excess. © 1995 John Wiley & Sons, Inc.
Effect of different cell culture conditions on the polypeptide integrity and N‐glycosylation of a recombinant model glycoprotein
Authors: Gawlitzek Martin , Conradt Harald S. , Wagner Roland
Published: December 2004

The effect of different short‐term controlled cell culture conditions on the product quality of a genetically engineered human interleukin‐2 N‐glycosylation variant protein expressed from a baby hamster kidney cell line (BHK‐21) has been investigated. A perfused 2‐L stirred tank reactor was used. Products purified from the culture supernatant of cells grown under experimentally initiated nutrient limitations (glucose, amino acids, pO2) were characterized by their HPLC‐elution profile, SDS‐PAGE and western blotting, amino acid sequencing as well as for their N‐linked carbohydrates, using “HPAEC‐PAD fingerprinting” and methylation analysis. The glycoprotein products secreted from cells under the different culture conditions (kept for 24 h, after an adaption time period of 48 h) showed an almost identical oligosaccharide pattern. By contrast, short‐term changes of the culture condition led to considerable differences in the ratio of glycosylated to unglycosylated protein forms. Significant amounts of NH2‐terminally truncated polypeptide forms were observed. They lacked proponderantly the first two amino acids; however, under certain culture conditions forms lacking up to eight NH2‐terminal amino acids were detected. © 1995 John Wiley & Sons, Inc.
The influence of electrostatic interactions on partition in aqueous polyethylene glycol/dextran biphasic systems: Part II
Authors: Schluck A. , Maurer G. , Kula M.‐R.
Published: December 2004

In aqueous polyethylene glycol/dextran two‐phase systems, the hydrophobicity, free volume, surface tension, and interfacial tension of the phases in equilibrium were measured as a function of pH and ionic strength. These parameters were found to change with pH, but the pattern and magnitude cannot explain the unusual partition of charged macromolecules, observed previously. The electrostatic potential difference was determined by a new experimental approach based on the measurement of the pH difference between the phases at equilibrium. In polyethylene glycol/dextran systems containing sodium chloride as ionized species, the electrostatic potential is not constant in the pH range 2 to 11. The partition behavior of charged macromolecules and its dependence on pH can be explained by the combined action of charge and phase potential. This conclusion was tested with poly‐L‐glutamate, which partitioned as predicted and in a pattern opposite to positively charged macro‐ molecules. © 1995 John Wiley & Sons, Inc.
Improvement of Escherichia coli microaerobic oxygen metabolism by Vitreoscilla hemoglobin: New insights from NAD(P)H fluorescence and culture redox potential
Authors: Tsai Philip S. , Rao Govind , Bailey James E.
Published: December 2004

On‐line NAD(P)H fluorescence and culture redox potential (CRP) measurements were utilized to investigate the role of Vitreoscilla hemoglobin (VHb) in perturbing oxygen metabolism of microaerobic Escherichia coli Batch cultures of a VHb‐synthesizing E. coli strain and the iso‐genic control under fully aerated conditions were subject to several high/low oxygen transitions, and the NAD(P)H fluorescence and CRP were monitored during these passages. The presence of VHb decreased the rate of net NAD(P)H generation by 2.4‐fold under diminishing oxygen tension. In the absence of aeration, the strain producing VHb maintained a steady NAD(P)H level 1.8‐fold less than that of the control, indicating that the presence of VHb keeps E. coli in a more oxidized state under oxygen‐limited conditions. Estimated from CRP, the oxygen uptake rates near anoxia were 25% higher for cells with VHb than those without. These results suggest that VHb‐expressing cells have a higher microaerobic electron transport chain turnover rate. To examine how NAD(P)H utilization of VHb‐expressing cells responds to rapidly changing oxygen tension, which is common in large‐scale fermentations, we pulsed air intermittently into a cell suspension and recorded the fluorescence response to the imposed dissolved oxygen (DO) fluctuation. Relative to the control, cells containing VHb had a sluggish fluorescence response to sudden changes of oxygen tension, suggesting that VHb buffers intracellular redox perturbations caused by extracellular DO fluctuations.© John Wiley & Sons, Inc.
Continuous monitoring of nitrogenase activity in Azotobacter vinelandii fermentation using off‐gas mass spectrometry
Authors: Prior John J. , Christie Patricia D. , Murray Robert J. , Orme‐Johnson William H. , Cooney Charles L.
Published: December 2004

This study evaluated the feasibility of monitoring nitro‐genase activity in situ through measurement of N2 uptake rate (NUR) using off‐gas mass spectrometry. Four 50‐L cultures of Azotobacter vinelandii were grown aer‐obically in nitrogen‐free medium to cell densities of 1.0‐1.3gL−1 magnetic‐sector mass spectrometer was used to monitor NUR along with other gas exchange rates. The small specific uptake rate (1.2 mmol g−1 h−1) and low cell density were found to lead to a NUR below the measurement accuracy limits under normal conditions. An operating strategy and feed gas mixture (40% O2, 45% N2 15% Ar) were designed to improve the signal‐to‐noise ratio while maintaining dissolved O2 and N2 levels in desired ranges. The fraction of N2 removed from the air stream was increased approximately 5‐fold from 0.2% to 1.0% and the measurement noise was reduced 25‐fold from a baseline of ±5to ±0.2 mmol L−1 h−1. The NUR measurements were compared against in vivo and in vitro acetylene reduction assays as well as on‐line cell growth rate measurements. While electron transfer requirements predict an NUR‐to‐acetylene reduction rate ratio of 0.33, measured ratios for the in vivo and in vitro assays were 0.8 and 0.44, respectively. This suggests that other rate‐limiting steps were present in the case of the in vivo assay. In accordance with reports in the literature, no concomitant hydrogen evolution was detected. This is the first reported continuous and direct measurement of NUR in fermentation and demonstrates a novel approach for improving measurement accuracy through rational adjustment of operating conditions. The technique has potential to provide useful insight for development and control of microbial nitrogen fixation processes.© John Wiley & Sons, Inc.