bluuebunny/crossref_metadata_2025 huggingface dataset:
A reagentless amperometric electrode based on carbon paste, chemically modified with D‐lactate dehydrogenase, NAD+, and mediator containing polymer for D‐lactic acid analysis. II. On‐line monitoring of fermentation process
Authors: Shu Hun‐Chi , Gorton Lo , Persson Björn , Mattiasson Bo
Published: December 2004

The production of D‐lactic acid by Lactobacillus delbrueckii (ATCC 9649) during fermentation was monitored on‐line with a reagentless D‐lactate dehydrogenase modified carbon paste electrode in a flow injection system integrated with a filtration sampling device. The time delay between sampling and detection was approximately 6 min. The use of an electropolymerized ortho‐phenylenediamine membrane on the elctrode resulted in a very selective sensor response with acceptable stability and sensitivity. The D‐lactate concentrations determined on‐line agreed well with those determined by a standard method, suggesting that this sensor system is suitable for on‐line monitoring of fermentation processes. © 1995 John Wiley & Sons, Inc.
Citric acid production by Candida lipolytica Y 1095 in cell recycle and fed‐batch fermentors
Authors: Rane Kishore D. , Sims Kevin A.
Published: December 2004

The effect of dissolved oxygen on citric acid production and oxygen uptake by Candida lipolytica Y 1095 was evaluated in cell recycle and fed‐batch fermentation systems. The maximum observed volumetric productivity, which occurred at a dilution rate of 0.06 h−1, a dissolved oxygen concentration of 80%, and a biomass concentration of 5% w/v, in the cell recycle system, was 1.32 g citric acid/L · h. At these same conditions, the citric acid yield was 0.65 g/g and the specific citric acid productivity was 24.9 mg citric acid/g cell · h. In the cell recycle system, citric acid yields ranged from 0.45 to 0.72 g/g. Both the volumetric and specific citric acid productivities were dependent on the dilution rate and the concentration of dissolved oxygen in the fermentor. Similar productivities (1.29 g citric acid/L · h) were obtained in the fed‐batch system operated at a cycle time of 36 h, a dissolved oxygen concentration of 80%, and 60 g total biomass. Citric acid yields in the fed‐batch fermentor were consistently lower than those obtained in the cell recycle system and ranged from 0.40 to 0.59 g/g. Although citric acid yields in the fed‐batch fermentor were lower than those obtained in the cell recycle system, higher citric:isocitric acid ratios were obtained in the fed‐batch fermentor. As in the cell recycle system, both the volumetric and specific citric acid productivities in the fed‐batch fermentor were dependent on the cycle time and dissolved oxygen concentration. © 1995 John Wiley & Sons, Inc.
Effect of the concentration of DODMAC and 1‐decanol on the behavior of reverse micelles in the extraction of amino acids
Authors: Wang W. , Weber M. E. , Vera J. H.
Published: December 2004

The concentrations of dioctyldimethyl ammonium chloride (DODMAC) and 1‐decanol in isooctane needed to form reverse micelles by phase contact have been determined. The behavior of these reverse micelles in the extraction of aspartic acid, glutamic acid, and threonine was studied by analyzing all of the ionic species in the aqueous phase. The amino acid is extracted from the aqueous phase by exchanging with the Cl− counterions of DODMAC in the reverse micelles. The ionic species in the reverse micelles tend toward their undissociated states as the water uptake by the reverse micelles decreases. The effect of 1‐decanol on the extraction of the amino acids with two negative charges is due to the change in the water uptake of the reverse micelles. The concentration of DODMAC has no effect on the ion exchange of the amino acid with one negative charge with the Cl− counterions of DODMAC in the reverse micelles. Higher molar ratios of decanol to DODMAC favor the selective separation of amino acids with different charge numbers. © 1995 John Wiley & Sons, Inc.
Monitoring of Saccharomyces cerevisiae in commercial bakers' yeast fermentation
Authors: Hatch Randolph T. , Veilleux Brendan G.
Published: December 2004

In the highly competitive market of commercial bakers' yeast, fermentations are operated for maximum efficiency and minimum production cost. In order to maintain competitiveness, the fermentations must be highly consistent with minimum variation in yeast performance, maximum yield on raw materials, and minimum production of undesirable side products. The use of advanced instrumentation is of critical importance to achieving these goals by the production engineer. An in situ optical density probe was used to determine the yeast cell density in full‐scale commercial bakers' yeast fermentations. The optical density probe results were compared with oxygen uptake rate analyses, packed cell volume, and off‐line measured cell dry weights. The most accurate measurement of cell density was found to be the optical density probe. This instrument allowed the on‐line determination of cell density with highly consistent results from fermentation batch to batch and with out the need for intermittent recalibration. © 1995 John Wiley & Sons, Inc.
Simultaneous production of H2 and O2 in closed vessels by marine Cyanobacterium anabaena sp. TU37‐1 under high‐cell‐density conditions
Authors: Kumazawa Shuzo , Asakawa Hidenori
Published: December 2004

A marine cyanobacterium, Anabaena sp. TU37‐1, exhibited stable production of hydrogen and oxygen in closed vessels. About 8.4 and 4.3 mL (at atmospheric pressure) of hydrogen and oxygen accumulated, respectively, in flasks with 20 mL gas phase during 48 h incubation. Thus, concentration of H2 and O2 became 26 and 13% of the gas phase, respectively. Duration of hydrogen production was prolonged by the periodic gas replacement in the reaction vessel. The conversion efficiencies of photosynthetically active radiation (fluorescent light, 22 W/m2) to hydrogen were 2.4 and 2.2% during the initial 12‐ and 24‐h incubation periods, respectively. © 1995 John Wiley & Sons, Inc.
Preparation of immobilized enzyme with high activity using affinity tag based on proteins A and G
Authors: Kondo Akihiko , Teshima Tadanaru
Published: December 2004

Affinity tag AG consisting of immunoglobulin G (lgG)‐binding domains of protein A from Staphylococcus aureus (EDABC) and those of protein G from Streptococcus strain G148 (C2C3) were used to facilitate immobilization of β‐galactosidase (βgal) from Escherichia coli. Poly(methylmethacrylate/N‐isopropylacrylamide/methacrylic acid) [P(MMA/NIPAM/MAA)] and poly(styrene/N‐isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] latex particles, which show thermosensitivity, were used as support materals to prepare affinity adsorbents. Human γ‐globulin (HγGb), whose major fraction is lgG, was used as an affinity ligand and was covalently immobilized onto the both latex particles by the carbodiimide method under various conditions. A fusion protein, AGβgal, was immobilized at pH 7.3 by the specific binding of affinity tag to these affinity adsorbents. The amount of adsorbed AGβgal per unit amount of immobilized HγGb, namely, efficiency of ligand utilization, was strongly affected by the type of latex particles and pH value for HγGb immobilization. The efficiency of ligand utilization was maximum in the affinity adsorbents prepared at pH 6.0 to 7.0, and that in the HγGb–P(MMA/NIPAM/MAA) latex particles was high. This result could be explained by the conformation and orientation of immobilized HγGb molecules. Immobilized AGβgal retained approximately 75% of its activity in solution and the binding is stable enough to allow repeated use. These results clearly demonstrate that combination of the affinity tag AG and the affinity adsorbents, based on the thermosensitive latex particles, offers a simple and widely applicable method for preparation of immobilized enzyme with high activity. © 1995 John Wiley & Sons, Inc.
Optimum autoregulator addition strategy for maximum virginiamycin production in batch culture of Streptomyces virginiae
Authors: Yang Young Kook , Shimizu Hiroshi , Shioya Suteaki , Suga Ken‐ichi , Nihira Takuya , Yamada Yasuhiro
Published: December 2004

Virginiae butanolides (VBs) are autoregulators of Streptomyces virginiae, which induce virginiamycin biosynthesis. Generally, autoregulators are synthesized by the microorganism itself during culture. Addition of chemically synthesized virginiae butanolide‐C (VB‐C), which is one of the VBs, can also control the induction time and the amount of virginiamycin production. The optimum concentration and shot‐feeding time of VB‐C for the maximum production of virginiamycins M and S were investigated in flasks and jar‐fermentor batch cultures. VB‐C addition later than 8 h from the start of culture induced not only virginiamycin M and S synthesis but also VB synthesis. Virginiamycin M and S production increased with the decrease of total VBs (produced VBs and added VB‐C) concentration. That is, although VBs are needed to induce virginiamycin M and S synthesis, the amount of VB‐C added should be such that as small an amount as possible of VBs is synthesized to achieve the maximum production of virginiamycins M and S. However, the VB‐C addition earlier than 8 h from the start of culture showed no clear relationship between the amounts of VBs and virginiamycins M and S produced. In conclusion, the maximum production of virginiamycins M and S was attained by the shot addition of 5 μg/L VB‐C at 8 h from the start of culture. The maximum value was about twofold that without VB‐C addition. The optimum addition strategy of VB‐C was confirmed by the jar‐fermentor experiments. © 1995 John Wiley & Sons, Inc.
Influence of electrostatic interactions on partitioning in aqueous polyethylene glycol/dextran biphasic systems: Part I
Authors: Schluck Andreas , Maurer Gerd , Kula Maria‐Regina
Published: December 2004

To study the influence of charges on the partition of solutes in aqueous two‐phase systems of polyethylene glycol and dextran, partition coefficients of dimethylaminoethyl‐dextran, trimethylamino‐dextran, and bis (α,ω)‐amino‐poly(ethylene glycol) were determined as a function of pH (range 2 to 12) and ionic strength. These polymers are derivatives of the phase forming components and carry ionizable groups that are charged or uncharged depending on the pH. Unexpectedly, the largest differences in the partition coefficients were found at high pH, where the modified polymers are uncharged. In addition, the partitioning of low‐molecular‐weight model compounds, ethylenediamine and iminodiacetic acid, as well as poly‐L‐lysine and poly(allylamine) was analyzed. A consistent pattern was observed in the partition of polyelectrolytes reflecting the influence of charge, but another property of aqueous phase systems unrelated to charge and changing with pH seems to be superimposed. © 1995 John Wiley & Sons, Inc.
Measuring enzyme hydration in nonpolar organic solvents using NMR
Authors: Parker M. C. , Moore B. D. , Blacker A. J.
Published: December 2004

A very sensitive NMR method has been developed for measuring deuterated water bound to proteins suspended in nonpolar solvents. This has been used to determine the amount of bound water as a function of water activity for subtilisin Carlsberg suspended in hexane, benzene, and toluene and for α‐chymotrypsin in hexane. The adsorption isotherms for subtilisin in the three solvents are very similar showing that water activity can be usefully employed to predict the amount of water bound to proteins in nonpolar organic media. Comparison of the degree of enzyme hydration reached in nonpolar solvents with that obtained in air shows that adsorption of strongly bound water is hardly affected by the low dielectric medium, but adsorption of loosely bound water is significantly reduced. This suggests that the hydrophobic regions of the protein surface are preferentially solvated by solvent molecules, and that in a nonpolar environment formation of a complete monolayer of water over the protein surface is thermodynamically unfavorable. © 1995 John Wiley & Sons, Inc.
Two‐step immobilized enzyme conversion of cephalosporin C to 7‐aminocephalosporanic acid
Authors: Conlon Hugh D. , Baqai Javed , Baker Kerry , Shen Yong Q. , Wong Bing L. , Noiles Ray , Rausch Carl W.
Published: December 2004

The first large‐scale production of 7‐aminocephalosporanic acid (7ACA) from cephalosporin C (CPC) using a wholly enzymatic synthesis method is reported here. We produced 7ACA from CPC in as high a molar yield as 85% using the immobilized enzymes D‐amino acid oxidase (D‐AOD) and glutaryl‐7‐ACA acylase (GL‐acylase). In the first reactor, CPC is converted to keto‐adipyl‐7‐aminocephalosporanic acid (keto‐7ACA) using an immobilized D‐AOD isolated from a yeast, Trigonopsis variabilis. The keto‐7ACA is then spontaneously converted to glutaryl‐7‐aminocephalosporanic acid (GL‐7ACA) via a chemical reaction with hydrogen peroxide. The hydrogen peroxide is also a product of the D‐AOD reaction. Near quantitative conversion of the keto‐7ACA to GL‐7ACA was observed. The second reactor converts GL‐7ACA to 7ACA using an immobilized GL‐acylase, which was isolated from a reconbinant Escherichia coli. The final 7ACA crystalline product is a high quality product. The reactions are conducted under very mild aqueous conditions: pH 8.0 and 20° to 25°C. The production of desacetyl side products is minimal. This process is currently being implemented on an industrial scale to produce 7ACA. © 1995 John Wiley & Sons, Inc.
Conversion of native lignin to a highly phenolic functional polymer and its separation from lignocellulosics
Authors: Funaoka M. , Matsubara M. , Seki N. , Fukatsu S.
Published: December 2004

An original reaction system (the phase separative reaction system) has been designed for derivatizing native lignins to highly phenolic, functional polymers. This system is composed of a phenol derivative and concentrated acid, which are not miscible at room temperature. The key point of the lignin functionalization process, including the phase separative system, is that lignin and carbohdrates, which are totally different in structures and reactivitie, are modified individually in the different phases: lignin is present in the organic phase and carbohydrates in the aqueous phase. Through the process, lignin was modified selectively at Cα‐positions of side chains, the most reactive sites, to give highly phenolic, light‐colored, diphenylmethane‐type materials which still retained original interunit linkages formed by the dehydrogenative polymerization during the biosynthesis. The carbohydrates were swollen, followed by partial hydrolysis and dissolution in the acid solution, resulting in the perfect decomposition of interpenetrating polymer network structures in the cell wall. Therefore, the functionalization of lignin and the separation of resulting lignin from carbohydrates were quickly achieved at room temperature, independent of wood species. This process would be a powerful tool for estimating strutures and reactivities of lignins as well as the functionalization of lignins, because of the selective structural modifications. © 1995 John Wiley & Sons, Inc.
Hyphal growth and fragmentation of Penicillium chrysogenum in submerged cultures
Authors: Nielsen Jens , Krabben Preben
Published: December 2004

A previously derived population model describing the average properties of hyphal elements in submerged cultures of filamentous fungi was revised, and a term for the influence fo spore germination on the average total hyphal length was added. The model was derived from a general balance for the distribution function for the hyphal elements. Based on experimental data and the derived model, simple kinetic expressions for spore germination, tip extension, branching, and hyphal break‐up were set up. It is concluded that spore germination can be quantified by three parameters: (1) the time at which spore germination is initiated, (2) the time at which spore germination terminates, and (3) the fraction of viable spores in a spore suspension. The frequency of spore germination can be described with the B‐distribution. For growth kinetics it is concluded that the branching frequency is closely correlated with the total hyphal length and that the average tip‐extension rate can be described with saturation kinetics with respect to the hyphal length. Finally, the rate of fragmentation is linearly related to the energy input to the bioreactor, and related to the effective hyphal length. © 1995 John Wiley & Sons, Inc.
Biocatalytic synthesis of acrylates in organic solvents and supercritical fluids: III. Does carbon dioxide covalently modify enzymes?
Authors: Kamat Sanjay , Critchley Glenn , Beckman Eric J. , Russell Alan J.
Published: December 2004

We have previously demonstrated that the activity of the lipase (Candida cylindracea) catalyzed transesterification reaction between methylmethacrylate and 2‐ethylhexanol in supercritical carbon dioxide is comparatively low. In this article, we have investigated the same reaction in supercritical carbon dioxide with a special emphasis on determining the extent of any interaction between the enzyme and carbon dioxide. Transesterification reaction rates in hexane and supercritical carbon dioxide are compared at different temperatures. In supercritical carbon dioxide, temperature was found to have no significant effect on reaction rate in the range of 40° to 55°C. Above 55°C, however, the reaction rate increased significantly as a function of temperature. It appears that carbon dioxide forms reversible complexes with the free amine groups on the surface of the enzyme. Direct evidence of modification was obtained using mass spectroscopy to detect the extent of modification of a pure protein. The kinetics of the reaction have been studied in hexane, and they obey a ping‐pong bi–bi mechanism with inhibition by 2‐ethylhexanol. The effect of bubbling carbon dioxide and/or fluoroform on the reaction rate in hexane at different temperatures suggests that the enzyme undergoes shear inactivation in hexane. © 1995 John Wiley & Sons, Inc.
Fundamental denitrification kinetic studies with Pseudomonas denitrificans
Authors: Wang J.‐H. , Baltzis B. C. , Lewandowski G. A.
Published: December 2004

Fundamental kinetic studies on the reduction of nitrate, nitrite, and their mixtures were performed with a strain of Pseudomonas denitrificans (ATCC 13867). Methanol served as the carbon source and was supplied in excess (2:1 mole ratio relative to nitrate and/or nitrite). Nitrate and nitrite served as terminal electron acceptors as well as sources of nitrogen for biomass synthesis. The results were explained under the assumption that respiration is a growth‐associated process. It was found that the sequence of complete reduction of nitrate to nitrogen gas is via nitrite and nitrous oxide.

It was found that the specific growth rate of the biomass on either nitrate or nitrite follows Andrews inhibitory kinetics and nitrite is more inhibitory than nitrate. It was also found that the culture has severe maintenance requirements which can be described by Herbert's model, i.e., by self‐oxidation of portions of the biomass. The specific maintenance rates at 30°C and pH 7.1 were found to be equal to about 28% of the maximum specific growth rate on nitrate and 23% of the maximum specific growth rate on nitrite. Nitrate and nitrite were found to be involved in a cross‐inhibitory noncompetitive kinetic interaction. The extent of this interaction is negligible when the presence of nitrite is low but is considerable when nitrite is present at levels above 15 mg/L.

Studies on the effect of temperature have shown that the culture cannot grow at temperatures above 40°C. The optimal temperature for nitrate or nitrite reduction was found to be about 38°C. Using an Arrhenius expression to describe the effect of temperature on the specific growth rates, it was found that the activation energy for the use of nitrate by the culture is 8.6 kcal/mol and 7.21 kcal/mol for nitrite. Arrhenius‐type expressions were also used in describing the effect of temperature on each of the parameters appearing in the specific growth rate expressions. Studies on the effect of pH at 30°C have shown that the culture reduces nitrate optimally at a pH between 7.4 and 7.6, and nitrite at a pH between 7.2 and 7.3. © 1995 John Wiley & Sons, Inc.
A comparison of lipase‐catalyzed ester hydrolysis in reverse micelles, organic solvents, and biphasic systems
Authors: Yang Fangxiao , Russell Alan J.
Published: December 2004

The performance of lipases from Candida rugosa and wheat germ have been investigated in three reaction media using three acetate hydrolyses as model reactions (ethyl acetate, allyl acetate, and prenyl acetate). The effect of substrate properties and water content were studied for each system (organic solvent, biphasic system, and reverse micelles). Not unexpectedly, the effect of water content is distinct for each system, and the optimal water content for enzyme activity is not always the same as that for productivity. A theoretical model has been used to simulate and predict enzyme performance in reverse micelles, and a proposed partitioning model for biphasic systems agrees well with experimental results. While the highest activities observed were in the micellar system, productivity in microemulsions is limited by low enzyme concentrations. Biphasic systems, however, support relatively good activity and productivity. The addition of water to dry organic solvents, combined with the dispersion of lyophilized enzyme powders in the solvent, resulted in significant enzyme aggregation, which not surprisingly limits the applicability of the “anhydrous” enzyme suspension approach. © 1995 John Wiley & Sons, Inc.
The effect of ammonia on the O‐linked glycosylation of granulocyte colony‐stimulating factor produced by chinese hamster ovary cells
Authors: Andersen Dana C. , Goochee Charles F.
Published: December 2004

Ammonium ion concentrations ranging from 0 to 10 mM are shown to significantly reduce the sialylation of granuiocyte colony‐stimulating factor (G‐CSF) produced by recombinant Chinese hamster ovary cells. Specifically, the degree of completion of the final reaction in the O‐linked glycosylation pathway, the addition of sialic acid in an α(2,6) linkage to N‐acetylgalactosamine, is reduced by NH concentrations of as low as 2 mM. The effect of ammonia on sialylation is rapid, sustained, and does not affect the secretion rate of G‐CSF. Additionally, the effect can be mimicked using the weak base chloroquine, suggesting that the effect is related to the weak base characteristics of ammonia. In support of this hypothesis, experiments using brefeldin A suggest that the addition of sialic acid in an α(2,6) linkage to N‐acetylgalactosamine occurs in the trans‐Golgi compartment prior to the trans‐Golgi network, which would be expected under normal conditions to have a slightly acidic pH in the range from 6.5 to 6.75. Ammonium ion concentrations of 10 mM would be expected to reduce significantly the differences in pH between acidic intracellular compartments and the cytoplasm. The pH‐activity profile for the CHO O‐linked α(2,6) sialytransferase using monosialylated G‐CSF as a substrate reveals a twofold decrease in enzymatic activity across the pH range from 6.75 to 7.0.Mathematical modeling of this sialylation reaction supports the hypothesis that this twofold decrease in sialyltransferase activity resulting from an ammoniainduced increase in trans‐Golgi pH could produce the observed decrease in G‐CSF sialylation. © 1995 John Wiley & Sons, Inc.
Production of a lipopeptide antibiotic, surfactin, by recombinant Bacillus subtilis in solid state fermentation
Authors: Ohno Akihiro , Ano Takashi , Shoda Makoto
Published: December 2004

Production of a lipopeptide antibiotic, surfactin, in solid state fermentation (SSF) on soybean curd residue, Okara, as a solid substrate was carried out using Bacillus subtilis MI113 with a recombinant plasmid pC112, which contains lpa‐14, a gene related to surfactin production cloned at our laboratory from a wild‐type surfactin producer, B. subtilis RB14. The optimal moisture content and temperature for the production of surfactin were 82% and 37°C, respectively. The amount of surfactin produced by MI113 (pC112) was as high as 2.0 g/kg wet weight, which was eight times as high as that of the original B. subtilis RB14 at the optimal temperature for surfactin production, 30°C. Although the stability of the plasmid showed a similar pattern in both SSF and submerged fermentation (SMF), production of surfactin in SSF was 4–5 times more efficient than in SMF. © 1995 John Wiley & Sons, Inc.
Studies of transcriptional state heterogeneity in sporulating cultures of bacillus subtilis
Authors: Chung John D. , Stephanopoulos Gregory
Published: December 2004

Using a flow cytometry‐based technique for detecting p‐galactosidase. lacZ fusions have been used to study the pattern of gene expression exhibited by stationary phase cultures of Bacillus subtilis that have initiated the developmental pathway of spore formation. We have found that within such cultures there exist two distinct cell types: one that has induced the developmental program of gene expression and one that has not. This heterogeneity among transcriptional states is shown to be established early during the stationary phase and plays a significant role in influencing later stationary phase events. Additionally, when this technique is used to study gene expression in mutants that display altered patterns of gene expression, we are able to conclude that the cellular apparatus responsible for sensing and transducing stationary phase developmental signals represents a bottleneck that controls the expression of certain early stationary phase genes. These findings provide a demonstration of the utility of single cell measurements of gene expression and indicate that unless culture heterogeneity is properly taken into account, standard measurements of gene expression may not provide information suitable for the analysis of events occurring at the cellular level. © 1995 John Wiley & Sons, Inc.
Nutrient enrichment and in‐situ waste removal through electrical means for hybridoma cultures
Authors: Chang Yu‐Hsiang David , Grodzinsky Alan J. , Wang Daniel I. C.
Published: December 2004

In‐situ dc electric fields were applied to remove ammonium and lactate from suspension hybridoma cultures (ATCC‐CRL‐1606) which used enriched media. Nutrient concentration was increased fourfold above the normal concentration of DMEM to study enhanced protein product formation in a dc electric field. In the presence of the electric field, hybridoma growth and antibody production were increased 1.5‐fold (from 3.7 × 106 to 9.1 × 106 viable cells/mL) and twofold (from 170 to 505 mg IgG/L), respectively, compared with the control. The effective removal of ammonium and lactate and increased concentrations of the various nutrients accounted for this enhancement. The enriched media caused the overflow metabolism of glucose, glutamine, and various essential amino acids. The overconsumption of glucose also produced substantial amounts of lactate, which in turn greatly increased the medium osmolarity. The increase in medium osmolarity is believed to be one of the causes of cell death in these culture systems.© 1995 John Wiley & Sons, Inc
Recombinant cyclin E expression activates proliferation and obviates surface attachment of chinese hamster ovary (CHO) cells in protein‐free medium
Authors: Renner Wolfgang A. , Lee Kelvin H. , Hatzimanikatis Vassily , Bailey James E. , Eppenberger Hans M.
Published: December 2004

Exogenous growth factors normally required in cell culture activate cell proliferation via the molecular controls of cell‐cycle progression. Highly differing influences of mitogenic stimulation of Chinese hamster ovary (CHO) cells by insulin and basic fibroblast growth factor(bFGF) have been clearly observed in a defined protein‐free medium. CHO K1 cells stimulated only with insulin grow with flattened cell morphology and extensive cell–cell contact, whereas stimulation with only bFGF or bFGF plus insulin results in loss of cell‐cell contact and a transformed and rounded‐up morphology. Compared with insulin‐stimulated cells, bFGF‐stimulated cells exhibit a relatively long G1, and short S phase, and contain higher levels of cyclin E. Observation of elevated levels of cyclin E in wild‐type CHO K1 cells mitogenically stimulated by basic fibroblast growth factor motivated transfection of these cells by a cyclin E expression vector. These transfectants grew rapidly in protein‐free basal medium and had similar cyclin b levels, distributions of nuclear cell‐cycle times, and cell morphologies as bFGF‐stimutated CHO K1 culture. Metabolic engineering of cell‐cycle regulation thus bypasses exogenous growth factor requirements, addressing a priority objective in economical, reproducible, and safe biopharmaceutical manufacturing. © 1995 John Wiley & Sons Inc.
The role of glucose in ajmalicine production by catharanthus roseus cell cultures
Authors: Schlatmann J. E. , Koolhaas C. M. A , Vinke J. L. , ten Hoopen H. J. G. , Heijnen J. J.
Published: December 2004

The role of glucose in ajmalicine production by Catharanthus roseus was investigated in the second stage of a two‐stage batch process. Activities of tryptophan decar‐boxylate (TDC) and anthranilate synthase (AS), two enzymes In the pathway leading to ajmalicine, were higher after induction with 40 g/L glucose than after induction with 60 or 80 g/L glucose. Experiments with different media containing mixtures of glucose and the nonpermeating osmotic agent xylose, and using an already induced culture as inoculum, revealed that a minimum amount of glucose is required to support ajmalicine production after enzyme induction. This requirement was not an osmotic effect. The relation between the glucose concentration and the specific ajmalicine production rate, qp, was investigated in seven (fed‐)batch cultures with constant glucose concentrations: 23, 29, 35, 53, 57, 75, and 98 g/L. In the cultures with a low glucose concentration (23, 29, and 35 g/L) the qp was 2.7‐times higher than the cultures with 53 and 57 g/L, and almost six times higher than the cultures with a high glucose concentration (75 and 98 g/L). A glucose perturbation experiment (from 53 to 32 g/L) demonstrated that the ajmalicine production rate was adjusted without much delay. A kinetic equation is proposed for the relationship between the glucose concentration and qp. Differences in enzyme induction and ajmalicine production at different glucose levels could not be explained by the intracellular concentrations of glucose, fructose, sucrose, or starch. © 1995 John Wiley & Sons Inc.
Surface IgG content of murine hybridomas: Direct evidence for variation of antibody secretion rates during the cell cycle
Authors: Charlet Marc , Kromenaker Sandra J. , Srienc Friedrich
Published: December 2004

Previous experiments have shown that population average surface lgG content is correlated with the specific antibody production rates of batch hybridoma cultures. Therefore, surface associated lgG content of single hybridoma cells might indicate antibody secretion rates of individual cells. Moreover, the surface lgG content should reflect the pattern of secretion rates during the cell cycle. To probe for lgG secretion rates during the cellcycle, a double staining procedure has been developed allowing simultaneousflow cytometric analysis of surface lgG content and DNA content of murine hybridoma cells. Crosslinking of the surface associated immunofluorescence with the cell by paraformaldehyde fixation permits subsequent DNA staining without loss of immunofluorescence. The optimized protocol has been used to determine the pattern of the surface lgG fluorescence as a function of the cell cycle position. It is highest during the G2+M cell cycle phase and the experimental data are in excellent agreement with the previously predicted secretion pattern during the cell cycle. © 1995 John Wiley & Sons Inc.
Kinetic model of disaccharide oxidation by Agrobacterium tumefaciens
Authors: Walter Jan , Jördening Hans‐Joachim
Published: December 2004

Disaccharides were microbaially transformed to their corresponding 3‐keto‐derivatives by resting cells of Agrobacterium tumefaciens NCPPB 396. The kinetics and yield of this highly specific oxidation depend on several factors. The oxygen concentration especially has a major influence on the production of 3‐keto‐derivatives and was investigated kinetically with respect to low stationary oxygen concentrations in solution. Experiments showed unconventional results that conflicted with normal Michaelis‐Menten kinetics. A kinetic model was developed and the kinetic constants were calculated. The model and experimental data for sucrose, maltose, iso‐maltulose (palatinose), and leucrose are in good agreement with each other. Initial reaction rates with different sugars using constant oxygen concentrations resulted in a Michaelis–Mentent type function. The complete kinetics, including the effect of disaccharide and oxygen concentrations, are presented. © 1995 John Wiley & Sons, Inc.
Characterization of an oligonucleotide‐binding fluorescent ligand for application in affinity purification of dsDNA
Authors: Haynes Charles A. , Sherwood Christopher S. , Turner Robin F. B.
Published: December 2004

The fluorescent probe PO‐PRO‐3 was investigated as a potential ligand for the affinity immobilization and purification of genomic or plasmid DNA fragments. Affinities and mechanisms for PO‐PRO‐3 binding to superhelical and linearized pUC 18 plasmid DNA were examined through measurement of binding isotherms, continuous‐variation analysis, and DNA titrations. In addition, the effects of DNA conformation, protein and RNA contaminants, solvent polarity, and ionic strength are examined with the aim of optimizing binding and elution conditions and of assisgning limits to the range of applicability of the affinity purification. © 1995 John Wiley & Sons, Inc.
Transesterification catalyzed by polystyrene‐supported chymotrypsin in toluene: The effect of neutralization of basic or acidic groups attaching to polystyrene resins
Authors: Ohtani Noritaka , Inoue Yukihiko , Kobayashi Atsushi , Sugawara Toshiki
Published: December 2004

Crosslinked polystyrene resins containing a low level of either basic or acidic groups were used for supports of α‐chymotrypsin (CT), which catalyzed the transesterification of N‐acetyl‐L‐phenylalanine ethyl ester (AcPheOEt) with propanol in toluene. With a minimal amount of water, CT was sorbed to the resins, basic or acidic groups of which were partly or fully neutralized by several soluble acids or bases. With an increasing degree of neutralization of basic resins by free acids, the rate of disappearance of AcPheOEt was decreased, whereas the by‐product formation of AcPheOH, due to hydrolysis, was considerably suppressed, compared with the ester‐exchange product, AcPheOPr. The pKa value of the neutralizing acid was also important for both CT activity and reaction selectivity. AcPheOPr was selectively produced at a certain range of pKa values. On the other hand, the neutralization of acidic resins with free amines enhanced the CT activity but a strong base promoted the formation of hydrolysis product. © 1995 John Wiley & Sons, Inc.
A combined cell‐cycle and metabolic model for the growth of hybridoma cells in steady‐state continuous culture
Authors: Martens D. E. , Sipkema E. M. , de Gooijer C. D. , Beuvery E. C. , Tramper J.
Published: December 2004

The Model presented in this work demonstrates the combination of cell‐cycle model with a model describing the growth and conversion kinetics of hybridoma cells in a steady‐state continuous culture. The cell‐cycle model is based upon a population balance model as described by Cazzador et al. and assumes the existence of a cycling‐and apoptotic‐cell population, which together form the viable‐cell population. In this part the fraction of apoptotic cells, the age distribution of the cycling and apoptotic‐cell population, the mean volume and biomass content per cell of the cycling, apoptotic, and viable cells, and the specific growth and death rates of the cells are calculated. The metabolic part consists of a Monod‐type growth equation, four elemental balances, an equation assuming a constant yield of ammonia on glutamine, an equation for product formation, and the relation of Glacken for energy production. Furthermore, a maintenance‐energy model for the consumption of glucose and glutamine is introduced, which combines the approaches of Herbert and Pirt into one model in a way similar to Beeftink et al. For energy consumption a Pirt model is assumed. The model is capable of predicting trends in steady‐state vaues of a large number of variables of interest like specific growth rate, specific death rate, viability, cell numbers, mean viable‐cell volume, and concentrations and conversion rates of product, glucose, glutamine, lactate, and ammonia. Also the concentrations and conversion rates of oxygen and carbon dioxide are qualitatively predicted. The values of the model predictions are generally close to experimental data obtained from literature. © 1995 John Wiley & Sons, Inc.
Studies on the synthesis of βhCG hormone in vero cells by recombinant vaccinia virus
Authors: Mukhopadhyay Asok , Mukhopadhyay S. N. , Talwar G. P.
Published: December 2004

Synthesis of the β‐subunit of human chorionic gonado‐tropin (βhCG) in Vero cells by the recombinant vaccinia virus has been studied. The yield of βhCG was a function of the multiplicity of infection (MOI), and was highest at 25 MOI. The kinetics of synthesis and initial secretion of βhCG, deduced from the pulse‐chase experiments were “zero order.” At 30 h postinfection, the relative values of net synthesis and secretion rates were 4.0 AU. mm2 βhCG/106 cells. h and 1.55 AU. mm2 βhCG/106 cells. h, respectively. The time required to secrete 50% of intracellular βhCG was 210 min. Pulse‐chase data also showed that 24% of βhCG was degraded intracelluiarly within 10 h, of which 17% was detected in the autoradiogram. Along with 30 kD βhCG, a satellite band of 28 kD was evident among the peptide synthesized in Vero cells. The molecular weight of vaccinia‐derived βhCG was 13 kD more that its nonglycosylated form, indicating extensive glycosylation in Vero cells. The mRNA levels in infected Vero cells at different postinfection times were quantified by excess DNA dot‐blot hybridization. It appears that the Vero cell possesses some host cell‐associated factor(s), which prevented the transcription of early βhCG–mRNA promoted by the early signal of the vaccinia P 7.5 promoter. The half‐life of βhCG–mRNA, as determined by follow‐up of decay after blocking transcription initiation, was found to be 6.4 h. The synthesized βhCG was immunoreactive as it reacted with monoclonal and polyclonal monospecific antibodies. The subunit was also biologically active, as it combined with native βhCG to form heterodimer βhCG, which competed with 125I‐hCG for radioreceptors and stimulated testosterone synthesis in Leydig cells. © 1995 John Wiley & Sons, Inc.
Sporulation: An alternative way to recover recombinant proteins from Bacillus subtilis
Authors: Maurizi Giovanni , Bossù Paola , Boraschi Diana , Ulisse Edy , Tagliabue Aldo , Ruggiero Paolo
Published: December 2004

A recombinant strain of Bacillus subtilis engineered for endocellular expression of human interleukin‐1 receptor antagonist (IL‐Ira) was subjected to sporulation. The recombinant protein was recovered from the sporulation supernatant in quantities, purity, and activity comparable with those obtained from a traditional cell lysate. Thus, exploitation of this natural mechanism of autolysis could overcome problems of intact protein recovery related to the cell disruption step. © 1995 John Wiley & Sons, Inc.
Real‐time monitoring of protein secretion in mammalian cell fermentation: Measurement of monoclonal antibodies using a computer‐controlled HPLC system (BioCad/RPM)
Authors: Ozturk S. S. , Thrift J. C. , Blackie J. D. , Naveh D.
Published: December 2004

On‐line, “real‐time” monitoring of product concentration is important for mammalian cell culture fermentation. The continuous measurement of monoclonal antibodies allows for instantaneous determination of cell productivity and effective manipulation of the fermentor operating conditions for optimal production. This article will present the evaluation and application of a BioCad/RPM system (Per Septive Biosystems) for rapid analysis of lgG concentration for hybridoma cell cultivation. Several commercial crossflow filtration devices are tested for low protein retention and fouling properties. A protein G column is used successfully for analyzing about 400 samples of lgG1, without significant loss in separation efficiency. The Immuno Detection system is integrated into a computer‐controlled 15‐L fermentor. This fermentor could be operated in batch and perfusion modes with cell densities up to 20 million cells/mL. A continuous cell‐free sample stream obtained by a hollow fiber filter system is introduced to the BioCad/RPM for analysis. The speed of this system allows for real‐time monitoring even at high densities with fast dynamics. A murine hybridoma cell (A10G10) is cultivated in batch and continuous reactors and antibody concentration is measured continuously with complete sterility. The results are compared to offline measurements with good agreement. © 1995 John Wiley & Sons, Inc.
Fed‐batch culture of Bacillus thuringiensis for thuringiensin production in a tower type bioreactor
Authors: Jong Jian‐Zhong , Hsiun Ding‐Yu , Wu Wen‐Teng , Tzeng Yew‐Min
Published: December 2004

Fed‐batch culture of Bacillus thuringiensis in a modified airlift reactor has been developed by using adaptive control of glucose concentration in the reactor. The glucose concentration was estimated via a correlation equation between carbon dioxide production rate and glucose consumption rate. The estimated glucose concentration as the output variable was fed back to computer for calculation of substrate addition. The modified reactor was an airlift reactor with a net draft tube. The airlift reactor had high oxygen transfer rate and low shear stress which were important factors for production of thuringiensin. Fed‐batch culture of Bacillus thuringiensis in the modified airlift reactor provided significant improvement of thuringiensin production. © 1995 John Wiley & Sons, Inc.
The effect of particle morphology and concentration on the directly measured yield stress in filamentous suspensions
Authors: Mohseni M. , Allen D. G.
Published: December 2004

The influence of concentration (mass and volume fraction) and particle morphology on the yielding properties of filamentous broths ofAspergillus niger and Strepto‐myces levoris was investigated using the rotating vane technique and compared with those of pulp suspensions. Two methods were applied to determine the volume fraction of the cells growing in filamentous form: the measurement of interstitial volume using a high molecular weight dye or dextran, and the measurement of dewatered broth filter cake volume by displacement using a pycnometer. The latter method provided the most reliable results. Cell concentrations ranging from 3 to 20 g dw/L, with corresponding volume fractions between 0.005 and 0.05, were obtained with broths generated in stirred tank and shake flask fermentations. The yield stress values obtained using the vane technique (0.1 ≤ τy ≤ 37 Pa) correlated with biomass concentration (X, grams dry weight per liter) using the equation τy = aXb with “b” ranging between 2 and 3. Increasing the length of organisms significantly increased the yield stress at a given cell concentration; this morphology effect was described using a “morphology factor” (range 0.016 to 0.11 Pa/[g dw/L]2.5), determined by setting the value of “b” to 2.5. The vane yield strain, which is the critical strain or deformation at the yield point, was independent of cell concentration, but increased with increasing length of organisms. Filamentous fermentation broths with highly branched organisms had a greater yield stress than pulp suspensions. © 1995 John Wiley & Sons, Inc.
Biomimetic‐dye affinity adsorbents for enzyme purification: Application to the one‐step purification of Candida boidinii formate dehydrogenase
Authors: Labrou N. E. , Karagouni A. , Clonis Y. D.
Published: December 2004

Formate dehydrogenase (FDH, EC 1.2.1.2) was purified from Candida boidinii cells in a single step by biomimetic‐dye affinity chromatography. For this purpose, seven' biomimetic analogues of the monochlorotriazine dye, Cibacron® Blue 3GA (CB3GA), and parent dichloro‐triazine dye, Vilmafix® Blue A‐R (VBAR), bearing a car‐boxylated structure as their terminal biomimetic moiety, were immobilized on crosslinked agarose gel, Ultrogel® A6R. The corresponding new biomimetic‐dye adsorbents, along with nonbiomimetic adsorbents bearing CB3GA and VBAR, were evaluated for their ability to purify FDH from extracts obtained after press‐disintegration of C. boidinii cells. Optimal conditions for maximizing specific activity of FDH in starting extracts (1.8 U/mg) were realized when cell growth was performed on 4% methanol, and press disintegration proceeded in four consecutive passages before the homogenate was left to stand for 1 h (4°C). When compared to nonbiomimetic adsorbents, biomimetic adsorbents exhibited higher purifying ability. Furthermore, one immobilized biomimetic dye, bearing as its terminal biomimetic moiety mercap‐topyruvic acid linked on the chlorotriazine ring (BM6), displayed the highest purifying ability. Adsorption equilibrium data which were obtained for the BM6 adsorbent in a batch system corresponded well to the Langmuir isotherm and, in addition, breakthrough curves were taken for protein and FDH adsorption in a fixed bed of BM6 adsorbent. The dissociation constant ( KD) of the complex between immobilized BM6 and FDH was found to equal 0.05 μM. Adsorbent BM6 was employed in the purification of FDH from a 18‐L culture of C. boidinii in a single step (60% overall yield of FDH). The purified FDH afforded a single‐band on sodium dodecyl sulphate poly‐acrylamide gel electrophoresis, and a specific activity of 7,0 U/mg (30°C). © 1995 John Wiley & Sons, Inc.
Recovery of insect cells using hollow fiber microfiltration
Authors: Trinh Loc , Shiloach Joseph
Published: December 2004

An efficient method was developed for media separation and cell collection for eukaryotic cells growing in suspension. The method is based on tangential flow microfiltration using an open channel arrangement in a hollow fiber configuration. Best results (highest processing flux rate) for polysulfone hollow fibers were obtained using fibers with internal diameter of 0.75 mm, 0.45 μm pore size, and a cell suspension flow at a shear rate of 14000 s−1 (0.032 L/min per fiber). A flux rate of 500 L/m2 h can be obtained by maintaining the surface area/cell ratio at 0.05 m2/10 L of cells at a concentration of 2.5 × 106 cells/mL. Forty liters of infected insect cells can be concentrated 10 times in 20 min without affecting cell viability. © 1995 John Wiley & Sons, Inc.