bluuebunny/crossref_metadata_2025 huggingface dataset:
α‐Factor directed expression of the human epidermal growth factor in Saccharomyces cerevisiae
Authors: Coppella Steven J. , Dhurjati Prasad
Published: May 2005

Expression kinetics of the human Epidermal Growth Factor (hEGF) from the α‐factor prepro region in a 2‐μm based plasmid was studied in Saccharomyces cerevisiae. Production of hEGF was highly medium de pendent as a chemically defined, nonenriched media had a significantly lower yield than did enriched media. Also cells grown on yeast nitrogen base without amino acids with casamino acids degraded the hEGF after cell growth as opposed to a yeast extract, peptone, and dextrose (YEPD) medium, which elicited no measurable extracellular proteolysis of the hEGF. α‐factor directed production kinetics of hEGF on the YEPD medium were growth associated, secretion limitations and extracellular degradation were negligible, and the hEGF was nearly 100% selectively secreted. With sufficient agitation, shake flask experiments were representative of aerated controlled batch fermentations. No effect of high cell density was observed on cell growth or hEGF production kinetics. The hollow fiber bioreactor had no direct effect on the substrate or protein yields of S. cerevisiae, however the low oxygen transfer capacity of the membrane was not sufficient to support respiration.
Transient kinetics of hybridoma growth and monoclonal antibody production in serum‐limited cultures
Authors: Dalili Mina , Ollis David F.
Published: May 2005

A quantitative study of the influence of initial serum concentration on hybridoma growth rate, maximum viable and total cell yield, and specific antibody production rate is presented. The specific growth rate varied in a Monod fashion with initial serum levels (2–10% FCS), giving Km = 1.6 v/v% and μmax = 0.92 d−1. The maximum cell yields (total and viable) were linear with initial serum level, indicating stoichiometric as well as kinetic limitation by serum component(s). The specific antibody production rate for each individual run fitted well to a non‐growth‐associated model. However, the non‐growth‐associated parameter varied monotonically with initial serum concentration, suggesting the catalytic role of serum component(s) in antibody production. Also, glutamine was utilized inefficiently, with at least a third of it secreted back into the culture supernate in the form of glutamate. While very simple model equations describe the specific growth and product formation rate for an individual batch run, the larger picture indicates need for a more detailed unstructured or structured model.
Induction of T4 DNA ligase in a recombinant strain of Escherichia coli
Authors: Whitney Gordon K. , Glick Bernard R. , Robinson Campbell W.
Published: May 2005

The kinetics of cell growth and foreign protein production, as well as factors affecting protein stability, were studied and optimized in batch and fed‐batch fermentations of a recombinant strain of Escherichia coli. The pL promoter from bacteriophage lambda under the control of a temperature‐sensitive cl represser, with the entire construct integrated into the E. coli chromosome through the use of a defective bacteriophage lambda lysogen, was used to direct the synthesis of T4 DNA ligase. The biphasic fermentations consisted of a primary growth phase at 30°C followed by an induction phase which was initiated by shifting the temperature to 42°C. In the fed‐batch fermentations, additional nutrients were added at the time of initiating induction. Maintenance of sufficiently high concentrations of the organic substrates (glucose and casamino acids) during the induction phase was required for continued cell growth at 42°C. Such growth was essential for T4 DNA ligase formation and in vivo stability. Hence, fed‐batch fermentations produced the highest yield of the foreign protein Commensurate with providing lower total amounts of substrates. In such cases, high cell densities (6 g dry wt/L) with substantial intracellular levels of T4 DNA ligase (4.6% total cellular protein, or 2.7% of the dry biomass) were achieved.
Adaptive on‐line optimizing control of bioreactor systems
Authors: Shi Zhongping , Shimizu Kazuyuki , Watanabe Norihiro , Kobayashi Takeshi
Published: May 2005

Several on‐line optimizing control strategies were proposed and tested by computer simulation for the efficient operation of bioreactors. The control task was divided into two, one of which was to search for the optimal operating point and passed the set point to the lower layer of which task was to make the process output follow the set point as soon as possible. It was shown to be effective for the upper layer to express the objective function as a polynomial with respect to the measurement variable and to make use of it for finding the optimum point. Noting that the major dynamic characteristics of bioreactor system is the time‐varying and nonlinear nature, the adaptive type control system is in evitable. It was shown to be quite effective to use discrete type self‐tuning PID controller and the optimal controller compensated for the interaction between the control loops.

Application was made to the cell recycle system for the production of lactic acid and baker's yeast cultivation. I was found from the former application that the control quality can be significantly improved by incorporating the decoupling strategy into the lower layer closed‐loop system. It was also found from the latter application that the initial startup period can be significantly reduced by making use of the rough mathematical model.
A mathematical model for the growth of a single cell of E. coli on a glucose/glutamine/ammonium medium
Authors: Shu J. , Shuler M. L.
Published: May 2005

Extension of a single cell model of E. coli B/r to make predictions of culture response to variations in glutamine/glucose/ammonium ion concentrations is described. A biphasic glutamine transport system, a nitrogen metabolism scheme that includes glutamate dehydrogenase (GDH), glutamine synthetase/glutamate synthase (GS/GOGAT), the glutaminase routes, and a transaminase mechanism for glutamine carbon usage are added to the prototype model. The predictions of the extended model with regard to nutrient concentrations and cell size compare well with the experimental data and the prototype model predictions, demonstrating the capability of the integrated kinetic model to illustrate important enzymological interactions in a biological system. The discrepancies between the experimental data and the model predictions on growth yield suggest that a more detailed regulatory system of the TCA cycle is required for a more accurate energy budget.
Batch and continuous 2‐heptanone production by Ca‐alginate/Eudragit RL entrapped spores of Penicillium roquefortii
Authors: Larroche Christian , Gros Jean‐Bernard
Published: May 2005

The synthesis of 2‐heptanone from octanoic acid by Caalginate/Eudragit RL entrapped spores of Penicillium roquefortii is performed in batch and continuous reactions. The measurement of the HCI solution needed to overcome the pH increase during the course of a reaction allows continuous monitoring of the reaction progress of batch as well as continuous processes without any aliquot sampling. The hydration of the biocatalyst prior to a bioconversion performance is of major importance to achieve reproducible results. A continuous reaction carried out at pH 6.5 for one month demonstrates the feasibility of such a process and shows that the reaction occurs with a Michaelian behavior, with KM = 1.82 mmol/L and rsm = 0.82 mmol/L h, and no intraparticle diffusion limitation is found. The complex behavior of the spores during batch reactions makes this kind of operation unsuitable for kinetic studies.
Purification of two immunologically distinct endoglucanases without affinity for microcrystalline cellulose from Cellulomonas sp. ATCC 21399
Authors: Poulsen O. M. , Petersen L. W.
Published: May 2005

Two endoglucanases (endoglucanase B and endoglucanase C) without affinity for cellulose were purified from the culture broth of Cellulomonas sp. ATCC 21399 using gelfiltration and ion exchange chromatography. Fused rocket immunoelectrophoresis was used to select the fractions with the highest content of endoglucanase and lowest content of contaminating proteins. The endoglucanases were purified to immunological homogeneity. In addition both endoglucanases were homogeneous when analyzed by sodium dodecyl sulfate—polyacrylamide gel electrophoresis (molecular weights of endoglucanase B and endoglucanase C were 67000 and 25000, respectively). Endoglucanase B was homogeneous when studied by isoelectric focusing showing one protein band at pl 4.3. Both endoglucanases lacked activity against microcrystalline cellulose (Avicel) and showed similar endo action on carboxymethylcellulose (CMC). Endoglucanase B had a high specific activity against CMC, H3PO4‐swollen Avicel and xylan, but showed no activity against galactomannan. In contrast, endoglucanase C showed activity against both CMC, xylan, and galactomannan all being polysaccharide substrates linked with β‐1‐4‐D‐glucoside bonds. The specific activity of endoglucanase C against H3PO4‐swollen Avicel was low.
Adaptive predictive control of a multistage fermentation process
Authors: Vigié P. , Goma G. , Renaud P. Y. , Chamilothoris G. , Dahhou B. , Pourciel J. B.
Published: May 2005

Theoretical and experimental studies concerning the application of modern adaptive techniques for the control of continuous alcoholic fermentation of glucose by a yeast strain conducted in a multistage reactor are reported. The practical control objective was the regulation of the substrate concentration in the process effluent. Mathematical expressions for the control scheme are given, with the underlying control engineering argumentation. Suitable feeding of sugar for stage 2 was controlled to minimize the effluent sugar concentration.
Optimal temperature control for a structured model of plant cell culture
Authors: Bailey C. M. , Nicholson H.
Published: May 2005

This article calculates optimal open‐loop temperature trajectories that maximize the average rate of product synthesis of a plant cell culture. It uses a previously published five‐state mathematical model which describes the growth and product synthesis of a batch plant cell suspension culture of Catharanthus roseus under temperature control. The optimal open‐loop temperatures maximize the final product concentration for predefined fermentation periods. A single switch in temperature is shown by computer simulation to be near optimal, with a 22% increase in final product yield over that obtained at the optimal constant temperature. Examination of the achieved final product yield as a function of fermentation period allows this period also to be chosen optimally. This time is reduced from 16 days in the constant temperature case to 12 days in the switched temperature case.
Effect of operating parameters on specific production rate of a cloned‐gene product and performance of recombinant fermentation process
Authors: Park Sunghoon , Ryu Dewey D. Y.
Published: May 2005

A two‐stage continuous system in combination with a temperature‐sensitive expression system were used as model systems to maximize the productivity of a cloned gene and minimize the problem associated with the plasmid instability for a high‐expression recombinant. In order to optimize the two‐stage fermentation process, the effects of such operational variables as temperature and dilution rate on productivity of cloned gene were studied using the model systems and a recombinant, Escherichia coli K12 ΔH1 Δtrp/pPLc23trp A1. When the expression of cloned gene is induced by raising the operating temperature above 38°C, a significant decrease in the colony‐forming‐units (CFU) of the plasmid‐harboring cell was observed, and the decrease was related to the product concentration. In order to describe this phenomenon, a new kinetic parameter related to the metabolic stress (metabolic stress factor) was introduced. It is defined as the ratio of the rate of change of pheno‐type from colony‐forming to non‐colony‐forming cells to the product accumulation per unit cell mass. At a fixed temperature of 40°C, the varying dilution rate D in the range of 0.35‐0.90 h−1 did not affect the metabolic stress factor significantly. At a fixed dilution rate of D = 0.35 h−1, this factor remained practically constant up to 41°C but increased rapidly beyond 41°C. The effects of temperature and dilution rate in the second stage on the specific production rate were also studied while maintaining the apparent specific growth rate (μ) of the second stage constant at or near μ = 0.26 h−1. Under a constant dilution rate, D2 = 0.35 h−1, the maximum specific production rate obtained was about qp, max = 38 units TrpA/mg cell/h at 41°C. At a constant temperature, T2 = 40°C, specific production rate increased with decreasing dilution rate with in the dilution rate range of D2 = 0.35‐0.90 h−1. Based on the results of our study, the optimal operating conditions found were dilution rate D2 = 0.35 h−1 and operating temperature T2 = 41°C at the apparent specific growth rate of 0.26 h−1. Under the optimal operating conditions, about threefold increase in productivity was achieved compared to the best batch culture result. In addition, the fermentation period could be extended for more than 100 h.
Cultivation of plant cells in a stirred vessel: Effect of impeller design
Authors: Hooker Brian S. , Lee James M. , An Gynheung
Published: May 2005

Suspension cultures of Nicotiana tabacum were grown in a batch fermentor using different agitation systems. The effects of the impeller type, size, and agitation speed on the productivity of cell mass and secondary metabolites (phenolics) have been investigated. The use of a large, flat‐bladed impeller (diameter 7.6 cm; width 14.0 cm) improved culture growth significantly over systems using a regular, flat‐bladed impeller (diameter 5.6 cm; width 1.5 cm). An impeller of the same dimensions as the 14.0‐cm‐wide, large, flat‐bladed impeller with sail cloth blades yielded a higher maximum growth rate in the exponential phase but resulted in a longer lag phase. Overall (intracellular and extracellular) phenolics concentration showed a direct relationship to culture growth rate whereas extracellular concentrations were a function of agitation conditions. Power consumption and flow pattern studies were also completed to further characterize the different impellers tested.
On‐line optimization of biotechnological processes: I. Application to open algal pond
Authors: Guterman H. , Ben‐Yaakov S.
Published: May 2005

A new on‐line optimization and control procedure applicable to biotechnological systems for which a precise mathematical model is unavailable has been developed and tested. The proposed approach is based on an online search for optimum operating conditions by an automatic system using a modified simplex algorithm to which several features have been added to permit real time operation. The simplex algorithm is the upper level of a hierarchical software package in which the other levels are cost evaluation, control, data acquisition, and signal processing. The optimization method was tested in a laboratory minipond for the cultivation of Spirulina platensis. The controlled parameters were light intensity, optical density, pH, and temperature. The proposed optimization method can be applied to other biological processes provided that the pertinent variables can be measured and controlled and the cost function can be defined mathematically.
Approximation of continuous fermentation by semicontinuous cultures
Authors: Westgate Paul J. , Emery Alden H.
Published: May 2005

Semicontinuous fermentations, in which a fraction of a culture is replaced with fresh media at regular intervals, have been previously used as a means of approximating continuous growth. In most cases deviations from continuous operation were erroneously estimated using Fencl's model, which is only valid when the specific growth rate is independent of the substrate concentration. An approach to modeling Semicontinuous growth that incorporates the same kinetics followed in batch and continuous growth was developed and tested for Monod's expression for the specific growth rate. A dimensionless form of the model was used to simulate Semicontinuous fermentations for comparison to continuous growth. Differences between Semicontinuous and continuous growth were found to depend on three dimensionless variables: feed concentration, replacement rate, and time between replacements. For given values of the dimensionless feed concentration and time between replacements, a range of dimensionless replacement rates can be determined over which semi‐continuous cultures are approximately continuous.
A thermodynamic model to predict Phanerochaete chrysosporium INA‐12 adhesion to various solid carriers in relation to lignin peroxidase production
Authors: Asther Marcel , Bellon‐Fontaine Marie‐Noëlle , Capdevila Cécile , Corrieu Georges
Published: May 2005

A thermodynamic model was used in this study to predict the adhesion of Phanerochaete chrysosporium INA‐12 as conidiospores or mycelium to various solid carriers. Theoretical predictions were closely reflected by experimental results. Amount of immobilized mycelium was higher for hydrophobic (polypropylene and polyurethane) than for hydrophilic carrier (stainless steel and grey). Lignin peroxidase production was stimulated in the same way. However, better results were obtained with polyurethane than with polypropylene and with grey than with stainless steel. These results were attributed to roughness effects of solid surfaces. Surface morphology characterization showed that the surface roughness parameter RA was higher for polyurethane and grey as compared to polypropylene and stainless steel, respectively. On the other hand, polyurethane is not simply rugous; it has an intraparticle porosity as well as a higher total surface area as compared to polypropylene.
Mathematical modeling of a single‐cell enzyme assay
Authors: Wittrup K. Dane , Bailey James E.
Published: May 2005

A quantitative assay of β‐galactosidase activity in single cells of Saccharomyces cerevisiae has been developed using a fluorogenic substrate and flow cytometry [reported in Wittrup & Bailey, Cytometry, 9,394 (1988)]. The β‐galactosidase activity is expressed in yeast from the Escherichia coli lacZ gene under the control of the yeast GAL10 promoter, and is used as a marker for multicopy plasmid content. A nonfluorescent fluorogenic substrate is enzymatically cleaved by intracellular β‐galactosidase to form a fluorescent product. The accumulation of fluorescent product in single cells was found to depend on bulk substrate concentration and single‐cell enzyme activity in a fashion that could not be described by a Michaelis–Menten kinetic rate form. It has been demonstrated that diffusion limitation rather than enzyme activity can determine the level of single‐cell fluorescence under certain assay conditions, and a mathematical model has; been formulated which accounts for substrate and product diffusion. Guided by the mathematical model, the assay conditions were modified to allow measurement of single‐cell enzyme activity rather than diffusion rates.
Propagation of an amplifiable recombinant plasmid in Saccharomyces cerevisiae: flow cytometry studies and segregated modeling
Authors: Wittrup K. Dane , Bailey James E. , Ratzkin Barry , Patel Avantika
Published: May 2005

Efficient expression of a foreign protein product by the yeastSaccharomyces cerevisiaerequires a stable recombinant vector present at a high number of copies per cell. A conditional centromere yeast plasmid was constructed which can be amplified to high copy number by a process of unequal partitioning at cell division, followed by selection for increased copy number. However, in the absence of selection pressure for plasmid amplification, copy number rapidly drops from 25 plasmids/cell to 6 plasmids/cell in less than 10 generations of growth. Copy number subsequently decreases from 6 plasmids/cell to 2 plasmids/cell over a span of 50 generations. A combination of flow cytometric measurement of copy number distributions and segregated mathematical modeling were applied to test the predictions of a conceptual model of conditional centromereplasmid propagation. Measured distributions of plasmid content displayed a significant subpopulation of cells with a copy number of 4–6, evenin a population whose mean copy number was 13.5. This type of copy number distribution was reproduced by a mathematical model which assumes that amaximum of 4‐6 centromere plasmids per cell can be stably partitionedat cell division. The model also reproduces the observed biphasic kinetics of plasmid number instability. The agreement between simulation and experimental results provides support for the proposed model and demonstrates the utility of the flow cytometry/segregated modeling approach for the study of multicopy recombinant vector propagation.
Oxygen transfer enhancement in aqueous/perfluorocarbon fermentation systems: II. theoretical analysis
Authors: Junker Beth H. , Wang Daniel I. C. , Hatton T. Alan
Published: May 2005

Two theoretical approaches were examined to quantify observed experimental oxygen transfer enhancements in aqueous/perfluorocarbon (pfc) fermentation systems. Steady‐state macroscopic balance results indicated that enhancements in the presence of reaction were a function of the gas bubble‐to‐droplet diameter ratio for pfc‐in‐water dispersions, but primarily a function of only the droplet diameter for water‐in‐pfc dispersions. The problem of unsteady‐state diffusion into a semi‐infinite composite medium was employed to predict enhancements in the absence of reaction based on the relative permeabilities of the perfluorocarbon and water phases. Since potential enhancements exceeded actual enhancements in the presence of an oxygen consuming reaction for water‐in‐pfc dispersions, the use of less expensive, lower oxygenpermeable oil phases is recommended in practice.
Critical assessment of the steady‐state Na2SO3 feeding method for kla measurement in fermentors
Authors: Linek V. , Beneš P. , Sinkule J.
Published: May 2005

Important aspects of kla measurement in agitated aerated vessels are briefly characterized from the standpoint of reliability of the measured data. It seems that most of the kla data, based on a number of variants of the steady‐state and dynamic methods in noncoalescent liquids, do not have a clear physical meaning, because they are affected by the differences between the actual driving force and the driving force assumed by the model used for its evaluation. A reliability test is given for the Na2SO3 feeding steady‐state method (FSM), by comparing the results of air and pure oxygen absorption in a noncoalescent liquid (0.5M Na2SO4 solution) with the results obtained by the independent pressure step dynamic method (RDM). The RDM is one of a few variants of the dynamic method which gives correct kla data unaffected by nonideal mixing of the gas phase in the reactor. It was found that the FSM yields correct kla values only when pure oxygen is used for absorption. When air is absorbed, the FSM gives kla values in the region of kla > 0.1 s−1 substantially (to 55%) lower than those for pure oxygen absorption.
Esterification reactions of lipase in reverse micelles
Authors: Hayes Douglas G. , Gulari Erdogan
Published: May 2005

The activities of lipase from Candida cylindracea and Rhizopus delemar have been investigated in water/AOT/iso‐octane reverse micellar media through the use of two esterification reactions: fatty acid‐alcohol esterification and glyceride synthesis. Such media promotes the occurrence of these two lipase‐catalyzed reactions due to its low water content. The effect of various parameters on the activity of lipase from C. cylindracea in reverse micelles was determined and compared to results where alternate media were employed. It was observed that the structure of the media, as dictated by the type and concentration of the substrates and products and by the water/AOT ratio, w0, had a strong impact on enzyme activity. Strong deactivation of both typase types occurred in reverse micelles, especially in the absence of substrates and for w0 values greater than 3.0. Glyceride synthesis was realized with lipase from R. delemar, but not with that from C. cylindracea; the temperature and concentration of substrates and water strongly dictated the reaction rate and the percent conversion.
Inhibition of Trichoderma reesei cellulase by sugars and solvents
Authors: Holtzapple Mark , Cognata Mona , Shu Yuancai , Hendrickson Christie
Published: May 2005

Inhibition of Trichoderma reesei cellulase by sugars (glucose, δ‐gluconolactone, and cellobiose) and solvents (ethanol, butanol, and acetone) was studied using cellulose azure. Glucose, cellobiose, ethanol, and butanol were noncompetitive inhibitors, δ‐gluconolactone was a mixed inhibitor, and acetone was a noncompetitive activator. Converting cellobiose to glucose reduces the effective inhibitor binding constant by 6 times and converting cellobiose to ethanol reduces it by 16 times.
Prediction of the profiles of the elution bands of proteins in preparative liquid chromatography
Authors: Katti Anita , Huang Jun‐Xiong , Guiochon Georges
Published: May 2005

A semiequilibrium model of chromatography has been developed to account for the elution profile of high concentration bands. The use of this model requires the prior determination of the equilibrium isotherms of the studied compounds in the chromatographic phase system. The model is applied here to a study of the isocratic elution bands of chicken albumin and conalbumin on a weak anion exchanger in aqueous buffered solutions. Satisfactory agreement is observed between the experimental elution profiles of large‐sample‐size bands of chicken albumin and conalbumin.
Chitin/chitosan transformation by thermo‐mechano‐chemical treatment including characterization by enzymatic depolymerization
Authors: Pelletier A. , Lemire I. , Sygusch J. , Chornet E. , Overend R. P.
Published: May 2005

Chitosan, the deacetylated derivative of chitin, was until recently produced by hydrolysis in 50% (w/v) NaOH. Application of thermo‐mechano‐chemical technology to chitin deacetylation was evaluated as an alternative method of chitosan production. This process consists of a cascade reactor unit operating under reduced alkaline conditions of 10% (w/v) NaOH. Prior mercerization of chitin at 4°C for 24 h was required for high deacetylation yields. Sudden decompression of the aqueous alkaline suspension of mercerized chitin resulted in near complete deacetylation of chitin. Reactor residence time was 90 s at 230°C prior to decompression. The chitosan produced was characterized by elemental analysis, 13C–NMR and enzymatic depolymerization. Enzymatic determination of the degree of acetylation of chitin/chitosan mixtures was also investigated. Relative chitinase and/or chitosanase digestibilities were shown to be strongly dependent on chitin deacetylation. Based on enzymatic digestibilities, the alkaline aqueous high shear process does not appear to produce significant secondary products. Correlation of chitosanase digestibility with percentage of deacetylation provides a simple biological assay to study chitosan composition.
Partitioning of host and recombinant cells in aqueous two‐phase polymer systems
Authors: LaMarca Concetta , Lenhoff Abraham M. , Dhurjati Prasad
Published: May 2005

The separation of host and recombinant Escherichia coli bacterial cells has been studied using the surface‐sensitive technique of partitioning in aqueous two‐phase polymer systems. Experiments were designed to probe charge‐and hydrophobicity‐related property differences of antibiotic‐resistant recombinant cells and their antibiotic‐sensitive hosts. Differential partitioning was observed in both charge‐sensitive and non‐charge‐sensitive phase systems for three host–recombinant cell systems, but the non‐charge‐related effects appear to have a greater impact on partitioning behavior. This result suggests that plasmid‐encoded products related to antibiotic resistance modify the surface hydrophobicity of the E. coli bacterial cell and that these differences can be exploited for cell separation.
Effect of cell growth rate on the performance of a two‐stage continuous culture system in a recombinant Escherichia coli fermentation
Authors: Park Sunghoon , Ryu Dewey D. Y. , Kim Jeong Yoon
Published: May 2005

As part of the process optimization of a two‐stage continuous culture system, the effect of growth rate μ on the performance of the second stage (production stage) was studied in a recombinant Escherichia coli K12 (ΔH1Δtrp/pPLc23trpA1). Important parameters considered were specific gene expression rate, plasmid content, and plasmid stability, all of which were closely related to the cell growth rate and the production rate of the cloned gene product (trpα). When operating conditions were maintained constant (T1 = 35°C, D1 = 0.9 h−1, T2 = 40°C, and D2 = 0.7 h−1) and μ was varied, plasmid content in the second stage showed its maximum at μ = 0.4 h−1 and decreased thereafter. Specific gene expression rate linearly increased with increasing μ, while plasmid stability decreased. Optimum cell growth rate giving the maximum value in overall productivity was observed at around μ = 0.4 h−1. The contribution or role of the three parameters, specific gene expression rate, plasmid content, and plasmid stability in exhibiting the maximum productivity at the optimal μ is discussed.
Trypsin purification by affinity binding to small unilamellar liposomes
Authors: Powers Johnny D. , Kilpatrick Peter K. , Carbonell Ruben G.
Published: May 2005

A novel protein purification process using affinity‐ligand‐modified liposomes and membrane ultrafiltration is described. The feasibility of the process was tested using trypsin as the model protein and p‐aminobenzamidine (PAB) as the affinity ligand for trypsin. The affinity liposomes were prepared by covalently attaching PAB to the surface of small unilamellar liposomes via the hydrophilic spacer arm diglycolic acid. The liposomes were comprised of dimyristoyl phosphatidyl choline, cholesterol, and dimyristoyl phosphatidyl ethanolamine to which the diglycolic acid was attached. The equilibrium binding constant between trypsin and immobilized PAB was shown to be dependent on the PAB density of the liposome surface. Bound trypsin was eluted from the liposomes by the trypsin inhibitor benzamidine. Trypsin was purified from a trypsin/chymotrypsin mixture and from one of its naturally occurring sources, porcine pancreatic extract. A recovery yield from the crude mixture of 68% was obtained with a trypsin purity of 98%. The affinity‐modified liposomes were stable in the complex mixture and retained their trypsin binding capacity after multiple adsorption/elution cycles over a 30‐day period.
On‐line monitoring of the viscosity in dextran fermentation using piezoelectric quartz crystal
Authors: Endo Hideaki , Sode Koji , Karube Isao , Muramatsu Hiroshi
Published: May 2005

A novel viscous sensor utilizing AT‐cut quartz crystal to monitor the viscosity of fermentation broth was developed. The sensor system was constructed from the piezoelectric quartz crystal fixed to the cell, exposing only one side of the quartz crystal electrode, an oscillating circuit, a peak level meter, and a personal computer. In order to investigate the characteristics of the sensor system, a sensor signal relating to the resonant resistance of the quartz crystal was measured using dextran solutions with different molecular weights. The linear relationship was obtained between the sensor signal and the (ρη)1/2 of the liquid, where ρ and η are the density and viscosity, respectively. The sensor signal was dependent not only on the viscosity of the liquid but also on the molecular weight of dextran, because dextran solution shows a non‐Newtonian property. The sensor system was applied for the on‐line monitoring of the viscosity in dextran fermentation. A good correlation was observed between the sensor signal and the viscosity value measured with a rotational viscometer for the fermentation broth. Little bubbling effect and agitation of the sensor signal were observed, showing that this system can be utilized for viscosity monitoring in a bioprocess.
Influence of whey protein on continuous acidogenic degradation of lactose
Authors: Kisaalita W. S. , Lo K. V. , Pinder K. L.
Published: May 2005

In previous studies on the acidogenic phase of anaerobic fermentation of lactose, a pathway for the reaction and a rate equation have been proposed. The question then remained as to the effect of the protein in whole whey on the mechanism and on the overall organic substrate conversion. In this study, it was found that as much as 70% of the protein was broken down in the acidogenic reactor. Radiotracer tests showed that the inclusion of protein had no effect on the reaction pathway for lactose degradation. Thus, the whole sweet cheese whey can be fermented as efficiently as whey from which the protein has been removed.
Ethanol production in a microporous hollow‐fiber‐based extractive fermentor with immobilized yeast
Authors: Kang Whankoo , Shukla Rajesh , Sirkar K. K.
Published: May 2005

Microporous‐membrane‐based extractive product recovery in product‐inhibited fermentations allows in situ recovery of inhibitory products in a nondispersive fashion. A tubular bioreactor with continuous strands of hydrophobic microporous hollow fibers having extracting solvent flowing in fiber lumen was utilized for yeast fermentation of glucose to ethanol. Yeast was effectively immobilized on the shell side in small lengths of chopped microporous hyrophilic hollow fibers. The beneficial effects of in situ dispersion‐free solvent ex (oleyl alcohol and dibutyl phthalate) were demonstrated for a 300 g/L glucose substrate feed. Outlet glucose concentration dropped drastically from 123 to 41 g/L as solvent/ substrate flow ratio was increased from 0 to 3 at 9 mL/h of substrate flow rate with oleyl alcohol as extracting solvent. The significant productivity increase with in situ solvent extraction became more evident as solvent/ substrate flow ratio increased. A model of the locally integrated extractive bioreactor describes the observed fermentor performance quite well.
Ethanol production from starch in a pervaporation membrane bioreactor using Clostridium thermohydrosulfuricum
Authors: Mori Yutaka , Inaba Toshiharu
Published: May 2005

To attain both high productivity and efficient recovery of ethanol from broth, a membrane bioreactor consisting of a jar fermentor and a pervaporation system was applied to the direct production of ethanol from uncooked starch with a thermophilic anaerobic bacterium, Clostridium thermohydrosulfuricum. From four types of ethanol‐selective membranes tested, microporous polytetrafluoroethylene (PTFE) membrane, the pores of which are impregnated with silicone rubber, was chosen for its large flux, high ethanol selectivity, and high stability. During fed‐batch fermentation with pervaporation in the membrane bioreactor, ethanol was continuously extracted and concentrated in two traps with concentrations at 5.6%–6.2% (w/w) in trap 1 (20 °C) and 27%–32% (w/w) in trap 2 (liquid N2), while the ethanol concentration in the broth was maintained at 0.85–0.9% (w/w). Due to the low ethanol concentration in the broth, and the immobilization of bacterial cells by the membrane, the number of viable cells, and, eventually, the ethanol productivity, increased in the membrane bioreactor.
A rapid and sensitive method for measurement of leukotrienes based on HPLC
Authors: Odlander Björn , Claesson Hans‐Erik
Published: May 2005

A rapid and sensitive method for simultaneous detection of leukotriene (LT) B4, LTC4, LTD4, LTE4, 5S,12S‐dihydroxy‐eicosatetraenoic acid, 6‐trans LTB4, 12‐epi‐6‐trans LTB4 and the ω‐metabolites of LTB4 has been developed. Leukocyte incubations were terminated by the addition of one volume of methanol, and the mixture was centrifuged. Analysis was then directly performed by reverse phase high performance liquid chromatography on a column packed with 3 μm Nucleosil C18 guarded by a precolumn. Quantitation of eluted compounds was carried out by UV‐monitoring at 280 nm and integration of the elution profile. The sensitivity of the method was approximately 1 ng for both LTB4 and LTC4. Accuracy was 101.6% (SD ± 9.8%) for LTB4 and 99.9% (SD ± 8.7%) for LTC4 in the interval 5–160 ng.
Comparison of iodinated [Nle15]‐ and [Met15]‐gastrin17 prepared by reversed‐phase HPLC
Authors: Seet Lin , Fabri Louis , Nice Edouard C. , Baldwin Graham S.
Published: May 2005

125I‐[Nle15]‐gastrin17 prepared by the iodogen method can be separated by reversed‐phase high performance liquid chromatography into two peaks, both of which elute after [Nle15]‐gastrin17. Direct determination of the specific activities of the two derivatives by microbore reversed‐phase HPLC indicated that they were the mono‐ and di‐iodinated species. In contrast the two peaks obtained with [Met15]‐gastrin17 iodinated under the same conditions eluted earlier, relative to the appropriate gastrin17 standard, than the [Nle15]‐gastrin derivatives. Treatment of either peak with 0.75 M dithiothreitol at 56°C or 95°C resulted in progressive conversion to compounds migrating in relative positions similar to the 125I‐[Nle15]‐gastrin17 derivatives. Direct determination of the specific activity of the earlier eluting [Met15]‐gastrin17 derivative before reduction indicated that it was the mono‐iodinated species. It thus appears likely that iodination of [Met15]‐gastrin17 by the iodogen method results predominantly in the formation of mono‐ and di‐125I‐[Met sulphoxide15]‐gastrin17. To avoid problems arising from oxidation of the methionine residue of gastrin during iodination, the use of 125I‐[Nle15]‐gastrin17 in binding studies is therefore recommended.
Determination of the enantiomeric purity of scopolamine isolated from plant extract using achiral/chiral coupled column chromatography
Authors: Stalcup Apryll M. , Faulkner James R. , Tang Yubing , Armstrong Daniel W. , Levy Luis W. , Regalado Edmundo
Published: May 2005

The optical purity of scopolamine derived from Datura sanguinea was determined using coupled column chromatography. A C18 column was used to separate scopolamine from the additional alkaloids and other biological material present in the vegetal extract. The C18 column was coupled through a six‐port switching valve to two β‐cyclodextrin columns in series which were used to resolve the scopolamine enantiomers. A single acetylated β‐cyclodextrin column gives equivalent results to the native cyclodextrin columns because of slightly higher enantioselectivity for scopolamine. A multistep extraction procedure is used to isolate scopolamine from the vegetal material. 4–6% of the scopolamine in the final extract was found to be the d enantiomer. Sample extracts as well as commercial scopolamine hydrobromide were treated under various conditions commonly encountered during typical commercial extraction procedures and analyzed in order to determine if the d enantiomer was present in the original material or if it was produced during the extraction process and, if so, determine which step and conditions contribute to acemization. Both the salt and the extract were found to be susceptible to racemization under basic conditions (≥pH 9) although the extract appeared to be more susceptible than the salt. Tropic acid formed from the hydrolysis of scopolamine seemed to be completely racemized even though the remaining scopolamine was only partially racemized. Within experimental error, no d enantiomer was found in the original fresh plant material.
High performance liquid chromatographic determination of theophylline metabolites in human liver microsomes
Authors: Sarkar Mohamadi A. , Karnes H. Thomas
Published: May 2005

A high performance liquid chromatographic (HPLC) technique for the determination of three metabolites of theophylline, 3‐methylxanthine (3‐MX), 1‐methylxanthine (1‐MX) and 1,3‐dimethyluric acid (1,3‐DMU) in human liver microsomes is described. The analytes were extracted from human liver microsomes with methylene chloride/isopropanol and stepwise gradient elution was employed for the resolution of peaks. The limits of quantitation were 15 ng/mL for 3‐MX, 20 ng/mL for 1‐MX and 20 ng/mL for 1,3‐DMU. The calibration range was linear for the three metabolites and the calibration ranges were 15–250 ng/mL for 3‐MX, 20–250 ng/mL for 1‐MX and 250–4000 ng/mL for 1,3‐DMU. The absolute recovery ranged from 63–84% for 3‐MX, 65–79% for 1‐MX and 77–89% for 1,3‐DMU over the calibration curve range. Accuracy for all three metabolites was within ± 10% and adequate seletivity was demonstrated by the lack of interfering peaks in blank chromatograms. The within‐run and interday precision were within 10% RSD for all three metabolites tested at two concentrations. The advantage of this method over previous methods is that the use of quaternary ammonium ion pair reagents in the mobile phase has been obviated. Also, unlike a previous radiometric HPLC method, the need for radiolabelled theophylline has also been eliminated. The method was used to characterize theophylline metasbolism in human liver microsomes for immunoinhibition studies and to investigate the interaction of theophylline with selected quinolone antibiotitics.
Evaluation of peroxyoxalate chemiluminescence postcolumn detection of fluphenazine in urine and blood plasma using high performance liquid chromatography
Authors: Mann B. , Grayeski M. L.
Published: May 2005

Peroxyoxalate chemiluminescence may be used for sensitive postcolumn detection of phenothiazine analytes separated by high performance liquid chromatography with appropriate optimization of measurement conditions such as solvent, pH and oxalate ester. Detectability of fluorescent analytes by chemical excitation varies greatly, but analytes with low oxidation potentials are generally more readily detected at low levels, as demonstrated for phenothiazines, an important class of fluorescent drugs. Some improvement in detection limits is observed for fluphenazine when chemiluminescence detection is compared to conventional fluorescence detection. Because of the specificity of chemical excitation, fewer interferences from fluorescent impurities in a urine matrix are observed.
Selective determination of secondary amines as their N‐diethylthiophosphoryl derivatives by gas chromatography with flame photometric detection
Authors: Kataoka Hiroyuki , Eda Michiko , Makita Masami
Published: May 2005

A selective and sensitive method has been developed for the determination of secondary amines by gas chromatography (GC). After removal of primary amines by the reaction with o‐phthaldialdehyde, secondary amines were converted into their N‐diethylthiophosphoryl derivatives and then measured by GC with flame photometric detection using a DB‐1701 capillary column. The derivatives were sufficiently volatile and stable to give single symmetrical peaks. The detection limits of secondary amines were ca. 0.05‐0.2 pmol per injection. N‐Methylcyclohexylamine was used as an internal standard. The calibration curves for secondary amines in the range 1–20 nmol were linear and sufficiently reproducible for quantitative determination. This method was successfully applied to small urine samples without prior clean‐up. Overall recoveries of secondary amines added to urine samples were 91–105%. By using this method, secondary amines in urine samples could be analysed without any influence from primary amines and other coexisting substances. The analytical results of secondary amine content in urine samples of normal subjects are presented.
Selective detection of terminally α 2–3 and α 2–6 sialylated neolacto‐series gangliosides by immunostaining on thin layer chromatograms
Authors: Müthing Johannes , Neumann Ulrich
Published: May 2005

A method for selective detection of terminally α 2–3 and α 2–6 sialylated neolacto‐series gangliosides has been developed. The procedure involves separation of gangliosides on high performance thin layer chromatography plates, fixation of the silica gel, treatment with Vibrio cholerae neuraminidase and incubation of the plates with nLcOse4 Cer‐specific antibodies. Alkaline phosphatase‐conjugated second antibodies were used to visualize bound first antibodies by generating a blue dye from 5‐bromo‐4‐chloro‐3‐indolyl phosphate. Neolacto‐series gangliosides from human granulocytes with terminally α 2–3 and α 2–6 linked N‐acetylneuraminic acid served as examples. Neuraminidase treatment of gangliosides with α 2–3 substituted sialic acid is necessary prior to immunostaining whereas α 2–6 sialylated gangliosides can be detected without enzyme treatment. Steric hindrance of sialic acid bound in position 3 to terminal galactose prevented binding of the antibody to the Galβ1‐4GlcNAc sequence whereas sialylation in position 6 to terminal galactose does not hinder recognition. The procedure is viable for the detection of amounts down to 10 ng of gangliosides. This method should be useful to screen gangliosides from different tissues or cell lines for the presence of such components, especially if only small quantities of material are available.
Assay of vitamin B6 in human plasma with graphitic carbon column
Authors: Kurioka Susumu , Ishioka Noriaki , Sato Junko , Nakamura Junta , Ohkubo Tatuo , Matsuda Makoto
Published: May 2005

A high performance liquid chromatographic (HPLC) procedure for measuring pyridoxal‐5′‐phosphate (PLP) and certain forms of B6 vitamers in plasma is presented here. This HPLC procedure consisted of a single graphitic carbon column with a fluorescence detector employing an isocratic eluent (15% acetonitrile:1% perchloric acid: 0.05% sodium bisulfite). The graphitic carbon column is useful in acidic eluent without deteriorization. The relatively low fluorescent intensity of PLP under acidic conditions is improved by its derivatization with bisulfite in the eluent during chromatographic separation. Using this procedure, the detection limit of PLP is 50 fmol, and an aliquot of 5–50 μL of human plasma is required giving satisfactorily precise results within 5 min. We applied this method to the determination of PLP and certain B6 vitamers in human plasma after oral supplementation of pyridoxine.
Lipid peroxidation and covalent binding in the early functional impairment of liver golgi apparatus by carbon tetrachloride
Authors: Poli G. , Cottalasso D. , Pronzato M. A. , Chiarpotto E. , Biasi F. , Corongiu F. P. , Marinari U. M. , Nanni G. , Dianzani M. U.
Published: May 2005

The onset of the lipoprotein secretory block provoked by CCl4 in the whole animal was monitored after purification of liver Golgi membranes. Both lipid transit through the apparatus the apparatus and hexosylation of the lipoprotein are markedly inhibited 5–15 min after poisoning. Pre‐treating the animal with alpha‐tocopherol, shown to prevent lipid peroxidation without modifying the covalent binding due to CCl4 metabolites, affords little protection against lipid accumulation in the Golgi, but total preservation of galactosyl transferase activity. While haloalkylation therefore appears to be the major mechanism of damage in the early phases of CCl4‐induced derangement of lipid secretion, lipid peroxidation is probably more involved later, this is indicated by the marked, though never complete, protection against fatty liver afforded at 24 h after CCl4 poisoning by supplementation of the membrane with alpha‐tocopherol.
Fructose‐1, 6‐diphosphate as an in vitro and in vivo anti‐alcohol agent in the rat
Authors: Galzigna L. , Bianchi M. , Rizzoli V. , Scuri R. , Giannetti P. , Paesano A.
Published: May 2005

Fructose‐1, 6‐diphosphate (FDP) decreases the effect of ethanol on Ca++ entry and inhibits the ethanol‐stimulated phosphate efflux in rat heart slices. FDP also inhibits the ethanol‐stimulated [36Cl−]‐uptake by rat brain microvesicles and affects the isolated GABA‐receptor in a way opposite to that of ethanol. The in vivo effects of FDP include a dose‐dependent decrease in ethanol‐induced gastric ulcers and a decrease in the serum transaminase levels raised by chronic ethanol administration. Other central actions of ethanol such as diuresis, narcosis, dependence and withdrawal symptoms are also counteracted by FDP.