bluuebunny/crossref_metadata_2025 huggingface dataset:
Nonlinear Dynamics of Eucaryotic Pyruvate Dehydrogenase Multienzyme Complex: Decarboxylation Rate, Oscillations, and Multiplicity
Authors: Zeng An‐Ping , Modak Jayant , Deckwer Wolf‐Dieter
Published: December 2002

Pyruvate conversion to acetyl‐CoA by the pyruvate dehydrogenase (PDH) multienzyme complex is known as a key node in affecting the metabolic fluxes of animal cell culture. However, its possible role in causing possible nonlinear dynamic behavior such as oscillations and multiplicity of animal cells has received little attention. In this work, the kinetic and dynamic behavior of PDH of eucaryotic cells has been analyzed by using both in vitro and simplified in vivo models. With the in vitro model the overall reaction rate (ν1) of PDH is shown to be a nonlinear function of pyruvate concentration, leading to oscillations under certain conditions. All enzyme components affect ν1 and the nonlinearity of PDH significantly, the protein X and the core enzyme dihydrolipoamide acyltransferase (E2) being mostly predominant. By considering the synthesis rates of pyruvate and PDH components the in vitro model is expanded to emulate in vivo conditions. Analysis using the in vivo model reveals another interesting kinetic feature of the PDH system, namely, multiple steady states. Depending on the pyruvate and enzyme levels or the operation mode, either a steady state with high pyruvate decarboxylation rate or a steady state with significantly lower decarboxylation rate can be achieved under otherwise identical conditions. In general, the more efficient steady state is associated with a lower pyruvate concentration. A possible time delay in the substrate supply and enzyme synthesis can also affect the steady state to be achieved and leads to oscillations under certain conditions. Overall, the predictions of multiplicity for the PDH system agree qualitatively well with recent experimental observations in animal cell cultures. The model analysis gives some hints for improving pyruavte metabolism in animal cell culture.
Operational Stability of High Initial Activity Protease Catalysts in Organic Solvents
Authors: Fernandes João F. Amorim , Halling Peter J.
Published: December 2002

The first studies on the operational stability of cross‐linked enzyme crystals (CLECs) in organic media are described. Although these catalysts display high initial specific activity, they inactivate rapidly, losing more than 50% of the initial activity within the first 4 h under continuous flow. Furthermore, the inactivation is not reversible when returned to an aqueous medium. The same rapid inactivation occurs with adsorbed protease preparations that show similar high initial specific activity (propanol‐rinsed enzyme preparations (PREPs) of subtilisin and α‐chymotrypsin).
Modulation of Phosphoenolpyruvate Synthase Expression Increases Shikimate Pathway Product Yields in E. coli
Authors: Yi Jian , Li Kai , Draths K. M. , Frost J. W.
Published: December 2002

Product yields in microbial synthesis are ultimately limited by the mechanism utilized for glucose transport. Altered expression of phosphoenolpyruvate synthase was examined as a method for circumventing these limits. Escherichia coli KL3/pJY1.216A was cultured under fed‐batch fermentor conditions where glucose was the only source of carbon for the formation of microbial biomass and the synthesis of product 3‐dehydroshikimic acid. Shikimate pathway byproducts 3‐deoxy‐d‐ arabino‐heptulosonic acid, 3‐dehydroquinic acid, and gallic acid were also generated. An optimal expression level of phosphoenolpyruvate synthase was identified, which did not correspond to the highest expression levels of this enzyme, where the total yield of 3‐dehydroshikimic acid and shikimate pathway byproducts synthesized from glucose was 51% (mol/mol). For comparison, the theoretical maximum yield is 43% (mol/mol) for synthesis of 3‐dehydroshikimic acid and shikimate pathway byproducts from glucose in lieu of amplified expression of phosphoenolpyruvate synthase.
Stabilization and Improvement of Catalytic Activity of a Low Molar Mass Cellobiase by Cellobiase‐Sucrase Aggregation in the Culture Filtrate of Termitomyces clypeatus
Authors: Saha Rina , Roy Sujata Burman , Sengupta S.
Published: December 2002

The extracellular cellobiase (EC 3.2.1.21) of Termitomyces clypeatus separated in two protein fractions when culture filtrate or ammonium sulfate precipitated proteins were chromatographed on BioGel P‐200 column. During purification of cellobiase (CBS) from the lower molar mass (LMM) protein fraction, the enzyme behaved like a low molecular weight multimeric protein. The purified enzyme gave a single 56 kDa band in SDS‐PAGE but ladderlike bands (14, 28, 42, and 56 kDa) on denaturation by reducing‐SDS and urea. The protein, however, dissociated on dilution and protomeric (14 kDa) and multimeric forms (28 and 60 kDa) were eluted separately during HPGPLC. Specific activity of CBS gradually decreased as the molar mass of the enzyme was lowered in different eluted peaks. Protein present in all CBS pool fractions had the same amino acid composition and all displayed the same, single protein peak in reverse‐phase HPLC and 56 kDa band in SDS‐PAGE. Thus, T. clypeatus CBS was a multimeric 14 kDa protein that was optimally active as a tetramer. CBS purified from the higher molar mass fraction (HMM) as a SDS‐PAGE homogeneous 110‐kDa protein did not dissociate on dilution or by SDS‐urea. The purified protein was a protein aggregate as CBS consistently contained 20 ± 5% sucrase (SUC) Units in the preparation. The aggregate resolved during reverse‐phase chromatography on a C4 column, and an additional protein peak other than CBS was detected. The aggregated CBS had a higher temperature optimum and was more stable toward thermal and chemical denaturations than SUC‐free CBS. Increase of stability and catalytic activity of CBS by aggregation with SUC was much higher than those by the multimerization of CBS itself. All of these observations for the first time suggested that the heterologous protein‐protein aggregation, observed for a long time for fungal enzymes, might have a significant role in modulating physicochemical properties of the extracellular enzyme.
Identification of Critical Batch Operating Parameters in Fed‐Batch Recombinant E. coli Fermentations Using Decision Tree Analysis
Authors: Buck Kristan K. S. , Subramanian Venkatanarayanan , Block David E.
Published: December 2002

To develop a useful fermentation process model, it is first necessary to identify which batch operating parameters are critical in determining the process outcome. To identify critical processing inputs in large databases, we have explored the use of Decision Tree Analysis with the decision metrics of Gain (i.e., Shannon Entropy changes), Gain Ratio, and a multiple hypergeometric distribution. The usefulness of this approach lies in its ability to treat “categorical” variables, which are typical of archived fermentation databases, as well as “continuous” variables. In this work, we demonstrate the use of Decision Tree Analysis for the problem of optimizing recombinant green fluorescent protein production in E. coli. A database of 85 fermentations was generated to examine the effect of 15 process input parameters on final biomass yield, maximum recombinant protein concentration, and productivity. The use of Decision Tree Analysis led to a considerable reduction in the fermentation database through the identification of the significant as well as insignificant inputs. However, different decision metrics selected different inputs and different numbers of inputs to classify the data for each output.
Kinetics of the Alkaline Phosphatase Catalyzed Hydrolysis of Disodium p‐Nitrophenyl Phosphate in Frozen Model Systems
Authors: Terefe Netsanet Shiferaw , Mokwena Kereilemang Khanah , Van Loey Ann , Hendrickx Marc E.
Published: December 2002

The alkaline phosphatase catalyzed hydrolysis of disodium‐ p‐nitrophenyl phosphate was studied in four model systems comprising sucrose, maltodextrin, carboxymethylcellulose (CMC), and CMC‐lactose in a temperature range of –28 to 20 °C. In the maltodextrin and CMC‐lactose model systems, the reaction rate decreased to a very low value as the glass transition temperature was approached. In the CMC and CMC‐lactose systems with low initial solute concentration, as a consequence of freeze‐concentration, a rate maximum around the initial freezing temperature was observed. The Arrhenius equation described the temperature dependence of the reaction rate both in the liquid and the glassy states in all systems studied, while a slightly curved Arrhenius plot was observed in the “rubbery” state of the CMC and CMC‐lactose systems. The WLF equation with system‐dependent coefficients described the kinetics in the rubbery state of all the model systems except sucrose, excluding the short temperature range where reaction rate enhancement with decreasing temperature was observed.
Comparison of Techniques for Monitoring Antibody Fragment Production in E. coli Fermentation Cultures
Authors: Bowering Leigh C. , Bracewell Daniel G. , Kesharvarz‐Moore Eli , Hoare Mike , Weir A. Neil C.
Published: December 2002

The use of an optical biosensor for monitoring antibody fragment accumulation following induction in a batch fermentation of recombinant E. coli is compared to the more traditional method of ELISA quantification. Using the biosensor, concentration data can be obtained within minutes of sample addition to the device, compared to an average assay time of 3–4 h for the ELISA. We describe two biosensor assays developed as an alternative to ELISA and compare them with ELISA in the ability to provide quantitative product accumulation profiles during fermentation. Discrepancies in titers recorded by the assays are explained by a combination of differences in product variants detected by each assay and interference from sample contaminants. Method of sample preparation is also shown to be important if accurate concentration data is required. Both biosensor assays are shown to be capable of providing product accumulation profiles comparable to those obtained by ELISA. The use of a rapid extraction technique would allow such data to be obtained during process operation, enabling improved fermentation control and more rapid process development.
Secretion of Cellobiase Is Mediated via Vacuoles in Termitomyces clypeatus
Authors: Mukherjee Sumana , Khowala Suman
Published: December 2002

The majority of cellobiase activity in Termitomyces clypeatus was localized in vacuolar fractions of the fungus under secretory and nonsecretory conditions of growth. Activities of marker proteins for subcellular organelles, e.g., vacuoles, cytosol, ER, and mitochondria, in mycelial extracts from the secreting conditions increased by approximately 20, 12, 5, and 2.5 times, respectively, as compared to those obtained from mycelium grown in nonsecreting conditions. The average size and concentration of vacuoles visualized by electron microscopy were also increased in secreting conditions in the fungus. The specific activity of cellobiase in vacuoles isolated in Ficoll‐sucrose gradient, as obtained from mycelial growth in secretory medium, was more than 40 times higher in comparison to that found from nonsecretory medium. The results indicated that subcellular localization of cellobiase in vacuoles is regulated by the cellular signaling prevailing in the fungus. Mycelial extraction of intracellular proteins by hand grinding and by bead‐beater from cells frozen in the presence or absence of liquid nitrogen was also compared. Maximum recovery of intracellular protein was obtained with the bead‐beater under aerobic conditions in the absence of nitrogen. Highest recovery of vacuoles up to 85% was obtained by single‐step ultracentrifugation of the mycelial extract of the fungus in Ficoll‐sucrose gradient. The method appeared to be useful for separation of other subcellular organelles in filamentous fungi.
Aqueous Extraction of β‐Glucuronidase from Transgenic Canola: Kinetics and Microstructure
Authors: Bai Yun , Nikolov Zivko L. , Glatz Charles E.
Published: December 2002

Aqueous extraction kinetics of recombinant β‐glucuronidase (rGUS) from transgenic canola ( Brassica napus) was investigated in terms of the particle size and microstructural characteristics resulting from canola seed processing. The canola had been transformed to express recombinant GUS intracellulary in the seed, and electron microscopy showed that the cells are distributed among (1) disrupted cells in a thin layer at or adjacent to the particle surface, (2) disrupted cells within the interior, and (3) intact cells within the interior. A simple compartmental model containing two extractable pools and a third nonextractable pool fitted the batch extraction results very well. Comparing the rate constants from the model to estimates of expected transport rates from the observed cell fractions showed that the two extractable pools roughly correspond to the two disrupted cell fractions. Both flaking, causing more extensive cell wall damage throughout the seed, and grinding, increasing the total surface area, increase the size of the first pool and, therefore, the extraction yield. Mass transfer from the same type of pool from two types of processed seed behaved similarly. GUS extraction from the first extractable pool is 10–20 times faster (<1 min) than from the second extractable pool.
On‐Line Determination of Biomass in a Microalga Bioreactor Using a Novel Computerized Flow Injection Analysis System
Authors: Meireles Luís A. , Azevedo José L. , Cunha João P. , Malcata F. Xavier
Published: December 2002

A flow injection analysis (FIA) device has been developed, which is able to assay successfully for biomass in a microalga bioreactor. The device is fully computerized and is operated via diluting small aliquots of the culture followed by measuring optical density (OD); this figure is then accurately correlated with biomass, in terms of both cell number and ash‐free dry weight, during the entire culture time. Furthermore, the device is not expensive, is highly versatile, and is easy to operate owing to specifically developed, user‐friendly software. The growth rate and biomass productivity of Pavlova lutheri, cultivated under batch and semicontinuous modes, were monitored as experimental testing model.
Microcarrier Culture of Lepidopteran Cell Lines: Implications for Growth and Recombinant Protein Production
Authors: Ikonomou Laertis , Drugmand Jean‐Christophe , Bastin Georges , Schneider Yves‐Jacques , Agathos Spiros N.
Published: December 2002

Several microcarrier systems were screened with Sf‐9 and High‐Five cell lines as to their ability to support cell growth and recombinant (β‐galactosidase) protein production. Growth of both cell lines on compact microcarriers, such as Cytodex‐1 and glass beads, was minimal, as cells detached easily from the microcarrier surface and grew as single cells in the medium. Cell growth was also problematic on Cytopore‐1 and ‐2 porous microcarriers. Cells remained attached for several days inside the microcarrier pores, but no cell division and proliferation were observed. On the contrary, insect cells grew well in the interior of Fibra‐Cel disks mainly as aggregates at points of fiber intersection, reaching final (plateau) densities of about 4 × 106 (Sf‐9) and 2.7 × 106 (High‐Five) cells mL‐1 (8 × 106 and 5.5 × 106 cells per cm2 of projected disk area, respectively). Their growth was described well by the logistic equation, which takes into account possible inhibition effects. β‐Galactosidase (β‐gal) production of Sf‐9 cells on Fibra‐Cel disks (infected at 3.3 × 106 cells mL‐1) was prolonged (192 h), and specific protein production was similar to that of high‐density free cell infection. Cultispher‐S microcarriers were found to be a very efficient system for the growth of High‐Five cells, whereas no growth of Sf‐9 cells took place for the same system. Concentrations of about 9 × 106 cells mL‐1 were reached within 120 h, with cell growth in both microcarriers and aggregates, appearance of cellular bridges between microcarriers and aggregates, and eventual formation of macroaggregates incorporating several microcarriers. Specific protein productions after β‐gal baculovirus infection at increasing cell concentrations were almost constant, thus leading to elevated volumetric protein production: final β‐gal titers of 946, 1728, and 1484 U mL‐1 were obtained for infection densities of 3.4, 7.2, and 8.9 × 106 cells mL‐1, respectively.
Design of Methanol Feed Control in Pichia pastoris Fermentations Based upon a Growth Model
Authors: Zhang Wenhui , Smith Leonard A. , Plantz Bradley A. , Schlegel Vicki L. , Meagher Michael M.
Published: December 2002

The methylotrophic yeast Pichia pastoris is an effective system for recombinant protein productions that utilizes methanol as an inducer, and also as carbon and energy source for a Mut+ (methanol utilization plus) strain. Pichia fermentation is conducted in a fed‐batch mode to obtain a high cell density for a high productivity. An accurate methanol control is required in the methanol fed‐batch phase (induction phase) in the fermentation. A simple “on‐off” control strategy is inadequate for precise control of methanol concentrations in the fermentor. In this paper we employed a PID (proportional, integral and derivative) control system for the methanol concentration control and designed the PID controller settings on the basis of a Pichia growth model. The closed‐loop system was built with four components: PID controller, methanol feed pump, fermentation process, and methanol sensor. First, modeling and transfer functions for all components were derived, followed by frequency response analysis, a powerful method for calculating the optimal PID parameters Kc (controller gain), θI (controller integral time constant), and θD (controller derivative time constant). Bode stability criteria were used to develop the stability diagram for evaluating the designed settings during the entire methanol fed‐batch phase. Fermentations were conducted using four Pichia strains , each expressing a different protein, to verify the control performance with optimal PID settings. The results showed that the methanol concentration matched the set point very well with only small overshoot when the set point was switched, which indicated that a very good control performance was achieved. The method developed in this paper is robust and can serve as a framework for the design of other PID feedback control systems in biological processes.
Preservation of Chitinolytic Pantoae agglomerans in a Viable Form by Cellular Dried Alginate‐Based Carriers
Authors: Zohar‐Perez C. , Ritte E. , Chernin L. , Chet I. , Nussinovitch A.
Published: December 2002

Improved viability of Gram‐negative bacteria during freeze‐dehydration, storage, and soil inoculation is of crucial importance to their efficient application. The chitinolytic Pantoae ( Enterobacter) agglomeransstrain IC1270, a potential biocontrol agent of soil‐borne plant‐pathogenic fungi, was used as a model organism to study the efficacy of freeze‐dried alginate‐based beads (macrocapsules) as possible carriers for immobilized Gram‐negative bacterial cells. These macrocapsules were produced by freeze‐dehydration of alginate gel spherical beads, in which different amounts of bacteria, glycerol, and colloidal chitin were entrapped. Subsequent drying produced different unexpected structures, pore‐size distributions, and changes in the outer and inner appearance of the resultant dried cellular solid. With increasing glycerol content, the proportion of larger pores increased. These structures can be related to changes in the slow‐release properties of the dried beads. The amount of glycerol in the beads differed from that in the alginate solution as a result of leakage during the beads' preparation and dehydration. Entrapping 109 cells per bead produced from alginate solution containing 30% glycerol and 1% chitin resulted in improved (in comparison to other studies) survival prospects (95%) during freeze‐drying. Moreover, immobilization of the bacterium sharply improved its survival in nonsterile irrigated and dry soils compared to bacteria in a water suspension. The results suggest that optimized conservation of Gram‐negative bacteria in dry glycerol‐containing alginate‐based cellular solids is not only possible but applicable for a variety of uses.
Facile and Statistical Optimization of Transfection Conditions for Secretion of Foreign Proteins from Insect Drosophila S2 Cells using Green Fluorescent Protein Reporter
Authors: Shin Hwa Sung , Cha Hyung Joon
Published: December 2002

Insect Drosophila melanogaster S2 cells were developed as a plasmid‐based and therefore nonlytic expression system for functional foreign proteins. Transfection is an important step to introduce foreign target DNA into cells and should be properly optimized to obtain maximum production yield. Single factor search (SFS) methodology is still generally used to determine optimal condition in a biological system. Although this method is relatively simple to perform, it has many disadvantages such as not considering interactions between several factors and not covering the entire region of the solution pool. Therefore, we approached this optimization problem statistically with response surface (RSM) and evolutionary operation (EVOP) methodologies and compared the transfection efficiencies with the traditional SFS method. We employed secreted green fluorescent protein (GFP) as a reporter for determination of optimal transfection condition and secreted human erythropoietin (hEPO) as a confirming foreign model protein. Consequently, we arrived at the best optimal transient transfection condition (1 μg of plasmid DNA, 5 μg of lipofectin, 2 × 106 cells of initial cell number, and 18 h of transfection duration time) through a systematic access in a series of SFS, RSM, and EVOP. The secreted hEPO yield using optimal transient transfection condition by EVOP methodology was enhanced by about 1.8‐fold compared to that of traditional SFS. This optimized transient transfection condition can be used as a basis for optimal stable transfections. A linear relationship between secreted GFP fluorescence intensity and secreted hEPO concentration indicated that facile and noninvasive determination of optimal transfection conditions for expression and secretion of foreign proteins in S2 cell cultures was made possible by simple measurement of GFP fluorescence.
On‐Line Purification of Monoclonal Antibodies Using an Integrated Stirred‐Tank Reactor/Expanded‐Bed Adsorption System
Authors: Ohashi Ryo , Otero Jose M. , Chwistek Adam , Yamato Ichiro , Hamel Jean‐François P.
Published: December 2002

While expanded‐bed adsorption (EBA) units have been used to recover proteins from whole cell cultures, the development of a more efficient, on‐line process could streamline the traditional multistep process. This study implements a bench‐scale on‐line purification system in which whole cell cultures are loaded directly into a chromatography column to capture a monoclonal antibody (mAb) in a single step. The on‐line purification system used here integrates a stirred‐tank reactor (STR) and an EBA unit into a new hybrid (STR‐EBA) system. To conduct this work, first, column and buffer conditions were optimized to capture immunoglobulin G from a hybridoma cell culture. A high cell removal (>95%) was achieved in part by removing the top flow distributor and mesh. Then, the 95% extent of removal was sustained for four successive cycles, each using PBS. With 20 mM phosphate buffer, however, the removal decreased from 95% to 75% stepwise. Next, the operational constraints of the EBA system were determined for the hybridoma cell culture, focusing on the effects of cell viability and density on cell removal. This study shows that the cell removal was not significantly different in the range of 80% to 0% viability. Cell density was also varied between 1 × 106 and 1 × 108 cells/mL. From 0.1 to 6 × 107 cells/mL, cell retention in the column was less than 5% and product recovery remained high, approximately 95%. After characterizing the working conditions of the EBA system, on‐line purification was performed. With 1.1 L of culture containing 3 × 106 cells/mL and 100 mg/L of IgG, repeated‐batch cultures were implemented. Half of the culture volume (550 mL) was directly sent to the EBA system every day, for 11 days, and the same amount of fresh medium was fed into the STR. During on‐line purification, productivity was 58 mg of IgG/cycle (day) and purity was greater than 95%. Simple batch culture alone produced 17 mg of IgG/day. This result suggests that the on‐line STR‐EBA system can achieve higher and faster production compared with STR batch and off‐line EBA purification. Overall, the STR‐EBA system with repeated‐batch mode was an effective and flexible system for bench‐scale mAb production.
Standing Wave Design and Experimental Validation of a Tandem Simulated Moving Bed Process for Insulin Purification
Authors: Xie Yi , Mun Sungyong , Kim Jinhyun , Wang Nien‐Hwa Linda
Published: December 2002

A tandem simulated moving bed (SMB) process for insulin purification has been proposed and validated experimentally. The mixture to be separated consists of insulin, high molecular weight proteins, and zinc chloride. A systematic approach based on the standing wave design, rate model simulations, and experiments was used to develop this multicomponent separation process. The standing wave design was applied to specify the SMB operating conditions of a lab‐scale unit with 10 columns. The design was validated with rate model simulations prior to experiments. The experimental results show 99.9% purity and 99% yield, which closely agree with the model predictions and the standing wave design targets. The agreement proves that the standing wave design can ensure high purity and high yield for the tandem SMB process. Compared to a conventional batch SEC process, the tandem SMB has 10% higher yield, 400% higher throughput, and 72% lower eluant consumption. In contrast, a design that ignores the effects of mass transfer and nonideal flow cannot meet the purity requirement and gives less than 96% yield.
Evaluation of Factors Promoting Astaxanthin Production by a Unicellular Green Alga, Haematococcus pluvialis, with Fractional Factorial Design
Authors: Choi Y. E. , Yun Y.‐S. , Park J. M.
Published: December 2002

Factors affecting the astaxanthin production by a unicellular green alga, Haematococcus pluvialis UTEX 16, were evaluated with sequential fractional factorial design. To simulate an actual production mode, a two‐stage process was adapted for astaxanthin production: the alga was first cultivated under vegetative growth conditions, and then astaxanthin production was induced by applying various induction methods. A high dose of irradiation was most effective for the production of astaxanthin both in weight (mg/g) and in cellular (pg/cell) contents. A combination of nitrogen deficiency and acetate addition also significantly increased the astaxanthin content of cells on a dry weight basis. Meanwhile, the acetate addition alone increased only the cellular content of astaxanthin. Although the addition of ferrous ion alone had a negative effect on the weight content of astaxanthin, simultaneous addition of ferrous ion and acetate was effective for increasing the cellular content of astaxanthin.
On‐Line Monitoring of Adhesion and Proliferation of Cultured Hepatoma Cells Using Optical Waveguide Lightmode Spectroscopy (OWLS)
Authors: Hug T. S. , Prenosil J. E. , Maier P. , Morbidelli M.
Published: December 2002

Monitoring of cell adhesion, cell spreading, and cell proliferation opens attractive perspectives in the on‐line control of monolayer cell cultures in toxicity tests, in bioreactors as used for the serial production of skin grafts, or in extracorporeal liver devices. In this study the hepatoma Hep G2 cell adhesion and proliferation was monitored using an integrated optical method, optical waveguide lightmode spectroscopy (OWLS). This method is based upon refractive index measurements within a 100‐nm thin layer above a Si(Ti)O2 surface on which the cells were cultured and exposed to cytotoxic and cytostatic agents. The OWLS signal was proportional to cell density during the spreading period (4 h), and in long‐term experiments (46 h) the OWLS signal correlated on a logarithmic scale with cell density. After administration of the protein synthesis inhibitor cycloheximide (4 μg/mL) to fully spread hepatoma cells, cell growth was arrested and change of the OWLS signal became noticeable within 6 h after drug administration. For exposure to increasing concentrations of the anticancer drug cyclophosphamide (2.5–20 mM) a concentration‐dependent reduction of the OWLS signal was found. For cycloheximide and cyclophospamide the OWLS signal was also confirmed by cell viability measurements using the neutral red assay, the thiazolylblue tetrazoliumbromide assay, total protein measurements, and cell morphology. It was demonstrated that the OWLS signal detects minor changes in cell adhesion, which serve as indicators of metabolic state and growth behavior. OWLS is thus a quantitative tool to characterize impaired cell growth mediated by culture medium, by extracellular matrix, or after exposure to a toxin.
Design of the pH Profile for Asymmetric Bioreduction of Ethyl 4‐Chloro‐3‐oxobutyrate on the Basis of a Data‐Driven Method
Authors: Chen Junghui , Wang Kuan‐Po , Houng Jer‐Yiing , Lee Shiou‐Ling
Published: December 2002

The goal of this paper was to design the optimal time‐varying operating pH profile in the asymmetric reduction of ethyl 4‐chloro‐3‐oxobutyrate by baker's yeast. Ethyl ( S)‐4‐chloro‐3‐hydroxybutyrate was produced to reach two important quality indices: reaction yield and product optical purity. The method integrated an orthogonal function approximation and an orthogonal array. The technique used a set of orthonormal functions as the basis for representing the possible profile. The optimal profile could be obtained if the orthogonal coefficients were properly adjusted. The orthogonal array was used to design and analyze the effect of each orthogonal coefficient in order to reach the optimal objective (quality) function. The performance based on the proposed strategy was significantly improved by over 10% compared with the traditional fixed pH or uncontrolled pH values during the reaction. The proposed method can be applied to the required dynamic profile in the bioreactor process to effectively improve the product quality, given good design directions and the advantage of the traditional statistical approach.
Electronic Speckle Pattern Interferometry: A Tool for Determining Diffusion and Partition Coefficients for Proteins in Gels
Authors: Karlsson David , Zacchi Guido , Axelsson Anders
Published: December 2002

The aim of this study was to demonstrate electronic speckle pattern interferometry (ESPI) as a powerful tool in determining diffusion coefficients and partition coefficients for proteins in gels. ESPI employs a CCD camera instead of a holographic plate as in conventional holographic interferometry. This gives the advantage of being able to choose the reference state freely. If a hologram at the reference state is taken and compared to a hologram during the diffusion process, an interferometric picture can be generated that describes the refraction index gradients and thus the concentration gradients in the gel as well as in the liquid. MATLAB is then used to fit Fick's law to the experimental data to obtain the diffusion coefficients in gel and liquid. The partition coefficient is obtained from the same experiment from the flux condition at the interface between gel and liquid. This makes the comparison between the different diffusants more reliable than when the measurements are performed in separate experiments. The diffusion and partitioning coefficients of lysozyme, BSA, and IgG in 4% agarose gel at pH 5.6 and in 0.1 M NaCl have been determined. In the gel the diffusion coefficients were 11.2 ± 1.6, 4.8 ± 0.6, and 3.0 ± 0.3 m2/s for lysozyme, BSA, and IgG, respectively. The partition coefficients were determined to be 0.65 ± 0.04, 0.44 ± 0.06, and 0.51 ± 0.04 for lysozyme, BSA, and IgG, respectively. The current study shows that ESPI is easy to use and gives diffusion coefficients and partition coefficients for proteins with sufficient accuracy from the same experiment.
Optimization of Allium sativum Solvent Extraction for the Inhibition of in Vitro Growth of Helicobacter Pylori
Authors: Cañizares Pablo , Gracia Ignacio , Gómez Luis A. , de Argila Carlos Martín , de Rafael Luis , García Antonio
Published: December 2002

Helicobacter pylori (Hp) is the bacterium responsible for serious gastric diseases such as ulcers and cancer. The work described here involved the study of the inhibitory power of Allium sativum extracts against the in vitro growth of Hp(Hp ivg). We used purple garlic of the “Las Pedroñeras” variety for this study. The effects of two different extraction methods (Soxhlet, stirred tank extractor) and four solvents with different characteristics (water, acetone, ethanol, and hexane) were investigated in terms of the efficiency of the extraction process. Satisfactory results were obtained in most cases in the activity tests, indicating that different extracts gave rise to good inhibitory activity against Hp ivg. The extracts that showed the highest bacteriostatic activities were selected to evaluate the influence of the most important operation variables on the extraction yield: stirring speed, operation time, garlic conditioning, and garlic storage time. The best results were obtained using ethanol and acetone as solvents in a stirred tank. The inhibitory powers of these extracts were compared to those shown by some commercial antibiotics used in the medical treatment of Hp infections. The results of this study show that garlic extracts produce levels of inhibition similar to those of the commercial materials. These extracts were also tested against other common bacteria, and equally satisfactory results were obtained. The research described here represents an important starting point in the fight against and/or prevention of peptic ulcers, as well as other pathologies associated with Hp infections such us gastric cancer. The extracted material can be used by direct application and involves a simple and economical extraction procedure that avoids isolation or purification techniques.
Enhancement of Enzyme Activity in Supercritical Carbon Dioxide via Changes in Acid‐Base Conditions
Authors: Harper Neil , Barreiros Susana
Published: December 2002

Enzyme performance is often impaired in supercritical carbon dioxide. We were able to enhance enzyme activity in this medium via changes in acid‐base conditions by using ion‐exchange materials (solid H+/Na+ buffer pairs and a zeolite), which were selected on the basis of the response of an organosoluble acid‐base indicator. The concentration of ion‐exchange materials had an important effect on the catalytic activity of subtilisin Carlsberg cross‐linked enzyme crystals (CLECs), and this was related to the protonation and hydration states of the enzyme. The buffer Na2CO3/NaHCO3 gave the highest enhancement in enzyme activity (by a factor of 54), probably as a result of its high basicity and capacity to counteract the deleterious effect of carbonic acid to a greater extent than the other materials tested.
Characterization of an Inducible Promoter System in Catharanthus roseus Hairy Roots
Authors: Hughes Erik H. , Hong Seung‐Beom , Shanks Jacqueline V. , San Ka‐Yiu , Gibson Susan I.
Published: December 2002

Transgenic hairy root cultures of Catharanthus roseus were established with a glucocorticoid‐inducible promoter controlling the expression of green fluorescent protein (GFP), and GFP expression was characterized. The inducible system shows a tightly controlled, reversible, and dosage‐dependent response to the glucocorticoid dexamethasone in C. roseus hairy roots. Full induction was noted after 12–18 h in the mature regions of the root tips and after 6 h in the meristem tissue. Upon removal of the inducing agent, GFP expression declined to undetectable levels in the mature tissues after 24 h and in the meristem after 48 h. Although no dosage‐dependent response was noted in the meristem region, such a response was apparent in the mature region of the tip and verified by quantitative GFP analysis. The inducible promoter system allowed quantitative control of GFP expression between 0.01 and 10 μM dexamethasone with saturation occurring at higher levels. Using GFP as a model system allowed demonstration of the ability to control temporal and quantitative gene expression with the glucocorticoid‐inducible promoter in transgenic C. roseus hairy roots.
Alterations in Taxol Production in Plant Cell Culture via Manipulation of the Phenylalanine Ammonia Lyase Pathway
Authors: Brincat Michelle C. , Gibson Donna M. , Shuler Michael L.
Published: December 2002

One approach to increasing secondary metabolite production in plant cell culture is to manipulate metabolic pathways to utilize more resources toward production of one desired compound or class of compounds, such as diverting carbon flux from competing secondary pathways. Since phenylalanine provides both the phenylisoserine side chain and the benzoyl moiety at C‐2 of Taxol, we speculated that blockage of the phenylpropanoid pathway might divert phenylalanine into Taxol biosynthesis. We used specific enzyme inhibitors to target the first enzyme in the phenylpropanoid pathway, phenylalanine ammonia lyase (PAL), the critical control point for conversion of l‐phenylalanine to trans‐cinnamic acid. Cinnamic acid acted quickly in reducing PAL activity by 40–50%, without affecting total protein levels, but it generally inhibited the taxane pathway, reducing Taxol by 90% of control levels. Of the taxanes produced, 13‐acetyl‐9‐dihydro‐baccatin III and 9‐dihydrobaccatin III doubled as a percentage of total taxanes in C93AD and CO93P cells treated with 0.20 and 0.25 mM cinnamic acid, when all other taxanes were lowered. The PAL inhibitor α‐aminooxyacetic acid (AOA) almost entirely shut down Taxol production at both 0.5 and 1.5 mM, whereas l‐α‐aminooxy‐β‐phenylpropionic acid (AOPP) had the opposite effect, slightly enhancing Taxol production at 1 μM but having no effect at 10 μM. The discrepancy in the effectiveness of AOA and AOPP and the lack of effect with addition of phenylalanine or benzoic acid derivatives further indicates that the impact of cinnamic acid on Taxol is related not to its effect on PAL but rather to a specific effect on the taxane pathway. On the basis of these results, a less direct route for inhibiting the phenylpropanoid pathway may be required to avoid unwanted side effects and potentially enhance Taxol production.
Metabolic Control Analysis of Eucaryotic Pyruvate Dehydrogenase Multienzyme Complex
Authors: Modak Jayant , Deckwer Wolf‐Dieter , Zeng An‐Ping
Published: December 2002

Metabolic control analysis (MCA) of pyruvate dehydrogenase multienzyme (PDH) complex of eucaryotic cells has been carried out using both in vitro and in vivo mechanistic models. Flux control coefficients (FCC) for the sensitivity of pyruvate decarboxylation rate to activities of various PDH complex reactions are determined. FCCs are shown to be strong functions of both pyruvate levels and various components of PDH complex. With the in vitro model, FCCs are shown to be sensitive to only the E1 component of the PDH complex at low pyruvate concentrations. At high pyruvate concentrations, the control is shared by all of the components, with E1 having a negative influence while the other three components, E2, X, and K, exert a positive control over the pyruvate decarboxylation rate. An unusual behavior of deactivation of the E1 component leading to higher net PDH activity is shown to be linked to the combined effect of protein X acylation and E1 deactivation. The steady‐state analysis of the in vivo model reveals multiple steady state behavior of pyruvate metabolism with two stable and one unstable steady‐states branches. FCCs also display multiplicity, showing completely different control distribution exerted by pyruvate and PDH components on three branches. At low pyruvate concentrations, pyruvate supply dominates the decarboxylation rate and PDH components do not exert any significant control. Reverse control distribution is observed at high pyruvate concentration. The effect of dilution due to cell growth on pyruvate metabolism is investigated in detail. While pyruvate dilution effects are shown to be negligible under all conditions, significant PDH complex dilution effects are observed under certain conditions. Comparison of in vitro and in vivo models shows that PDH components exert different degrees of control outside and inside the cells. At high pyruvate levels, PDH components are shown to exert a higher degree of control when reactions are taking place inside the cells as compared to the in vitro situation.
Complete Conversion of d,l‐5‐Monosubstituted Hydantoins with a Low Velocity of Chemical Racemization into d‐Amino Acids Using Whole Cells of Recombinant Escherichia coli
Authors: Martinez‐Rodriguez Sergio , Javier Las Heras‐Vazquez Francisco , María Clemente‐Jimenez Josefa , Mingorance‐Cazorla Lydia , Rodriguez‐Vico Felipe
Published: December 2002

A reaction system was developed for the production of d‐amino acids from d,l‐5‐monosubstituted hydantoins with a very slow rate of spontaneous racemization. For this purpose the d‐hydantoinase and d‐carbamoylase from Agrobacterium radiobacter NRRL B11291 were cloned in separate plasmids and expressed in Escherichia coli. The third enzyme, hydantoin racemase, was cloned from Agrobacterium tumefaciens C58. The hydantoin racemase amino acid sequence was significantly similar to those previously described. A reaction system consisting of recombinant Escherichia coli whole cell biocatalysts containing separately expressed d‐hydantoinase, d‐carbamoylase, and hydantoin recemase showed high substrate specificity and was effective toward both aliphatic and aromatic d,l‐5‐monosubstituted hydantoins. After optimizing reaction conditions (pH 8 and 50 °C), 100% conversion of d,l‐5‐(2‐methylthioethyl)‐hydantoin (15 mM) into d‐methionine was obtained in 30 min.
Laminar‐Flow‐Based Separations at the Microscale
Authors: Oakey John , Allely Josh , Marr David W. M.
Published: December 2002

The natural separation maintained by microfluidic flows is employed as the basis of a particle/cell sorting device. This method of separating particulate suspensions exploits the inherent laminar nature of microscale fluid dynamics and incorporates applied fields and image cytometry to enable sorting based upon any visually identifiable difference between colloid‐sized cells or particles. This technique may be used to easily isolate, separate, sort, or enrich virtually any suspension of microscale biological or colloidal particles within a microfluidic system. The entire footprint of the device described here is less than 0.01 mm2, allowing it to be readily incorporated within highly integrated micro total analysis systems (μTAS).
Integrated Process for the Enzymatic Synthesis of the Octapeptide PhAcCCK‐8
Authors: Fité Mercè , Clapés Pere , López‐Santín Josep , Benaiges M. Dolors , Caminal Glòria
Published: December 2002

A process for the enzymatic synthesis of PhAcCCK‐8 is presented. The CCK‐8 (CCK26‐33) peptide fragment is the minimum sequence with biological activity of the cholecystokinin hormone. A synthetic convergent strategy has been developed starting from amino acid derivatives as raw materials, employing proteases as biocatalysts for each peptide coupling. The enzymes have been immobilized by deposition onto solid supports in order to be employed in organic media at low water activity. N‐Terminal protecting groups such as PhAc, which can be introduced and removed enzymatically, have been employed. The synthesis process has been set up at preparative level with focus in the integration of reaction and separation steps with an overall yield of 15%.
High Initial Activity but Low Storage Stability Observed for Several Preparations of Subtilisin Carslberg Suspended in Organic Solvents
Authors: Martínez Susimar González , Alvira Edgardo , Cordero Liz Vergara , Ferrer Amaris , Montañés‐Clemente Ileana , Barletta Gabriel
Published: December 2002

Colyophilization with methyl‐β‐cyclodextrin activates subtilisin Carlsberg by more than 200‐fold in organic solvents, though this is a short‐lived effect. About 93% of the enzyme's high initial activity observed in THF (at 45 °C) decreases exponentially with a t1/2 of 1.8 h, until it reaches a residual activity (of 7%) that remains constant throughout the 4 days duration of the experiment. A further study of this enzyme reveals a general trend: the initial activities of the lyophilized powder and the cross‐linked enzyme crystals are also greatly reduced upon incubation in this solvent, although these preparations retain 50% of their activity after about 20 h of incubation. All of the preparations studied retained some residual activity (which persisted throughout the duration of the experiments) after the initial exponential decay. The data here presented suggest that the mode of enzyme preparation is an important issue to consider when planning lengthy reactions.
Strawberry Pectin Methylesterase (PME): Purification, Characterization, Thermal and High‐Pressure Inactivation
Authors: Ly‐Nguyen Binh , Van Loey Ann M. , Fachin Diana , Verlent Isabel , Duvetter Thomas , Vu Son T. , Smout Chantal , Hendrickx Marc E.
Published: December 2002

Pectin methylesterase (PME) was extracted from strawberries ( Fragaria ananassa, cv Elsanta) and purified by affinity chromatography on a CNBr‐Sepharose 4B‐PME‐inhibitor column. A single protein and PME activity peak was obtained. A biochemical characterization in terms of molecular mass, pI, and kinetic parameters of strawberry PME was performed. In a second step, the thermal and high‐pressure stability of the enzyme was studied. Isothermal and combined isothermal‐isobaric inactivation of purified strawberry PME could be described by a fractional‐conversion model. Purified strawberry PME is much more stable toward high‐pressure treatments in comparison to those from oranges and bananas.
Renaturation and Interaction of Ribonuclease A with AOT Surfactant in Reverse Micelles
Authors: Chen Wen‐Yih , Lee Ya‐Wen , Lin Shin‐Chun , Ho Ching‐Wen
Published: December 2002

This study extended works on effects of solute on the percolation of reverse micelles to the effects of interactions between protein and surfactants on protein refolding by reverse micelles. The changes in percolation behavior were identified and attributed to the position of solutes in the core aqueous phase and the interaction between the solute and the surfactants. The percolation behavior of reverse micelles with solutes was related to protein renaturation and the reverse micelle. This study aims to highlight the involvement of the interface and the interaction of the protein with the surfactant during protein refolding. Ribonuclease A and AOT reverse micelles together constitute a model system considered here. The systemic parameters of the reverse micelle, water content ( Wo) and pH value, were applied to modify the interaction between the denatured protein molecules and the surfactant interface. The interactions and the locations of the protein molecules were determined from changes in percolation temperature measured by conductivity. The percolation and protein activity show that a stronger interaction of the protein molecules with surfactant corresponds to superior recovery of protein activity. The investigation concludes that the refolding of protein by reverse micelles is not only facilitated by the isolation of reverse micelles but also by the interaction due to the interface of the reverse micelle.