bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modeling the performance of pilot‐scale countercurrent chromatography: Scale‐up predictions and experimental verification of erythromycin separation
Authors: Booth A.J. , Sutherland I. A. , Lye G. J. 
Published: January 2003

AbstractBiosynthesis of polyketide antibiotics, such as erythromycin A (EA), can result in the formation of analogues of the main compound that are chemically and structurally extremely similar. The large‐scale purification of these antibiotics by conventional high‐performance liquid chromatography (HPLC) can be prohibitively expensive due to the large volume of both solvent and adsorbent required. This study examines the feasibility of using a novel pilot‐scale countercurrent chromatography (CCC) machine as an alternative to HPLC. CCC is a low‐pressure (typically <4000 kN m−2) liquid–liquid chromatographic technique that allows the separation of solutes on the basis of their partitioning between two immiscible liquid phases. The effects of mobile phase flow rate, column rotational speed, and sample injection volume on the attainable yield and purity of EA were investigated. Our results show that, at a mobile phase flow rate of 40 mL min−1, a rotational speed of 1200 rpm, and an injection volume of 100 mL (10 g total erythromycin), EA could be satisfactorily fractionated with a purity of ∼92% (w/w) and a recovery yield of ∼100% (w/w). The total solute throughput was estimated to be 0.41 kg day−1. More importantly, we demonstrated simple and predictive linear scale‐up of the CCC separation based on data obtained from a single laboratory‐scale CCC chromatogram, and verified this experimentally. The retention time and peak width of the target compound at the pilot scale could be predicted to within 4% for operation at a range of mobile‐phase flow rates and injection volumes. This predictable nature of CCC separations, unlike HPLC methods, can greatly reduce process development times and enable a complete process‐scale operating scenario to be planned. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 640–649, 2003.
Parameter scanning ultrafiltration: Rapid optimisation of protein separation
Authors: Ghosh Raja , Wan Yinhua , Cui Zhanfeng , Hale Geoff 
Published: January 2003

AbstractHigh‐resolution fractionation of proteins using ultrafiltration is feasible only at highly optimised conditions. Conventional process optimisation methodology demands both time and material. Pulsed sample injection ultrafiltration has been suggested as a rapid process optimisation technique. In the present work the scope of this technique is further extended by “parameter scanning ultrafiltration,” which involves continuous change of a process parameter (e.g., pH, salt concentration). The time and material consumption are thus further reduced. The technique was validated using different proteins and membranes. Sieving coefficients at different pH and salt concentration were compared to those obtained in fixed parameter ultrafiltration experiments. As fractionation case studies the separation of monoclonal antibody from bovine serum albumin and separation of human IgG from human serum albumin were examined. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 673–682, 2003.
Cell affinity separations using magnetically stabilized fluidized beds: Erythrocyte subpopulation fractionation utilizing a lectin‐magnetite support
Authors: Putnam David D. , Namasivayam Vijay , Burns Mark A. 
Published: January 2003

AbstractA magnetically stabilized fluidized bed is used to separate erythrocyte subpopulations. Binding specificity was obtained by immobilizing the lectin Helix pomatia Agglutinin (HpA) or Griffonia simplicifolia I (GSI) onto a magnetite‐containing support. Separation of type A and type O erythrocytes with the lectin HpA was particularly effective, leading to a 94% purity of retained type A erythrocytes. A 3.1 ± 0.6 log removal of type A erythrocytes was also accomplished leading to a 99.7% ± 0.4% purity and 95% ± 7% yield of type O erythrocytes in the collected effluent. Elution of the purified cells was accomplished using fluidization in the presence of a sugar competing for the lectin–erythrocyte binding site. A mathematical model based on the depth filtration model of Putnam and Burns (Chem Eng Sci 1997;52(1):93–105) was extended to include multicomponent cell adhesion. This filtration model is the first to take into account the finite binding capacity of the chromatographic support and is used to characterize the cell binding behavior and to determine optimal parameters and conditions that lead to high capacities and selectivities. Model parameter values and observations from in situ adsorption studies suggest that the non‐spherical shape of the magnetite‐based support allows for a more efficient utilization of the support surface area than the spherical shape. Using a 1.5‐cm diameter laboratory column and realistic parameter values, the processing rates of the system are predicted to be at least an order of magnitude greater than the 108/h cells that can typically be processed in packed bed cell affinity chromatography (CAC) systems. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 650–665, 2003.
Use of the two‐liquid phase concept to exploit kinetically controlled multistep biocatalysis
Authors: Bühler Bruno , Bollhalder Irene , Hauer Bernhard , Witholt Bernard , Schmid Andreas 
Published: January 2003

AbstractThe two‐liquid phase concept was used to develop a whole cell biocatalytic system for the efficient multistep oxidation of pseudocumene to 3,4‐dimethylbenzaldehyde. Recombinant Escherichia coli cells were employed to express the Pseudomonas putida genes encoding xylene monooxygenase, which catalyzes the multistep oxygenation of one methyl group of toluene and xylenes to corresponding alcohols, aldehydes, and acids. A fed‐batch based two‐liquid phase bioconversion was established with bis(2‐ethylhexyl)‐ phthalate as organic carrier solvent and a phase ratio of 0.5; the product formation pattern, the impact of the nutrient feeding strategy, and the partitioning behavior of the reactants were studied. On the basis of the favorable conditions provided by the two‐liquid phase system, engineering of the initial pseudocumene concentration allowed exploiting the complex kinetics of the multistep reaction for the exclusive production of 3,4‐dimethyl‐ benzaldehyde. Further oxidation of the product to 3,4‐dimethylbenzoic acid could be inhibited by suitable concentrations of pseudocumene or 3,4‐dimethylbenzyl alcohol. The optimized biotransformation setup includes a completely defined medium with high iron content and a nutrient feeding strategy that avoids severe glucose limitation as well as high inhibitory glucose levels. Using such a system on a 2‐liter scale, we were able to produce, within 14.5 h, 30 g of 3,4‐dimethylbenzaldehyde as predominant reactant in the organic phase and reached a maximal productivity of 1.6 g per liter liquid volume per hour. The present study implicates that the two‐liquid phase concept is an efficient tool to exploit the kinetics of multistep biotransformations in general. © 2003 Wiley Periodicals. Biotechnol Bioeng 81: 683–694, 2003.
Preparation and characterization of reactive and stable glucose oxidase‐containing liposomes modulated with detergent
Authors: Yoshimoto Makoto , Wang Shaoqing , Fukunaga Kimitoshi , Walde Peter , Kuboi Ryoichi , Nakao Katsumi 
Published: January 2003

AbstractGlucose oxidase‐containing liposomes (GOL) as well as detergent‐modulated glucose oxidase‐containing liposomes were prepared and characterized, focusing not only on the reactivity of the liposomes upon external addition of glucose but also on the leakage of the entrapped glucose oxidase (GO) from the liposomes with the aim of developing a reactive and stable liposomal GO system. The membranes of the GOL prepared were composed of 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC) and modulated with either Triton X‐100 or cholate. In the absence of added detergent, no GO leakage from the GOL was observed while its enzymatic activity was very low (low glucose permeability). As detergent‐modulated liposomes, mixed POPC/Triton X‐100 and mixed POPC/cholate liposomes (abbreviated as TL and CL, respectively) were prepared at different effective detergent/POPC molar ratios (Re) ranging from Re = 0 to Re = Resat (Resat is the critical value of Re at which the liposome membrane is saturated with detergent). The reactivity of GO‐loaded TL (abbreviated as GOTL) or GO‐loaded CL (GOCL) increased drastically with increase in the respective detergent content in the liposomes. In the case of GOTL, at Resat = 0.40, a high reactivity was measured with a simultaneous high extent of GO leakage, suggesting that the observed enzymatic reaction was catalyzed mainly by leaked GO, caused by the interaction of Triton X‐100 with the POPC membrane. On the other hand, GOCL prepared at Resat = 0.43 showed relatively high reactivity with only a small extent of GO leakage, suggesting that most of the enzyme reaction was limited by the glucose permeation across the bilayers of GOCL. The GO leakage from GOCL was found to occur mostly during the rearrangement of the liposomal membrane during the preparation of the GOCL (mixing the GOL and cholate). Fluorescence polarization measurements of membrane‐associated DPH (1,6‐diphenyl‐1,3,5‐hexatriene) indicated that CL prepared by modifying POPC with cholate did not lead to a drastic change in membrane fluidity, indicating that the interacting cholate molecules did not penetrate deeply into the POPC bilayers. In summary, it was clearly shown that the membrane permeability of GOL can be quite simply modulated by mixing it with a certain amount of cholate to form highly reactive and stable GOCL with minimal enzyme leakage. © 2003 Wiley Periodicals. Biotechnol Bioeng 81: 695–704, 2003.
Immobilization of D‐ribulose‐1,5‐bisphosphate carboxylase/oxygenase: A step toward carbon dioxide fixation bioprocess
Authors: Chakrabarti Subhra , Bhattacharya Sumana , Bhattacharya Sanjoy K. 
Published: January 2003

AbstractImmobilization of D‐ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) from spinach leaves is described. This enzyme enables the fixation of carbon dioxide on a five‐carbon sugar D‐ribulose‐1,5‐bisphosphate (RuBP). Two different immobilization methods were employed: dicyclohexylcarbodiimide coupling on nylon membrane matrix and dimethylpimelimidate immobilization on protein A agarose. The reusability of immobilized enzymes, coupling efficiency, and temperature–activity relationship of soluble and immobilized Rubisco are presented. The immobilization imparted greater thermal and storage stability. The thermal deactivation rates of the immobilized enzymes were considerably lower than those of the soluble enzyme. © 2003 Wiley Periodicals. Biotechnol Bioeng 81: 705–711, 2003.
Propagation of viruses on micropatterned host cells
Authors: Endler Elizabeth E. , Duca Karen A. , Nealey Paul F. , Whitesides George M. , Yin John 
Published: January 2003

AbstractWe have developed a technique to characterize the in vitro propagation of viruses. Microcontact printing was used to generate linear arrays of alkanethiols on gold surfaces, which served as substrates for the patterned culture of baby hamster kidney (BHK‐21) cells. Vesicular stomatitis virus (VSV) was added to unpatterned cell reservoirs adjacent to the patterned cells and incubated, setting in motion a continuously advancing viral infection into the patterned cells. At different incubation times, multiple arrays were chemically fixed to stop the viral propagation. Viral propagation distances into the patterned cells were determined by indirect immunofluorescent labeling and visualization of the VSV surface glycoprotein (G). The infection spread at approximately 50 μm/h in the 140‐μm lines. Moreover, different temporal stages of the infection process were simultaneously visualized along individual lines. These stages included initiation of infection, based on G protein expression; cell–cell fusion, based on virus‐induced clustering of cell nuclei; and cytoskeletal degradation, based on localized release of cells from the surface. This work sets a foundation for parallel, high‐throughput characterization of viral and cellular processes. © 2003 Wiley Periodicals Inc. Biotechnol Bioeng 81: 719–725, 2003.
Stability of protein production from recombinant mammalian cells
Authors: Barnes Louise M. , Bentley Catherine M. , Dickson Alan J. 
Published: January 2003

AbstractOne of the most important criteria for successful generation of a therapeutic protein from a recombinant cell is to obtain a cell line that maintains stability of production. If this is not achieved it can generate problems for process yields, effective use of time and money, and for regulatory approval of products. However, selection of a cell line that sustains stability of production over the required time period may be difficult to achieve during development of a therapeutic protein. There are several studies in the literature that have reported on the instability of protein production from recombinant cell lines. The causes of instability of production are varied and, in many cases, the exact molecular mechanisms are unknown. The production of proteins by cells is modulated by molecular events at levels ranging from transcription, posttranscriptional processing, translation, posttranslational processing, to secretion. There is potential for regulation of stability of protein production at many or all of these stages. In this study we review published information on stability of protein production for three industrially important cell lines: hybridoma, Chinese hamster ovary (CHO), and nonsecreting (NS0) myeloma cell lines. We highlight the most likely molecular loci at which instability may be engendered and indicate other areas of protein production that may affect stability from mammalian cells. We also outline approaches that could help to overcome the problems associated with unpredictable expression levels and maximized production, and indicate the consequences these might have for stability of production. © 2003 Wiley Periodicals Inc. Biotechnol Bioeng 81: 631–639; 2003.
Application of a gratuitous induction system in Kluyveromyces lactis for the expression of intracellular and secreted proteins during fed‐batch culture
Authors: Panuwatsuk Wannana , Da Silva Nancy A. 
Published: January 2003

AbstractA gratuitous induction system in the yeast Kluyveromyces lactis was evaluated for the expression of intracellular and extracellular products during fed‐batch culture. The Escherichia coli lacZ gene (β‐galactosidase; intracellular) and MFα1 leader‐BPTI cassette (bovine pancreatic trypsin inhibitor; extracellular) were placed under the control of the inducible K. lactis LAC4 promotor, inserted into partial‐pKD1 plasmids, and transformed into a ga1‐209 K. lactis strain. To obtain a high level of production, culture conditions for growth and expression were initially evaluated in tube cultures. A selective medium containing 5 g/L glucose (as carbon source) and 0.5 g/L galactose (as inducer) demonstrated the maximum activity of both β‐galactosidase and secreted BPTI. This level of expression had no significant effect on the growth of the recombinant cells; growth rate dropped by approximately 11%, whereas final biomass concentrations remained the same. In shake‐flask culture, biomass concentration, β‐galactosidase activity, and BPTI secreted activity were 4 g/L, 7664 U/g dry cell, and 0.32 mg/L, respectively. Fed‐batch culture (with a high glucose concentration and a low galactose [inducer] concentration feed) resulted in a 6.5‐fold increase in biomass, a 23‐fold increase in β‐galactosidase activity, and a 3‐fold increase in BPTI secreted activity. The results demonstrate the success of gratuitous induction during high‐cell‐density fed‐batch culture of K. lactis. A very low concentration of galactose feed was sufficient for a high production level. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 712–718, 2003.
Enzymatic hydrolysis of water‐soluble wheat arabinoxylan. 1. Synergy between α‐L‐arabinofuranosidases, endo‐1,4‐β‐xylanases, and β‐xylosidase activities
Authors: Sørensen Hanne R. , Meyer Anne S. , Pedersen Sven 
Published: January 2003

AbstractHydrolysis of arabinoxylan is an important prerequisite for improved utilization of wheat hemicellulose in the ethanol fermentation industry. This study investigates the individual and combined efficiencies of three commercial, cellulytic and hemicellulytic enzyme preparations, Celluclast 1.5 L, Ultraflo L, and Viscozyme L, in catalyzing the liberation of arabinose and xylose from water‐soluble wheat arabinoxylan. Ultraflo L was the best enzyme preparation for releasing arabinose, liberating 53 wt% of the theoretical maximum after 48 h of reaction (10 wt% enzyme/substrate ratio, 40°C, pH 6). Celluclast 1.5 L was superior to the other enzyme preparations in releasing xylose, liberating 26 wt% of the theoretical maximum after 48 h of reaction (10 wt% enzyme/substrate ratio, 50°C, pH 5). The 50:50 mixtures of the enzyme preparations showed no synergistic cooperation in arabinose release, but a synergistic interaction in xylose release was found between Ultraflo L and Celluclast 1.5 L. On the basis of high‐performance anion exchange chromatography (HPAEC) analysis of the hydrolysates after enzymatic reaction, we propose that the observed synergism between Celluclast 1.5 L and Ultraflo L is the result of positive interaction between α‐L‐arabinofuranosidase and endo‐1,4‐β‐xylanase activities present in Ultraflo L that released arabinose, xylobiose and xylotriose, and β‐xylosidase activities in Celluclast 1.5 L, capable of catalyzing the hydrolysis of xylobiose and xylotriose to xylose. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 726–731, 2003.
Transglutaminase‐catalyzed synthesis of trypsin–cyclodextrin conjugates: Kinetics and stability properties
Authors: Villalonga Reynaldo , Fernández Michael , Fragoso Alex , Cao Roberto , Di Pierro Prospero , Mariniello Loredana , Porta Raffaele 
Published: January 2003

AbstractBovine pancreatic trypsin was modified by the mono‐6‐amino‐6‐deoxy derivatives of α‐, β‐, and γ‐cyclodextrin through a transglutaminase‐catalyzed reaction. The trypsin–cyclodextrin conjugates, containing about 3 mol of oligosaccharide per mole of protein, were tested for their catalytic and stability properties. The specific esterolytic activity and the kinetics constants of trypsin were significantly improved following the transglutaminase‐induced structural modifications. Trypsin–cyclodextrin conjugates were also found markedly (sixfold) more resistant to autolytic degradation at alkaline pH, and their thermal stability profile was improved by about 16°C. Moreover, they were particularly resistant to heat inactivation when treated at different temperatures ranging from 45°C to 70°C for different periods of time. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 732–737, 2003.
Intracellular Ca2+ imaging for micropatterned cardiac myocytes
Authors: Kaji Hirokazu , Takoh Kimiyasu , Nishizawa Matsuhiko , Matsue Tomokazu 
Published: January 2003

AbstractThe patterning of cardiac myocytes on a micron scale (∼5 μm) was achieved by microcontact printing of fibronectin onto a hydrophobically pretreated glass substrate. The patterned cardiac myocytes conjugated with each other by forming a gap junction, as judged from the synchronized Ca2+ transition over the pattern, and thus simultaneously contracted. The dynamic change of the Ca2+ concentration within the patterned tissue was analyzed quantitatively during successive contraction and relaxation using a Nipkow‐type high‐speed confocal microscope. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 748–751, 2003.
Protein phosphatase 2C is involved in the cAMP‐dependent ciliary control in Paramecium caudatum
Authors: Noguchi Munenori , Sasaki Jun‐Ya , Kamachi Hiroyuki , Inoue Hiroshi 
Published: January 2003

AbstractForward swimming of the Triton‐extracted model of Paramecium is stimulated by cAMP. Backward swimming of the model induced by Ca2+ is depressed by cAMP. Cyclic AMP and Ca2+ act antagonistically in setting the direction of the ciliary beat. Some ciliary axonemal proteins from Paramecium caudatum are phosphorylated in a cAMP‐dependent manner. In the presence of cAMP, axonemal 29‐ and 65‐kDa polypeptides were phosphorylated by endogenous A‐kinase in vitro. These phosphoproteins, however, were not dephosphorylated after in vitro phosphorylation, presumably because of the low endogenous phosphoprotein phosphatase activity associated with isolated axonemes. We purified the protein phosphatase that specifically dephosphorylated the 29‐ and 65‐kDa phosphoproteins from Paramecium caudatum. The molecular weight of the protein phosphatase was 33 kDa. The protein phosphatase had common characteristics as protein phosphatase 2C (PP2C). The characteristics of the protein phosphatase were the same as those of the PP2C from Paramecium tetraurelia (PtPP2C) [Grothe et al., 1998: J. Biol. Chem. 273:19167–19172]. We concluded that the phosphoprotein phosphatase is the PP2C from Paramecium caudatum (PcPP2C). The PcPP2C markedly accelerated the backward swimming of the Triton‐extracted model in the presence of Ca2+. On the other hand, the PcPP2C slightly depressed the forward swimming speed. This indicates that the PP2C plays a role in the cAMP‐dependent regulation of ciliary movement in Paramecium caudatum through dephosphorylation of 29‐ and/or 65‐kDa regulatory phosphoproteins by terminating the action of cAMP. Cell Motil. Cytoskeleton 54:95–104, 2003. © 2003 Wiley‐Liss, Inc.
Hemidesmosome protein dynamics in live epithelial cells
Authors: Tsuruta Daisuke , Hopkinson Susan B. , Jones Jonathan C.R. 
Published: January 2003

AbstractHemidesmosomes mediate stable anchorage of epithelial cells to laminin‐5 in the basement membrane zone and have been likened to spot‐welds. Indeed, it has been assumed that hemidesmosomes are not dynamic, at least when compared to other matrix adhesion sites including focal contacts. We tested this notion by monitoring the fate of green fluorescent protein (GFP)‐tagged human integrin β4 subunit (GFP‐hβ4) and GFP‐tagged 180‐kD human bullous pemphigoid (BP) autoantigen (GFP‐BP180) in live cultures of 804G cells that assemble numerous mature hemidesmosomes. In subconfluent 804G cells, both GFP‐hβ4 and GFP‐BP180 protein clusters are not stable but assemble into and disassemble out of cat paw–like arrays at a relatively rapid rate. In confluent populations of 804G cells, although some cat paw–like clusters of both GFP‐hβ4 and GFP‐BP180 are stable over periods of >60 min, other GFP‐hβ4 and GFP‐BP180 protein arrays form and/or disappear during the same time period. Moreover, individual labeled particles show considerable motility in the plane of the membrane. Fluorescence recovery after photobleaching analyses provide a further indication of the dynamics of hemidesmosome proteins. In particular, bleached GFP‐hβ4 protein clusters in confluent cells recover signal within about 30 min, indicating that there is a relatively rapid turnover of hemidesmosome components in protein arrays clustered along the substratum attached surface of a cell. The rate of recovery is dependent on an intact microfilament system. In sharp contrast, bleached GFP‐BP180 protein clusters in confluent cells fail to recover signal even when observed for longer than 60 min. To evaluate hemidesmosome protein dynamics in motile cells, we monitored GFP‐hβ4 and GFP‐BP180 in 804G cells populating scrape wound sites in vitro. In these migratory cells, which lack mature hemidesmosomes, integrin β4 subunit and BP180 protein clusters progressively assemble and disassemble into linear and cat‐paw arrays. In summary, hemidesmosome protein clusters, like their counterparts in focal contacts, are dynamic. We discuss these results in relation to hemidesmosome functions. Cell Motil. Cytoskeleton 54:122–134, 2003. © 2003 Wiley‐Liss, Inc.