bluuebunny/crossref_metadata_2025 huggingface dataset:
Modeling of hollow‐fiber capillary reactor for the production of L‐Alanine with coenzyme regeneration
Authors: Fujii Tomoyuki , Miyawaki Osato , Yano Toshimasa
Published: December 2004

Continuous production of L‐alanine with conjugated enzyme systems of alanine dehydrogenase (AlaDH) and lactate dehydrogenase (LDH) or alcohol dehydrogenase (ADH) was carried out with NAD regeneration in an ultrafiltration hollow‐fiber capillary reactor (HFCR) which was proposed as a test bioreactor with very small scale. In the AlaDH/LDH system, pyruvate is the intermediate product for L‐alanine so that an optimal point existed in pyruvate concentration for the production rate of L‐alanine. NAD cycling number of 4850 and L‐alanine productivity of 61.7 mmol/L h were obtained at the best condition. In the AlaDH/ADH system, however, the substrate inhibition in the AlaDH reaction by pyruvate should be considered and the best results of NAD cycling number and L‐alanine productivity were 2700 and 13.5 mmol/L h, respectively. In consideration of concentration distribution and mixing in the axial direction on an HFCR, performance of the reactor was theoretically analyzed with a multistage stirred tank reactor model combined with the kinetic model based on all the elementary reactions involved. Although quantitative discrepancy existed in some cases, the present theoretical model could explain experimental results and is expected to be generally applicable to standard hollow fiber reactors.
High‐density photoautotrophic algal cultures: Design, construction, and operation of a novel photobioreactor system
Authors: Javanmardian Minoo , Palsson Bernhard O.
Published: December 2004

A photobioreactor system has been designed, constructed and implemented to achieve high photosynthetic rates in high‐density photoautotrophic algal cell suspensions. This unit is designed for efficient oxygen and biomass production rates, and it also can be used for the production of secreted products. A fiber‐optic based optical transmission system that is coupled to an internal light distribution system illuminates the culture volume uniformly, at light intensities of 1.7 mW/cm2 over a specific surface area of 3.2 cm2/cm3. Uniform light distribution is achieved throughout the reactor without interfering with the flow pattern required to keep the cells in suspension. An on‐line ultrafiltration unit exchanges spent with fresh medium, and its use results in very high cell densities, up to 109 cells/mL [3% (w/v)] for eukaryotic green alga chlorella vulgaris. DNA histograms obtained form flow cytometric analysis reveal that on‐line ultrafiltration influences the growth pattern. Prior to ultrafiltration the cells seem to have at a particular point in the cell cycle where they contain multiple chromosomal equivalents. Following ultrafiltration, these cells divide, and the new cells are committed to division so that cell growth resumes. The Prototype photobioreactor system was operated both in batch and in continuous mode for over 2 months. The measured oxygen production rate of 4‐6 mmol/L culture h under continuous operation is consistent with the predicted performance of the unit for the provided light intensity.
A novel continuous centrifugal bioreactor for high‐density cultivation of mammalian and microbial cells
Authors: Van Wie Bernard J. , Brouns Thomas M. , Elliot Michael L. , Davis William C.
Published: December 2004

A continuous centrifugal bioreactor (CCBR), developed to study the growth and productivity of dense suspensions cultures, has been applied to both fermentation and mammalian cell cultivation processes. With this approach, high‐density nonflocculent cultures are maintained in a tapered fluidized bed by balancing the drag forces on the cells due to following substrate with the centrifugal forces. The Sysyem was first used to produce ethanol by fermentation with Saccharomyces cerevisiae; then with H21A1 mouse hybridoma cells secreting monoclonal antibody (MoAb), lgM. Results of this research show the feasibility of using the CCBR for both production of secreted products and as a research tool for studying cell metabolism and production kinetics. Media recycle may be used to modify the behavior of the system form a plug flow apparatus to a continuous stirred reactor (CSTR).
Solute exclusion from cellulose in packed columns: Process modeling and analysis
Authors: Neuman R. P. , Walker L. P.
Published: December 2004

In this investigation, process modeling and analysis were used to explore the behavior of solute exclusion from cellulose in packed columns. The study focused on modeling the effects of dispersion, mass transport, and pore diffusion. Three mathematical models were used to predict the behavior of the columns: an equilibrium model, a mass transfer model, and a combined mass transfer and pore diffusion model. Computer implementations of these models were tested against experimental conditions where cellulose particle size and solution velocity were used to either amplify or minimize dispersion or skewness in the elution curves. For small cellulose particles (200–300 mesh), all three models accurately predicted the shape of the elution curve and the particle porosity. For larger particles (45‐60 mesh), the mass transfer model and the combined mass and pore diffusion model best represented the behavior of the column. At high solution velocities (0.63 cm3 min−1) and large particles, only the combined mass transfer and pore diffusion model accurately represent the column behavior. Sensitivity analysis revealed that the mass transfer coefficient had little effect on the elution curves for the range of values (10−6–10−3 cm s−1) calculated from the experimental data. The combined mass transfer and pore diffusion model presented in this article can be used to design solute exclusion measurement experiments for the larger cellulose particles found in a commercial cellulose‐to‐ethanol plant.
Emulsion stability and demulsification during the direct extraction of penicillin broth
Authors: Szabó Csaba
Published: December 2004

In this article, we present the factors which influence the stability of the emulsion forming under the circumstances of the direct extraction of penicillin fermentation broth and the conditions of demulsification. A laboratory method has been developed for testing demulsifiers and a quantitative evaluation of the properties determining the separation of phases has been worked out as well. It has been found that an emulsion of oil in water (o/w) forms and that this is very stable; it can be broken only by the presence of demulsifier and by using a field of centrifugal force. We have discovered that the most effective demulsifiers are cation‐active surfactants for 1–4 g/L broth concentration. Furthermore, it is advantageous if the demulsifier contains a nonionic surfactant and wetting agent as well. From the 19 surface‐active compounds studied in the laboratory, we found that the NADAR 107 A and ARMOGARD D‐5306 demulsifiers contained the best properties for the extraction of the penicillin broth.
Use of glucose starvation to limit growth and induce protein production in Escherichia coli
Authors: Tunner Joseph R. , Robertson Channing R. , Schippa Serena , Matin Abdul
Published: December 2004

The use of glucose starvation to uncouple the production of recombinant β‐galactosidase from cell growth in Escherichia coli was investigated. A lacZ operon fusion to the carbon starvation‐inducible cst‐1 locus was used to control β‐galactosidase synthesis. β‐Galactosidase induction was observed only under aerobic starvation conditions, and its expression continued for 6 h following the onset of glucose starvation. The cessation of β‐galactosidase expression closely correlated with the exhaustion of acetate, an overflow metabolite of glucose, from the culture medium. Our results suggest the primary role of acetate in cst‐1‐controlled protein expression is that of an energy source. Using this information, we metered acetate to a glucose‐starved culture and produced a metabolically sluggish state, where growth was limited to a low linear rate and production of recombiant β‐galactosidase occurred continuously throughout the experiment. The cst‐1 controlled β‐galactosidase synthesis was also induced at low dilution rates in a glucose‐limited chemostat, suggesting possible applications to high‐density cell systems such as glucose‐limited recycle reactors. This work demonstrates that by using an appropriate promoter system and nutrient limitation, growth can be restrained while recombinant protein production is induced and maintained.
Enzymatic reaction kinetic: Comparison in an organic solvent and in supercritical carbon dioxide
Authors: Dumont T. , Barth D. , Corbier C. , Branlant G. , Perrut M.
Published: December 2004

Myristic acid esterification has been performed by an immobilized lipase from Mucor Miehei both in n‐hexane and in supercritical carbon dioxide (SCCO2). The enzyme is stable in SCCO2 at 15 MPa and 323 K. The reaction rate is influenced by the concentration of water and by the reaction medium composition. A reaction mechanism is proposed, and kinetic parameters are determined at 12.5 MPa and 313 K. Maxium velocity appears 1.5‐fold higher in SCCO2 than in n‐hexane; however, as solubility of myristic acid is greater in n‐hexane, it is not yet definitively clear that the supercritical medium is more favorable than the classical organic solvent for this type of enzyme reaction.
Changes in yeast amino acid pool with respiratory versus fermentative metabolism
Authors: Martíez‐force Enrique , Benítez Tahía
Published: December 2004

The precursors of the amino acid yeast pool are intermediates of either the glycolytic or the tricarboxilic acid pathway (TCA). Accordingly, the influence of the metabolism (fermentative or respiratory) on the internal amino acid pool of the yeast Saccharmyces cerevisiae was established by measuring the intracellular amino acid concentration of the “grande” strain IF1256 and its “petite” mutant either in steady‐state or when shifting from fermentative to respiratory conditions. Under steady‐state conditions, when the cells only respire, there is a decrease in nearly all the amino acids whose precursors are intermediates of the glycolytic pathway. When the metabolism is exclusively fermentative, the opposite change takes place. This effect is not observed in those amino acids whose precursors come from the TCA cycle. However, in continuous culture and at dilution rates lower than 0.06 h−1, there is an enormous increase in the concentration of all the amino acids in both strains, whatever their precursor, whereas, in batch cultures, both strains undergo variations in the concentration of most amino acids, when entering stationary growth phase. Results therefore indicate that, the fact that the cells ferment or respire effectively affect their amino acid pool according to their precursors coming from the glycolytic or the TCA pathway, but other parameters, such as growth rate or exponential versus stationary phase, have a much stronger effect on yeast amino acid concentration. © 1992 John Wiley & Sons, Inc.
Computer‐assisted modeling of subtilisin enantioselectivity in organic solvents
Authors: Fitzpatrick Paul A. , Ringe Dagmar , Klibanov Alexander M.
Published: December 2004

In order to rationalize our discovery of a marked dependence of subtilisin's enantioselectivity on the organic solvent used as the reaction medium, we empolyed the X‐ray crystal structure of the enzyme and the means of interactive computer modeling to construct the structures of the reactive enzyme‐substrate complexes. For subtilisin‐catalyzed transesterifications between vinyl butyrate and S and R enantiomers of chiral secondary alcohols XCH(OH)Y, the computer simulation data clearly explain a higher reactivity of the former enantiomer on the basis of severe steric hindrances experienced by the latter enantiomer in the active site of subtilisin. The models of binding derived by computer modeling also successfully predicted changes in subtilisin enantioselectivity as a function of the sizes of the X and Y substituents in the nucleophile and upon addition of certain inhibitors. © 1992 John Wiley & Sons, Inc.
Continuous hydrolysis of olive oil by immobilized lipase in organic solvent
Authors: Yang Dongsoo , Rhee Joon Shick
Published: December 2004

Lipase (EC 3.1.1.3) from Candida rugosa was immobilized with DEAE‐Sephadex A50, Sephadex G50, Sephadex LH‐20, Amberlite IRA94, and Amberlite XAD‐7. The enzye immobilized with DEAE‐Sephadex A50 was found to be most effective for continuous hydrolysis of olive oil in isooctane. For the continuous reaction, 0.2 g of dry immobilized enzyme was swollen with predetermined amount of water, and packed in a glass column reactor. When the organic solvent (Isooctane) containing olive oil substrate was cocurrently fed with aqueous buffer, the two phases were evenly distributed throughout the packed bed without surfactant supplement or prior mixing of the two phases. A small amount of the surfactant (AOT) was used only in packing procedure, and no additional surfactant was necessary thereafter. Effects of initial water content of the swollen gel, buffer types, and strength were examined in the continuous reaction. Our results suggest that the operational half‐life was affected by desorption of the bound enzyme. Under the conditions of 20% olive oil in isooctane and 25 mM triethanolamine buffer (pH 7.0), operational half life was 220 h at 30°C. The reactor was also operable with n‐hexane, but the operational stability of the immobilized enzyme in n‐hexane was only half of that in isooctane. Our results indicate that various enzyme carrier having hydrophilic or amphiphilic properties could be used for two‐phase continuous reaction in packed‐bed column, reactor without any surfactant supply or prior dispersion of the two immiscible phases. © 1992 John Wiley & Sons, Inc.
Stimulation of acid phosphatase induction in Saccharomyces cerevisiae by electrochemical modulation of effector concentration
Authors: Haruyama Tetsuya , Kobatake Eiry , Ikariyama Yoshihito , Aizawa Masuo
Published: December 2004

A novel modulating method of the expression of Saccharomyces PHO 5 gene, responsible for acid phosphatase (APase), is proposed. The method is based on electrochemical modulation of an effector (inorganic phosphate) concentration, as the gene expression is initiated below a threshold concentration of phosphate and is terminated above the threshold value. By positioning the yeast in the close neighborhood of a conducting polymer, the authors show the effectiveness of the electrochemical approach toward PHO 5 induction. Based on the approach, phosphate concentration is easily modulated at the boundary concentration by taking advantage of anion doping–undoping at a conducting polymer and the resulting anion localization–delocalization in the polymer, as the local enrichment of phosphate in the polymer results in the lowering of phosphate in the vicinity of polypyrrole. External phosphate concentration is thus electrochemically modulated when the conducting polymer is positioned in the close neighborhood of the yeast cells; thereby the PHO gene is induced. Here an electrochemical approach for the APase expression as a strategy of selective induction of specific genetic information is described. © 1993 John Wiley & Sons, Inc.
Kinetic study of lipase catalyzed esterification reactions in water‐in‐oil microemulsions
Authors: Stamatis Haralambos , Xenakis Aristotelis , Menge Ulrich , Kolisis Fragiskos N.
Published: December 2004

The kinetics of the esterification of lauric acid by (‐)menthol, catalyzed by Penicillium simplicissimum lipase, was studied in water/bis‐(2‐ethylhexyl)sulfosuccinate sodium salt (AOT)/isooctane microemulsions. Due to their low water content, microemulsions assist in reversing the direction of lipase activity, favoring synthetic reactions. The kinetics of this synthesis follows a Ping‐Pong BiBi mechanism. The values of all apparent kinetic parameters were determined. The theoretical model for the expression of enzymic activity in reverse micelles, proposed by Verhaert et al. (Verhaert, R., Hilhorst, R., Vermüe, M., Schaafsma, T. J., Veeger, C. 1990. Eur. J. Biochem. 187: 59–72) was extended to express the lipase activity in an esterification reaction involving two hydrophobic substrates in microemulsion systems. The model takes into account the partitioning of the substrates between the various phases and allows the calculation of the intrinsic kinetic constants. The experimental results showing the dependence of the initial velocity on the hydration ratio, Wo = [H2O]/[AOT], of the reverse micelles, were in accordance with the theoretically predicted pattern. © 1993 John Wiley & Sons, Inc.
Estimation of disruption of animal cells by turbulent capillary flow
Authors: Zhang Z. , Al‐Rubeai M. , Thomas C. R.
Published: December 2004

Disruption of animal cells in turbulent capillary flows has been predicted from a model of cell–hydrodynamic interactions using cell mechanical properties determined by micromanipulation. Eddies of sizes similar to or smaller than the cells are presumed to interact with those cells, causing local surface deformations. The proposed mechanism of cell damage is that such deformations result in an increase in membrane tension and surface energy and that a cell disrupts when its bursting membrane tension and bursting surface energy are exceeded. The surface energy of the cells is estimated from the kinetic energy of appropriately sized eddies. To test the model, cells were disrupted in turbulent flows in capillaries at mean energy dissipation rates up to 2 × 104 m2/s3. In all cases the model underestimated the cell disruption by about 15%. Such good agreement implies that the approach of the model to the complicated phenomena of cell turbulence interactions is reasonable. © 1993 John Wiley & Sons, Inc.
Engineering cellulase mixtures by varying the mole fraction of Thermomonospora fusca E5 and E3, Trichoderma reesei CBHI, and Caldocellum saccharolyticum β‐glucosidase
Authors: Walker L. P. , Belair C. D. , Wilson D. B. , Irwin D. C.
Published: December 2004

In this study, different mole fractions of pure Thermomonospora fusca E5 and E3, plus Trichoderma reesei CBHI were studied for reducing sugar production at 2 h, degree of synergism, and cellulose binding. In addition, the effects of introducing the Caldocellum saccharolyticum β‐glucosidase into this cellulase system were investigated. The cellulases used were purified to homogeneity. Avicel PH 102 (4% w/w solution in 0.05 sodium acetate pH 5.5 buffer) was the substrate. Reactions were run at 50°C for 2 h using total cellulase concentrations of 8.3 or 12.2 μM. A bimixture of T. fusca E3 and T. reesei CBHI was very effective in hydrolyzing microcrystalline cellulose (9.1% conversion). The addition of endoglucanase E5 to the mixture only increased conversion to 9.8%. However, when both E5 and β‐glucosidase were added, conversion increased to 14%. It was also observed that increasing total cellulase concentration beyond 8.3 μM did little to increase percent conversion of cellulose into glucose. The results of the binding studies indicate no competition for binding sites between the endo‐ and exocellulases. © 1993 John Wiley & Sons, Inc.
Study on separation of conalbumin and lysozyme from high concentration fresh egg white at high flow rates by a novel ion‐exchanger
Authors: Ming Fang , Howell John , Acosta Fernando , Hubble John
Published: December 2004

In this report, we show that it is possible to separate valuable proteins from egg‐white using a ProductivTM CM ion‐exchanger column operated at flow rates significantly higher than those than can be achieved using traditional particulate adsorbents. In the approach taken, sample pretreatment is restricted to a simple dilution of the egg‐white, which can then be applied to the column at superficial velocities (Vs) of up to 13.8 m/h. Under a loading of 220 mg total protein per milliliter of ion‐exchanger, the resolution (Rs) between the eluted conalbumin and lysozyme fractions was found to be almost constant during nine consecutive adsorption/desorption cycles. For all nine consecutive batches, the column average adsorption capacity was greater than 30 mg/mL, with 90% recovery of adsorbed protein being achieved in each run. The overall productivity achieved was 12.6 kg/m3 h for lysozyme and 31.2 kg/m3 h for conalbumin. © 1993 John Wiley & Sons, Inc.
Stability of antibody productivity is improved when hybridoma cells are entrapped in calcium alginate beads
Authors: Lee Gyun Min , Palsson Bernhard O.
Published: December 2004

Loss of monoclonal antibody (MAb) productivity in long‐term, free‐suspended cell culture is often attributed to the appearance of a nonproducing population of hybridoma cell (NP) in the culture which has a growth advantage over the producing population (P). However, when an NP appears in long‐term culture of entrapped cells, it may not be able to take over the whole culture in a short period of time due to the limited growth of the entrapped cells. In order to examine the hypothesis that entrapped cells can have improved stability of MAb productivity due to limited cell growth, free‐suspended cell culture and calcium alginate–entrapped cell culture with inocula consisting of a P and an NP were compared with regard to stability of MAb productivity in a repeated fed‐batch culture. In free‐suspended cell culture, the NP appeared to take over the whole culture within three batches, and thereby MAb production completely disappeared. In entrapped cell culture, an NP appeared to outgrow the P rapidly only during an exponential growth phase, resulting in a significant decrease in specific MAb productivity, qMAb, from 11.58 μg/106 cell/day to 2.76 μg/106 cell/day. However, when the cell growth was limited in entrapped cell culture, the NP no longer outgrew the P rapidly, as indicated by the stable value of qMAb. In addition, when the cells recovered from the alginate beads by citrate buffer treatment were subcultured in free‐suspended cell culture, MAb production rapidly deteriorated and completely disappeared within two batches. Thus, the P present at a small fraction of viable cell concentration in the beginning of the free‐suspended cell culture, which were previously entrapped in alginate beads, seemed to be outgrown rapidly by the NP. Taken together, the results obtained from these experiments support the hypothesis that the limited cell growth in entrapped cell culture, which keeps an NP from taking over the whole culture, is responsible, in part, for the improved stability of MAb productivity. © 1993 John Wiley & Sons, Inc.
The influence of vessel height and top‐section size on the hydrodynamic characteristics of airlift fermentors
Authors: Russell A. B. , Thomas C. R. , Lilly M. D.
Published: December 2004

Fermentations of the yeast Saccharomyces cerevisiae were carried out in a 90 to 250‐L working volume concentric tube airlift fermentor. Measurements of liquid circulation velocity, gas hold‐up, and liquid mixing were made under varying conditions of gas flowrate, vessel height, and top‐section size. Both liquid circulation velocity and mixing time increased with vessel height. Liquid velocity varied approximately in proportion to the square root of column height, supporting a theoretically based relationship. The effect of vessel height on gas hold‐up was negligible. The height of the top‐section had a significant effect on liquid mixing. Mixing time decreased with increasing size of the top‐section up to a critical height. As the top‐section was expanded beyond this height, little improvement in mixing was seen. This indicated the presence of a two‐zone flow pattern in the top‐section. Liquid velocity and gas hold‐up were essentially independent of top‐section height. © 1994 John Wiley & Sons, Inc.
Cell aggregation and neurite growth in gels of extracellular matrix molecules
Authors: Krewson Christine E. , Chung Sonia W. , Dai Weiguo , Mark Saltzman W.
Published: December 2004

Components of the extracellular matrix are believed to guide both nerve cells and neurites to their targets during embryogenesis and, therefore, might be useful for controlling regeneration of nervous tissue in adults. To study the influence of extracellular conditions on neurite outgrowth and cell motility, PC12 cells were suspended in three‐dimensional gels containing (i) collagen (0.4 to 2 mg/mL), (ii) collagen (1 mg/mL) with added fibronectin or laminin (1 to 100 μg/mL), and (iii) agarose (7 mg/mL) with added collagen (0.001 to 1 mg/mL). Neurite outgrwoth was stimulated with nerve growth factor (NGF) and both the extent of neurite outgrowth ad cell aggregation were quantitated over 10 to 12 days in culture. The extent of neurite outgrowth was greatest at the lowest collagen concentration tested (0.4 mg/mL) and decreased with increasing concentration. The addition of laminin or fibronectin altered the extent of neurite outgrowth in collagen gels, but the differences were small. Although no neurite growth was observed in pure agarose gels, considerable neurite outgrowth occurred with the addition of small amounts (≥0.01 mg/mL) of collagen. Mean aggregate size increased more quickly in gels with lower concentrations of collagen. For cells in 1.0 mg/mL collagen, a four‐ to fivefold increase in aggregate volume was seen between days 2 and 10 o the culture period, whereas the increase in DNA content during this same period was less than twofold, suggesting that the cells were aggregating, not multiplying. These results suggest that the composition of the matrix supporting nerve cells has a significant effect on both neurite outgrowth and cell motility. © 1994 John Wiley & Sons, Inc.
Analysis of endothelial cell locomotion: Differential effects of motility and contact inhibition
Authors: Lee Y. , McIntire L. V. , Zygourakis K.
Published: December 2004

Video microscopy and digital time‐lapse recording were used to monitor locomotion and proliferation of bovine pulmonary artery endothelial (BPAE) cells cultured with varying concentrations of basic fibroblast growth factor (bFGF). Cell trajectories were reconstructed using a generalized nearest‐neighbor algorithm and analyzed to determine how cell motility is affected by cell‐cell collisions, cell divisions, and increasing cell density. The temporal evolution patterns of the average speed of locomotion for all cells in a culture were computed and the effects of varying bFGF concentrations were analyzed. Intermediate concentrations of bFGF (30 and 50 ng/mL) significantly increased the speed of locomotion above the levels we observed with 0 and 100 ng/mL concentrations of bFGF. Increases in cell density due to proliferation were immediately accompanied by a decrease in the average speed of locomotion of the cell population. Finally, the effect of bFGF concentration on the overall cell proliferation rates was assessed. With the addition of 30 or 50 ng/mL of bFGF to the culture media, the observed cell proliferation rates increased significantly. The proliferation rates decreased when the bFGF concentration increased to 100 ng/mL. These results show that bFGF concentrations that increase the motility of BPAE cells also increase the observed cell proliferation rates. © 1994 John Wiley & Sons, Inc.
Isolated hepatocytes in a bioartificial liver: A single group view and experience
Authors: Rozga Jacek , Morsiani Eugenio , Lepage Elaine , Moscioni Albert D. , Demetriou Achilles A. , Giorgio Todd
Published: December 2004

Despite recent advances in medical supportive therapy, patients with severe fulminant hepatic failure (FHF) have mortality rate approaching 90%. Investigators have attempted to improve survival by using various extracorporeal liver support systems loaded with sorbents and liver tissue preparations. None of them succeeded in gaining clinical acceptance and orthotopic liver transplantation (OLT) remains a primary therapeutic option for patients with FHF. In this study, authors discuss the systems which utilize isolated hepatocytes. Most of these devices were tested in vitro and in animals with chemically and surgically induced liver failure. In some studies, signficant levels of detoxification and liver functions were achieved. The authors describe their own hepatocyte‐based artificial liver (BAL). It is based on plasma perfusion through a hollow‐fiber module seeded with matrix‐anchored porcine hepatocytes. The BAL was used 14 times to treat 9 patients with acute liver failure. On 10 occasions, a charcoal column was included in the plasma circuit. Each treatment lasted 7 ± 1 h. All procedures were tolerated well and 8 patients (including 6 patients with FHF) underwent OLT. Five patients with increased intracranial pressure (ICP) and evidence of decerebration had normalization of ICP and enjoyed full neurologic recovery after OLT. Laboratory data showed evidence for bilirubin conjugation, decrease in blood ammonia, maintenance of low lactic acid levels, and increase in the ration between the branched chain and aromatic amino acids. No allergic reactions to xenogeneic hepatocytes were observed. The authors conclude that BAL treatment with porcine hepatocytes appears to be safe and can help maintain patients alive and neurologically intact until a liver becomes available for transplantation. © 1994 John Wiley & Sons, Inc.
Mini‐review: Islet transplantation to create a bioartificial pancreas
Authors: Mikos Antonios G. , Papadaki Maria G. , Kouvroukoglou Stylianos , Ishaug Susan L. , Thomson Robert C.
Published: December 2004

Donor scarcity precludes the use of pancreatic transplantation to treat type I diabetes. Xenogeneic islet transplantation offers the possibility of overcoming this problem; however, it entails the use of immunoisolation devices to prevent immune rejection of the transplanted islets. These devices consist of a semipermeable membrane, which surrounds the islets and isolates them from the host's immune system, while allowing the passage of insulin and essential nutrients, including glucose. Problems associated with proposed device designs include diffusion limitations, biocompatibility, device retrieval in the event of failure, and mechanical integrity. Microencapsulation appears to be the most promising system of immunoisolation, however, the design of a device suitable for human clinical use remains a challenge. © 1994 John Wiley & Sons, Inc.
Tissue engineering of a bioartificial kidney
Authors: Cieslinski Deborah A. , David Humes H.
Published: December 2004

Tissue engineering is a rapidly growing field in biotechnology. The use and packaging of synthetic materials, biologic compounds, and cellular components of specific tissues can be envisioned to replace physiologic function of diseased organs. Long‐term ex vivo therapy for kidney failure has been achieved, so that the kidney may be the first solid organ in which tissue engineering concepts can produce an implantable device for long‐term in vivo replacement therapy. To replace the kidney's excretory function, an implantable bioartificial kidney requires both a device to replace blood ultrafiltration performed by renal glomeruli and a device to replace transport regulatory function of the renal tubule. The initial concepts for these devices are just beginning to be considered and developed. © 1994 John Wiley & Sons, Inc.
A near‐infrarod spectroscopy technique for the control of fermentation processes: An application to lactic acid fermentation
Authors: Vaccari Giuseppe , Dosi Elisabetta , Campi Anna L. , Mantovani Giorgio , González‐Vara y R. Antonio , Matteuzzi Diego
Published: December 2004

A near‐infrared (NIR) spectroscopy technique for the control of lactic acid fermentation process has been proposed. Lactic acid, glucose, and biomass concentrations were determined by the NIR spectroscopy method. The three parameters examined were closely correlated to the results obtained with classical laboratory procedures. Moreover, the conditions for the on‐line utilization of the NIR spectroscopy measurement system were pointed out. The great versatility of the NIR spectroscopy should permit its use for other fermentation processes. © 1994 John Wiley & Sons, Inc.
Polymer–polymer organic solvent two‐phase system: A new type of reaction medium for bioorganic synthesis
Authors: Otamiri Marina , Adlercreutz Patrick , Mattiasson Bo
Published: December 2004

Mixing solutions of polymers dissolved in chloroform resulted in turbid solutions that parted into two separate phases upon standing. Each phase consisted primarily of one of the two polymers and contained only small amounts of the other. An enzyme (α‐chymotrypsin) added to the two‐phase system partitioned preferentially to one of the phases; this was observed with native enzyme and with enzyme associated with one of the polymers through non‐convalent interactions. Under the conditions studied, α‐chymotrypsin was active and expressed even higher activity and stability than native enzyme added to the organic solvent without polymer. An emulsion was easily formed on mixing with small droplets of one of the phases suspended in the other phase. By operating with the enzyme in the emulsion, a very attractive system for carrying out enzyme‐catalyzed conversions was created. Short diffusion distances and minimized steric hindrance are two characteristics of such systems. At the conclusion of the reaction, stirring/mixing was ceased and, after phase separation, it was possible to recover the enzyme as well as the product, under ideal conditins, from different phases. The enzyme was then reused. © 1994 John Wiley & Sons, Inc.
Purification of monoclonal antibodies from whole hybridoma fermentation broth by fluidized bed adsorption
Authors: Thömmes Jörg , Halfar Markus , Lenz Suzanne , Kula Maria‐Regina
Published: December 2004

To achive the coarse purification of a monoclonal antibody from whole hybridoma fermentation broth a fluidized bed cation exchange process was used. The procedure consisted of application of the crude sample and washing of the bed in a fluidized mode and elution in a fixed bed mode. A completely clarified eluate was obtained with purification factors between 4 and 8 and a concentration of the desired product (monoclonal antibody) by a factor of more than 3 was achived. Thus, a combination of the three early steps of the downstream process clarification, concentration and coarse purification was possible. Two different materials were tested: a commercially available agarose‐based matrix (Stream‐line‐SP), and a self‐derivatized material based on controlled‐pore glass (Bioran). Initial experiments were performed to describe the fluidization of the glass material. Comparison with the agarose material showed several differences, the agarose matrix allowing liquid flow closer to plug flow than the glass material. Increased backmixing in the liquid phase was detected when fluidizing the glass adsorbent compared with the agarose‐based matrix. Despite this fact, comparison of the two materials with respect to antibody binding and elution demonstrated a similar performance. © 1995 John Wiley & Sons, Inc.
Modeling of anaerobic formate kinetics in mixed biofilm culture using dynamic membrane mass spectrometric measurement
Authors: Dornseiffer P. , Meyer B. , Heinzle E.
Published: December 2004

The dynamics of the anaerobic conversion of formate in a microbial mixed culture taken from an anaerobic fluidized bed reactor was studied using a new stirred micro reactor equipped with a membrane mass spectrometer. The microreactor with a toroidally shaped bottom and pitched blade turbine and a cylindrical flow guide was thermostated and additionally equipped with a pH electrode and pH control. During fed‐batch experiments using formate, the dissolved gases (methane, hydrogen, and carbon dioxide), as well as the acid consumption rates for pH control were monitored continuously. Initially and at the end of each experiment, organic acids were analyzed using ion chromatography (IC). It was found that about 50% of the formate was converted to methane via hydrogen and carbon dioxide, 40% gave methane either directly or via acetate. This was calculated from experiments using H13CO3− pulses and measurement of 12CH4 and 13CH4 production rates. About 10% of the formate was converted to lactate, acetate, and propionate, thereby increasing the measured CO2/CH4 production ratio. The nondissociated formic acid was shown to be rate determining. From the relatively high Ks value of 2.5 mmol m−3, it was concluded that formate cannot play an important role in electron transfer. During dynamic feeding of formate, hydrogen concentration always increased to a maximum before decreasing again. This peak was found to be very discriminative during modeling. From the various models set up, only those with two‐stage degradation and double Monod kinetics, both for CO2 and hydrogen, were able to describe the experimental data adequately. Additional discrimination was possible with the IC measurement of organic acids. © 1995 John Wiley & Sons, Inc.
Effect of surfactant and particle size reduction on hydrolysis of deinking sludge and nonrecyclable newsprint
Authors: Duff Sheldon J. B. , Moritz John W. , Casavant Tracy E.
Published: December 2004

Disposal of sludge from deinking mills represents a significant proportion of operating costs. Bioconversion of the cellulosic fraction of deinking sludge (DIS) to ethanol greatly reduces disposal costs while producing an environmentally friendly fuel. In this study, the cellulosic fraction of newsprint and deinking sludge was hydrolysed to produce fermentable sugars. For newsprint, a particle size of 1 to 1.5 mm provided optimal reaction rates in batch reactors over practical hydrolysis times, and reducing sugar concentrations as high as 35 g/L could be achieved using a fed‐batch reactor configuration. For both newsprint and DIS, the hydrolysis rate increased nonlinearly with enzyme loading. Tween‐80 only marginally improved sugar production but was able to release sugars from cellulosic substrates in the absence of lytic enzymes, in an amount proportional to the surfactant concentration and the substrate particle size. DIS was relatively recalcitrant to enzymatic hydrolysis, possibly due in part to inhibition by hydrophobic constituents. © 1995 John Wiley & Sons, Inc.
Enzymic peptide synthesis in microaqueous, solvent‐free systems
Authors: Kuhl P. , Elchhorn U. , Jakubke H.‐D.
Published: December 2004

Thermolysin‐catalyzed (EC 3.4.24.4) and chymotrypsin‐catalyzed (EC 3.4.21.1) peptide synthesis reactions were accomplished without any organic solvent in the presence of low amounts of water under sonication and fluidization. The systems used are considered to be microaqueous solvent‐free ones. The influence of several reaction parameters, such as time, the amount of enzyme, the amount of water in free form or bound as hydration water, and the N/C component ratio, on the vield of the thermolysin‐catalyzed synthesis of Z–Phe–Leu–NH2 (up to 87% yield) was investigated in a sonicated system. Besides Z–Phe–Leu–NH2, the tripeptide derivatives Ac–Xaa–Trp–Leu–NH2, (Xaa = Gly, Ala) were also obtained in good yields of 79 and 71% respectively. In the latter case, no hydrolytic side reactions were observed. Using a fluidized‐bed reactor, chymotrypsin‐ and thermolysin‐catalyzed syntheses of N‐protected di‐ and tripeptide amides could be perfromed with yields in the range of 10 to 40%. © 1995 John Wiley & Sons, Inc.
Polyphenoloxidases immobilized in organic gels: Properties and applications in the detoxification of aromatic compounds
Authors: Crecchio C. , Ruggiero P. , Pizzigallo M. D. R.
Published: December 2004

Gelatine gels originate from water in oil microemulsions in which the ternary system consists of isooctane/ sulfosuccinic acid bis [2‐ethyl hexyl] ester/water; the solubilization of gelatin in the water pool of these microemulsions transforms them into viscous gels in which it is possible to cosolubilize various reactive molecules. These gels were used to immobilize two phenoloxidases, a laccase from Trametes versicolor and a tyrosinase from mushroom. The best balance between gel retention and catalytic activity was reached at a gelatine concentration of 2.5% (w/v) in the case of tyrosinase, while laccase immobilization was independent of gelatine concentration. Both enzymes kept the same optimum pH as the corresponding soluble controls, while a partial loss of activity was observed when they were immobilized. Immobilized enzymes showed an increased stability when incubated for several days at 4°C with a very low release from the gels in the incubation solutions. The immobilization of tyrosinase and of laccase enhanced stability to thermal inactivation. Furthermore, gel‐entrapped tyrosinase was almost completely preserved from proteolysis: more than 80% of the activity was maintained, while only 25% of the soluble control activity was detected after the same proteolytic treatments. A column packed with gel‐immobilized tyrosinase was used to demonstrate that enzymes immobilized with this technique may be reused several times in the same reaction without loosing their efficiency. Finally, gel‐entrapped tyrosinase and laccase were capable of removing naturally occurring and xeno‐biotic aromatic compounds from aqueous suspensions with different degrees of efficiency. © 1995 John Wiley & Sons, Inc.
N‐glycans of recombinant human interferon‐γ change during batch culture of chinese hamster ovary cells
Authors: Hooker Andrew D. , Goldman Merlin H. , Markham Nicola H. , James David C. , Ison Andrew P. , Bull Alan T. , Strange Philip G. , Salmon Ian , Baines Anthony J. , Jenkins Nigel
Published: December 2004

A recombinant Chinese hamster ovary (CHO) cell line making human interfron‐γ (IFN‐γ) was grown in 12‐L stirred tank fermentors in three batch fermentations under conditions of constant temperature, pH, and dissolved oxygen tension. In addition to cell growth, metabolite, and productivity data, a detailed analysis of the carbohydrate structures attached to each glycosylation site of IFN‐γ was achieved using matrix‐assisted laser desorption mass spectrometry (MALDI‐MS) in combination with exoglycosidase array sequencing. Complex biantennary oligosaccharides (particularly Gal2GlcNAc4Man3which was core ℵl‐6 fucosylated at Asn25but not at Asng97) were most prevalent at both glycosylation sites. However, considerable microheterogeneity arising from the presence of triantennary and truncated glycan structures was also observed. The proportion of the dominant core glycan structure (Gal2GlcNAc4Man3± Fuc1) decreased by 15–26% during batch culture, with increases in the proportion of oligomannose and truncated glycans over the same time period. Prolonged culture resulting from an extended lag phase led to further accumulation of oligomannose and truncated structures, reaching up to 52% of total glycans attached to Asng97by 240 h of culture. The implications of these glycosylation changes for optimizing the time for harvesting cell cultures, and for the clearance of recombinant therapeutic products in vivo are discussed. © 1995 John Wiley & Sons, Inc.
Less Symmetrical Dicopper(II) Complexes as Catechol Oxidase Models—An Adjacent Thioether Group Increases Catecholase Activity
Authors: Merkel Michael , Möller Niclas , Piacenza Manuel , Grimme Stefan , Rompel Annette , Krebs Bernt
Published: December 2004

Three new unsymmetrical compartmental dinucleating ligands, 4‐bromo‐2‐(4‐methylpiperazin‐1‐ylmethyl)‐6‐[{2‐(1‐piperidyl)ethyl}aminomethyl]phenol (HL1), 4‐bromo‐2‐(4‐methylpiperazin‐1‐ylmethyl)‐6‐[{2‐(morpholin‐4‐yl)ethyl}aminomethyl]phenol (HL2), and 4‐bromo‐2‐(4‐methylpiperazin‐1‐ylmethyl)‐6‐[{2‐(thiomorpholin‐4‐yl)ethyl}aminomethyl]phenol (HL3), have been synthesized in order to model the active site of type 3 copper proteins. The dicopper(II) complexes of these ligands give first hints about the influence of a thioether group close to the metal site. The bromophenol‐based ligands have one piperazine arm and one other bidentate arm in positions 2 and 6 of the phenolic ring, respectively. With each ligand a dinuclear copper(II) complex was prepared and structurally characterized. The copper ions were found to have square pyramidal environments and a mixture of endogenous phenoxo and exogenous acetate bridging. The influence of a heteroatom in one arm of the ligand on catecholase activity and speciation in solution was studied by UV/Vis spectroscopy, ESI‐MS experiments and, DFT calculations.
Bilayer Vesicles of Amphiphilic Cyclodextrins: Host Membranes That Recognize Guest Molecules
Authors: Falvey Patrick , Lim Choon Woo , Darcy Raphael , Revermann Tobias , Karst Uwe , Giesbers Marcel , Marcelis Antonius T. M. , Lazar Adina , Coleman Anthony W. , Reinhoudt David N. , Ravoo Bart Jan
Published: December 2004

A family of amphiphilic cyclodextrins (6, 7) has been prepared through 6‐S‐alkylation (alkyl=n‐dodecyl and n‐hexadecyl) of the primary side and 2‐O‐PEGylation of the secondary side of α‐, β‐, and γ‐cyclodextrins (PEG=poly(ethylene glycol)). These cyclodextrins form nonionic bilayer vesicles in aqueous solution. The bilayer vesicles were characterized by transmission electron microscopy, dynamic light scattering, dye encapsulation, and capillary electrophoresis. The molecular packing of the amphiphilic cyclodextrins was investigated by using small‐angle X‐ray diffraction of bilayers deposited on glass and pressure–area isotherms obtained from Langmuir monolayers on the air–water interface. The bilayer thickness is dependent on the chain length, whereas the average molecular surface area scales with the cyclodextrin ring size. The alkyl chains of the cyclodextrins in the bilayer are deeply interdigitated. Molecular recognition of a hydrophobic anion (adamantane carboxylate) by the cyclodextrin vesicles was investigated by using capillary electrophoresis, thereby exploiting the increase in electrophoretic mobility that occurs when the hydrophobic anions bind to the nonionic cyclodextrin vesicles. It was found that in spite of the presence of oligo(ethylene glycol) substituents, the β‐cyclodextrin vesicles retain their characteristic affinity for adamantane carboxylate (association constant Ka=7.1×103 M−1), whereas γ‐cyclodextrin vesicles have less affinity (Ka=3.2×103 M−1), and α‐cyclodextrin or non‐cyclodextrin, nonionic vesicles have very little affinity (Ka≈100 M−1). Specific binding of the adamantane carboxylate to β‐cyclodextrin vesicles was also evident in competition experiments with β‐cyclodextrin in solution. Hence, the cyclodextrin vesicles can function as host bilayer membranes that recognize small guest molecules by specific noncovalent interaction.
Chemo‐ and Regioselective Intermolecular Cyclotrimerization of Terminal Alkynes Catalyzed by Cationic Rhodium(I)/Modified BINAP Complexes: Application to One‐Step Synthesis of Paracyclophanes
Authors: Tanaka Ken , Toyoda Kazuki , Wada Azusa , Shirasaka Kaori , Hirano Masao
Published: December 2004

A highly regioselective intermolecular cyclotrimerization of terminal alkynes has been developed based on the use of the cationic rhodium(I)/DTBM‐Segphos complex. This method can be applied to a variety of terminal alkynes to provide 1,2,4‐trisubstituted benzenes in high yield and with high regioselectivity. A chemo‐ and regioselective intermolecular crossed‐cyclotrimerization of dialkyl acetylenedicarboxylates with a variety of terminal alkynes has also been developed based on the use of the cationic rhodium(I)/H8‐BINAP complex, furnishing 3,6‐disubstituted phthalates in high yields. It constitutes a highly efficient new method for intermolecular crossed‐cyclotrimerization of two different monoynes in terms of catalytic activity, chemo‐ and regioselectivity, scope of substrates, and ease of operation. The versatility of this new crossed‐alkyne cyclotrimerization procedure is demonstrated through its application to one‐step synthesis of a [6]metacyclophane and [7]–[12]paracyclophanes from the corresponding terminal α,ω‐diynes. Mechanistic studies have revealed that the chemo‐ and regioselectivity of this crossed‐alkyne cyclotrimerization are determined by the preferential formation of a specific rhodium metallacycle derived from a terminal alkyne and a dialkyl acetylenedicarboxylate.