bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and application of thermo‐sensitive immunomicrospheres for antibody purification
Authors: Kondo Akihiko , Kaneko Tetsuya , Higashitani Ko
Published: December 2004

The latex particles composed of poly(styrene/N‐isopropylacrylamide/glycidyl methacrylate) [P(St/NIPAM/GMA)] and poly(styrene/N‐isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] were prepared by emulsifier‐free emulsion polymerization. These latex particles with submicrometer size showed the thermosensitivity originated from the thermo‐sensitive nature of NIPAM. That is, the minimum NaCI concentration for flocculation of these latex particles [critical flocculation concentration (CFC)] decreased significantly with increasing temperature and reached constant values at above the critical temperature [critical flocculation temperature (CFT)]. At a certain NaCl concentration, the thermo‐sensitive latex particles were flocculated by raising temperature, and conversely, the flocculated thermo‐sensitive latex particles were completely dispersed by lowering temperature. Bovine serum albumin (BSA) was covalently immobilized onto the P(St/NIPAM/GMA) and P(St/NIPAM/MMA) latex particles with high efficiency. The BSA‐immobilized P(St/NIPAM/GMA) and P(St/NIPAM/MAA) latex particles (immunomicrospheres) showed the similar dependencies of CFC on temperature to the bare latex particles. These thermo‐sensitive immunomicrospheres were successfully used for the immunoaffinity purification of anti‐BSA antibodies from antiserum. © 1994 John Wiley & Sons, Inc.
Genetically engineered charge modifications to enhance protein separation in aqueous two‐phase systems: Electrochemical partitioning
Authors: Luther John R. , Glatz Charles E.
Published: December 2004

We have examined the effect of genetically engineered charge modifications on the partitioning behavior of proteins in dextran/polyethylene glycol two‐phase systems containing potassium phosphate. By genetically altering a protein's charge, the role of charge on partitioning can be assessed directly without the need to modify the phase system. The charge modifications used are of two types: Charged tails of polyaspartic acid fused to β‐galactosidase and charge‐change point mutations of T4 lysozyme which replace positive lysine residues with negative glutamic acids. The partition coefficient Kp for these proteins was related to measured interfacial potential differences Δϕ using the simple thermodynamic model, In Kp = In Ko + (F/RT)Zp δϕ. The protein net charge Zp was determined using the Henderson–Hasselbalch relationship with modifications based on experimentally determined titration and isoelectric point data. It was found that when the electropartitioning term Zp δϕ was varied by changing the pH, the partitioning of T4 lysozyme was quantitatively described by the thermodynamic model. The β‐galactosidase fusions displayed qualitative agreement, and although less than predicted, the partitioning increased more than two orders of magnitude for the pH range examined. Changes in the partitioning of lysozyme due to the various mutations agreed qualitatively with the thermodynamic model, but with a smaller than expected dependence on the estimated charge differences. The β‐galactosidase fusions, on the other hand, did not display a consistent charge based trend, which is likely due either to the enzyme's large size and complexity or to nonelectrostatic contributions from the tails. The lack of quantitative fit with the model described above suggests that the assumptions made in developing this model are oversimplified. © 1994 John Wiley & Sons, Inc.
Microfiltration of streptococcal fermentation broths: Study of the factors affecting the concentration effect
Authors: Hernandez‐Pinzon Immaculada , Bautista Juan
Published: December 2004

Streptokinase (SK) recovery from streptococcal fermentation broth by cross‐flow microfiltration has been studied. Recovery of SK in the filtrate, independent of the volumetric concentration factor, is approximately two‐fold lower than the initial SK activity in the fermentation broth; moreover, the SK activity in the retentate increase during the process, reaching a concentration factor of 2.73. These results show that the membrane works more as an ultrafiltration membrane, with rejection of S = 0.6, than as a microfiltration membrane. Under filtration conditions, the membrane permeation rate decreased with time. This decreased could be explained by deposition and interaction of material onto/with the membrane resulting in the concentration of permeable products. Studies of the individual concentration factors for the main streptococcal exocellular proteins, indicate clearly that the concentration of the proteins during the microfiltration process is independent of the size of the proteins, suggesting that other factors, such as charge and hydrophobicity, along with concentration‐polarization, should be taken also into account for the understanding of this phenomenon. © 1994 John Wiley & Sons, Inc.
Chemical and enzymatic extraction of heavy metal binding polymers from isolated cell walls of Saccharomyces cerevisiae
Authors: Brady D. , Stoll A. D. , Starke L. , Duncan J. R.
Published: December 2004

Isolated cell walls of the yeast Saccharomyces cerevisiae were treated by either chemical (alkali and acid) or enzymatic (protease, mannanase or β‐glucuronidase) processes to yield partially purified products. These products were partially characterized by infrared analysis. They were subsequently reacted with heavy metal cation solutions and the quantity of metal accumulated by the cell wall material determined. The Cu2+ ion (0.24, 0.36, 1.12, and 0.60 μmol/mg) was accumulated to a greater extent than either Co2+ (0.13, 0.32, 0.43, and 0.32 μmol/mg) or Cd2+ (0.17, 0.34, 0.39, and 0.32 μmol/mg) by yeast cell walls, glucan, mannan, and chitin, respectively The isolated components each accumulated greater quantities of the cations than the intact cell wall. Removal of the protein component of the yeast cell walls by Pronase caused a 29.5% decrease in metal accumulation by yeast cell walls per mass, indicating the protein is a heavy metal accumulating component. The data indicate that the outer mannan–protein layer of the yeast cell wall is more important than the inner glucan‐chitin layer in heavy metal action accumulation. © 1994 John Wiley & Sons, Inc.
Modification of islet of langerhans surfaces with immunoprotective poly(ethylene glycol) coatings via interfacial photopolymerization
Authors: Sawhney Amarpreet S. , Pathak Chandrashekhar P. , Hubbell Jeffrey A.
Published: December 2004

Poly(ethylene glycol) (PEG) has been used previously to alter immune interactions and systemic clearance of therapeutic proteins. We present herein chemical approaches for the conceptually similar treatment of therapeutic cells and tissues whereby immune and cell adhesive interactions may be reduced or interrupted, in the context of the transplantation of xenogeneic islets of Langerhans for the treatment of insulin‐dependent diabetes mellitus. Visible‐light‐initiated interfacial photopolymerization of multifunctional PEG‐based macromers was performed directly upon the surface of rat islets of Langerhans to produce conformal barrier hydrogel coatings with thickness of order 10 μ;m. The islets continued to be normal in ultrastructure and function as reflected by response to a glucose challenge in vitro. © 1994 John Wiley & Sons, Inc.
Effects of water activity on reaction rates and equilibrium positions in enzymatic esterifications
Authors: Svensson Ingemar , Wehtje Ernst , Adlercreutz Patrick , Mattiasson Bo
Published: December 2004

A technique of continuous water activity control was used to examine the effects of water activity on enzyme catalysis in organic media. Esterification catalyzed by Rhizopus arrhizus lipase was preferably carried out at a water activity of 0.33, which resulted in both maximal initial reaction rate and a high yield. When Pseudomonas lipase was used as catalyst it was beneficial to start the reaction at high water activity (giving the optimal reaction rate with this enzyme) and then shift to a lower water activity toward the end of the reaction to obtain a high yield. The apparent equilibrium constant of the reaction was influenced by the water activity of the organic solvent. © 1994 John Wiley & Sons, Inc.
Formation and growth of heterotrophic aerobic biofilms on small suspended particles in airlift reactors
Authors: Tijhuis L. , van Loosdrecht M. C. M. , Heijnen J. J.
Published: December 2004

In this article, the conditions for aerobic biofilm formation on suspended particles, the dynamics of biofilm formation, and the biomass production during the start‐up of a Biofilm Airlift Suspension reactor (BAS reactor) have been studied. The dynamics of biofilm formation during start up in the biofilm airlift suspension reactor follows three consecutive stages: bare carrier, microcolonies or patchy biofilms on the carrier, and biofilms completely covering the carrier. The effect of hydraulic retention time and of substrate loading rate on the formation of biofilms were investigated. To obtain in a BAS reactor a high biomass concentration and predominantly continuous biofilms, which completely surround the carrier, the hydraulic retention time must be shorter than the inverse of the maximum growth rate of the suspended bacteria. At longer hydraulic retention times, a low amount of attached biomass can be present on the carrier material as patchy biofilms. During the start‐up at short hydraulic retention times the bare carrier concentration decreases, the amount of biomass per biofilm particle remains constant, and biomass increase in the reactor is due to increasing numbers of biofilm particles. The substrate surface loading rate has effect only on the amount of biomass on the biofilm particle. A higher surface load leads to a thicker biofilm.

A strong nonlinear increase of the concentration of attached biomass in time was observed. This can be explained by a decreased abrasion of the biofilm particles due to the decreasing concentration of bare carriers. The detachment rate per biofilm area during the start‐up is independent of the substrate loading rate, but depends strongly upon the bare carrier concentration.

The Pirt‐maintenance concept is applicable to BAS reactors. Surplus biomass production is diminished at high biomass concentrations. The average maximal yield of biomass on substrate during the experiments presented in this article was 0.44 ± 0.08 C‐mol/C‐mol, the maintenance value 0.019 ± 0.012 C‐mol/(C‐mol h). The lowest actual biomass yield measured in this study was 0.15 C‐mol/C‐mol. © 1994 John Wiley & Sons, Inc.
The release of virus‐like particles from recombinant Saccharomyces cerevisiae: Effect of freezing and thawing on homogenization and bead milling
Authors: Milburn P. T. , Dunnill P.
Published: December 2004

Recombinant cells of Saccharomyces cerevisiae, expressing virus‐like particles (Ty‐VLPs), can be readily disrupted in a high pressure homogenizer and show identical disruption kinetics to the untransformed host strain. When the cells are freeze/thawed before disruption, they become about four times more resistant to homogenization. This effect increases with the number of freeze/thaw cycles, but is independent of the time the cells remain frozen. The freeze/thaw effect is observed with cells harvested during both the logarithmic and stationary phase of growth, and occurs with the untransformed host strain as well as the transformed one. Freeze/thawed cells are twice as resistant to disruption in the bead mill as fresh cells. © 1994 John Wiley & Sons, Inc.
Recovery of proteins and amino acids from reverse micelles by dehydration with molecular sieves
Authors: Gupta Ram B. , Han Chae J. , Johnston Keith P.
Published: December 2004

A new method is presented to precipitate proteins and amino acids from reverse micelles by dehydrating the micelles with molecular sieves. Nearly complete precipitation is demonstrated for α‐chymotrypsin, cytochromec, and trytophan from 2‐ethylhexyl sodium sulfosuccinate (AOT)/isooctane/water reverse micelle solutions. The products precipitate as a solid powder, which is relatively free of surfactant. The method does not require any manipulation of pH, ionic strength, temperature, pressure, or solvent composition, and is applicable over a broad range of these properties. This general approach is compared with other techniques. This general approach is compared with other techniques for the recovery of biomolecules from reverse micelles. © 1994 John Wiley & Sons, Inc.
Stoichiometric model of the aerobic metabolism of the biological phosphorus removal process
Authors: Smolders G. J. F. , van der Meij J. , van Loosdrecht M. C. M. , Heijnen J. J.
Published: December 2004

In the aerobic phase of the biological phosphorus removal process, poly‐β‐hydroxybutyrate, produced during anaerobic conditions, is used for cell growth, phosphate uptake, and glycogen formation. A metabolic model of this process has been developed. The yields for growth, polyphosphate and glycogen formation are quantified using the coupling of all these conversions to the oxygen consumption. The uptake of phosphate and storage as polyphosphate is shown to have a direct effect on the observed oxygen consumption in the aerobic phase. The overall energy requirements for the P‐metabolism are substantial: 25% of the acetate consumed during anaerobic conditions and 60% of the oxygen consumptions is used for the synthesis of polyphosphate and glycogen. © 1994 John Wiley & Sons, Inc.
New catalytic properties of monoamine oxidase immobilized in Langmuir–blodgett films with amphiphilic polyelectrolytes
Authors: Barmin Anatoli V. , Eremenko Arkadi V. , Kurochkin llya N. , Moskvitina Tatyana A.
Published: December 2004

Langmuir‐Blodgett (LB) films of monoamine oxidase (MAO) have been formed on the surface f a polypropylene membrane using amphiphilic polyelectrolytes. The enzyme activity of such protein‐polyelectrolyte films was measured by a Clark electrodes. It was shown that in LB films thus formed the use of amphiphilc polyelectrolytes, MAO activity was higher than in polyelectrolyte‐free LB films. Immobilization of MAO with branched polyethylenimine modified on 12% by laurylchain led to pronounced changes in its catalytic properties. The dependence of the enzyme's kinetic parameters on amphiphilic polyelectrolyte structures was discussed. © 1994 John Wiley & Sons, Inc.
Continuous, real‐time monitoring of the oxygen uptake rate (OUR) in animal cell bioreactors
Authors: Yoon Sung‐jin , Konstantinov Konstantin B.
Published: December 2004

A new method for real‐time monitoring of the oxygen uptake rate (OUR) in bioreactors, based on dissolved oxygen (DO) measurement at two points, has been developed and tested extensively. The method has several distinct advantages over known techniques.It enables the continuous and undisturbed monitoring of OUR, which is conventionally impossible without gas analyzers. The technique does not require knowledge of kLa. It provides smooth, robust, and reliable signal. The monitoring scheme is applicable to both microbial and mammalian cell bioprocesses of laboratory or industrial scale. The method was successfully used in the cultivation of NSO‐derived murine myeloma cell line producing monoclonal antibody. It was found that while the OUR increased with the cell density, the specific OUR decreased to approximately one‐half at cell concentrations of 16 × 106 cells/mL, indicating gradual reduction of cell respiration activity. Apart from the laboratory scale cultivation, the method was applied to industrial scale perfusion culture, as well as to processes using other cell lines. © 1994 John Wiley & Sons, Inc.
Hyperosmotic hbridoma cell cultures: Increased monoclonal antibody production with addition of glycine betaine
Authors: Øyaas Karin , Ellingsen Trond E. , Dyrset Nils , Levine David W.
Published: December 2004

When mouse hybridoma cells were grown in culture media which were made hyperosmotic through the addition of NaCl or sucrose, the specific rate of antibody production increased with medium osmolality, reaching approx. 1.9 times the level obtained at physiological osmolality. However, due to a simultaneous reduction of the maximal cell density in the hyperosmotic media, the effect of the increased production rate did not give significant increases in the maximum antibody titer obtained in the cultures. When the osmoprotective compound, glycine betaine, was included in the NaCl‐ or sucrose‐stressed cultures, the specific antibody production rate wasincreased up to 2.6‐fold and maximum antibody titer up to twofold over that obtained in the control culture (physiological osmolality). A similar pattern of response was observed when other osmoprotective compounds (sarcosine, proline, glycine) were added to NaCl‐stressed hybridoma cell cultures. For the present experiments, the results suggest that medium osmolality, rather than growth rate, will determine the specific antibody production rate by hybridoma cell line 6H11 growing in hyperosmotic culture media. © 1994 John Wiley & Sons, Inc.
On‐line estimation of biological variables during pH controlled lactic acid fermentations
Authors: Acuña Gonzalo , Latrille Eric , Béal Catherine , Corrieu Georges , Chéruy Arlette
Published: December 2004

Indirect measurement of lactose, galactose, lactic acid, and biomass concentration from on‐line sodium hydroxide weight measurements have been obtained for pure and mixed batch cultures of Streptococcus salivarius ssp. thermophilus 404 and Lactobacillus delbrueckii subsp. bulgaricus 398 conducted at controlled pH and temperature. Linear correlations were established between the equivalent sodium hydroxide concentration and the lactose (substrate), galactose and lactic acid (products) concentrations while nonlinear relationships were developed between biomass and lactic acid concentrations. These nonlinear relationships took into account the inhibitory effect of lactic acid on growth and acidification. The indirect measurements of biomass concentration were introduced into a nonlinear estimator of the state variables and of the specific growth and lactic acid production rates. Good agreement was found between estimated and measured biomass concentrations (error index ranging from 10.8% to 12.6%). The results showed the feasibility of on‐line estimation of biomass concentration and of the specific kinetics from NaOH addition weight measurements and its applicability for monitoring lactic acid fermentations. Using off‐line measurements of L(+) and D(‐) lactic acid concentrations, the evolution of the concentration of each strain in mixed cultures was obtained from the relationships proposed for the mixed cultures. © 1994 John Wiley & Sons, Inc.
A facile transphosphatidylation reaction using a culture supernatant of actinomycetes directly as a phospholipase D catalyst with a chelating agent
Authors: Nakajima Jan , Nakashima Toshimitsu , Shima Yu , Fukuda Hideki , Yamane Tsuneo
Published: December 2004

An attempt was made to use the phospholipase D (PLD)‐ containing culture supernatants of actinomycetes directly as catalysts for the transphosphatidylation reaction of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) in a biphasic system. Of the five actinomycetes (three Streptomyces sp. and two Streptoverticillium sp.) examined, three (St. mediocidicus, Stv. cinnamoneum and Stv. hachijoense) exhibited good PLD production performance, but the selectivity (ratio of transphosphatidylation to hydrolysis) of the PLDs in the culture supernatant of all three actinomycetes were significantly low. However, the addition of EDTA to the reaction mixture as a chelating agent remarkably improved the selectivity of the PLDs, which approached 100% in all the culture supernatants. Commercially available PLDs were also investigated and classified into two types. The PLDs of one type had high selectivity and no metal was required for the enzyme activity, while those of the other type showed low selectivity and a metal was necessary for the enzyme to be activated. From this finding, it was considered that the culture supernatants used in this study contained several PLDs of both types. When the chelating agent was added to the reaction mixture, the hydrolysis due to PLDs with low selectivity was suppressed by removal of the essential metal, resulting in an increased in the overall selectivity of the PLDs in the culture supernatant. Repeated batch transphosphatidylation reactions were performed 20 times, reusing the PLDs in the aqueous phase by centrifugation; the reaction rate gradually decreased to 60% of that of batch 1 by batch 20. This suggests that the transphosphatidylation reaction using a culture supernatant has potential for industrial application. © 1994 John Wiley & Sons, Inc.
Comparison of recombinant Escherichia coli strains for synthesis and accumulation of poly‐(3‐hydroxybutyric acid) and morphological changes
Authors: Lee Sang Yup , Lee Kyung Mi , Chan Ho Nam , Steinbüchel Alexander
Published: December 2004

A stable high‐copy‐number plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoic acid (PHA) biosynthesis genes was constructed. This plasmid was transferred to seven Escherichia coli strains (K12, B, W, XL1‐Blue, JM109, DH5α, and HB101), which were subsequently compared for their ability to synthesize and accumulate ploy‐ (3‐hydroxybutyric acid) (PHB). Growth of recombinant cells and PHB synthesis were investigated in detail in Luria‐Bertani (LB) medium containing 20 g/L glucose. Cell growth, the rate of PHB synthesis, the extent of PHB accumulation, the amount of glucose utilized, and the amount of acetate formed varied from one strain to another. XL1‐Blue (pSYL105) and B (pSYL105) synthesized PHB at the fastest rate, which was ca. 0.2 g PHB/g true cell mass‐h, and produced PHB up to 6‐7 g/L. The yields of cell mass, true cell mass, and PHB varied considerably among the strains. The PHB yield of XL1‐Blue (pSYL105) in LB plus 20 g/L glucose was as high as 0.369 g PHB/g glucose. Strains W (pSYL105) and K12 (pSYL105) accumulated the least amount of PHB with the lowest PHB yield at the lowest synthesis rate. JM109 (pSYL105) accumulated PHB to the highest extent (85.6%) with relatively low true cell mass (0.77 g/L). Considerable filamentation of cells accumulating PHB was observed for all strains except for K12 and W, which seemed to be due either to the overexpression of the foreign PHA biosynthesis enzymes or to the accumulation of PHB. © 1994 John Wiley & Sons, Inc.
Immiscible organic solvent inactivation of urease, chymotrypsin, lipase, and ribonuclease: Separation of dissolved solvent and interfacial effects
Authors: Ghatorae Amreek S. , Guerra Maria J. , Bell George , Halling Peter J.
Published: December 2004

A new technique with controlled interface generation allows separation and quantitation of enzyme inactivation by both solvent/aqueous interface and dissolved solvent. This has now been used in n‐butanol, isopropylether, 2‐octanone, n‐hexane, n‐butylbenzene, and n‐tridecane. Ribonuclease was stable with all the solvent/aqueous interfaces studied. Chymotrypsin was mainly inactivated by the more hydrophobic solvent/aqueous interfaces, whereas lipase was only inactivated by the less hydrophobic solvent/aqueous interfaces. Urease was inactivated by some interfaces, but not all, without an obvious trend. Thus, the commonly expected simple relationship with solvent polarity (e.g., log P) does not apply when interfacial inactivation is determined specifically. Greater dissolved solvent inactivation occurred with the more polar solvents, though only a general trend was apparent with log P. A better correlation was noted with the Hilde‐brand solubility parameter. Interfacial effects are discussed with reference to enzyme molecular weight, denaturation temperature, hydrophobicity, and adiabatic compressibility. © 1994 John Wiley & Sons, Inc.
The use of hollow fiber cross‐flow microfiltration in bioaccumulation and continuous removal of heavy metals from solution by Saccharomyces cerevisiae
Authors: Brady D. , Rose P. D. , Duncan J. R.
Published: December 2004

Cross‐flow microfiltration was shown to retain Saccharomyces cerevisiae biomass utilized for heavy metal bioaccumulation. The passage of metal‐laden influent through a series of sequential bioaccumulation systems allowed for further reductions in the levels of copper, cadmium, and cobalt in the final effluent than that afforded by a single bioaccumulation process. Serial bioaccumulation systems also allowed for partial separation of metals from dual metal influents. More than one elemental metal cation could be accumulated simultaneously and in greater quantities than when a single metal was present in the effluent (Cu2+ 0.43 mmol, Cu2+ + Cd2+ 0.67 mmol, and Cu2+ + Co2+ 0.83 mmol/g yeast dry mass when the initial concentration of each of the metal species was 0.2 mmol·L−1). Co‐accumulation of two different metal cations allowed higher total levels of bioaccumulation than found with a single metal. The flux rate was 2.9 × 102 L·h−2μm−2 using a polypropylene microfiltration membrane (0.1 μm pore size) at 25°C. © 1994 John Wiley & Sons, Inc.
Parametric sensitivity of stoichiometric flux balance models applied to wild‐type Escherichia coli metabolism
Authors: Varma Amit , Palsson Bernhard O.
Published: December 2004

Stoichiometrically based flux balance models provide a method to quantify the metabolic pathway fluxes within a living cell. Predictions of flux balance models are expected to have applications in pathway engineering as well as in bioprocess design and control. These models utilize optimality principles applied to metabolic pathway stoichiometry along with the metabolic requirements for growth to determine the flux distribution in a metabolic network. A flux balance model has been developed for Escherichia coli W3110 using five experimentally determined strain‐specific parameters. In this report, we determine the sensitivity of the predictions of the flux balance model to these five strain‐specific parameters. Model predictions are shown to be sensitive to the two parameters describing metabolic capacity, while they are relatively insensitive to the three parameters that describe the metabolic requirements for growth. Thus, when stoichiometrically based models are formulated for additional strains one needs to measure the metabolic capacity (maximum rates of nutrient and oxygen utilization) accurately. Determination of metabolic capacity from batch experiments is relatively easy to perform. On the other hand, the harder to determine maintenance parameters need not be as accurately determined. © 1995 John Wiley & Sons, Inc.
Working at controlled water activity in a continuous process: The gas/solid system as a solution
Authors: Lamare Sylvain , Legoy Marie Dominique
Published: December 2004

Fusarium solani cutinase and Candida cylindracea lipase were used to catalyze a transesterification reaction in a continuous gas/solid bioreactor. In this system, a solid phase composed of a packed enzymatic preparation was continuously percolated with carrier gas which fed substrate and removed reaction products simultaneously. Different conditions of immobilization were used and compared to the results obtained with a nonsupported enzyme. The enzymatic activity was found to be highly dependent of a key parameter: water activity (aw). Biocatalyst stability was greatly influenced by water activity and the choice of immobilization technique for the enzymatic material. For free and adsorbed enzymes, water requirements exhibited optima which corresponded to the complete hydration coverage of the protein. These optima presented a good correlation with the isotherm sorption curves obtained for the different preparations. In this work are reported the results concerning the possibility of using a continuous system able to operate at controlled water activity in a heterogeneous medium. Lipolytic enzyme in such a system appears to be a new process for the biotransformation of volatile esters. © 1995 John Wiley & Sons, Inc.
The protective effect of specific medium additives with respect to bubble rupture
Authors: Chattopadhyay Devamita , Rathman James F. , Chalmers Jeffrey J.
Published: December 2004

A significant degree of cell damage is observed during suspension cell culture with air sparging. Protective agents can be added to the culture medium to protect the cells from damage. It has been observed that cells tend to adhere to air–medium interfaces and cell damage is mainly due to this cell–bubble interaction; protective additives have been found to prevent this cell adhesion to the bubble surfaces. In this article, it is demonstrated that the interfacial tension between the air and medium is related to the effectiveness of the protective additives to prevent adhesion of cells to this interface. Five different types of additives (Pluronic F‐68, Methocels, dextran, Polyvinyl alcohol, and polyethylene glycols) were studied in an effort to determine their protective characteristics. Liquid–vapor interfacial tensions of the culture medium, with and without the additives, were measured by two different techniques (maximum bubble pressure method and Wilhelmy plate method). In addition, visualization techniques showed that in the presence of certain protective additives cells do not adhere to the bubble surface. Results obtained from these experiments indicate that the additives which rapidly lower the liquid–vapor interfacial tension of the culture medium also prevent adhesion of cells to the bubble surface. Experiments have also been conducted to determine the number of cells killed due to bubble rupture, and it was observed that this number is related to the amount of cells adhering to the bubble surface. © 1995 John Wiley & Sons, Inc.

This article is a US Government Work and, as such, is in the public domain in the United States of America.
Continuously coupled transcription‐translation system for the production of rice cytoplasmic aldolase
Authors: Tulin Edgardo E. , Tsutsumi Ken‐ichi , Ejiri Shin‐ichiro
Published: December 2004

A continuously coupled cell‐free transcription‐translation system was developed for the production of rice cytoplasmic aldolase, an enzyme involved in both glycolytic and gluconeogenic pathways in eukaryotic cells. The system works with a continous flow of feeding solution containing nucleoside triphosphates and amino acids into a 1‐mL reactor containing wheat‐germ extract, plasmid DNA, and transcription enzyme, and continuous removal of translation product through an ultrafilatration membrane fitted in the reactor. Addition of free nucleotide primer, m7G(5′)ppp(5′)G, to this reactor was necessary for efficient transcription, thus producing biologically active mRNA for translation. The rate of aldolase synthesis was constant during the continuous translation reaction. It was observed that from 3 h onward only aldolase was synthesized by the system. After 30 h, the total amount of protein synthesized reached 205.6 μg, which is comparable with the amount sysnthesized (255.6 μg) in the translation system only where separetaly prepared capped mRNAs were added to the reactor for translation. Autoradiogram and Western blot analyses of the translated product showed a distinct band corresponding to the calculated molecular weight of the protein. These results have shown the establishment of a continuously coupled eukaryotic transcription‐translation system for the expression of genes from eukaryotic cells. © 1995 John Wiley & Sons, Inc.
Occurrences at mineral–bacteria interface during oxidation of arsenopyrite by Thiobacillus ferrooxidans
Authors: Fernandez Marcos G. Monroy , Mustin Christian , de Donato Philippe , Barres Odile , Marion Philippe , Berthelin Jacques
Published: December 2004

The combination of an improved bacterial desorption method, scanning electron microscopy (SEM), diffuse reflectance and transmission infrared Fourier transform spectroscopy, and a desorption‐leaching device like high‐pressure liquid chromatography (HPLC) was used to analyze bacterial populations (adhering and free bacteria) and surface‐oxidized phases (ferric arsenates and elemental sulfur) during the arsenopyrite biooxidation by Thiobacillus ferrooxidans. The bacterial distribution, the physicochemical composition of the leachate, the evolution of corrosion patterns, and the nature and amount of the surface‐oxidized chemical species characterized different behavior for each step of arsenopyrite bioleaching. The first step is characterized by a slow but strong adhesion of bacteria to mineral surfaces, the appearance of a surface phase of elemental sulfur, the weak solubilization of Fe(II), As(III), and As(V), and the presence of the first corrosion patterns, which follow the fragility zones and the crystallographic orientation of mineral grains. After this short step, growth of the unattached bacteria begins, while ferrous ions in solution are oxidized by them. Ferric ions produced by the bacteria can oxidize the sulfide directly and are regenerated by Fe(II) bacterial oxidation. At this time, a bioleaching cycle takes place and a coarse surface phase of ferric arsenate (FeAsO4 · xH2O where x ≈ 2) and deep ovoid pores appear. At the end of the bioleaching cycle, the high concentration of Fe(III) and As(V) in solution promotes the precipitation of a second phase of amorphous ferric arsenate (FeAsO4 · xH2O where x ≈ 4) in the leachate. Then the biooxidation process ceases: The bacteria adhering to the mineral sufaces are coated by the ferric arsenates and the concentration of Fe(III) on the leachate is found to have decreased greatly. Both oxidation mechanisms (direct and indirect oxidation) have been stopped. © 1995 John Wiley & Sons, Inc.
Cell cycle and cell size dependence of susceptibility to hydrodynamic forces
Authors: Al‐Rubeai Mohamed , Singh R. P. , Emery A. N. , Zhang Z.
Published: December 2004

Exposure of animal cells to intense hydrodynamic forces exerted in turbulent capillary flow, and by controiled agitation and aeration, resulted in preferential destruction of S and G2 cells and the extent of destruction of these cells was dependent upon the intensity of the action. The loss of these cells was possibly due to their larger size. However, the appearance of large numbers of membrane‐bound vesicular structures similar to apoptotic bodies as well as cells with low DNA stainability (in a sub‐G1 peak) suggested that the action of adverse hydrodynamic forces on these large cells may at least in part be to induce an apoptotic response. © 1995 John Wiley & Sons, Inc.
Enzymatic glucosylation of hydrophobic alcohols in organic medium by the reverse hydrolysis reaction using almond‐β‐D‐glucosidase
Authors: Vic Gabin , Biton Jacques , Le Beller Dominique , Michel Jean‐Marc , Thomas Daniel
Published: December 2004

Alkyl β‐D‐glucosides were synthesized from D‐glucose and alcohols by reverse hydrolysis using the commercially available almond β‐D‐glucosidase in 9:1 (v/v) acetonitrile–water medium. The main characteristics of this enzyme‐catalyzed glucosylation were established by using 2‐hydroxybenzyl alcohol. The reaction is entirely regio‐ and stereoselective. The solvent plays a fundamental role because, by decreasing the water concentration in the medium, the shift of the reaction equilibrium toward synthesis is realized without using an excessive amount of alcohol. Nevertheless, a minimum amount of water is necessary to maintain the enzyme activity. In contrast to the use of the enzyme in aqueous medium, the pH of the added water in acetonitrile did not influence the synthesis. Using this procedure, we have conducted systematic glucosylation of numerous alcohols and we have investigated enzyme specificity and alcohol reactivity. The enzyme has a pronounced affinity for the alcohols containing a phenyl group, and enantioselectivity for the aglycon is obtained with 1‐phenylethyl alcohol. Moreover, by using almond β‐D‐glucosidase it was also possible to synthesize alkyl β‐D‐galactosides. © 1995 John Wiley & Sons, Inc.
A strategy for construction of industrial strains of distiller's yeast
Authors: Ibragimova S. I. , Kozlov D. G. , Kartasheva N. N. , Sutsov N. I. , Efremov B. D. , Benevolensky S. V.
Published: December 2004

A procedure was developed for construction of industrial strains of distiller's yeast (Saccharomyces cerevisiae). It includes several steps: construction of congenic genetically marked haploid strains of opposite mating types starting from an industrial strain of hybrid nature, integrative transformation of the above haploid strains with a DNA fragment containing an expression cassette responsible for new technological facilities, and hybridization of transformants and isolation of final industrial homozygous strains under experimental conditions simulating commercial fermentation processes. This strategy permits the generation of strains that have desirable characteristics of traditional races of distiller's yeast along with new technological facilities determined by the particular expression cassette. Using this procedure, we have constructed an industrial strain with improved amylolytic activity. © 1995 John Wiley & Sons, Inc.
A model that links growth and secondary metabolite production in plant cell suspension cultures
Authors: Guardiola J. , Iborra J. L. , Cánovas M.
Published: December 2004

Plant cell suspensions of grape cells (Vitis vinifera L. cv. Gamay Fréaux) were grown in shake flasks operated both in the batch and semicontinuous mode. A mathematical model was developed to describe grape cell growth, sucrose uptake, and secondary metabolite (anthocyanin) production. Parameters were estimated from batch studies data. The model was able to predict results for semicontinuous experiments by only modifying the value of four of these parameters. The modified parameters (maximum specific rate of biomass production, maximum specific rate of substrate consumption for maintenance, maximum specific rate of anthocyanin production, and degradation constant of anthocyanins) were related to the kinetics rather than to the yield of the process. The model introduces the concept of primary and secondary metabolism substrate concentration–dependent competition for precursors. Further, the model was able to predict the evolution of the cell system when substrate is scarce, as the value of the different kinetic constants determines the portion of substrate that is used for biomass production, secondary metabolite production, and cell maintenance. © 1995 John Wiley & Sons, Inc.
Removal of water produced from lipase‐catalyzed esterification in organic solvent by pervaporation
Authors: Kwon Seok Joon , Song Kyu Min , Hong Won Hi , Rhee Joon Shick
Published: December 2004

Esterification of oleic acid with n‐butanol in the presence of Lipozyme® was carried out at 25°C in isooctane with various initial water activities. Initial reaction rate as well as equilibrium conversion decreased at high initial water activity. Therefore, removal of water present in the reaction mixtures was essential. A pervaporation process was applied to the lipase‐catalyzed synthesis of n‐butyloleate to remove water. Pervaporation selectively separated water from the reaction mixture using a nonporous polymeric membrane, cellulose acetate. Therefore, pervaporation is potentially applicable to remove the water produced from various enzymatic processes, such as synthesis of various esters, peptides, and glycosides in a solvent system as well as in a solvent‐free system. © 1995 John Wiley & Sons, Inc.
Fluorophore‐labeled carbohydrate analysis of immunoglobulin fusion proteins: Correlation of oligosaccharide content with in vivo clearance profile
Authors: Flesher Alan R. , Marzowski JoAnne , Wang Wei‐Chun , Raff Howard V.
Published: December 2004

CTLA4 is a membrane receptor on cytotoxic T cells whose interaction with the B7 counterreceptor on B cells is important in alloantigen responses. Soluble recombinant human and murine CTLA4 were produced using either Chinese hamster ovary or NS‐0 cell lines. Expression vectors were constructed containing the gene coding for the extracellular domain of CTLA4 fused to either human lgG1 hinge, CH2, and CH3 domains or murine lgG2a hinge, CH2, and CH3 domain genes. These glycoproteins were produced in hollow‐fiber or packed‐bed‐type bioreactors and purified from conditioned media by protein A affinity chromatography. Batches of purified CTLA4lg were analyzed for size, composition, and isoelectric point (pl) patterns by standard protein methods; oligosaccharide and monosaccharide profiles using several carbohydrate specific techniques; and in vivo clearance profiles using a murine model. Significant differences were observed between lots in their pl, clearance, and crbohydrate profiles. Higher overall pl values correlated with accelerated α‐phase clearance and changes in oligosaccharide composition as determined by lectin binding analysis and electrophoresis of fluorophore‐conjugated carbohydrates. Preparations exhibiting slower clearance profiles had oligosaccharides with higher quantities of N‐acetylneuraminic acid and were predominantly of an N‐linked biantennary complex‐type. Conversely, batches with accelerated clearance profiles had less detectable N‐acetylneuraminic acid. Oligosaccharides from murine CTLA4lg produced in NS‐0 cells had terminal N‐glycolylneuraminic acid but no detectable N‐acetylneuraminic acid and had concomitant accelerated clearance. These data suggest that the presence and quantity of N‐acetylneuraminic acid is an important component in predicting CTLA4lg plasma clearance rates and that production lots can be analyzed for oligosaccharide heterogeneity and sialic acid content by electrophoresis of fluorophore‐conjugated carbohydrates. © 1995 John Wiley & Sons, Inc.
Influence of operating variables on liquid circulation in a 10.5‐m3 jet loop bioreactor
Authors: García‐Salas Sergio , Flores‐Cotera Luis B.
Published: December 2004

A 10.5‐m3 concentric tube jet loop reactor was used to study the influence of the working liquid volume, mean superficial air velocity, operating pressure, downcomer aeration, liquid jet velocity, and two ratios of draft tube/reactor diameter (Dt/D) on liquid circulation time (Tc). The experiments were carried out in a water–air system with the use of the acid pulse method. Results showed that circulation time was independent of the working liquid volume over a certain minimum liquid level, whereas downcomer aeration and Dt/D ratio appeared as amenable parameters to achieve a high degree of control over liquid circulation and mixing efficiency, and to optimize the overall reactor performance. Increasing the operating pressure caused a reduction of the liquid circulation rate. However, ionger residence times of the air bubbles and the higher mass transfer driving force that result at higher pressures improve oxygen utilization. The relationship between Tc and air load was independent of the operating pressure, provided the correlation is given as a function of the mean superficial air velocity. Neither liquid circulation nor gas holdup were significantly affected by liquid jet velocity. © 1995 John Wiley & Sons, Inc.
An experimental study on a β‐cyclodextrin carbonate membrane reactor in PNPA hydrolysis
Authors: Drioli Enrico , Natoli Mariapina , Koter Izabel , Trotta Francesco
Published: December 2004

Flat sheet membranes made of polyetheretherketone, known as PEEK‐WC, and O‐octyloxycarbonyl β‐cyclodextrins were prepared by the phase inversion method. The cyclodextrins were entrapped in the polymeric membranes easily and simply in a single operation. Such functionalized membranes were tested for their catalytic activity. PNPA hydrolysis in a cyclodextrin membrane reactor was carried out as a model reaction. A significant improvement of reaction rate in comparison with the batch hydroxide ions catalyzed reaction was observed. © 1995 John Wiley & Sons, Inc.
A mathematical model for the growth of mycelial pellet populations
Authors: Tough A. J. , Pulham J. , Prosser J. I.
Published: December 2004

In liquid culture, filamentous organisms often grow in the form of pellets. Growth result in an increase in radius, whereas shear forces result in release of hyphal fragments which act as centers for further pellet growth and development. A previously published model for pellet growth of filamentous microorganisms has been examined and is found to be unstable for certain parameter values. This instability has been identified as being due to inaccuracies in estimating the numbers of fragments which seed the pellet population. A revised model has been formulated, based on similar premises, but adopting a finite element approach. This considers the population of pellets to be distributed in a range of size classes. Growth results in movement to classes of increasing pellet size, while fragments enter the smallest size class, from which they grow to form further pellets. The revised model is stable and predicts changes in the distribution of pellet sizes within a population growing in liquid batch culture. It considers pellet growth and death, with fragmentation providing new centers of growth within the pellet population, and predicts the effects of shear forces on pellet growth and size distribution. Predictions of pellet size distributions are tested using previously published data on the growth of fungal pellets and further predictions are generated which are suitable for experimental testing using cultures of filamentous fungi or actinomycetes. © 1995 John Wiley & Sons, Inc.
Polymerized liposome as ligand carrier for affinity precipitation of proteins
Authors: Sun Yan , Yu K. , Jin X. H. , Zhou X. Z.
Published: December 2004

A polymerized liposome (PLS) was prepared using a synthesized phospholipid with a diacetylene moiety in the hydrophobic chain and an amino group in the hydrophilic head. The PLS was used as a novel ligand carrier for affinity precipitation of proteins because it showed a reversibly precipitable property on salt addition and removal. Soybean trypsin inhibitor (STI) was easily immobilized on the PLS by a one‐step carbodiimide reaction. The PLS showed no nonspecific adsoprtion of proteins. It had a large ligand coupling capacity, and then a large adsorption capacity for trypsin after STI immobilization. The PLS with immpbilized STI was recycled three times for the purification of trypsin from a crude pancreatic extract. Although the degree of purification was compromised by the impurity of the STI employed, in each run the purification factor reached about 6 and more than 80% of trypsin activity was recovered. The results indicated that the PLS was a potential ligand carrier for affinity precipitation of proteins. © 1995 John Wiley & Sons, Inc.
Thermal isoelectric precipitation of α‐lactalbumin from a whey protein concentrate: Influence of protein–calcium complexation
Authors: Bramaud C. , Aimar P. , Daufin G.
Published: December 2004

The selective precipitation of α‐lactalbumin (α‐LA) at a pH around its isoelectric point (4.2) under heat treatment is the basis for a fractionation process of whey proteins. As precipitation is a phenomenon dependent on the protein hydrophobicity, and as the release of the tightly bound calcium occurring at pH around 4 modifies the α‐LA hydrophobicity, the specific role of calcium on isoelectric precipitation is investigated. A study of the extent of α‐LA precipitation in a whey protein concentrate under various operating conditions of pH, temperature, protein concentration, and calcium content is presented. We propose a mechanism for this phenomenon as a combination of a complexation equilibrium and of an irreversible precipitation, to account for the influence of temperature, α‐LA concentration total ionic content, and calcium concentration, and also to estimate the complexation equilibrium constant. © 1995 John Wiley & Sons, Inc.