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Product Formation (product + formation)
Terms modified by Product Formation Selected AbstractsInteresting Product Formation During O-Alkylation and Subsequent Rearrangement of Two Building Blocks of Dibromotyrosine Based Natural Products.CHEMINFORM, Issue 42 2002Uppuluri V. Mallavadhani Abstract For Abstract see ChemInform Abstract in Full Text. [source] Dynamic modelling of bacterial cellulose formationENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 4 2009Michael Hornung Abstract The interest in cellulose produced by bacteria from surface cultures has increased steadily in recent years because of its potential for use in medicine and cosmetics. Unfortunately, the low yield of this production process has limited the commercial usefulness of bacterial cellulose. The aim of this paper is to show the effect of substrate mass transfer on the growth of the bacteria and on their physiological potential for product formation by means of a dynamic mathematical model. [source] Solvolysis of Some Arenediazonium Salts in Binary EtOH/H2O Mixtures under Acidic ConditionsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 17 2003Román Pazo-Llorente Abstract We have determined the product distribution, the rate constants for dediazoniation product formation, and the solvolytic rate constants for 2-, 3-, and 4-methylbenzenediazonium ions (2-, 3-, and 4-MBD, respectively) loss in acidic ethanol/water mixtures over the whole composition range by a combination of spectrophotometric (UV/Vis) and high performance liquid chromatography (HPLC) measurements. The observed rate constants (kobs) for substrate loss are equal to those for product formation, and they remain essentially constant (2-MBD) with changing solvent composition but increase by a factor of ,2 (4MBD) on going from water to 100% EtOH. Up to four dediazoniation products , cresols (ArOH), chlorotoluene (ArCl), methylphenetole (ArOEt), and toluene (ArH) , were detected, depending on the solvent composition; the major dediazoniation products were the ArOH and ArOEt derivatives. The product selectivity (S) of the reaction towards nucleophiles is low and essentially constant with changing solvent composition, and good linear correlations between log kobs and YCl (solvent ionizing power) were observed for the three ArN2+ ions. All data are consistent with the rate-determining formation of an aryl cation, which reacts immediately with available nucleophiles. The data suggest that the distribution of neutral and anionic nucleophiles in the neighborhood of the ground state arenediazonium ion remains essentially unchanged upon dediazoniation, the observed product distribution reflecting the concentrations of nucleophiles in their immediate environment (i.e., in the first solvation shells of the arenediazonium ions). (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003) [source] Kinetics of inhibition of acetylcholinesterase in the presence of acetonitrileFEBS JOURNAL, Issue 8 2009Markus Pietsch The hydrolysis of acetylthiocholine by acetylcholinesterase from Electrophorus electricus was investigated in the presence of the inhibitors tacrine, gallamine and compound 1. The interaction of the enzyme with the substrate and the inhibitors was characterized by the parameters KI, ,,, b or ,, Km and Vmax, which were determined directly and simultaneously from nonlinear Michaelis,Menten plots. Tacrine was shown to act as a mixed-type inhibitor with a strong noncompetitive component (,, , 1) and to completely block deacylation of the acyl-enzyme. In contrast, acetylcholinesterase inhibition by gallamine followed the ,steric blockade hypothesis', i.e. only substrate association to as well as substrate/product dissociation from the active site were reduced in the presence of the inhibitor. The relative efficiency of the acetylcholinesterase,gallamine complex for the catalysis of substrate conversion was determined to be 1.7,25% of that of the free enzyme. Substrate hydrolysis and the inhibition of acetylcholinesterase were also investigated in the presence of 6% acetonitrile, and a competitive pseudo-inhibition was observed for acetonitrile (KI = 0.25 m). The interaction of acetylcholinesterase with acetonitrile and tacrine or gallamine resulted in a seven- to 10-fold increase in the KI values, whereas the principal mode of inhibition was not affected by the organic solvent. The determination of the inhibitory parameters of compound 1 in the presence of acetonitrile revealed that the substance acts as a hyperbolic mixed-type inhibitor of acetylcholinesterase. The complex formed by the enzyme and the inhibitor still catalysed product formation with 8.7,9.6% relative efficiency. [source] Batch kinetics and modelling of ethanolic fermentation of wheyINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 6 2005Salman Zafar Summary The fermentation of whey by Kluyveromyces marxianus strain MTCC 1288 was studied using varying lactose concentrations at constant temperature and pH. The increase in substrate concentration up to a certain limit was accompanied by an increase in ethanol formation, for example, at a substrate concentration of 10 g L,1, the production of ethanol was 0.618 g L,1 whereas at 50 g L,1 it was 3.98 g L,1. However, an increase in lactose concentration to 100 g L,1 led to a drastic decrease in product formation and substrate utilization. The maximum ethanol yield was obtained with an initial lactose concentration of 50 g L,1. A method of batch kinetics was utilized to formulate a mathematical model using substrate and product inhibition constants. The model successfully simulated the batch kinetics observed at S0 = 10 and 50 g L,1 but failed in case of S0 = 100 g L,1 because of strong substrate inhibition. [source] Imidazolium-2-Carboxylate as an Efficient, Expeditious and Eco-Friendly Organocatalyst for Glycerol Carbonate SynthesisADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 11-12 2009Prashant Abstract An improved and greener approach towards the synthesis of glycerol carbonate, via transesterification, using 1- n -butyl-3-methylimidazolium-2-carboxylate as catalyst is described. The catalyst loading as low as 1% was sufficient to yield quantitative conversions. A plausible mechanism is proposed for the catalytic cycle leading to product formation. [source] Browning Prevention by Ascorbic Acid and 4-Hexylresorcinol: Different Mechanisms of Action on Polyphenol Oxidase in the Presence and in the Absence of SubstratesJOURNAL OF FOOD SCIENCE, Issue 9 2007E. Arias ABSTRACT:, We have investigated the mechanism of action of 4-hexylresorcinol (4-HR) and ascorbic acid (AA) on the polyphenol oxidase (PPO) catalyzed oxidation of phenolic substrates. Incubation of PPO with 4-HR diminishes strongly PPO activity. This effect can be erroneously interpreted, due to the high affinity of 4-HR for PPO, as irreversible inactivation of PPO. However, PPO activity can be recovered by dialysis after incubation with 4-HR. 4-hexylresorcinol is a canonical enzyme inhibitor that binds preferentially to the oxy form of PPO. It is a mixed-type inhibitor, because it influences both apparent Vmax (1.26 compared with 0.4 units in the absence and presence of 4-HR, respectively) and Km values (0.28 mM compared with 0.97 mM in the absence and in the presence of 4-HR, respectively) of PPO. AA can prevent browning by 2 different mechanisms: In the absence of PPO substrates it inactivates PPO irreversibly, probably through binding to its active site, preferentially in its oxy form. In the presence of PPO substrates, AA reduces PPO oxidized reaction products, which results in a lag phase when measuring PPO activity by monitoring dark product formation but not when monitoring O2 consumption. The simultaneous use of both 4-HR and AA on PPO results in additive prevention of browning. [source] A Survey on the Potential Mode of Inhibition for Oxalic Acid on Polyphenol OxidaseJOURNAL OF FOOD SCIENCE, Issue 8 2003R. Yoruk ABSTRACT: The potential mode of inhibition for xalic acid on polyphenol oxidase (PPO) was investigated. The extent of inhibition was influenced not only by oxalic acid concentration but also by pH. Inhibition was most prominent at pH 4.0 where complete inhibition occurred at the 4-mM oxalic acid concentration and was less evident at higher pH values. Inhibition of PPO by oxalic acid was due to its binding with copper to form an inactive complex, and the inhibition was characterized as noncompetitive. Oxalic acid diminished the catechol-quinone product formation, and no quinone bleaching was observed. Oxalic acid was a more potent inhibitor of PPO compared with other structurally related acids. [source] Collision-induced dissociation studies of protonated ether,(H2O)n (n = 1,3) clustersJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2006Daniel J. Goebbert Abstract We have studied the protonated ether,(H2O)n (n = 1,3) complexes containing tetrahydrofuran, dimethyl, diethyl, dibutyl, and butylmethyl ethers using a flowing afterglow triple-quadrupole mass spectrometer. Collision-induced dissociation, CID, of all clusters with n = 1, 2 shows sequential water loss. The n = 3 cluster of dimethyl ether shows sequential water loss, while all other ether clusters display selective product formation. The CID spectra are interpreted based on known energetics, and theoretical studies of the dimethyl and diethyl ether systems. Copyright © 2006 John Wiley & Sons, Ltd. [source] The role of the cyclic imide in alternate degradation pathways for asparagine-containing peptides and proteinsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 10 2007Michael P. DeHart Abstract Peptides and proteins exhibit enhanced reactivity at asparagine residues due to the formation of a reactive succinimide intermediate that produces normal and isoaspartyl deamidation products along with significant racemization. This study examines the potential for attack of amine nucleophiles at the succinimide carbonyls to generate alternate decomposition products, depending on the nucleophile involved in the reaction. The reactions of the model peptides Phe-Asn-Gly (FNG) and Phe-isoAsn-Gly (FisoNG) were explored as a function of pH (8.5,10.5) in the presence and absence of ammonia buffer (0.2,2 M) using an isocratic HPLC method to monitor reactant disappearance and product formation. In addition to deamidation to form isoAsp and Asp peptides, two additional types of reactions were found to occur via the succinimide intermediate under these conditions. Back-reaction of the succinimide with ammonia led to peptide backbone isomerization while intramolecular attack by the amino terminus produced diketopiperazines. A kinetic model assuming a central role for the succinimide intermediate was derived to fit the concentration versus time data. These studies implicate the cyclic imide as a key intermediate in the formation of alternate peptide and protein degradants, including possible covalent amide-linked aggregates that may form from intermolecular attack of the cyclic imide by neighboring amino groups. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 2667,2685, 2007 [source] Butanolysis of 2-methylbenzenediazonium ions: product distribution, rate constants of product formation, and activation parametersJOURNAL OF PHYSICAL ORGANIC CHEMISTRY, Issue 5 2009M. José Pastoriza-Gallego Abstract We have determined the product distributions, the rate constants of product formation and substrate loss, and the activation parameters for the butanolysis of 2-methylbenzenediazonium, 2MBD, tetrafluoroborate in aqueous 1-Butanol (BuOH) solutions by combining UV,VIS spectroscopy, high performance liquid chromatography (HPLC), and a derivatization protocol that traps unreacted 2MBD as a stable azo dye. BuOH/H2O solutions are miscible over a narrow composition range, but in reverse micelles composed of sodium dodecyl sulfate, SDS, BuOH, and water, are miscible between 45,80%. Two major and two minor dediazoniation products are observed, 2-cresol, ArOH, 2-butyl-tolyl-ether, ArOBu, and small amounts of 2-chlorobenzene, ArCl (from HCl added to control solution acidity) and toluene, ArH (a reduction product). Product yields depend on experimental conditions, but quantitative conversion to products is achieved over the entire composition ranges investigated. The observed rate constants, kobs, obtained by monitoring 2MBD loss or by monitoring ArOH or ArOBu formation, are the same and they are only modestly affected by changes in the solution composition. The activation parameters obtained from the effect of temperature on kobs show that the enthalpy of activation is relatively high compared to those found in bimolecular reactions and the entropy of activation is small but positive. The results suggest that 2MBD is mainly sampling in the BuOH-H2O rich interfacial region of the reverse micelle and are consistent with 2MBD decomposing through a DN,+,AN mechanism, i.e., a rate limiting formation of an aryl cation that reacts immediately with nucleophiles. Copyright © 2008 John Wiley & Sons, Ltd. [source] Applicability of laser-induced Raman microscopy for in situ monitoring of imine formation in a glass microfluidic chipJOURNAL OF RAMAN SPECTROSCOPY, Issue 10 2003Moonkwon Lee Abstract Laser-induced Raman microscopy has been used to illustrate its applicability for the in situ monitoring of imine formation reaction in a glass microfluidic chip. In order to monitor the diffusion process in a micro channel, the Raman spectra were measured at various points along the channel with a constant flow rate of 2.7 µl min,1. Time-dependent Raman spectra were also measured without flow in order to monitor the variation of Raman peaks to a complete conversion. The disappearance of the CO stretching peak at 1700 cm,1 of the reactant, benzaldehyde, and the appearance of the Raman peak for the product, an imine, at 1628 cm,1 were successfully monitored. In addition, the intensity increases of three phenyl stretching modes in the 1550,1630 cm,1 region were also observed. The increase in Raman intensity for this vibrational mode is caused by an effective ,-electron conjugation between two phenyl rings through the ,CN,bridging group of the product. Laser-induced Raman microscopy enables us to monitor in situ product formation and to obtain detailed structural information in a glass microfluidic chip. Copyright © 2003 John Wiley & Sons, Ltd. [source] Scale-up of natural product formation and isolationMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 8 2005Ralf G. Berger [source] P450-catalyzed vs. electrochemical oxidation of haloperidol studied by ultra-performance liquid chromatography/electrospray ionization mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 9 2010Tove Johansson Mali'n The metabolites formed via the major metabolic pathways of haloperidol in liver microsomes, N -dealkylation and ring oxidation to the pyridinium species, were produced by electrochemical oxidation and characterized by ultra-performance liquid chromatography/electrospray ionization mass spectrometry (UPLC/ESI-MS). Liver microsomal incubations and electrochemical oxidation in the presence of potassium cyanide (KCN) resulted in two diastereomeric cyano adducts, proposed to be generated from trapping of the endocyclic iminium species of haloperidol. Electrochemical oxidation of haloperidol in the presence of KCN gave a third isomeric cyano adduct, resulting from trapping of the exocyclic iminium species of haloperidol. In the electrochemical experiments, addition of KCN almost completely blocked the formation of the major oxidation products, namely the N -dealkylated products, the pyridinium species and a putative lactam. This major shift in product formation by electrochemical oxidation was not observed for the liver microsomal incubations where the N -dealkylation and the pyridinium species were the major metabolites also in the presence of KCN. The previously not observed dihydropyridinium species of haloperidol was detected in the samples, both from electrochemical oxidation and the liver microsomal incubations, in the presence of KCN. The presence of the dihydropyridinium species and the absence of the corresponding cyano adduct lead to the speculation that an unstable cyano adduct was formed, but that cyanide was eliminated to regenerate the stable conjugated system. The formation of the exocyclic cyano adduct in the electrochemical experiments but not in the liver microsomal incubations suggests that the exocyclic iminium intermediate, obligatory in the electrochemically mediated N -dealkylation, may not be formed in the P450-catalyzed reaction. Copyright © 2010 John Wiley & Sons, Ltd. [source] Dynamic gene expression regulation model for growth and penicillin production in Penicillium chrysogenumBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Rutger D. Douma Abstract As is often the case for microbial product formation, the penicillin production rate of Penicillium chrysogenum has been observed to be a function of the growth rate of the organism. The relation between the biomass specific rate of penicillin formation (qp) and growth rate (µ) has been measured under steady state conditions in carbon limited chemostats resulting in a steady state qp(µ) relation. Direct application of such a relation to predict the rate of product formation during dynamic conditions, as they occur, for example, in fed-batch experiments, leads to errors in the prediction, because qp is not an instantaneous function of the growth rate but rather lags behind because of adaptational and regulatory processes. In this paper a dynamic gene regulation model is presented, in which the specific rate of penicillin production is assumed to be a linear function of the amount of a rate-limiting enzyme in the penicillin production pathway. Enzyme activity assays were performed and strongly indicated that isopenicillin-N synthase (IPNS) was the main rate-limiting enzyme for penicillin-G biosynthesis in our strain. The developed gene regulation model predicts the expression of this rate limiting enzyme based on glucose repression, fast decay of the mRNA encoding for the enzyme as well as the decay of the enzyme itself. The gene regulation model was combined with a stoichiometric model and appeared to accurately describe the biomass and penicillin concentrations for both chemostat steady-state as well as the dynamics during chemostat start-up and fed-batch cultivation. Biotechnol. Bioeng. 2010;106: 608,618. © 2010 Wiley Periodicals, Inc. [source] The effect of heating rate on Escherichia coli metabolism, physiological stress, transcriptional response, and production of temperature-induced recombinant protein: A scale-down studyBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Luis Caspeta Abstract At the laboratory scale, sudden step increases from 30 to 42°C can be readily accomplished when expressing heterologous proteins in heat-inducible systems. However, for large scale-cultures only slow ramp-type increases in temperature are possible due to heat transfer limitations, where the heating rate decreases as the scale increases. In this work, the transcriptional and metabolic responses of a recombinant Escherichia coli strain to temperature-induced synthesis of pre-proinsulin in high cell density cultures were examined at different heating rates. Heating rates of 6, 1.7, 0.8, and 0.4°C/min were tested in a scale-down approach to mimic fermentors of 0.1, 5, 20, and 100 m3, respectively. The highest yield and concentration of recombinant protein was obtained for the slowest heating rate. As the heating rate increased, the yield and maximum recombinant protein concentration decreased, whereas a larger fraction of carbon skeletons was lost as acetate, lactate, and formate. Compared to 30°C, the mRNA levels of selected heat-shock genes at 38 and 42°C, as quantified by qRT-PCR, increased between 2- to over 42-fold when cultures were induced at 6, 1.7, and 0.8°C/min, but no increase was observed at 0.4°C/min. Only small increases (between 1.5- and 4-fold) in the expression of the stress genes spoT and relA were observed at 42°C for cultures induced at 1.7 and 6°C/min, suggesting that cells subjected to slow temperature increases can adapt to stress. mRNA levels of genes from the transcription,translation machinery (tufB, rpoA, and tig) decreased between 40% and 80% at 6, 1.7 and 0.8°C/min, whereas a transient increase occurred for 0.4°C/min at 42°C. mRNA levels of the gene coding for pre-proinsulin showed a similar profile to transcripts of heat-shock genes, reflecting a probable analogous induction mechanism. Altogether, the results obtained indicate that slow heating rates, such as those likely to occur in conventional large-scale fermentors, favored heterologous protein synthesis by the thermo-inducible expression system used in this report. Knowledge of the effect of heating rate on bacterial physiology and product formation is useful for the rational design of scale-down and scale-up strategies and optimum recombinant protein induction schemes. Biotechnol. Bioeng. 2009;102: 468,482. © 2008 Wiley Periodicals, Inc. [source] Aldehyde,alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentationsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Ryan Sillers Abstract Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acetone, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently developed ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alcohol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA, respectively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of butyryl-CoA. In order to increase then the flux towards butyryl-CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl-CoA to acetoacetyl-CoA, the first step of the pathway from acetyl-CoA to butyryl-CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpression in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular butyryl-CoA pool and reduce the acetyl-CoA pool in order to achieve improved butanol titers and selectivity. Biotechnol. Bioeng. 2009;102: 38,49. © 2008 Wiley Periodicals, Inc. [source] A hidden square-root boundary between growth rate and biomass yieldBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Wilson W. Wong Abstract Although the theoretical value of biomass yield can be calculated from metabolic network stoichiometry, the growth rate is difficult to predict. Since the rate and yield can vary independently, no simple relationship has been discovered between these two variables. In this work, we analyzed the well-accepted enzyme kinetics and uncovered a hidden boundary for growth rate, which is determined by the square-root of three physiological parameters: biomass yield, the substrate turnover number, and the maximum synthesis rate of the turnover enzyme. Cells cannot grow faster than the square-root of the product of these parameters. This analysis is supported by experimental data and involves essentially no assumptions except (i) the cell is not undergoing a downshift transition, (ii) substrate uptake enzyme activity is proportional to its copy number. This simple boundary (not correlation) has escaped notice for many decades and suggests that the yield calculation does not predict the growth rate, but gives an upper limit for the growth rate. The relationship also explains how growth rate is affected by the yield and sheds lights on strain design for product formation. Biotechnol. Bioeng. 2009;102: 73,80. © 2008 Wiley Periodicals, Inc. [source] Development of a minimal defined medium for recombinant human interleukin-3 production by Streptomyces lividans 66BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2008Keyvan Nowruzi Abstract A systematic approach was developed to identify and optimize the essential amino acids in defined minimal medium for the production of recombinant human interleukin 3 (rHuIL-3) by Streptomyces lividans. Starvation trials were carried out initially to narrow down the number of probable essential amino acids from an initial number of 20 to 8. Then a screening mixture experiment was designed and performed with the eight identified amino acids and distance-based multivariate analysis was employed to rank the probable essential amino acids regarding both growth and product formation. Following this procedure, the search was narrowed to four amino acids (Asp, Leu, Met, and Phe). Finally, a mixture design experiment known as the simplex lattice design was carried out and the composition of the optimum minimal medium was found (Asp 53%, Met 5%, and Phe 42%). Biotechnol. Bioeng. 2008;99: 214,222. © 2007 Wiley Periodicals, Inc. [source] A perspective of metabolic engineering strategies: moving up the systems hierarchyBIOTECHNOLOGY & BIOENGINEERING, Issue 7 2003Thomas Bulter Abstract Metabolic engineering has been established as an important field in biotechnology. It involves the analysis, design, and alteration of the stoichiometric network using sophisticated mathematical and molecular biology techniques. It allows for improvement of pathway kinetics by removing flux bottlenecks, balancing precursors, and recycling cofactors used to increase product formation. The next step in the systems hierarchy is the constructive manipulation of regulatory networks. As our understanding of regulation continues to expand rapidly, engineering of intracellular regulation will become an integral aspect of metabolic engineering. © 2003 Wiley Periodicals, Inc. [source] Characterization of flow conditions in 2 L and 20 L wave bioreactors® using computational fluid dynamicsBIOTECHNOLOGY PROGRESS, Issue 1 2010Alper A. Öncül Abstract Characterization of flow conditions is of great importance to control cell growth and cell damage in animal cell culture because cell viability is influenced by the flow properties in bioreactors. Alternative reactor types like Wave Bioreactors® have been proposed in recent years, leading to markedly different results in cell growth and product formation. An advantage of Wave Bioreactors® is the disposability of the Polyethylenterephthalet-bags after one single use (fast setup of new production facilities). Another expected advantage is a lower shear stress compared to classical stirred-tank reactors, due to the gentle liquid motion in the rocking cellbag. This property would considerably reduce possible cell damage. The purpose of the present study is to investigate in a quantitative manner the key flow properties in Wave Bioreactors®, both numerically and experimentally. To describe accurately flow conditions and shear stress in Wave Bioreactors® using numerical simulations, it is necessary to compute the unsteady flow applying Computational Fluid Dynamics (CFD). Corresponding computations for two reactor scales (2 L and 20 L cellbags) are presented using the CFD code ANSYS-FLUENT®. To describe correctly the free liquid surface, the present simulations employ the Volume of Fluid (VOF) method. Additionally, experimental measurements have been carried out to determine liquid level, flow velocity and liquid shear stress, which are used as a validation of the present CFD simulations. It is shown that the obtained flows stay in the laminar regime. Furthermore, the obtained shear stress levels are well below known threshold values leading to damage of animal cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Effects of glucose and nitrogen source concentration on batch fermentation kinetics of Lactococcus lactis under hemin-stimulated respirative conditionBIOTECHNOLOGY PROGRESS, Issue 4 2008Azher Razvi Abstract Analytical solutions to the ordinary differential equations governing the kinetics of cell growth, substrate utilization, and product formation of batch fermentation processes were derived and used to study the kinetics of the hemin-stimulated respiratory cultivation of Lactococcus lactis at varied initial glucose concentrations and nitrogen source concentrations. Studies revealed that initial glucose concentration varying in the range of 60 to 90 g/L had no significant substrate inhibitive effect. Furthermore, elevating the concentration of complex nitrogen sources while maintaining glucose concentration at 60% led to a high final biomass concentration of 6.6 g/L, substantially higher than that obtained with the basic medium, which was 4.1 g/L. [source] Dynamic Metabolic Modeling for a MAB BioprocessBIOTECHNOLOGY PROGRESS, Issue 1 2007Jianying Gao Production of monoclonal antibodies (MAb) for diagnostic or therapeutic applications has become an important task in the pharmaceutical industry. The efficiency of high-density reactor systems can be potentially increased by model-based design and control strategies. Therefore, a reliable kinetic model for cell metabolism is required. A systematic procedure based on metabolic modeling is used to model nutrient uptake and key product formation in a MAb bioprocess during both the growth and post-growth phases. The approach combines the key advantages of stoichiometric and kinetic models into a complete metabolic network while integrating the regulation and control of cellular activity. This modeling procedure can be easily applied to any cell line during both the cell growth and post-growth phases. Quadratic programming (QP) has been identified as a suitable method to solve the underdetermined constrained problem related to model parameter identification. The approach is illustrated for the case of murine hybridoma cells cultivated in stirred spinners. [source] Defined Protein-Free NS0 Myeloma Cell Cultures: Stimulation of Proliferation by Conditioned Medium FactorsBIOTECHNOLOGY PROGRESS, Issue 1 2005Erika Spens A chemically defined, protein-free, and animal-component-free medium, designated RITM01, has been developed for NS0 myeloma cells. The basal medium used was a commercial serum-free and protein-free hybridoma medium, which was supplemented with phosphatidylcholine, cholesterol, ,-cyclodextrin, and ferric citrate. Increasing the amino acid concentration significantly improved cell growth. An NS0 cell line, constitutively producing a human IgG1 antibody, reached a peak cell density of 3 × 106 cells mL,1 in this medium. The antibody yield was 195 mg L,1 in batch culture, which is a 3-fold increase compared to that of a standard serum-supplemented medium, even though the cell yield was the same. The increase in antibody yield was a consequence of a longer growth phase and a slight increase in specific antibody production rate at low specific proliferation rates. Adaptation of the NS0 myeloma cell line to the protein-free conditions required about 3 weeks before viability and cell densities were stabilized. Most probably, changes in gene expression and phenotypic behavior necessary for cell survival and proliferation occurred. We hypothesize that mitogenic factors produced by the cells themselves are involved in autocrine control of proliferation. To investigate the presence of such factors, the effect of conditioned (spent) medium (CM) on cell growth and proliferation was studied. Ten-fold concentrated CM, harvested at a cell density of 2 × 106 cells mL,1, had a clear positive effect on proliferation even if supplied at only 2.5% (v/v). CM was found to contain significant amounts of extracellular proteins other than the antibody. Fractionation of CM on a gel filtration column and subsequent supplementation of new NS0 cultures with the individual fractions showed that factors eluting at 20,25 kDa decreased the lag phase and increased the peak cell density as compared to control cultures. Identification of autocrine factors involved in regulation of proliferation may lead to completely new strategies for control of growth and product formation in animal cell processes. [source] Monitoring of Recombinant Protein Production Using Bioluminescence in a Semiautomated Fermentation ProcessBIOTECHNOLOGY PROGRESS, Issue 4 2003I. Trezzani On-line optimization of fermentation processes can be greatly aided by the availability of information on the physiological state of the cell. The goal of our "BioLux" research project was to design a recombinant cell capable of intracellular monitoring of product synthesis and to use it as part of an automated fermentation system. A recombinant plasmid was constructed containing an inducible promoter that controls the gene coding for a model protein and the genes necessary for bioluminescence. The cells were cultured in microfermenters equipped with an on-line turbidity sensor and a specially designed on-line light sensor capable of continuous measurement of bioluminescence. Initial studies were done under simple culture conditions, and a linear correlation between luminescence and protein production was obtained. Such specially designed recombinant bioluminescent cells can potentially be applied for model-based inference of intracellular product formation, as well as for optimization and control of recombinant fermentation processes. [source] Improving Glucose and Glutamine Metabolism of Human HEK 293 and Trichoplusiani Insect Cells Engineered To Express a Cytosolic Pyruvate Carboxylase EnzymeBIOTECHNOLOGY PROGRESS, Issue 1 2003Cynthia B. Elias Metabolic engineering has been defined as a directed improvement of product formation or cellular properties by modification of specific biochemical pathways or introduction of new enzymatic reactions by recombinant DNA technology. The use of metabolic flux analysis (MFA) has helped in the understanding of the key limitation in the metabolic pathways of cultured animal cells. The MFA of the major nutrients glucose and glutamine showed that the flux of glucose to the TCA cycle and its subsequent utilization is limited as a result of the lack of certain key enzymes in the pathway. One of the key enzymes controlling this flux is pyruvate carboxylase. Introduction of this enzyme into mammalian cells has been shown to improve the utilization of glucose and limit the production of lactate and ammonia, which are deleterious to cell growth. In the present work a yeast pyruvate carboxylase gene has been introduced into mammalian (HEK 293) and insect ( Trichoplusiani High-Five) cells, resulting in the cytosolic expression of the enzyme. In both cases the resulting transfected cells were able to utilize glucose and glutamine more efficiently and produce lower amounts of lactate and ammonia. Differences in the amino acid utilization pattern were also observed, indicating changes in the basic metabolism of the cells. The performance of the transfected cells as expression systems for adenovirus and baculovirus vectors, respectively, has also been examined. The results obtained and their impact on the process development for protein and viral vector production are discussed. [source] Improved Paclitaxel and Baccatin III Production in Suspension Cultures of TaxusmediaBIOTECHNOLOGY PROGRESS, Issue 3 2002Rosa M. Cusidó A cell suspension culture of Taxus media was established from a stable callus line of this species. The growth rate and production of paclitaxel and baccatin III of this cell suspension were significantly increased during the shake flask culture in its respective optimum media for cell growth and product formation, which were selected after assaying 24 different culture media. The highest yields of paclitaxel (2.09 mg L,1) and baccatin III (2.56 mg L,1) in the production medium rose (factors of 7.0 and 3.0, respectively) in the presence of methyljasmonate (220 ,g g,1 FW). When the elicitor was added together with mevalonate (0.38 mM) and N -benzoylglycine (0.2 mM), the increase in the yields of paclitaxel and baccatin III was even higher (factors of 8.3 and 4.0, respectively). Thereafter, a two-stage culture for cell suspension was carried out using a 5,l stirred bioreactor running for 36 days, the first stage being in the cell growth medium until cells entered their stationary growth phase (12 days) and the second stage being in the production medium supplemented with the elicitor and two putative precursors in the concentrations indicated above. Under these conditions, 21.12 mg L,1 of paclitaxel and 56.03 mg L,1 of baccatin III were obtained after 8 days of culture in the production medium. [source] Effects of Cloned Gene Dosage on the Response of Recombinant CHO Cells to Hyperosmotic Pressure in Regard to Cell Growth and Antibody ProductionBIOTECHNOLOGY PROGRESS, Issue 6 2001Joon Soo Ryu The effect of cloned gene dosage on growth and product formation under hyperosmotic conditions has been studied using recombinant Chinese hamster ovary (rCHO) cell lines producing chimeric antibody. Batch cultures of four rCHO cell lines carrying different numbers of antibody gene copies were carried out using the hyperosmolar medium. Depending on cloned gene dosage, hyperosmotic pressure decreased specific growth rate (,) and increased specific antibody productivity (qAb) to a different degree. The cell line with lower cloned gene dosage displayed more significant enhancement in qAb and less reduction in , at hyperosmolalities. However, the cell line with higher cloned gene dosage still yielded higher maximum antibody concentration at hyperosmolality up to 469 mOsm/kg. Northern blot analysis showed a positive relationship between immunoglobulin mRNA level per cell and qAb, indicating that transcriptional regulation was involved in the response of rCHO cells to hyperosmotic pressure. Cell cycle analysis showed that hyperosmotic pressure induced G1 -phase arrest, suggesting that the increase of cell population in G1 -phase may contribute in part to enhanced qAb at hyperosmolality. Taken together, although the cell line with lower cloned gene dosage displayed more significant enhancement in qAb at hyperosmolality, the factor that determined the maximum antibody concentration in hyperosmotic rCHO cell cultures was almost exclusively the gene dosage. [source] Measurement and Control of Dissolved Carbon Dioxide in Mammalian Cell Culture Processes Using an in Situ Fiber Optic Chemical SensorBIOTECHNOLOGY PROGRESS, Issue 5 2000Robert N. Pattison At high viable cell concentrations in large-scale mammalian cell culture processes, the accumulation of dissolved carbon dioxide (dCO2, typically quantified as an equilibrium gas-phase concentration) becomes problematic as a result of low CO2 removal rates at reduced surface-to-volume ratios. High dCO2 concentrations have previously been shown to inhibit cell growth and product formation in mammalian cells and to alter the glycosylation pattern of recombinant proteins. Therefore, reliable monitoring and control of dCO2 are important for successful large-scale operation. Off-line measurements by instruments such as blood gas analyzers (BGA) are constrained by the low frequency of data collection and cannot be used for on-line control. In a preliminary evaluation of the YSI 8500 in situ sensor, a response time (t90%) of 6 min, sensitivity of 0.5% CO2 (3.6 mmHg), and linearity of measurement (R2 = 0.9997) between the equivalent gas-phase partial pressure of 0,180 mmHg (0% and 25% CO2) were established. Measurements were found to be unaffected by culture pH and typical mammalian cell culture concentrations of glucose, glutamine, glutamate, lactate, and ammonium. The sensor withstood repeated sterilization and cleaning cycles. The reliability of this sensor was demonstrated in microcarrier-based Chinese hamster ovary (CHO) cell perfusion cultures at reactor scales of 30, 40, 340, and 2000 L and was successfully implemented in a dCO2 control strategy using N2 sparging. [source] Replacement of Natural Cofactors by Selected Hydrogen Peroxide Donors or Organic Peroxides Results in Improved Activity for CYP3A4 and CYP2D6CHEMBIOCHEM, Issue 6 2006Amandine Chefson P450 enzymes made easier to use in synthesis. The use of cumene hydroperoxide with CYP2D6 or CYP3A4 leads to as much as a twofold increase in the rate of product formation compared to the use of the natural cofactors. [source] | |||||||||||||