bluuebunny/crossref_metadata_2025_split huggingface dataset:
Papain in organic solvents: Determination of conditions suitable for biocatalysis and the effect on substrate specificity and inhibition
Authors: Stevenson David E. , Storer Andrew C. 
Published: December 2004

AbstractSynthesis of N‐CBZ‐(N‐Carbobenzoxy)‐1‐amino‐acid methyl esters from N‐CBZ‐amino acids and methanol has been used as an assay to examine the properties of papain in organic solvents containing small amounts of water. Papain is active in solvents ranging in polarity from acetonitrile to tetrachloromethane. The optimal activity in each solvent varied only about three to four fold, but the amount of added water required to achieve it varied from 4% (v/v) in acetonitrile to 0.05% (v/v) in tetrachloromethane. The enzyme was generally more stable in hydrophobic solvents and at lower water contents. The apparent Km value of CBZ‐glycine was 26 times higher in acetonitrile than in toluene due to differential partitioning of the substrate between aqueous and organic phases. The substrate specificity of the enzyme was qualitatively little different from that in aqueous solution, with amino acid derivatives still the best substrates. Nitrile analogs of substrates inhibited the enzyme, as they do in aqueous solution, and inhibition by a variety of substituted aromatic hydrocarbons showed that the main specificity of papain for hydrophobic side chains at its S2 subsite, was little affected. The results show that papain can catalyze reactions under a variety of conditions in organic solvents but its substrate specificity is little changed from that in aqueous media.
Kinetics and modeling of temperature effects on batch xanthan gum fermentation
Authors: Shu Chin‐Hang , Yang Shang‐Tian 
Published: December 2004

AbstractBatch fermentation kinetics of xanthan gum production from glucose by Xanthomonas campestris at temperatures between 22°C and 35°C were studied to evaluate temperature effects on cell growth and xanthan formation. These batch xanthan fermentations were modeled by the logistic equation for cell growth, the Luedeking–Piret equation for xanthan production, and a modified Luedeking–Piret equation for glucose consumption. Temperature dependence of the parameters in this model was evaluated. Growth‐associated rate constants increased to a maximum at ∼30°C and then decreased to zero at ∼35°C. This temperature effect can be modeled using a square‐root model. On the contrary, non‐growth‐associated rate constants increased with increasing temperature, following the Arrhenius relationship, in the entire temperature range studied. The model developed in this work fits the experimental data very well and can be used in a simulation study. However, due to the empirical nature of the model, the parameter values need to be reevaluated if the model is to be applied to different growth conditions.
Hydrolysis of inulin: A kinetic study of the reaction catalyzed by an inulinase from Aspergillus ficuum
Authors: Carniti Paolo , Beltrame Pier Luigi , Guardione Donatella , Focher Bonaventura , Marzetti Annamaria 
Published: December 2004

AbstractA kinetic study of the hydrolysis of inulin was performed by using as catalyst a commercial inulinase from Aspergillus ficuum. The reaction was studied carrying out initial rate as well as time course measurements. Both inulinase and invertase activities of the enzyme were taken into account, and the corresponding kinetic parameters were determined in the temperature range 30‐50°C. The activation energies of the turnover constant for inulinase and invertase activities were found to be similar (56–57 kJ · mol−1). The ratio S/I of invertase to inulinase activity was 1.6 regardless of temperature. The thermal degradation of the enzyme was also investigated up to 70°C, and an activation energy of 350‐370 kJ · mol−1 was evaluated.
Transient response method for evaluating the rate constants of reactions over immobilized enzymes
Authors: Wu Jau‐Yann , Weng Hung‐Shan 
Published: December 2004

AbstractThe transient response method was utilized to evaluate the rate constants of reaction over immobilized enzyme. Glucose oxidation catalyzed by the immobilized glucose oxidase in a fixed‐bed reactor was selected as an example. A theoretical model including the effects of axial dispersion, film diffusion, and intraparticle diffusion was established for the reactor. The individual rate constant of each elementary step of this enzymatic reaction was determined through direct fitting of the experimental response data to the model.
A novel technique for the measurement of disruption in high‐pressure homogenization: Studies on E. coli containing recombinant inclusion bodies
Authors: Middelberg Anton P. J. , O'Neill Brian K. , L. Bogle I. David , Snoswell Mark A. 
Published: December 2004

AbstractThe high‐pressure homogenization of Escherichia coli, strain JM101, containing inclusion bodies of recombinant porcine somatotropin was investigated. A novel technique employing an analytical disc centrifuge was used to monitor the disruption. This a direct technique which measures cell disintegration rather than soluble protein release. The technique is particularly suited to measurements where the disruption approaches 100%. The disk centrifuge provides a size distribution of the homogenate, and furnishes evidence for the preferential disruption of larger cells. For E. coli containing inclusion bodies, and increase in the cell feed concentration from 145 g/L (wet weight) to 330 g/L resulted is poorer homogenization. Poorer disruption was also obtained by lowering the feed temperature from 20°C to 5°C. Only slight variations in performance were obtained by increasing the feed pH from 7.5 to 9.0 or by storing the feed at 4°C for 24 h prior to disruption. Comparison with uninduced E. coli strain JM101, showed that the disruption obtained is higher for bacteria containing a recombinant inclusion body.
Routine determination of enzyme kinetics using Plate reader
Authors: Galgani F. , Cadiou Y. , Bocquene G. 
Published: December 2004

AbstractA method is described for the routine determination of kinetics parameters from colorimetric enzyme reactions using a 96‐well microtiter plate reader commonly used in immunoassay. A program in Turbo Pascal have been developed for the analysis of results. The system performs the description of plate format, the definition of conditions of assays, the measurement of enzyme reactions and collection of data, analysis, visualization of results and modification of data. Measurement of Km and Vmax is made with different methods, including iterative determination, Lineweaver‐Burk transformation, and Hofster transformation, to give the final values of kinetic parameters with their associated standards errors. This approach should be applicable to routine determination of kinetic parameters.
Diffusion in gels containing immobilized cells: A critical review
Authors: Westrin Bengt A. , Axelsson Anders 
Published: December 2004

AbstractEleven experimental investigation of diffusion in gels containing immobilized cells are reviewed. The experimental data, which quantitatively express the diffusion coefficient as a function of the cell concentration, are compared with a number of well‐known equations developed for mass transfer in heterogeneous media. Based on this comparison, a procedure for the theoretical prediction of effective diffusion coefficients in cell‐containing gels is recommended.
Affinity partitioning of vancomycin
Authors: Godbole Pradyumna P. , Tsai Ruey‐S. , Clark William M. 
Published: December 2004

AbstractThe use of the Flanagan and Barondes model14 describing affinity partitioning as an aid in designing separation systems is discussed. Experimental studies are described for affinity partitioning of vancomycin, a glycopeptide antibiotic, in a water–methoxypolyethylene glycol‐dextran system using methoxypolyethylene glycol‐dextran system using methoxypolyethylene glycol bound D‐alanyl‐Dalanyl‐D‐alanine or D‐alanyl‐D‐alanine as the reversible affinity ligand. Even for this ideal case of 1:1 binding interaction, the model only qualitatively predicts the affinity effect when all model parameters are measured independently. The discrepancy between measured and predicted values can be attributed to a difference in exposed surface of the free antibiotic and ligand compared to that in the bound state.The effect of experimentally varying model parameters is also described. It was determined that a polymers‐ligand which partitions more strongly to the top phase would provide the most significant enhancement to this affinity partitioning system. Such an improvement can be made by increasing the molecular weight of the hydrophobicity of the polymer‐ligand. A process for vancomycin recovery from fermentation broth using D‐‐alanyl‐D‐alanine sepharose as affinity ligand is described.
Factors controlling cell proliferation and antibody production in mouse hybridoma cells: I. Influence of the amino acid supply
Authors: Duval D. , Demangel C. , Munier‐Jolain K. , Miossec S. , Geahel I. 
Published: December 2004

AbstractTo investigate the influence of medium amino acid resources on hybridoma cell proliferation and monoclonal antibody secretion, we first determined, in two different cell lines, the pattern of amino acid consumption. We then showed that complementation of each cell line by an amino acid solution prepared to cover its needs led in both cases to a marked increase in cell density and monoclonal antibody production. This observation suggests that the amino acid supply is one of the factors limiting cell growth and productivity in typical batch processes, and we demonstrated that a similar limitation occurs in a semicontinuous culture. Finally, we showed that the cell decline observed after a period of amino acid deficiency is not irreversible and that proliferation could be resumed if the required amino acids were added.
Cell‐cycle‐dependent protein accumulation by producer and nonproducer murine hybridoma cell lines: A population analysis
Authors: Kromenaker Sandra J. , Srienc Friedrich 
Published: December 2004

AbstractSingle‐cell rates of accumulation of cellular protein have been determined as a function of total protein content using flow cytometry and population balance equations for exponentially growing murine hybridoma cells in the individual G1, S1 and G2 + M cell cycle phases. A novel flow cytometric technique for the identification of hybridoma cells in mitosis was developed and implemented. The data were obtained from a producer cell line which synthesizes and secretes high levels of monoclonal antibodies, and from a nonproducer clone which does not synthesize and secrete substantial amounts of antibody. The results indicate that the kinetics of single‐cell protein accumulation in these two cell lines are considerably different. In particular, low protein content G1 phase producer cells were characterized by a rate of protein accumulation which was approximately five times higher than the mean rate observed for higher protein content producer cells cycle phase. In contrast, the rate of accumulation of protein increased continuously with totalprotein content for the G1 phase nonproducer cells. S phase hybridoma cells were characterized by a considerably lower rate of protein accumulation which did not vary much with protein content for either cell line. Finally, G2 + M phase producer cells demonstrated a negative rate of protein accumulation which indicates that the rates of protein synthesis. It was hypothesized that these differences in total protein accumulation are caused by differences in monoclonal antibody accumulation. The distribution of rates suggests the need for a segregated approach to the modeling of the kinetics of antibody production in hybridomas.
Molecular design of expression systems: Comparison of different control configurations using molecular mechanism models
Authors: Chen Wilfred , Bailey James E. , Lee Sun Bok 
Published: December 2004

AbstractMolecular‐level mathematical models have been used to evaluate the effectiveness of eight different configurations of repressor synthesis control on the regulation of cloned gene expression initiated from a promoter‐operator sequence. Both single and dual‐repressor situations were considered, employing genetically structured models for the lac and λPR promoter‐operators in example calculations. Simulation results suggest that the most effective mode of cloned gene expression control is a cross‐regulation configuration carried on a multicopy plasmid. This system was able to control cloned gene transcription in the uninduced state over a broad range of plasmid copy number and also provided the highest overall transcription rate in the induced state. The general strategies suggested by these simulations should be applicable for other repressor‐operator‐promoter systems in diverse hosts.
Kinetic study of hybridoma cell growth in continuous culture: II. Behavior of producers and comparison to nonproducers
Authors: Frame Kelly K. , Hu Wei‐Shou 
Published: December 2004

AbstractA hybridoma cell line, AFP‐27‐P, was cultivated in continuous culture under glucose‐limited conditions. The viable cell concentration, dead‐cell concentration, and cell volume all varied with the dilution rate. A model previously developed for a nonproducing clone of the same cell line, AFP‐27‐NP, was extended to describe the behavior of the cells. The relationship between the specific growth rate and glucose concentration is described by a function similar to the Monod model. A threshold glucose concentration and a minimum specific growth rate are incorporated; the model is meaningful only at glucose concentration and a minimum specific growth rate are incorporated; the model is meaningful only at glucose concentrations and specific growth rates above these levels. The relationship between the death rate and the glucose concentration is described by an inverted Monod‐type function. Furthermore, the yield coefficient based on glucose is constant in the lower range of specific growth rates and changes to a new constant value in the upper range of specific growth rates. No maintenance term for glucose consumption is used; in the plot of specific glucose consumption rate vs. specific growth rate, the line intercepts the specific growth rate at a value close to the minimum growth rate. The productivity of antibody as a function of the specific growth rate is described by a mixed type model with a noon‐growth‐associated term and a negative‐growth‐associated term. The values for the model parameters were determined from regression analysis of the steady state data.
Simple structured model for α‐amylase synthesis by Bacillus amyloliquefaciens
Authors: Ponzo John H. , Weigand William A. 
Published: December 2004

AbstractA predictive, simple, structured model describing the synthesis of α‐amylase by Bacillus amyloliquefaciens was formulated. Three key intracellular processes were identified (i.e, translation, and excretion) along with two key intracellular components (i.e., mRNA and the intracellular form of the α‐amylase enzyme). Nearly all the model parameters were estimated by means of performing independent experiments, primarily fed‐batch experiments. The model was shown to predict transient system behavior in batch and in fed‐batch operation with some limitation and minor model parameter revisions. Since a principal objective was to demonstrate that independent experimental parameter determination can be used to construct the predictive model, further fine‐tuning of the parameters may be necessary before application for optimization and control purposes.
Assessment of virus production and chloramphenicol acetyl transferase expression by insect cells in serum‐free and serum‐supplemented media using a temperature‐sensitive baculovirus
Authors: King G. , Kuzio J. , Daugulis A. , Faulkner P. , Allen B. , Wu J. , Goosen M. 
Published: December 2004

AbstractSpodoptera frugiperda insect cells were grown in Sf‐900 serum‐free medium and two kinds of serum‐supplemented media (IPL −41 and Grace's). The specific growth rates of uninfected cells were found to be 0.024, 0.35, and 0.034 h−1 respectively, at 33°C. The IPL −41 medium supported to highest maximum cell density (10.6 × 106 cells/mL) compared to 3.5 × 106 and 8.7 × 106 cells/mL with the Grace's and serum‐free media, respectively. In temperature shifdown experiments with a temperature‐sensitive baculo‐virus (acts10YM1CAT), virus titer and chloramphenicol acetyl transferase (CAT) expression were highest in the IPL −41 (5.1 × 107 PFU/mL and 20000 U/mL). Use of Grace's medium gave higher virus titers than the serum‐free medium (4.4 × 106 vs 4.1 × 105 PFU/mL) as well as higher CAT titers (7050 vs 1980 U/mL). Interestingly, in the three media used, the highest virus and CAT titers were obtained at MOI (multiplicity of infection) of 0.02 At MOI of 2.0 virtually no increase in virus of CAT titer was observed. This result is contrary to those obtained at constant‐temperature (27°C) infection and cell culture, in which higher virus titers and recombinant protein expression and obtained at higher MOI.
Effects of Glutamate, Glucose, Phosphate, and Alkali metal ions on cephamycin C production by Nocardia lactamdurans in defined media
Authors: Kirpekar A. C. , Kirwan D. J. , Stieber R. W. 
Published: December 2004

AbstractA High cephamycin C producing strain of Nocardia lactam‐durans was used to study cell growth and antibiotics production in defined media. Batch fermentations in shake flasks and stirred tanks showed that antibiotic production occurred during cell growth and the production rate rapidly decline as the growth slowed. Glutamate served as a primary substrate during this phase. Later, ammonia was utilized along with a remainder of the glucose. Rapid antibiotic production occurred in this phase. Increased glutamate promoted higher growth, a rise in ammonium ion concentration, and a marked reduction in antibiotic titers. An increase of the glucose concentration along with the glutamate concentration balanced to the medium; no ammonium ion rise occurred and a peak specific antibiotic titer comparable to the control medium was obtained. In a phosphate‐limited medium, cell growth equivalent to the control medium and increased antibiotic titers were obtained. In these experiments, adjustment of Na+ and K+ ion concentration equal to that in the control medium was found to be important. Based on carbon and nitrogen balances, the activity of the key nitrogen metabolism enzymes, and the published literature, a two‐stage model of regulation is suggested.
Electrically promoted protein production by mammalian cells cultured on the electrode surface
Authors: Kojima Junichiro , Shinohara Hiroaki , Ikariyama Yohihito , Aizawa Masuo , Nagaike Kazuhiro , Morioka Satoshi 
Published: December 2004

AbstractProtein production of mammalian cells has been promoted by applying a small constant potential to the surface of an electrode on which cells are cultured. Human carcinoma line of MKN45 cells were cultured on the surface of a platinum‐coated plastic plate electrode. Low d.c. voltage of constant potential was applied to the electrode during 4‐day culture to modulate the production of carcinoembryonic antigen (CEA). The amounts of both secreted and membrane‐bound CEA were dependent on the applied potential during culture. Secreted CEA was more than twice in amount in the potential range from 0.2 V to 0.6 V vs. Ag/Agcl as compared with that of normal culture. In the potential range, CEA was also increased in membrane‐bound form. The potential‐controlled cell culture may have an enhanced effect on protein production.
A study on enzymatic reaction using a liquid emulsion membrane technique
Authors: Ha Heung Yong , Hong Seong‐Ahn 
Published: December 2004

AbstractAn enzymatic reaction using a liquid emulsion membrane technique was studied to investigate the effects of some experimental variables on the stability of liquid membrane, enzyme deactivation, and transport of substrates and products. The hydrolysis of L‐phenylalanine methyl ester by α‐chymotrypsin was selected as a model reaction system. First, a transport mechanism for the substrates and products across the membrane was qualitatively identified. Second, it was found that the pH of the internal phase was one of the most important variables to determine the enzyme activity in a liquid membrane. Third, the effect of membrane phase which consists of surfactant, carrier, and organic solvent on the emulsion stability was investigated. It was found that the properties of the organic solvents greatly affect the emulsion stability. For an optimum condition, it was possible to reuse the emulsion which consists of membrane phase and internal phase without further separation. It was finally concluded that the enzyme in a liquid membrane retained 60% of its native activity in spite of vigorous mixing during the emulsification step.
A two‐step enzymatic synthesis of dipeptides
Authors: Schwarz A. , Wandrey C. , Steinke D. , Kula M. R. 
Published: December 2004

AbstractA simple system is introduced to produce dipeptides continuously by enzyme catalyzed condensation of amino acid esters and amino acid amides. Synthesis of N‐terminal free dipeptide‐amides is achieved by means of carboxypeptidase Y. The peptide‐amide is deamidated utilizing a newly isolated peptide‐amide is deamidated utilizing a newly isolated peptide‐amidase. Separation of substrates and products is accomplished by anion‐exchange chromatography. Modeling of the reactions shows that the two reactions have to be carried out in a cascade of two reactors in order to prevent hydrolysis of the peptide by the carboxypeptidase. Continuous production of Kyotorphin (H‐TyrArg‐OH) with a space–time yield of 257 g/L · d shows the feasibility of this concept.
Stabilization of substilisin E in organic solvents by site‐directed mutagenesis
Authors: Martinez Pascal , Van Dam Mariana E. , Robinson Amy C. , Chen Keqin , Arnold Frances H. 
Published: December 2004

AbstractSubtilisin E was rationally engineered to improve its stability in polar organic solvents such as dimethylformamide (DMF). A charged surface residue, Asp248, was substituted by three amino acids of increasing hydrophobicity, Asn, Ala, and Leu; all three variants were stabilized with respect to wild type in 80% DMF. This stabilization was only observed in the presence of high concentrations of the organic solvent: no stability enhancements were observed in 40% DMF. In contrast, the mutation Asn218 → Ser alters internal hydrogen bonding interactions and stabilizes subtilisin E in both 40% and 80% DMF. This study provides additional evidence that substitution of surface‐charged residues is a generally useful mechanism for stabilizing enzymes in organic media and that the stabilizing effects of such substitutions are unique to highly altered solvent environments. The effects of the single amino acid substitutions on free energies of stabilization are additive in the Asp248 → Asn + Asn218 → Ser combination variant, yielding an enzyme that is 3.4 times more stable than wild type in 80% DMF.