bluuebunny/crossref_metadata_2025 huggingface dataset:
Continuous production of kyotorphin
Authors: Flörsheimer Andreas , Kula Maria‐Regina , Schütz Hans‐Jürgen , Wandrey Christian
Published: December 2004

The continuous α‐chymotrypsin‐catalyzed peptide synthesis of kyotorphin, tyrosyl‐arginine, via the Nα formyltyrosyl‐arginine propyl ester is described. For continuous process development, two reaction systems were studied: immobilized α‐chymotrypsin covalently bound to Eupergit C packed in a column, and soluble α‐chymotrypsin utilizing an enzyme membrane reactor. Selectivities and kinetic parameters are discussed. The use of soluble enzyme in an enzyme membrane reactor proved superior to the covalently immobilized enzyme. A significant loss of enzyme activity and a certain decrease of selectivity was observed during immobilization. It was shown that the addition of organic solvent, in this case n‐propanol, causes a severe diminuation of the enzyme activity.
Solubilization of soybean mitochondria in AOT/isooctane water‐in‐oil microemulsions
Authors: Hochkoeppler Alejandro , Luisi Pier Luigi
Published: December 2004

The water‐in‐oil microemulsion system bis(2 ethyl‐hexyl‐sodium‐succinate (AOT)/isooctane/water is able to solubilize soybean nodules mitrochrondria. Transparent and thermodynamically stable hydrocarbon solutions are obtained, which can be assayed for mitochondrial activity just as aqueous solutions. Malate dehydrogenase (MDH) activity was measured in vivo and gave in reverse micelles very similar results as in water. However the kinetic behavior of this reaction in AOT/isooctane reverse micelles shows some differences with respect to water. Mitochondria in reverse AOT micelles are able to retain about 70% of their initial MDH activity after three days. Mitochondria can be back‐transferred from reverse micelles to water and show respiratory activity almost identical to the native organelles. Electron microscopy studies show that the dimensions of mitochondria back‐transferred into water from AOT micelles are comparable to the dimensions of the native organelles.
Evidence for nitrogen catabolite repression during citric acid production by Aspergillus niger under phosphate‐limited growth conditions
Authors: Dawson M. W. , Maddox I. S. , Brooks J. D.
Published: December 2004

Experiments performed in batch fermentation under phosphate‐limited growth conditions showed that the citric acid yield was inversely related to the excess nitrogen concentration in the medium. Results from chemostat culture confirmed a negative relationship between the citric acid yield and both the specific growth rate and the nitrogen consumption rate. This is evidence for nitrogen catabolite repression. A fed‐batch fermentation performed under dual phosphate/nitrogen limitation produced results very similar to those from a culture limited by nitrogen alone. There is no advantage in maintaining an excess of phosphate during citric acid production and the process will therefore be more economic when operated under dual limitation conditions.
Evaluation of thermotolerant yeasts in controlled simultaneous saccharifications and fermentations of cellulose to ethanol
Authors: Spindler Diane D. , Wyman Charles E. , Grohmann Karel
Published: December 2004

Simultaneous saccharification and fermentation (SSF) experiments were performed at selected temperatures (37, 41, and 43°C) to obtain comprehensive material balance and performance data for several promising strains of thermotolerant yeast. Parameters measured were ethanol concentration, yeast cell density, and residual sugar and cellulose concentrations. The three yeasts Saccharomyces uvarum, Candida brassicae, and C. lusitaniae and two mixed cultures of Brettanomyces clausenii with S. cerevisiae (mixed culture I) and C. Iusitaniae with S. uvarum (mixed culture II) exhibited rapid rates of fermentation, high ethanol yields, strong viability, or high cellobiase activity. Overall, mixed culture II at 41°C performed better than either component yeast by themselves because it combined a cellobiose fermenting capability with the high ethanol tolerance and rapid glucose fermentation of conventional industrial yeasts. Thus, the mixed cultures provide good initial rates by preventing buildup of cellobiose (a strong inhibitor of enzyme activity) while attaining high ultimate yields of ethanol for high cellulase concentrations. However, C. brassicae and S. uvarum gave similar results to mixed culture II at 37°C.
Desorption of Trichoderma reesei cellulase from cellulose by a range of desorbents
Authors: Otter D. E. , Munro P. A. , Scott G. K. , Geddes R.
Published: December 2004

The desorption of Trichoderma reesei cellulase from Avicel by a wide range of desorbents was measured. Emphasis was placed on desorption at alkaline pH. A maximum desorption of 65–68% Avicelase activity was achieved by contact with NaOH, pH 10.0, at 40°C for 5 min in the presence of 0.005% Triton X‐100 or Tween 80. The design of a suitable desorption process using these conditions is discussed. Glycerol was also effective as a desorbent either alone or in combination with alkali and detergent. However, relatively high concentrations of glycerol were needed and the maximum desorption achieved, 68%, was not significantly greater than that with only alkali and detergent.
Purification of wheat germ agglutinin using affinity flocculation with chitosan and a subsequent centrifugation or flotation step
Authors: Senstad C. , Mattiasson B.
Published: December 2004

Affinity purification is by tradition almost equivalent to affinity chromatography. In this report, a purification process is described in which the affinity interaction is performed in free solution. A precipitation step follows, thereby separating the affinity bound material from free. Chitosan is used as a natural polyligand rich in N‐acetyl‐D‐glucosamine and is, because of its specificity, used in affinity precipitation of wheat germ agglutinin (WGA). The polyligand is soluble at pH below 6.5 and precipitates at higher values, thus coprecipitating associated WGA. A process was developed where the overall yield of WGA was 70% and the final product gave a single band on SDS‐PAGE. The process was also run on a larger scale, where even affinity flotation was included in the study as an alternative way to harvest the precipitate. Scaling up the affinity precipitation was a very easy task.
Effects of temperature on the hydrolysis of lactose by immobilized β‐galactosidase in a capillary bed reactor
Authors: Peterson R. S. , Hill C. G. , Amundson C. H.
Published: December 2004

The effects of temperature on the hydrolysis of lactose by immobilized beta‐galactosidase were studied in a continuous flow capillary bed reactor. Temperature affects the rates of enzymatic reactions in two ways. Higher temperatures increase the rate of the hydrolysis reaction, but also increase the rate of thermal deactivation of the enzyme. The effect of temperature on the kinetic parameters was studied by performing lactose hydrolysis experiments at 15, 20, 25, 30, and 40°C. The kinetic parameters were observed to follow an Arrhenius‐type temperature dependence. Galactose mutarotation has a significant impact on the overall rate of lactose hydrolysis. The temperature dependence of the mutarotation of galactose was effectively modelled by first‐order reversible kinetics. The thermal deactivation characteristics of the immobilized enzyme reactor were investigated by performing lactose hydrolysis experiments at 52, 56, 60, and 64°C. The thermal deactivation was modelled effectively as a first order decay process. Based on the estimated thermal deactivation rate constants, at an operating temperature of 40°C, 10% of the enzyme activity would be lost in one year.
Protein partitioning in two‐phase aqueous polymer systems
Authors: Baskir Jesse N. , Hatton T. Alan , Suter Ulrich W.
Published: December 2004

Theories of protein partitioning in two‐phase polymer systems which account for the effects of different aspects of system composition—such as the choice of materials, protein size, polymer molecular weight, polymer concentration, salt concentration, and affinity ligands—are reviewed. Although the present models provide some information about specific aspects of partitioning, a comprehensive and fundamental theory which can be used to predict protein partitioning behavior has not yet been developed. Some recommendations for future work are given.
Effects of pH and acetic acid on homoacetic fermentation of lactate by Clostridium formicoaceticum
Authors: Tang I‐Ching , Okos Martin R. , Yang Shang‐Tian
Published: December 2004

Clostridium formicoaceticum homofermentatively converts lactate to acetate at 37°C and pH 6.6–9.6. However, this fermentation is strongly inhibited by acetic acid at acidic pH. The specific growth rate of this organism decreased from a maximum at pH 7.6 to zero at pH 6.6. This inhibition effect was found to be attributed to both H+ and undissociated acetic acid. At pH values below 7.6, the H+ inhibited the fermentation following non‐competitive inhibition kinetics. The acetic acid inhibition was found to be stronger at a lower medium pH. At pH 6.45–6.8, cell growth was found to be primarily limited by a maximum undissociated acetic acid concentration of 0.358 g/L (6mM). This indicates that the undissociated acid, not the dissociated acid, is the major acid inhibitor. At pH 7.6 or higher, this organism could tolerate acetate concentrations of higher than 0.8M, but salt (Na+) became a strong inhibitor at concentrations of higher than 0.4M. Acetic acid inhibition also can be represented by noncompetitive inhibition kinetics. A mathematical model for this homoacetic fermentation was also developed. This model can be used to simulate batch fermentation at any pH between 6.9 and 7.6.
Diosgenin formation by freely suspended and entrapped plant cell cultures of Dioscorea deltoidea
Authors: Robertson G. H. , Doyle L. R. , Sheng P. , Pavlath A. E. , Goodman N.
Published: December 2004

Altered phosphorus concentration, oxygen supply rate, and programmed addition of sucrose and phosphorus were applied to Dioscorea deltoidea plant cells in airlift suspension to increase the formation of the secondary metabolite diosgenin. A low oxygen supply rate (kla′ of 3.9 h−1) completely inhibited formation of diosgenin. A high oxygen supply rate (kla′ of 17.1 h−1) led to the greatest formation of diosgenin in 30 g/L sucrose when the sucrose‐to‐phosphorus mole ratio was 42.5:1. Programmed addition of nutrients over a 15‐day period reduced growth of cell mass relative to diosgenin mass. Intentional aggregation by entrapment of virtually all cells in reticulated polyurethane foam led to reduced cell‐mass yield, diosgenin yield and concentration relative to suspended cells at the same conditions. Entrapment of a small fraction of the cells led to a delayed development of a suspension culture and to formation of significantly higher concentrations of diosgenin. Nearly all of this increase was attributable to the suspended cells. This result suggested sequestering of nonproductive cells by the matrix or sequestering of important nutrients by the matrix‐bound cells. Entrapped cells attained densities of 40 g/L in the matrix.
Localization of cloned invertase in Saccharomyces cerevisiae directed by the SUC2 and MFα1 signal sequences
Authors: Marten Mark R. , Seo Jin‐Ho
Published: December 2004

Protein localization in Saccharomyces cerevisiae was studied with two plasmid systems used as a model: one containing the SUC2 structural gene fused with the MFα1 (α‐factor) promoter and signal‐sequence, the other containing the entire SUC2 gene. Special emphasis was placed on the effect of promoter/signal‐sequence (SUC2 vs. MFα1) on the efficiency of invertase transport. The MFα 1 and SUC2 signal sequences were capable of transporting, respectively, 83% and 77% of cloned invertase out of the cytoplasm. However, the SUC2 promoter was easier to control since a six‐fold enhancement of the transported invertase activity associated with derepression was achieved in response to a glucose concentration change from 10 to 2 g/L Cloning on a multicopy plasmid resulted in a four‐fold increase in total specific invertase activity over the wild type yeast strain (which harbors a single copy of the SUC2 gene on the chromosome), whereas the chromosomal site was more efficient for invertase localization yielding over 90% of the invertase transported out of the cytoplasm. Transient experiments done with the SUC2 signal‐sequence‐containing plasmid showed that the specific invertase activity in the periplasmic space reached a maximum three hours after derepression, then decreased very slowly with an accompanying gradual increase in invertase activity in the growth medium.
The use of reverse micelles for the simultaneous extraction of oil and proteins from vegetable meal
Authors: Leser Martin E. , Luisi Pier Luigi , Paimieri Sandro
Published: December 2004

A method for the simultaneous extraction of oil and proteins from vegetable meals is presented. The method uses hydrocarbon reverse micelles, so that the oil is extracted directly into the hydrocarbon phase and the proteins are solubilized in the water pools of the reverse micelles. The surfactant used is bis (2‐ethylhexyl) sodium sulfosuccinate (AOT) in isooctane at variable w0 values (w0 measures the amount of water in the system, where w0 = [H2O]/[AOT]). A comparison with the usual extraction methods is offered. It is shown that with the micelle system the extraction of oil is as large as with the usual methods, and it is independent of w0. However the amount and type of proteins extracted depends strongly on w0. At w0 values below 6, no protein and only low molecular weight compounds (i.e. chlorogenic acid) are extracted, at larger water content (i.e. by increasing the dimension of the micelle water pool), also proteins are solubilized in a significant amount and with a molecular weight which increases by increasing W0. The protein solubilized in the microemulsion system can be recovered into an aqueous phase with a back‐transfer step.
Syntheses and applications of water‐soluble reactive polymers for purification and immobilization of biomolecules
Authors: Nguyen A. L. , Luong J. H. T.
Published: December 2004

Reactive polymers have been prepared by copolymeriz‐ing N‐isopropyl acrylamide (NIPAM) with N‐acryloxy‐succinimide (NASI) or glycidyl methacrylate (GMA). The amino groups of ligands could react with the residues of NASI or GMA and the polymers could be precipitated by temperature and/or salinity variation, since they contained the NIPAM residues. As a model, p‐aminobenza‐midine, a trypsin inhibitor, was attached to the polymers to form water‐soluble macroligands, capable of selectively binding trypsin from a trypsin–chymotrypsin solution. After precipitation of the macroligand–trypsin complex, followed by dissociation, approximately 82% trypsin was isolated. The NIPAM‐GMA copolymer was also reacted with immunogammaglobulin (IgG) and alkaline phosphatase (AP). It was demonstrated that the IgG bearing polymer was able to bind protein A and the whole complex was precipitable. The reactive polymer was also used for direct immobilization of AP which was active in repeated reactions.
Production of ethanol by Clostridium thermosaccharolyticum: I. Effect of cell recycle and environmental parameters
Authors: Mistry Firoz R. , Cooney Charles L.
Published: December 2004

The direct microbial conversion (DMC) process for the production of ethanol from lignocellulosic biomass is limited by low volumetric ethanol production rates due to the low cell densities of Clostridium thermosaccharolyticum which is a key organism for ethanol production in this process. Hence, this study focuses on the use of a continuous‐ culture cell recycle system to improve the volumetric ethanol productivity and yield of the fermentation of xylose by C. thermosaccharolyticum. Early experiments with the continuous‐culture cell recycle system showed a two‐fold improvement in volumetric ethanol productivity. However, the ethanol yield at the higher dilution rates suffered because of the large amount of lactate produced. The manipulation of two environmental parameters—iron concentration in the nutrient medium and the N2 purge rate of the fermentor headspace—allowed a dramatic reduction in the lactate production and a simultaneous improvement in the ethanol titer and yield. Under the improved conditions of increased iron concentration (12.5 mg/L FeSO4 · 7H2O) and decreased N2 purge rate (0.1 L/min), a continuous culture of C. thermosaccharolyticum operating at a dilution rate of 0.24 h−1 and 50% cell recycle produced 8.6 g/L ethanol and less than 1 g/L each of acetate and lactate. The volumetric ethanol productivity was 2.2 g/L/h, which is 8 times larger than obtained for a continuous culture operated with no cell recycle and the same specific growth rate.
Immobilized enzyme cellulose hollow fibers: I. Immobilization of heparinase
Authors: Comfort Ann R. , Albert Elizabeth C. , Langer Robert
Published: December 2004

The immobilization of heparinase to tresyl‐chloride‐activated cellulose hollow fibers for the removal of heparin from the bloodstream was examined. Whole blood can be circulated through cellulose hollow fibers without hemolysis and the tresyl chloride chemistry provides a strong linkage which limits the release of the enzyme from the support. The tresylation and immobilization methods were modified and optimized to improve the heparinase activity retained by cellulose. Pretreatment of the hollow fibers with 0.05/V sodium hydroxide increased the degree of tresylation and the immobilization yield by a factor of five. The use of triethylamine as the organic base in the tresyl chloride activation resulted in threefold greater activity retention by the support than when pyridine was used. Together, sodium hydroxide pretreatment and triethylamine enhanced the activity retained by cellulose to 26.2 ± 7.0% of that bound to the support. The activity retention was also a function of the technique used for immobilization. The best results were achieved when the enzyme was applied to the activated fibers once every 12 to 24 h for a total of four times. The active enzyme loading on the fibers was 0.3 mg heparin degraded/h cm2 when 4.5 μg protein/cm2 was bound to the fibers.
Immobilized enzyme cellulose hollow fibers: II. Kinetic analysis
Authors: Comfort Ann R. , Albert Elizabeth , Langer Robert
Published: December 2004

Immobilized enzyme hollow fibers may be useful in the purification or treatment of whole blood under clinical conditions. In this study, catalytically pure heparinase was immobilized to cellulose to analyze the feasibility for the removal of heparin's anticoagulant activity from whole blood. The kinetics of catalytically pure heparinase immobilized to regenerated cellulose hollow fibers were quantified with respect to mass transfer coefficient and enzyme loading. The kinetic analysis showed that increases in the mass transfer coefficient of heparin in the fiber lumen decreased the apparent Michaelis constant while increases in enzyme activity immobilized to the fiber lumen increased the apparent Michaelis constant. The apparent Michaelis constant was an order of magnitude greater than the intrinsic Km value for the system. The intrinsic Km value for heparinase–cellulose is 0.4 ± 0.3 mg/mL (N = 6) and it is the same order of magnitude as the Km value for soluble heparinase.
Separation of L‐valine from fermentation broths using a supported liquid membrane
Authors: Deblay P. , Minier M. , Renon H.
Published: December 2004

A carrier‐mediated counter transport process is proposed to separate and to purify an amino acid produced by microbial fermentation. The case of L‐valine permeation through a liquid membrane, constituted by a solution of Aliquat 336 in decanol and supported by a hydrophobic microporous membrane, is reported. A mathematical model was developed to estimate distribution coefficients and permeabilities and to predict the influence of hydrodynamic and pH conditions on supported liquid membrane (SLM) performances. Optimum conditions for the transport and the concentration of valine were achieved with synthetic aqueous valine solutions. Series of experiments on fermentation broths, where molasses and biomass contents were varied, permitted pointing out the role of the broth composition on the kinetics and yields of separation. The selectivity of transport of valine by an Aliquat 336/decanol liquid membrane was about 10 toward molasses dyes, 100 toward glucose, and beyond 1000 toward sucrose. This allowed us to achieve the recovery and one step of purification of the product in a single operation. The stability of the Aliquat 336/decanol liquid membrane was sufficient to ensure a selective transport of valine during a continuous run lasting 18 days.
Chemometric optimization of an asymmetric reduction catalyzed by baker's yeast
Authors: Boccù E. , Ebert C. , Gardossi L. , Gianferrara T. , Linda P.
Published: December 2004

The stereoselective reduction of ethyl acetoacetate to (+)‐(S)‐ethyl 3‐hydroxybutyrate catalyzed by Saccharomyces cerevisiae was optimized by means of chemometric methods. The quantitative effects of temperature, time of incubation, and concentrations of yeast and substrate on the optical purity and on the percent of reduced substrate were investigated using a factorial design at two levels. This approach gave information about the chemical behavior of the catalyst. The variability of the two responses was expressed by means of their corresponding response surfaces. Use of desirability functions allowed the overall optimization of the process, also taking into account the importance of economic factors. The investigation showed that it is possible to reduce the substrate completely obtaining (+)‐(S)‐ethyl 3‐hydroxybutyrate with percent of enantiomeric excess>98% and, at the same time, to operate in more convenient experimental conditions than those previously reported.
Electrochemical regeneration of NAD in a plug‐flow reactor
Authors: Fassouane Aziz , Laval Jean‐Marc , Moiroux Jacques , Bourdillon Christian
Published: December 2004

Electrochemical regeneration of NAD was performed at a laboratory preparative scale to illustrate both the efficiency and intrinsic simplicity of the electrochemical method. A powerful plug‐flow reactor was realized with a flow through graphite felt electrode, the ratio of the effective area of electrode/volume of reactor increased to 380 cm2/cm3. This graphite‐felt electrode was able to oxidize NADH coenzyme at a very low overvoltage. On the example of the gluconic acid production catalyzed by glucose dehydrogenase, current as high as 0.1 A was obtained in experience where enzymatic activity was the main limitation. In confirmation of our previous work, the results show that the yield of NADH electrochemical oxidation is better than 99.95%.
Studies on the deactivation of immobilized urease
Authors: Vasudevan P. T. , Ruggiano L. , Weiland R. H.
Published: December 2004

The results of experiments in a fixed‐bed reactor and a CSTR containing urease immobilized on a nonporous support and conducted in the absence of diffusional limitations are reported. Kinetic parameters were established by separate batch experiments. The key observation was that the product ammonia attacked the free form of the enzyme and thereby illustrates the importance of mechanism in determining deactivation kinetics.
Production of recombinant human growth hormone in Escherichia coli: Expression of different precursors and physiological effects of glucose, acetate, and salts
Authors: Jensen E. Bech , Carlsen S.
Published: December 2004

The constitutive cytoplasmic expression in E. coli of human growth hormone (hGH) with different N‐terminal extensions (3 or 4 amino acids) has been studied. These hGH precursors were used for in vitro cleavage to obtain the mature, authentic hormone. Small changes in the amino acid extensions of the hGH precursors led to three‐fold differences in specific expression rates. The specific expression rate of the hGH precursors was inversely proportional to the ratios of the specific growth rates of plasmid containing and plasmid free cells (μ+/μ−) and also to the genetic stability. To ensure a satisfactory genetic stability in production fermentors, an hGH precursor with a moderate expression efficiency was chosen.

The medium composition and growth conditions were studied, resulting in the choice of a glucose fed batch fermentation process using a complex medium. In this process a yield of 2000 mg/L of met‐ala‐glu‐hGH (MAE‐hGH) was obtained. The fermentation process comprised a glucose‐limited growth phase followed by a second phase with increased glucose feed and exhaustion of phosphate from the medium. The second phase is characterized by an MAE‐hGH production, whereas further biomass formation is blocked. High concentrations of glucose led to reduced specific expression of MAE‐hGH—the specific and total yield in batch glucose fermentations is only about 30% of the yield in optimized fed batch fermentations. The physiological background for this was investigated. Chemostat experiments showed that the glucose concentration and the metabolic condition of the cells—i.e. with or without formation of acetate—was not critical per se in order to obtain a high specific yield of MAE‐hGH. Therefore it is unlikely that formation of MAE‐hGH is catabolite repressed by glucose. Furthermore it was shown that the specific production rate of MAE‐hGH was independent of the specific growth rate and it was further demonstrated that the decrease in expression efficiency in glucose batch fermentation was a result of an inhibitory effect of acetic acid. In batch fermentations this inhibitory effect was enhanced by a salt effect caused by increased consumption of acid and base used to control pH. The identity of the acid and the base used are not important in this context.

From studies of the expression of other proteins in E. coli. with constitutive as well as inducible promoters we conclude that glucose fed batch processes are often superior to batch processes in the production of heterologous proteins E. coli.
Oscillatory behavior of Saccharomyces cerevisiae in continuous culture: I. Effects of pH and nitrogen levels
Authors: Chen Ching‐I , McDonald Karen A. , Bisson Linda
Published: December 2004

The appearance of sustained oscillations in bioreactor variables (biomass and nutrient concentrations) in continuous cultures of Saccharomyces cerevisiae indicates the complex nature of microbial systems, the inadequacy of current growth kinetic models, and the difficulties which may arise in bioprocess control and optimization. In this study we investigate continuous bioreactor behavior over a range of operating conditions (dilution rate, feed glucose concentration, feed ammonium concentration, dissolved oxygen, and pH) to determine the process requirements which lead to oscillatory behavior. We present new results which indicate that high feed ammonium concentrations may eliminate oscillations and that under oscillatory conditions ammonium levels are generally low and oscillatory as well. The effects of pH are complex and oscillations were only observed at pH values 5.5 and 6.5; no oscillations were observed at a pH of 4.5. Under our nominal operating conditions (feed glucose concentration 10 g/L, dilution rate 0.145 h−1, feed ammonium concentration 0.0303M, dissolved oxygen level 50%, pH 5.5, and T = 30°C) we found two possible final bioreactor states depending on the transient used to reach the nominal operating conditions. One of the states was oscillatory and characteristic of oxidative metabolism and the other was nonoscillatory and fermentative.
Comparative study of production of dextransucrase and dextran by cells of Leuconostoc mesenteroides immobilized on Celite and in calcium alginate beads
Authors: El‐Sayed Abdel‐Halim M. M. , Mahmoud Wafaa M. , Coughlin Robert W.
Published: December 2004

In fed‐batch fermentation, cells of L. mesenteroides immobilized on three types of Celite were used to produce dextransucrase (DS) followed by production of dextran. A layer of calcium alginate on the porous Celite R630 particles improved their mechanical stability, increased the amount of soluble DS produced and decreased the cell leakage from the highly porous support. Enzyme production with the immobilized cell cultures was significantly affected by both pore and particle size. Immobilized cultures using Celite R648 (average particle radius of 200 μm and pore size of 0.14 μm) produced the highest total enzymatic activity, followed by Celite R633, alginate‐coated Celite R630, Celite R630, and then calcium alginate beads. Culture of free cells produced about 18% more total enzymatic activity than immobilized cells in calcium alginate beads, but about 64% less than immobilized cells on Celite R630. It is expected that larger amounts of enzymatic activity than measured are immobilized inside the alginate‐coated Celite R630 and calcium alginate beads due to the mass transfer limitation conferred by the dextran product formed therein. The dextran yield from conversion of sucrose to dextran and fructose with all such enzyme‐enriched, immobilized‐cell cultures was higher than that obtained from free‐cell culture under similar conditions.
Ethanol extraction by supported liquid membrane during fermentation
Authors: Christen P. , Minier M. , Renon H.
Published: December 2004

A supported liquid membrane system was developed for the extraction of ethanol during semicontinuous fermentation of Saccharomyces bayanus. it consisted of a porous Teflon sheet as support, soaked with isotridecanol. This assembly permitted combining biocompatibility, permeation efficiency, and stability. The removal of ethanol from the cultures led to decreased inhibition and, thus, to a gain in conversion of 452 g/L glucose versus 293 g/L glucose without extraction. At the same time, the ethanol volumetric productivity was enhanced 2.5 times, due to an improvement of yeast viability, while the substrate conversion yield was maintained above 95% of its theoretical value. Besides these improvements in fermentation performances, the process resulted in ethanol purification, since the separation was selective towards microbial cells and carbon substrate, and likely selective to mineral ions present in the fermentation broth. For pervaporation, a concentration of ethanol four times greater was obtained in the collected permeate.
Sorbitol production in charged membrane bioreactor with coenzyme regeneration system: II. Theoretical analysis of a continuous reaction with retained and regenerated NADPH
Authors: Ikemi Masahisa , Ishimatsu Yoshiaki , Kise Shoichi
Published: December 2004

A theoretical model was constructed in order to study charged membrane bioreactors (CMBRs). In this model, it was postulated that a native nicotinamide coenzyme NADP(H) can be partially retained by a charged membrane in continuous operation. A multienzyme system composed of NADPH‐dependent aldose reductase (AR) and glucose dehydrogenase (GDH) was used for the production of sorbitol and gluconic acid from glucose and for the conjugated enzymatic regeneration of NADP(H). Both enzymes were studied with respect to their reaction kinetics. AR was determined to obey the Theorell–Chance mechanism. GDH reaction was approximated by the initial velocity equation of the sequential Bi–Bi mechanism since the reverse reaction could be neglected. Significant inhibitions of both enzymes by sorbitol, gluconic acid, and glucose were observed, and the mode of inhibition was estimated to modify the velocity equations. The differential equation system for each component was derived and numerically analyzed according to the model. The theoretical model elucidated several features of the CMBR. (1) When compared at the same productivity, higher retainment was found to bring about a higher coenzyme turnover number, indicating that the feed coenzyme concentration can be reduced. (2) Under constant conversion, a contradictory relationship between turnover number and residence time arises if the feed concentration of a coenzyme varies. The theoretical model predicts that there is a practically optimal concentration for using NADP(H) efficiently. This concentration was consistent with that yielding the estimated minimum total cost. (3) In this system, excess‐GDH‐to‐AR activity was required because of differences in their kinetic constants. The amount of regeneration enzyme required can be reduced by the accumulation of excels NADPH due to coenzyme retainment. (4) Comparison with an ideal repeated batch reaction revealed that the continuously operated CMBR was vastly superior with respect to productivity as well as operation ability.
Chemostat‐cultivated Escherichia coli at high dilution rate: Multiple steady states and drift
Authors: Majewski R. A. , Domach M. M.
Published: December 2004

The representation of metabolic network reaction kinetics in a scaled, polynomial form can allow for the prediction of multiple steady states. The polynomial formalism is used to study chemostat‐cultured Escherichia coli which has been observed to exhibit two multiple steady states under ammonium ion‐limited growth conditions: a high cell density–low ammonium ion concentration steady state and a low cell density–high ammonium ion concentration steady state. Additionally, the low‐cell‐density steady state has been observed to drift to the high‐cell‐density steady state. Inspection of the steady‐state rate expressions for the ammonium ion transport/assimilation network (in polynomial form) suggests that at low ammonium ion concentrations, two steady states are possible. One corresponds to heavy use of the glutamine synthetase‐glutamate synthase (GLNS–GS) branch and the second to heavy use of the glutamate dehydrogenase (GDH) branch. Realization of the predicted intracellular steady states is also found to be dependent on the parameters of the transport process. Moreover, the two steady states differ in where their energy intensity lies. To explain the drift, GLNS, which is inducible under low ammonium ion concentrations, is suggested to be a “memory element.” A chemostat‐based model is developed to illustrate that perturbations in dilution rate can lead to drift between the two steady states provided that the disturbance in dilution rate is sufficiently large and/or long in duration.
Production of dextransucrase by Leuconostoc mesenteroides immobilized in calcium‐alginate beads: I. Batch and fed‐batch fermentations
Authors: El‐Sayed Abdel‐Halim M. M. , Mahmoud Wafaa M. , Coughlin Robert W.
Published: December 2004

In batch fermentation Leuconostoc mesenteroides immobilized in calcium alginate beads produced a total dextransucrase activity equal to about 93% of that by free, suspended bacterial cells under comparable conditions in a bubble column reactor. Continuous sucrose feeding (5 g/L h) to the immobilized‐cell culture in the airlift bioreactor increased production of enzymatic activity by about 107% compared with ordinary batch operation of this reactor. About 14% of the enzymatic activity produced by the immobilized cells appears as soluble activity in the cell‐free broth compared with about 40% in case of free cells. In an airlift bioreactor, both the soluble and the intact (sorbed and entrapped) enzymatic activity produced by the immobilized bacterial cells was about 34% greater under automatic pH control, compared to that produced in a bubble column reactor with only manual pH control. During formation of dextran by intact enzyme within cells and beads, declines are observed in apparent enzymatic activity.
A new biochemical engineering approach to the fractional precipitation of proteins
Authors: Richardson P. , Hoare M. , Dunnill P.
Published: December 2004

A biochemical engineering framework for optimizing the design and operation of fractional protein precipitation has been developed. The method utilizes a fractionation diagram to represent the purification of a product protein relative to total contaminating protein. The purification factor for a single or double‐cut fractional precipitation is obtained as the gradient of an appropriate operating tie‐line. A computer algorithm has been devised to maximize the tie‐line gradient for a given yield enabling a plot of optimum purification factor versus yield to be constructed. The recovery of the enzyme alcohol dehydrogenase from clarified bakers homogenate using saturated ammonium sulphate has been examined. Fractionation and purification versus yield diagrams were used to investigate the effects of such process parameters as pH, temperature, and initial total protein concentration on fractionation efficiency. The results are discussed in terms of the underlying solubility and mixing phenomena and the industrial application of fractional precipitation.
Growing Saccharomyces cerevisiae in calcium‐alginate beads induces cell alterations which accelerate glucose conversion to ethanol
Authors: Galazzo Jorge L , Bailey James E.
Published: December 2004

Nongrowing Saccharomyces cerevisiae cells previously grown in alginate exhibit ethanol production rates 1.5 times greater than cells previously grown in suspension. Analysis of glucose, ethanol, and glycerol formation data using quasi‐steady‐state pathway stoichiometry shows that alginate‐grown cells possess phosphofructokinase (PFK), ATPase, and polysaccharide synthesis maximum activities which are approximately two‐, two‐, and ninefold larger, respectively, than in suspension‐grown cells. The estimated change in PFK maximum velocity is consistent with in vitro assays of PFK activity in extracts of suspension‐ and alginate‐grown yeast. Estimation of ethanol production flux control coefficients using in vivo nuclear magnetic resonance (NMR) spectroscopy measurements of intracellular metabolite concentrations and a previously proposed detailed kinetic model of ethanol fermentation in yeast shows that glucose uptake dominates flux control in alginate‐grown cells in suspension while earlier research revealed that PFK and ATPase exert significant flux control in suspension‐grown cells. When placed in a calcium alginate matrix, alginate‐grown cells produced ethanol 1.8 times more rapidly and accumulated substantially more polyphosphate than suspension‐grown cells placed in alginate. Cells growing in alginate elicit responses at the genetic level which substantially alter pathway rates and flux control when these cells are used as either a suspended or an immobilized biocatalyst. These responses in gene expression to growth in alginate serve to reconfigure flux controls in alginate to a pattern which is similar to that obtained for suspended‐grown cells in suspension.
The use of a metabolically structured model in the study of growth, nitrification, and denitrification by Thiosphaera pantotropha
Authors: Geraats S. G. M. , Hooijmans C. M. , van Niel E. W. J. , Robertson L. A. , Heijnen J. J. , Luyben K. Ch. A. M. , Kuenen J. G.
Published: December 2004

Thiosphaera pantotropha is capable of aerobic heterotrophic nitrification and both aerobic and anaerobic denitrification. These phenomena have been studied in acetate‐limited aerobic and anaerobic continuous cultures supplied with ammonia and nitrate. The internal reaction rates were defined, based on biochemical knowledge. The observable external conversion rates are related through a linear equation on the basis of the specified internal reaction rates. The linear equation is a Pirt relation extended for microbial systems with multiple electron donors (acetate and ammonia) and electron acceptors (oxygen and nitrate). The coefficients in this equation were estimated from the continuous culture measurements, and are composed of parameters involved in ATP production and consumption by the microorganism. It is shown that with realistic values for these parameters, the metabolically structured model describes the aerobic as well as the anaerobic experiments.
Physiological, biochemical, and mathematical studies of micro‐aerobic continuous ethanol fermentation by Saccharomyces cerevisiae. II: Intracellular metabolite and enzyme assays at steady state chemostat cultures
Authors: Grosz Ron , Stephanopoulos Gregory
Published: December 2004

Intracellular metabolite concentration and enzyme activity measurements were made to explain the new metabolic and growth phenomena seen in the micro‐aerobic, continuous yeast cultures described in Part I. The results of these assays suggested mechanisms for the observed maximum in the specific ethanol productivity as a function of the oxygen feed rate, changing ATP yields, the effects of antifoam, and the sharp changes in the biomass concentration with small changes in the oxygenation. Measured were the intracellular concentrations of ATP, NADH, glucose 6‐phosphate, pyruvate, glycerol, and ethanol, and the activities of hexokinase and alcohol dehydrogenase. Rate‐limiting steps were identified by the accumulation of metabolites upstream and the depletion of metabolites downstream of the step.

A potential mechanism for the stimulation of fermentation with decreasing oxygenation was an activation of glucose transport by an accumulating intracellular ATP concentration. The inhibition of fermentation at yet lower oxygenation rates may have been caused by the continued accumulation of ATP to the point that the glycolytic kineses were inhibited. A mechanism for the changing ATP yields and intracellular ATP concentration proposed the existence of ATPases or ATP waste reactions stimulated by both oxygen and ATP. Antifoam had the effect of decreasing the resistance for glycerol transport out of the cell. The resulting stimulation of glycerol production and inhibition of ethanol production decreased the intracellular ATP content. Finally, intracellular ethanol was found not to accumulate to levels of higher than the extracellular concentration.
Immobilization can improve the stability of hybridoma antibody productivity in serum‐free media
Authors: Lee Gyun Min , Palsson Bernhard O.
Published: December 2004

Hybridoma cells (S3H5/γ2bA2) were cultivated in spinner flasks with 1% serum media and serum‐free media. Monoclonal antibody productivity was maintained in 1% serum media. However, cells in serum‐free media showed a decrease in antibody productivity, and it completely disappeared in IMDM‐based low protein medium. This loss of antibody productivity was not observed when the cells were immobilized in alginate beads. In fact, immobilization enhanced the specific MAb productivity.
A completely automated system for on‐line monitoring of the production of a growth factor secreted during fermentation of Escherichia coli
Authors: Lundström Håkan , Brobjer Michael , Österlöf Björn , Moks Tomas
Published: December 2004

The production of IGF‐1 (insulin‐like growth factor 1), expressed in Escherichia coli as a secreted fusion protein with affinity for the Fc region of IgG, was monitored automatically during fermentations. A sampling device was used to automatically inject filtered culture medium from the fermentor onto a small affinity column (IgG Sepharose® Fast Flow) connected to a chromatographic system. The area of the eluted peak was proportional to the concentration of the fusion protein. The relationship was linear over the range 25–630 μg/mL with relative standard deviation of around 1% at the higher concentrations. Samples could be monitored automatically every half hour during fermentation (48 h). The method of analysis is nondestructive, allowing further analysis of product quality. A complete evaluation of the monitoring system is described. With this system, fermentations based on the described expression system can be optimized on the basis of product concentration; this will lead to more effective fermentations and higher product yields. It should also be possible to monitor other secreted products with this system by using other affinity gels.
Investigation of binding site density: Effects on the interaction between cibacron blue–dextran conjugates and lysozyme
Authors: Mayes A. G. , Moore J. D. , Eisenthal R. , Hubble J.
Published: December 2004

Cibacron‐blue–dextran conjugates have been produced with a range of ligand loadings using a dextran preparation of average molecular weight of 2 × 106. The equilibrium binding capacity of these ligand conjugates for lysozyme was determined using a gel permeation procedure to separate bound from free protein. The results obtained give clear evidence for at least two types of binding showing a marked difference in affinity. For the higher‐affinity interaction the half‐saturation constant decreases with increasing ligand loading. The number of dye molecules participating in binding is proportional to loading up to 154 mol dye/mol dextran but is reduced at the highest loading used (315 mol dye/mol dextran). This may be due to steric interference or to dye stacking reducing the number of dye molecules available for binding.
An approach for the stable immobilization of proteins
Authors: Leckband Deborah , Langer Robert
Published: December 2004

An approach is presented for the stable covalent immobilization of proteins with a high retention of biological activity. First, chemical modification studies were used to establish enzyme structural and functional properties relevant to the covalent immobilization of an enzyme to agarose based supports. Heparinase was used as a model enzyme in this set of studies. Amine modifications result in 75–100% activity loss, but the effect is moderated by a reduction in the degree of derivatization. N‐hydroxysuccinimide, 1,1,1‐trifluoroethanesulfonic acid, and epoxide activated agarose were utilized to determine the effect of amine reactive supports on immobilized enzyme activity retention. Cysteine modifications resulted in 25–50% loss in activity, but free cysteines were inaccessible to either immobilized bromoacetyl or p‐chloromercuribenzoyl groups. Amine reactive coupling chemistries were therefore utilized for the covalent immobilization of heparinase. Second, to ensure maximal stability of the immobile protein–support linkage, the identification and subsequent elimination of the principal sources of protein detachment were systematically investigated. By using high‐performance liquid chromatography (HPLC), electrophoresis, and radiolabeling techniques, the relative contributions of four potential detachment mechanisms—support degradation, proteolytic degradation, desorption of noncovalently bound protein, and bond solvolysis—were quantified. The mechanisms of lysozyme, bovine serum albumin, and heparinase leakage from N‐hydroxysuccinimide or 1,1,1‐trifluoroethanesulfonic acid activated agarose were elucidated. By use of stringent postimmobilization support wash procedures, noncovalently bound protein loss. An effective postimmobilization washing procedure is presented for the removal of adsorbed protein and the complete elimination of immobilized protein loss.