bluuebunny/crossref_metadata_2025 huggingface dataset:
Recycle use of immobilized glucoamylase by tangential flow filtration unit
Authors: Uttapap Dudsadee , Koba Yojiro , Ishizaki Ayaaki
Published: December 2004

Various properties of glucoamylase immobilized onto corn stover supporting material and separation of immobilized enzyme by tangential flow filtration unit were studied. Optimum pH and temperature of immobilized enzyme were 3.5 and 60°C, respectively. Enzyme stability was studied in a packed‐bed column. The starch conversion rate was attained at 81% for 15 days; after that, the hydrolysis rate gradually decreased. Size of supporting material proved to be an important factor, with higher activity and good loading yield resulting from smaller supporting material. Glucoamylase immobilized onto supporting material less than 44 μm was used for hydrolysis of 10% soluble starch at pH 3.5 and 40°C for 3 h. Then immobilized glucoamylase was separated from the product by means of a tangential flow filtration unit using a 0.2‐μm pore size Nylon 66 membrane filter. This operation was continued until 180 ml filtrate was obtained from a 260‐mL starting volume. Then, the next batch was started by adding 180 mL starch substrate into the reactor. The batchwise experiments were repeated 20 times. The average filtration rate of each batch was determined and found to sharply decline during the first four batches. Thereafter, it gradually decreased from batch to batch. The cause of decreasing filtration rate appeared to be due to retrogradation of starch. The percentage of starch hydrolysis within 20 batches was in the range 89–96%. The filtration rate becomes higher if the hydrolyzation time is extended to 14 h. Resistance to filtration was also investigated. Almost all of the total resistance is related to insoluble materials, with the significant part of this from the resistance due to insoluble materials deposited on a surface of membrane and boundary layer resistance. Using a microscopic method, no microorganisms were found in the filtrate.
Drag reducing properties of microalgal exopolymers
Authors: Ramus J. , Kenney B. E. , Shaughnessy E. J.
Published: December 2004

Dilute aqueous solutions of polymers released by marine phytoplankton (microalgae) were shown to effectively reduce drag in capillary pipe flow. Tests were performed in a capillary turbulent flow viscometer which extruded small samples under high pressures. In all, 22 species were screened, and the products of one chlorophyte and four rhodophyte species proved especially effective. The viscoelastic polymers produced by these species delayed the transition from laminar to turbulent flow to significantly higher Re. In general, polymeric regime segments come off the maximum drag reduction asymptote at characteristic retro‐onset points, and come to lie approximately parallel to, but displaced upwards from the Prandtl–von Karman line. The delay to transition was shown to be dependent on additive polymer concentration, capillary diameter, and temperature. Ionic concentration, ionic composition, or pH had little effect on drag reducing properties.
Liquid residence time distributions in immobilized cell bioreactors
Authors: Swaine Donald E. , Daugulis Andrew J.
Published: December 2004

Previous work has demonstrated that high ethanol productivities can be achieved using yeast or bacterial cells adsorbed onto the surface of ion exchange resin in vertical packed bed bioreactors. The present work quantitatively characterizes the overall degree of backmixing in such reactors at two scales of operation: 2.0 and 8.0 L. Stimulus‐response experiments, using two solvents (2,3‐butanediol and 2‐ethoxyethanol) as tracers, were performed to measure the liquid phase residence time distribution (RTD) during continuous ethanol fermentations using the yeast Saccharomyces cerevisiae and the bacterium Zymomonas mobilis at the 2‐L scale, and with S. cerevisiae at the 8‐L scale. In order to separately determine the effects of liquid flow rate and gas evolution on the degree of mixing, stimulus–response experiments were also performed in the systems without microbial cells present. The evolution of CO2 was found to dramatically increase the extent of mixing; however, the tanks‐in‐series model for non‐ideal flow represented the systems adequately. The packed beds were equivalent to over 70 tanks‐in‐series during abiotic operation while during fermentations, with similar liquid flow rates, they ranged in equivalence from 35 to 15 tanks‐in‐series. This increased knowledge of the overall degree of mixing in packed bed, immobilized cell bioreactors will allow for more accurate kinetic modelling and efficient scale up of the process.
Fermentation data analysis and state estimation in the presence of incomplete mass balance
Authors: Liao James C.
Published: December 2004

A method is developed for identifying measurement errors and estimating fermentation states in the presence of unidentified reactant or product. Unlike conventional approaches using elemental balances, this method employs an empirically determined basis, which can tolerate unidentified reaction species. The essence of this approach is derived from the concept of reaction subspace and the technique of singular value decomposition. It is shown that the subspace determined via singular value decomposition of multiple experimental data provides an empirical basis for identifying measurement errors. The same approach is applied to fermentation state estimation. Via the formulation of the reaction subspace, the sensitivity of state estimates to measurement errors is quantified in terms of a dimensionless quantity, maximum error gain (MEG). It is shown that using the empirically determined subspace, one can circumvent the problem of unidentified reaction species, meanwhile reducing the sensitivity of the estimates.
Kinetics of whey–lactose acidogenesis
Authors: Kissalita W. S. , Lo K. V. , Pinder K. L.
Published: December 2004

The mixed‐culture anaerobic conversion of lactose to organic acids in a bench‐scale continuous‐flow stirredtank fermentor is considered. The major acidogenic end‐product distribution with respect to the dilution rate are presented. A Monod chemostat model is employed to describe a microbial growth, and the influence on pH of the estimated model parameters is discussed.
Plasmid maintenance and protein overproduction in selective recycle bioreactors
Authors: Ogden Kimberly L. , Davis Robert H.
Published: December 2004

A new plasmid construct has been used in conjunction with selective recycle to successfully maintain otherwise unstable plasmid‐bearing E. coli cells in a continuous bioreactor and to produce significant amounts of the plasmid‐encoded protein β‐lactamase. The plasmid is constructed so that pilin expression, which leads to bacterial flocculation, is under control of the tac operon. The plasmid‐bearing cells are induced to flocculate in the separator, whereas cell growth and product synthesis occur in the main fermentation vessel without the inhibiting effects of pilin production. Selective recycle allows for the maintenance of the plasmid‐bearing cells by separating flocculent, plasmid‐bearing cells from nonflocculent, segregant cells in an inclined settler, and recycling only the plasmid‐bearing cells to the reactor. As a result, product expression levels are maintained that are more than ten times the level achieved without selective recycle. All experimental data agree well with theoretical predictions.
Ion exchange and affinity chromatography in the scaleup of the purification of α‐galactosidase from soybean seeds
Authors: Porter Jill E. , Ladisch Michael R. , Herrmann Klaus M.
Published: December 2004

Soybeans (Glycine max) contain an α‐galactosidase that makes up a small fraction of the total protein of the seed. The properties of this enzyme are of interest because of its potential to convert the galactooligosaccharides, stachyose and raffinose, in soybean meal to sugars digestible in the human gastro intestinal tract and thereby increase potential uses of this vegetable protein source in human and animal foods. Study of this enzyme required the isolation of milligram quantities of electrophoretically pure protein from ground soybeans and therefore, scaleup of laboratory procedures by a factor of 300 times. Large scale acid precipitation, ammonium sulfate precipitation, and centrifugal recovery of the precipitated protein allowed α‐galactosidase to be isolated from 45.5 kg soybean meal containing 17.1 kg protein, to obtain an enzyme extract with a specific activity of 90 to 100. A novel combination of strong anion exchange and cation exchange chromatography followed by Concanavalin‐A affinity chromatography with a methyl α‐D mannoside gradient gave α‐galactosidase with an average specific activity of 56,000. Ion exchange chromatography preceding Concanavalin‐A affinity chromatography allowed elimination of a relatively costly melibiose affinity chromatography step (which followed the Concanavalin‐A column In the laboratory procedure) thereby making scaleup practical.
Enzymatic synthesis of a sucrose‐containing linear polyester in nearly anhydrous organic media
Authors: Patil Damodar R. , Rethwisch David G. , Dordick Jonathan S.
Published: December 2004

A variety of enzymes have been found to acylate sucrose in anhydrous pyridine. The enzymic reaction is highly selective; with trifluoroethylbutyrate as ester donor, enzyme‐catalyzed transesterification of sucrose yielded sucrose 1′‐butyrate and sucrose 6, 1′‐dibutyrate. No sucrose–tributyrates were formed. Using a similar technique, a long‐chain linear sucrose polyester has been prepared using Proleather, an alkaline protease from a Bacillus sp. This protease catalyzes the esterification of sucrose with bis(2, 2, 2‐trifluoroethyladipate) in a 1:1 ratio to yield a sucrose‐containing polyester with Mw = 2100 and Mn = 1600 for a polydispersity of 1. 31. Polymers with molecular weights in excess of 13, 000 have been prepared by this enzymic approach, indicating that molecules containing over 30 sucrose units have been produced. The polyester is extremely water soluble and soluble in polar organic solvents. As with the sucrose dibutyrate, the polyester has ester linkages at the C6 and C1′ positions on the sucrose. The polyester can be depolymerized using Proleather in aqueous buffer, pH7. After 9 days in aqueous buffer, Proleather catalyzed the breakdown of the polyester to an Mw of ca. 900. This sucrose‐containing polyester may have applications as a water‐absorbent, biodegradable plastic for use as diapers and hygienic products, water‐treatment chemicals, and components of drug delivery systems.
Retention of enzyme by electropolymerized film: A new approach in developing a hypoxanthine biosensor
Authors: Nguyen An‐Lac , Luong John H. T. , Yacynych Alexander M.
Published: December 2004

An amperometric biosensor for hypoxanthine was constructed by forming a layer of crosslinked xanthine oxidase on a platinum electrode, followed by electropolymerization of a submonolayer film of resorcinol and para‐diaminobenzene. The fabricated electrodes were evaluated for speed of response, sensitivity, and reusability. Optimal performance was obtained with enzyme‐based electrodes sparsely covered with film which was formed by electropolymerization in less than 6 min. The resulting electrodes exhibited linear response to hypoxanthine in the. range 5–300 μM with a response time of 2 min. Application of the biosensor in monitoring hypoxanthine content of fish extracts yielded results which agreed well with spectrophotometric assays using soluble xanthine oxidase. The biosensor was stable for 60 days when stored at 4°C in phosphate buffer and it could be used continuously for 6 h with over 50 assays.
A fiber‐bed bioreactor for anchorage‐dependent animal cell cultures: Part I. Bioreactor design and operations
Authors: Chiou Tzyy‐Wen , Murakami Sei , Wang Daniel I. C. , Wu Wen‐Tung
Published: December 2004

A concentric‐cylinder airlift reactor, in which the annulus is a packed bed of glass fibers, has been developed in order to facilitate the scaleup and enhance the volumetric productivity of anchorage‐dependent animal cell cultures. In this bio‐reactor, oxygen‐containing gas is sparged through the inner draft tube, causing bubble‐free medium to flow through the fiber bed in the outer cylinder and providing both oxygenation and convective nutrient transfer to the cells. Several other desirable features for reactor operation are also provided by this design. Cell cultivations in this bioreactor have been successfully carried out and provide data for the feasibility of the large‐scale cell cultivation.
A fiber‐bed bioreactor for anchorage‐dependent animal cell cultures: Part II. Scaleup potential
Authors: Murakami Sei , Chiou Tzyy‐Wen , Wang Daniel I. C.
Published: December 2004

A simple hydrodynamic model is introduced to describe the airlift fiber‐bed bioreactor, which can enhance the volumetric productivity of anchorage‐dependent animal cell cultures. By applying the model, liquid flow rates and volumetric mass transfer coefficients are predicted and are in agreement with experimental measurements. Consequently, the optimal reactor configuration giving the maximal oxygen supply is derived. Also, theoretical scaleup potential of this concentric internal loop reactor is considered for volumes ranging from 10 to 67,000 L with which cell densities of 5.1 × 107 and 1.2 × 107 cells/cm3, respectively, can be maintained.
Enzymatic production of cyclodextrins from unliquefied corn starch in an attrition bioreactor
Authors: Lee Yun‐Dong , Kim Hak‐Sung
Published: December 2004

A novel enzymatic process for the production of cyclodextrins from unliquefied starch was developed. Cyclodextrins were produced in an attrition bioreactor in which simultaneous hydrolysis of starch and synthesis of cyclodextrins by cyclodextrin glycosyltransferase (CGTase) occur. The CGTase was obtained from isolated Bacillus sp. BE101, and maximum activity of the enzyme was observed at pH 6.0 and a temperature of 45°C. The effect of milling media size and material on the performance of the attrition bioreactor was investigated, and operational parameters such as agitation speed, volume of milling media, ratio of enzyme to starch, and starch concentration were optimized. The production yield of cyclodextrins from unliquefied corn starch of 15% reached 35% at 24 h under optimized conditions. Energy consumption for the production of cyclodextrins in the attrition bioreactor system was estimated to be about 25% of that required for the liquefaction of starch in the conventional process.
Optimization of pro‐urokinase secretion from recombinant Saccharomyces cerevisiae
Authors: Turner Brian G. , Avgerinos George C. , Melnick Laurence M. , Moir Donald T.
Published: December 2004

Secretion of a nonglycosylated form of human pro‐urokinase, also known as single‐chain urinary plasminogen activator (scu‐PA), from Saccharomyces cerevisiae is described. A “supersecreting” yeast strain harboring multiple copies of integrated plasmids was grown batchwise and at constant respiratory quotient (RQ) in 20‐L fermenters. Because the promoters used to drive expression of the pro‐urokinase genes are not tightly regulated, secretion into the culture supernatant was growth associated. Although the final cell density achieved in the perturbed‐batch fermentation (45 g dry wt/L) was less than that observed in the RQ‐controlled culture (77 g dry wt/L), the scu‐PA titer in the perturbed‐batch fermentation (1863 IU/mL) was nearly twice that attained at constant RQ (1108 IU/mL). The effects on cell growth and scu‐PA titer of other process variables (pH, temperature, phosphate concentration, and medium composition) are also discussed.
Structured modeling of a microbial system: I. A theoretical study of lactic acid fermentation
Authors: Nielsen Jens , Nikolajsen Karin , Villadsen John
Published: December 2004

Most fermentation models presented in the literature are unstructured, i.e., the biomass composition is assumed constant during all operating conditions. These models are unable to simulate experiments carried out at widely different operating conditions. It is therefore interesting to examine simple structured models where knowledge of the cell physiology is taken into account in the modeling phase. In this article, a simple structured model is presented. The model is based on experimental work with the lactic acid bacteria Streptococcus cremoris, but due to the similarities in basic metabolism for many microorganisms it is applicable also for other fermentation system. The basic assumption in the model is that the biomass can be divided into two parts (compartments)–an active part and a mainly inactive structural part. The size of the active part has a pivotal role in the model.
Characterization of a pilot plant airlift tower loop bioreactor. I: Evaluation of the phase properties with model media
Authors: Fröhlich S. , Lotz M. , Korte T. , Lübbert A. , Schügerl K. , Seekamp M.
Published: December 2004

Investigations were carried out in a 9 m high, 4 m3 volume, pilot plant airlift tower loop bioreactor with a draft tube. The reactor was characterized by measuring residence time distributions of the gas phase using pseudostochastic tracer signals and a mass spectrometer and by evaluating the mixing in the liquid phase with single‐pulse tracer inputs. The local gas holdup and the bubble size (piercing length) were measured with two‐channel electrical conductivity probes. The mean residence times and the intensities of the axial mixing in the riser and downcomer and the circulation times of the phases as well as the fraction of the recirculated gas phase were evaluated. The gas holdup in the riser is nearly uniform along the reactor. In the downcomer, it diminishes from top to bottom. The liquid phase dispersion coefficients, DL, are smaller than those measured in the corresponding bubble columns. In the pilot plant with tap water the following relationship was found: DLr = cw; with c = 203.4; n = 0.5;DLr(cm2 s−1;) and WSG(cm s−1) where DLr is the longitudinal dispersion coefficient in the riser and WSG is the superficial gas velocity. The gas phase dispersion coefficients in the riser of the pilot plant, DGr, are also enlarged with increasing superficial gas velocity, WSG, however, no simple relationship exists. Parameter DGr is the highest in the presence of antifoam agents, intermediate in tap water, and the smallest in ethanol solution.
Dilution rate as a determinant of mycelial morphology in continuous culture
Authors: Wiebe Marilyn G. , Trinci Anthony P. J.
Published: December 2004

The morphology of mycelial fungi in liquid culture effects culture rheology and this in turn may affect product yield. It is therefore important to understand how environmental factors influence mycelial morphology and this paper describes the effect of dilution rate on two strains ofFusarium graminearum,the relatively sparsely branched parental strain (A3/5) and a relatively highly branched “colonial” variant (C106). At any given dilution rate, the concentration of mycelial fragments present at steady state of both strains remained approximately constant with time, suggesting that mycelial fragmentation occurred in a regular manner. However, for both strains fragment concentration decreased with increasing dilution rate. The strains had a similar morphology at a dilution rate of 0.07 h−1. The length of the hyphal growth unit of A3/5 increased with increase in dilution rate, while that of C106 decreased with increase in dilution rate. At all dilution rates, C106 produced up to ten times more macroconidia than A3/5.
Protection mechanisms of freely suspended animal cells (CRL 8018) from fluid‐mechanical injury. Viscometric and bioreactor studies using serum, pluronic F68 and polyethylene glycol
Authors: Michaels James D. , Petersen Jonathan F. , Mclntire Larry V. , Papoutsakis Eleftherios T.
Published: December 2004

We use bioreactor and viscometric studies to examine the mechanism by which three additives, fetal bovine serum (FBS), pluronic F68, and polyethylene glycol (PEG), protect the freely suspend CRL‐8018 cells from damage due to interactions with bubbles in agitated bioreactors. In bioreactor studies, the protective effect of an addictive could be due to either changes in the ability of the cell resist shear (biological mechanism) or to changes in the medium properties that effect the level or frequency of forces experienced by the cells (physical mechanism). Bioreactor studies show that protection by all three addictives occurs whether the cells are grown in the presence of the addictives (long exposure) or the addictives are added to medium after the cells were exposed to detrimental agitation intensity (short exposure). In the viscometric studies, exposure of cells to laminar shear in the absence of gas‐liquid interfaces assesses only the ability of the cells to resist a constant level of shear in a medium with or without an additive. Viscometric studies show that prolonged exposure to FBS makes the cells more shera tolerant, but that short (30–120 min) exposure to FBS does not affect their shear tolerance. We thus conclude that the protective effect of FBS in bioreactors id of both physical and biological nature. The biological contribution is metabolic in nature rather than fast acting. Viscometric studies show that either long or short exposure of the cells to either F68 or PEG does not make the cells more shear tolerant. WE therefore conclude that the protective effect of F68 and PEG does not make the cells more shear tolerant. We therefore conclude that the protective effect of F68 and PEG in bioreactors is physical in nature.
Kinetics, mechanism, and time course analysis of lipase‐catalyzed hydrolysis of high concentration olive oil in AOT–isooctane reversed micelles
Authors: Tsai Shau‐Wei , Chiang Chen‐Li
Published: December 2004

Candida rugosa lipase has been used to investigate the hydrolysis of high concentration olive oil in the AOT–isooctane reversed micellar system at Wo = 10, pH 7.1, and 37°C. Results from this work show the hydrolytic reaction obeys Michaelis–Menten kinetics up to the initial substrate concentration of 1.37M, with turnover number kcat and Michaelis constant KM of 67.1 μmol/min mg enzyme and 0.717M, respectively. A competitive inhibition by the main product, oleic acid, has been found with a dissociation constant KI for the complex EP* of 0.089M. The rate equation was further analyzed in the time course reaction and was found in agreement with the experimental results for lower substrate concentrations, up to 0.341M. Large deviation occurred at high substrate concentrations, which may be due to the effects of large consumption of water on kinetics, on the formation of glycerol, and on the deactivation of lipase in the hydrolysis reaction as well.
Process development with nitrogenase‐producing Escherichia coli C‐M74 (pUS1) CellS
Authors: Ahlmann N. , Schügerl K.
Published: December 2004

A process for the continuous fermentation of the genetically modified, nitrogenase‐producing Escherichia coli C‐M74 (pUS1)‐strain has been developed. This strain, which is able to fix molecular nitrogen, has the nifgenes of the bacterium Klebsiella pneumoniae. Cell growth and nitrogenase activity of the enzyme have been optimized both in batch and continuous fermentations. For the fermentations, trial runs were performed by cultivating the E. coli cells in 50‐ml culture bottles. The medium composition was varied in order to provide high biomass production and nitrogenase activity. For an effective fermentation control, an on‐line analysis was built up for the substrates ammonium and glucose. Other medium components such as ampicillin, citric acid, acetic acid, nitrogenase activity, and protein were measured by using different off‐line methods. Modern optical methods like in‐line microfluorometry for monitoring the culture fluorescence and laser flow cytometry for the estimation of DNA and protein content were also employed. Plasmid stability was also determined.
The Effect of sorbitol on acid phosphatase deactivation
Authors: Gianfreda Liliana , Toscano Giuseppe , Pirozzi Domenico , Greco Guido
Published: December 2004

Acid phosphatase thermal deactivation follows a complex path: an initial decay toward an equilibrium distribution of at least two intermediate structures, mutually at the equilibrium, followed by a final breakdown toward a completely inactive enzyme configuration. The results obtained in the presence of sorbitol have been compared to those produced in the course of purely thermal deactivation of the native enzyme. For any sobitol concentration, an equivalent temperature is calculated that results in exactly the same activity‐versus‐time profile. This suggests enzyme deactivation to be controlled by a single, unchanging step. Immobilized enzyme runs have been performed, as well, by entrapping acid phosphates within a polymeric network formed onto the upstream surface of an ultrafiltration membrane. The stabilizing effect of entrapment cumulates with that produced by sorbitol. In this case, however, an equivalent temperature cannot be determined, thus indicating that a different deactivation mechanism is followed.
Steroid bioconversion in a microemulsion system
Authors: Smolders A. J. J. , Pinheiro H. M. , Noronha P. , Cabral J. M. S.
Published: December 2004

The Δ1,2‐dehydrogenation of high concentrations of the steroid ‐methyl‐Reichstein's compound S‐21‐acetate (16MRSA) in a microemulsion system was studied using heat‐dried and thawed Arthrobacter simplex cells as biocatalyst. The microemulsion system consists of an organic phase [75−95% (v/v)] with steroid (1−60 g/Ltot), an aqueous phase [5−25% (v/v)] containing the cells (5−30 g/Ltot), and a neutral surfactant (5−20 g/L organic solvent). Benzene derivatives, which solubilize 16MRSA up to 94 g/L, and phospholipids were used as organic solvents and surfactants, respectively, and menadione was added as an external electron acceptor. Factors affecting the dehydrogenation rate in the microemulsion system were studied. The influences of the 16MRSA and the menadione concentration on the dehydrogenation rate were described by Michaelis‐Menten kinetics, apparent V′max and K′m values of 2.06 g/g dry weight h and 18.9 g/L for 16MRSA and 4.97 g/g dry weight h and 1.91 g/L for menadione being obtained. Optimal menadione concentration was dependent on the steroid concentration was dependent on the steroid concentration used. The reaction was strongly inhibited by high product concentrations. Much higher activities were obtained with the thawed cells than with the dried cells, conversions of 98% being reached within 14−16 h. for 16MRSA and cell dry weight concentrations of 40 and 10 g/L, respectively. Activity retention in a batch stirred tank reactor remained constant during the first 16−24 h of operation and then decreased, depending on the stirring rate; 22 to 65% of the initial reaction rate was obtained after 48 h at stirring rates of 650 and 2000 rpm, respectively.
Enzymatic preparation of organometallic compounds with second‐order Optical nonlinearities
Authors: Yamazaki Yoshimitsu , Hosono Kuniaki , Matsuda Hiro , Minami Nobutsugu , Asai Michihiko , Nakanishi Hachiro
Published: December 2004

Twelve species of optically active metallocene derivatives having a 4‐nitrophenyl group were prepared with horse liver alcohol dehydrogenase‐ or lipase‐catalyzed optical resolution as the key step. The second harmonic generation (SHG) efficiently of these products was measured by the power method using the fundamental light from the Nd:YAG laser. (−)‐1‐ (4‐Nitrophenylthio)ethylferrocene (9), (+)‐1‐(4‐nitrophenylthio)ethylruthenocene (16), (+)‐1‐(4‐nitrophenylthio)ethylosmocene (19), (+) ‐1‐(5‐nitro‐2‐pyridylthio)ethylruthenocene (21), and (+) ‐1‐[(4‐nitrophenylhydrazono)methyl] ‐2‐methylferrocene (12) showed SHG signals. The highest SHG efficiency was found with (+) ‐16, being 27 times more intense than the commonly used urea standard.
Protein extraction by reverse micelles: A study of the factors affecting the forward and backward transfer of α‐chymotrypsin and its activity
Authors: Marcozzi Giordana , Correa Nancy , Luisi Pier Luigi , Caselli Maurizio
Published: December 2004

α‐chymotrypsin is taken as a model protein to investigate three aspects of the protein extraction by reverse micelles: (1) the comparison between the two forward transfer techniques, i.e., the liquid‐liquid and the solid state‐liquid transfer; (2)the back‐transfer, i.e., the capability of the protein to be recovered from the micellar solution; and (3) the maintainance of the enzyme activity at the end of the extraction cycle. Concerning the forward transfer from the liquid phase, we study first the effect of salt initially present in the aqueous phase on the equilibrium concentration of the extracted species; further, we study the forward protein extraction from the solid state, and the effect of pH, salt, and protein concentration on the transfer efficiency. Concerning the back transfer, we find the somewhat surprising result, that the percentage of protein back‐extraction depends on the type and concentration of salt used for the forward transfer. Preliminary data concerning an alternative method for the back‐transfer using silica gel to liberate the protein from the micellar environment, are presented. Finally, it is found that the enzyme activity depends again on the type and concentration of salt used for the forward transfer.
Characteristics of batch culture of a recombinant bacillus brevis excreting a foreign esterase
Authors: Tulin Edgardo E. , Takagi Hiroaki , Ueda Shunsaku , Yamagata Hideo , Yamane Tsuneo , Udaka Shigezo
Published: December 2004

The productivity of extracellular enzyme was evaluated in batch culture using a protein hyperexcreting host, Bacillus brevis HPD315 harboring pHSC131, which carried a gene (est) encoding esterase activity from Bacillus stearother mophilus. Optimum temperature and pH for the bacterial growth and the production of extracellular esterase were found to be 35°C and pH 6.5, by using the standard medium (GPY) containing neomycin as a selective pressure, Under the cultivation condition employed, cell growth reached 5 g dry cell weight/L, while the extracellular esterase activity amounted to 4.5 U/mL. Most (79%‐92%) of the esterase produced was excreted into the medium. pHSC131 was stably retained in the host cell during cultivation in the presence of neomycin. However, in the absence of neomycin, the plasmid was completely lost from the host after 12‐h cultivation accompanied by decreases in both esterase activity and production of total extracellular protein. The copy number of the plasmid was estimated to be approximately 7 throughout the cultivation. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the excreted proteins showed the presence of a protein having an apparent molecular weight of 32,000, which equals to the value predicted from the DNA sequence of the est gene.
The effects of plasmid content, transcription efficiency, and translation efficiency on the productivity of a cloned gene protein in Escherichia coli
Authors: Kim Jeong‐Yoon , Ryu Dewey D. Y.
Published: December 2004

In order to investigate how plasmid content, transcription efficiency, and translation efficiency affect the productivity of a cloned gene protein, a new vector (pPLc‐RP4.5) was constructed. The vector has PL promoter and lacZ as a structure gene 4.5S RNA gene between PL promoter and lacZ gene. We took advantage of the characteristic that the 4.5S RNA is accumulated inside E. coli cells and can be quantitatively measured. A two‐stage continuous culture system in combination with a temperature‐sensitive gene switching system was used to study the performance of the recombinant fermentation. It was found that the plasmid content as varied by the dilution rate in the production stage showed a different pattern from that in the growth stage. The result showed that promoter strength had a greater influence on the overall gene expression efficiency of a cloned gene than the plasmid content, and the overall gene expression efficiency was largely dependent upon translation efficiency when a multicopy plasmid (pBR322 derivative and rop‐) and a strong promoter (PL) were used to express a heterologous protein in E. coli.
Extractive bioconversions in aqueous two‐phase systems: Enzymatic hydrolysis of casein proteins
Authors: Mukataka S. , Haynes C. A. , Prausnitz J. M. , Blanch H. W.
Published: December 2004

Partition coefficients in poly(ethylene glycol)/dextran aqueous two‐phase systems are reported for mixed‐casein and its components, α, β and κ casein. Rates of casein proteolysis by α‐chymotrypsin and by trypsin are reported in single‐phase and aqueous two‐phase reactor systems. The advantages resulting from selective partitioning of substrates, enzymes, and products are examined in terms of relative volumetric reaction rates.
Optimization of the production of full‐length rCD4 in baculovirus‐infected Sf9 cells
Authors: Lazarte Jaime E. , Tosi Pierre‐Francois , Nicolau Claude
Published: December 2004

The baculovirus‐insect cell system is reliable in expressing a variety of recombinant proteins. A recombinant baculovirus encoding the full length human CD4 has been used to infect Spodoptera frugiperda 9 cells in 6‐L‐airlift fermentors. The procedured described in this report permitted a 6.5‐fold enhancement of rCD4 expression as compared to standard procedures previously published. The increase of rCD4 expression on the cell surface was achieved by using the following steps: (1) Optimal seeding density of 0.8 × 106 cells/mL used to multiply cells at a maximum exponential growth of 4.5 × 106; (2) high multiplicity of infection (MOI) of 580 PFU/cell; (3) addition of medium at time of infection. In addition to full‐length rCD4, a “short” rCD4 with largely deleted cytoplasmic sequence (last 31 C‐terminal amino acids) was also efficiently expressed.
Performance of mammalian cell culture bioreactor with a new impeller design
Authors: Shi Yuan , Ryu Dewey D. Y. , Park Sun Ho
Published: December 2004

To improve the oxygen transfer in a mammalian cell bioreactor, a new type of impeller consisting of a double‐screen concentric cylindrical cage impeller (annular cage impeller in short) was designed and its mass transfer rate evaluated. This new impeller design increases the specific screen area, and the convective mass transfer rate through the annular cage was significantly increased. The oxygen transfer rates with the new impeller and the commercially available cell‐lift impeller (CelliGen by New Brunswick Scientific Co.) were evaluated and their performance compared at various rates of aeration and agitation. The results showed that with the new impeller, the oxygen transfer rate was increased by 19% in water and 21% in cell‐free culture medium supplemented with 10% horse serum, the total hybridoma cell concentration was increased to 3.4 × 107 cells/mL, and the IgG1 subtype monoclonal antibody (MAb) product concentration was also increased to 512 mg/L in perfusion culture of murine hybridoma cell line 62′D3. These improvements in oxygen transfer rate, cell concentration, and MAb product concentration are all very significant. The mass transfer resistance in the cell‐lift impeller system was found to be mainly due to the surface area of the single‐screen cage impeller. The new annular cage impeller not only provided the increased surface area for convective oxygen transfer but also protected cells from hydrodynamic shear damage, thereby achieving a significant bioprocess improvement in terms of higher viable cell concentration, higher product concentration, and higher oxygen transfer rate in the mammalian cell bioreactor system.
Dependence on growth phase and temperature of the composition of a nitrogen‐fixing cyanobacterium
Authors: Fontes Agustín G. , Moreno José , Vargas M. Angeles , Rivas Joaquín
Published: December 2004

The effects of growth pahase and temperature on the molecular and elemental composition of Anabaena variabilis cells grown under nitrogen‐fixing conditions have been studied in batch cultures. Irrespective of the growth temperature, this cyanobacterium underwent a gradual increase in its protein and lipid contents in the transition from exponential to deceleration pahse that was accompanied by a parallel decrease in carbohydrates and nucleic acids. Also in response to this, transition increases in carbon, nitrogen, and hydrogen contents with a concomitant decline in oxygen content was a common pattern for all growth temperatures tested. Whereas temperature rise did not affect significantly the protein and carbohydrate contents in exponentially growing cells, for cells in the deceleration phase proteins declined and carbohydrates increased with increasing temperature. From growth and elemental composition data several bioenergetic parameters were derived for A. variabilis cells. Both aging of cultures and rise in temperature resulted in increases of both biomass degree of reduction and heat of combustion. Nevertheless, biomass energetic yield was only slightly affected by variations in growth temperature and the maintenance coefficient was virtually constant within the range of temperatures tested. © 1992 John Wiley & Sons, Inc.
Oxygen transfer in liquid‐impelled loop reactors using perfluorocarbon liquids
Authors: van Sonsbeek H. M. , De Blank H. , Tramper J.
Published: December 2004

Oxygen transfer in the liquid‐impelled loop reactor is described for a setup in which the perfluorochemical FC40 is aerated externally. Two sizes of reactors are investigated. The mass‐transfer coefficient k appears to be lower with a factor of about 0.6 compared to gas liquid systems. the specific exchange area in the present experimental setup is found to be favorable when compared with gas liquid bioreactors at the same superficial dispersed‐phase velocities. However, slow coalescence of the dispersed‐phase drops in the phase separation section limits the dispersed‐phase flow rate seriously. In Case this become crucial from the point of view of oxygen supply, special measures need to be found or alternatives such as combined sparging of air and solvent. © 1992 John Wiley & Sons, Inc.
Activity of mushroom polyphenol oxidase in organic medium
Authors: Burton Stephanie G. , Duncan John R. , Kaye Perry T. , Rose Peter D.
Published: December 2004

A kinetic study of the activity of mushroom polyphenol oxidase in an organic system was carried out to obtain detailed enzyme kinetic data in relation to optimization of reaction conditions and substrate specificity. A simple method for consistent measurement of reaction rates in the heterogeneous enzyme/organic solvent system (consisting of immobilized polyphenol oxidase and a hydrated solution of the substrate in chloroform) was designed. The aqueous content of the system was optimized using p‐cresol as the substrate. With this system, a crude extract of Agaricus bisporus was used to hydroxylate and oxidize a range of selected p‐substituted phenolic substrates, yielding o‐quinone products. Michaelis–Menten kinetics were used to obtain apparent KM and Vmax values with respect to each of these substrates. Results from this analysis indicated a correlation between the enzymic kinetic parameters obtained and the steric requirements of the substrates, which could be rationalized in terms of the restricted flexibility of the enzyme when it is in chloroform and also in terms of substrate and solvent hydrophobicity. In the course of the investigation UV molar absorption coefficients of several o‐quinones were measured by a novel method: 1H nuclear magnetic resonance (NMR) spectroscopy was employed to determine component concentrations in reaction mixtures resulting from the transformation of phenols by polyphenol oxidase in chloroform. Thus the UV molar absorption coefficients could be obtained directly, avoiding the necessity to isolate the water‐sensitive, unstable o‐quinones. © 1993 John Wiley & Sons, Inc.
Construction and evaluation of a metal ion biosensor
Authors: Tescione Lia , Belfort Georges
Published: December 2004

Escherichia coli, genetically engineered with a mercury(II)‐sensitive promoter and the lux genes from Vibrio fischeri, were used as microbial bioluminescent sensors for the detection of mercury. Evaluation of this genetic construction was carried out by determining the effects of various parameters on cell suspensions maintained at constant conditions in a small 100‐mL vessel. The strongest light intensities and quickest induction times occurred with cells in the midexponential growth phase maintained at 28°C, concentrated to 1 × 109 cells/mL, mixed at very fast speeds, and aerated at 2 vvm (volume of air per volume of culture per minute) during light measurement in the small vessel. The cells were sensitive to the mercuric ion in the range of 20 nM to 4 μM (4 to 800 ppb), and the total response time was on the order of 1 hour, depending on the above parameters. The cells exhibited great specificity for mercury. The cells had almost equal specificity for organic and inorganic forms of the mercuric ion and responded more weakly to the mercurous ion. A simple, inexpensive, durable miniature probe (3 mL) was constructed and operated using the optimum parameters found in the small vessel as a guide. The range of sensitivity to the mercuric ion detected in the probe was 10 nM to 4 μM when aeration was provided. © 1993 John Wiley & Sons, Inc.
Temperature control of growth and productivity in mutant Chinese hamster ovary cells synthesizing a recombinant protein
Authors: Jenkins Nigel , Hovey Alison
Published: December 2004

The use of a temperature switch to control the growth and productivity of temperature‐sensitive (ts) mutants was investigated to extend the productive life span of recombinant Chinese hamster ovary (CHO) cells in batch culture. Bromodeoxyuridine was used at 39°C to select mutagenized CHO‐K1 cells, which resulted in the isolation of 31 temperature‐sensitive mutants that were growth inhibited at 39°C. Two of these mutants were successfully transfected with the gene for tissue inhibitor of metalloproteinases (TIMP) using glutamine synthetase amplification, and a permanent recombinant cell line established (5G1‐B1) that maintains the ts phenotype.

Continuous exposure to the nonpermissive temperature (npt) of 39°C led to a rapid decline in cell viability. However, a temperature regime using alternating incubations at 34°C and 39°C arrested the 5G1‐B1 cells while retaining a high cell viability for up to 170 h in culture. The specific production rate of the growth‐arrested cells was 3–4 times that of control cultures maintained at a constant 34°C over the crucial 72–130‐h period of culture, which resulted in a 35% increase in the maximum product yield. Glucose uptake and lactate production both decreased in arrested cells. Flow cytometric analysis indicated that 5G1‐B1 cells arrested in the G1 or G0 phase of the cell cycle, and no major structural damage was caused to these cells by the alternating temperature regime.

These results demonstrate that growth‐arrested ts CHO cells have increased productivity compared to growing cultures and maintain viability for longer periods. The system offers the prospect of enhancing the productivity of recombinant mammalian cells grown in simple batch fermentors. © 1993 John Wiley & Sons, Inc.
Effect of shear on the inactivation kinetics of the enzyme dextransucrase
Authors: Lencki Robert W. , Tecante Alberto , Choplin Lionel
Published: December 2004

An inactivation model previously developed to characterize the rate of enzyme activity loss in unstirred solutions was extended to take into account orthokinetic interactions resulting from convective mixing. A synergistic relationship between shear rate and temperature was observed; the rate of inactivation of the enzyme dextransucrase was unaffected by the action of shear below 25°C, but was increased by the shear rate at 30°C. Shear rate does not appear to influence the equilibrium between native and denatured dextransucrase either directly in solution or indirectly by augmenting the turnover of the gas–liquid interface. However, a second‐order plot of the inverse of relative activity (AO/A) versus Gt (shear rate × time) of dextransucrase at a constant temperature was linear because of the influence of shear on the coagulation of the denatured enzyme. The addition of 0.01 g L−1 of polyethylene glycol (MW 20,000) blocked this coagulation reaction, thereby completely inhibiting the shear‐induced inactivation of dextransucrase at 30°C. © 1993 John Wiley & Sons, Inc.
A simple model for the optimization of the extraction yield of antibiotics isolated from fermented broths by direct crystallization
Authors: Cardoso Joaquim Pereira
Published: December 2004

This article is concerned with the development of a model to maximize the overall yield of isolation of antibiotics recovered from fermented broths by the so‐called direct precipitation method. In this process, as in most antibiotics isolation processes, a second filtration of the semiexhaust mycellium from the first filtration, after slurrying it in water at the suitable pH, is required. The maximization of the overall yield of isolation implies the use of an optimal amount of water, measured as a volume to fermented broth mass ratio, which can be calculated by using the model derived here. The model also allows for the calculation of the partial and overall yields of the isolation process, and its validity is demonstrated by its ability to describe reasonably well the isolation data of two different tetracycline‐fermented broths produced according to two different technologies.

The application of the model requires only the knowledge of easily obtainable fermented broth parameters and is illustrated for two different types of tetracycline‐fermented broths. Although the model had been derived for the optimization of the overall yield of isolation of antibiotics recovered by direct precipitation, it can easily be adapted to be used for the optimization of the overall yield of isolation of antibiotics recovered by other isolation processes. © 1993 John Wiley & Sons, Inc.
Production of Bacillus thuringiensis spores in total cell retention culture and two‐stage continuous culture using an internal ceramic filter system
Authors: Kang Byung Chul , Lee Sang Yup , Chang Ho Nam
Published: December 2004

The production of Bacillus thuringiensis spores was investigated in a bioreactor incorporating a ceramic membrane filter to improve spore concentration and volumetric productivity. Two cultivation methods were used in this study: a total cell retention culture (TCRC), and a two‐stage continuous culture with partial cell bleeding. In the TCRC, fed by 50 g/L of glucose, a spore concentration of 1.6 × 1010 CFU/mL was obtained with a spore percentage of greater than 95% and a maximum cell mass of 82.2 g/L. The volumetric productivity was four times higher than that obtained from batch cultivation. In the two‐stage continuous culture with partial cell bleeding spore concentration was strongly dependent on the bleed ratio. The spore concentration of 1.8 × 109 CFU/mL and the spore percentage of 70% were obtained at the second stage when a bleed ratio of 0.33 and a dilution rate of 0.23 h−1 were used. © 1993 John Wiley & Sons, Inc.