摘要:

AbstractExperiments are reported here on the equilibrium partitioning of lysozyme and ribonuclease‐a between aqueous and reversed micellar phases comprised of an anionic surfactant, sodium di‐2‐ethylhexyl sulfosuccinate (AOT), in isooctane. A distinct maximum, [P]rm,maxwas found for the quantity of a given protein that can be solubilized in the reverse micelle phase by the phase‐transfer method. This upper limit depended upon the size of the protein, the surfactant concentration, and the aqueous phase ionic strength, and was determined by complex formation between protein and surfactant molecules to form an insoluble interfacial precipitate at high values of [P]rm. In this work, it was found to be possible to dissociate the protein–surfactant complex and recover the precipitated protein. The kinetics of protein–surfactant complex formation depended upon the nature and concentration of the solubilized protein and on the surfactant concentration. Calculations of micellar occupancy and the relative surface areas of protein molecules and surfactant head‐groups suggested that it was the exposure of the solubilized protein to the bulk organic solvent which promoted protein–surfactant complex formation as [P]rm→ [P]rm,max. In the light of the experimental results and calculations described above, a mechanistic model is proposed to account for the observed phenomena. This is based upon the competing effects of increasing the solubilized protein concentration and the corresponding increase in the rate of protein‐surfactant complex formation. The dynamic nature of the reverse micelles is inherent in the model, explaining the formation of the interfacial precipitate with time and its dependence on the internal phase volume of the micellar phase. Experiments on the co‐partitioning of water and measurement ofthe AOT concentration in both phases verified the loss of protein, water, and surfactant from the organic phase at high values of [P]rm. © 19

ISSN:0006-3592    

DOI:10.1002/bit.260470502

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractThe feasibility of continuous production of proteins in chemostat cultures ofBacillus subtiliswas investigated. An expression system consisting of the bacteriumB. subtilisBR151 carrying plasmid p602/19 was used. The plasmid contains thecat(chioramphenicol acetyltrans‐ferase) gene downstream of a strong vegetative T5 promoter. It was found that, at a dilution rate of 0.2 h−1production of relatively high levels of CAT protein (about 4% ofcellular protein) can be sustained. But, experiments at a higher dilution rate of 0.4 h−1were unproductive because of high acidformation and washout. Combination of low cell yield, which results from excessive acid formation, and low dilution rate led to a low volumetric CAT productivity. Our recent work with the nonrecombinant cells, has demonstrated that uptake of small amounts of citrate significantly reduces or entirelyeliminates the acid formation. This superior performance in the presence ofcitrate was hypothesized, based on strong experimental evidence, to be the result of a reduction in glycolysis flux through a sequence of events leading to a reduction in pyruvate kinase and phosphof‐ ructokinase activities, the regulatory enzymes of glycol‐ysis. In this study, it is demonstrated that cofeeding of glucose and citrate substantially reduces theorganic acid formation and significantly increases the recombinant culture productivity. The combination of high specific CAT activity and cell density resulted in a total of six‐ to tenfold higher culture productivitywhen citrate and glucose were cometabolized than when glucose was the only carbon source. © 1995 John Wi

ISSN:0006-3592    

DOI:10.1002/bit.260470503

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractThe role of glucose in ajmalicine production byCatharanthus roseuswas investigated in the second stage of a two‐stage batch process. Activities of tryptophan decar‐boxylate (TDC) and anthranilate synthase (AS), two enzymes In the pathway leading to ajmalicine, were higher after induction with 40 g/L glucose than after induction with 60 or 80 g/L glucose. Experiments with different media containing mixtures of glucose and the nonpermeating osmotic agent xylose, and using an already induced culture as inoculum, revealed that a minimum amount of glucose is required to support ajmalicine production after enzyme induction. This requirement was not an osmotic effect. The relation between the glucose concentration and the specific ajmalicine production rate,qp, was investigated in seven (fed‐)batch cultures with constant glucose concentrations: 23, 29, 35, 53, 57, 75, and 98 g/L. In the cultures with a low glucose concentration (23, 29, and 35 g/L) theqpwas 2.7‐times higher than the cultures with 53 and 57 g/L, and almost six times higher than the cultures with a high glucose concentration (75 and 98 g/L). A glucose perturbation experiment (from 53 to 32 g/L) demonstrated that the ajmalicine production rate was adjusted without much delay. A kinetic equation is proposed for the relationship between the glucose concentration andqp. Differences in enzyme induction and ajmalicine production at different glucose levels could not be explained by the intracellular concentrations of glucose, fructose, sucrose, or starch. © 1995 John Wiley&

ISSN:0006-3592    

DOI:10.1002/bit.260470504

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractPrevious experiments have shown that population average surface lgG content is correlated with the specific antibody production rates of batch hybridoma cultures. Therefore, surface associated lgG content of single hybridoma cells might indicate antibody secretion rates of individual cells. Moreover, the surface lgG content should reflect the pattern of secretion rates during the cell cycle. To probe for lgG secretion rates during the cellcycle, a double staining procedure has been developed allowing simultaneousflow cytometric analysis of surface lgG content and DNA content of murine hybridoma cells. Crosslinking of the surface associated immunofluorescence with the cell by paraformaldehyde fixation permits subsequent DNA staining without loss of immunofluorescence. The optimized protocol has been used to determine the pattern of the surface lgG fluorescence as a function of the cell cycle position. It is highest during the G2+M cell cycle phase and the experimental data are in excellent agreement with the previously predicted secretion pattern during the cell cycle. © 1995 John Wiley&Sons Inc

ISSN:0006-3592    

DOI:10.1002/bit.260470505

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractOxygen transfer characteristics of self‐immobilizedSolanum avicularecells were measured using aggregates 3.0 to 12.5 mm in diameter. Apparent specific oxygen uptake rates in the absence of external boundary layers varied from 5.9 × 10−11to 8.5 × 10−7kg kg−1s−1dry weight, but did not decline continuously with increasing particle size. The effective diffusivity of oxygen in deactivated aggregates increased with particle diameter, varying from 5.0 × 10−11to 1.0 × 10−9m2s−1or between 2% and 40% of the molecular diffusivity in water at the same temperature. Gas spaces detected in the larger aggregates were confined to the central core and were not distributed throughout the tissue to facilitate oxygen transfer. Oxygen consumption rates in the absence of diffusional limitations were estimated using the relationship between the observable Thiele modulus and effectiveness factor for zero‐order reaction. The calculated results indicated severe oxygen limitations in the aggregates, but were inconsistent with the observation that relatively largeS. aviculareaggregates contained a high fraction of viable cells and were capable ofactive growth and steroidal alkaloid synthesis. This work suggests that oxygen delivery is facilitated in living plant cell aggregates by mechanisms which depend on metabolic activity and which do not function in deactivated cells. © 19

ISSN:0006-3592    

DOI:10.1002/bit.260470506

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractThe influence of the microcarrier type on the performance of a controlled release process used to produce a recombinant glycosyl‐phosphatidylinositol anchored protein was investigated. Chinese hamster ovary (CHO) cells expressing the human melanoma tumor antigen (p97) were cultured in 10% serum on Cultispher‐GH porous microcarriers and then, for protein production, maintained in 2% serum. Cells were harvested every 48 h and p97 was recovered at 90 μg/mL and 40% purity. Harvested p97 concentrations were increased by harvestingfrom spheroid (241 μg/mL) and smaller porous microcarrier, Cultispher‐G (167 μg/mL) cultures. The low total cell specific p97 production of cells cultured on Cultispher‐GH was due to necrosis of cells within the beads, decreased p97 expression of the immobilized cells, dilution by the liquid (up to 40% volume) associated with settled beads, and incomplete recovery of p97 from within the beads. Cells cultured on solid microcarriers, Cytodex‐1, had the highest cell viability and cell specific p97 production, It is recommended that a two‐stage cyclic harvesting process of cells cultured on small Cultispher‐G or on Cytodex‐1 beads would minimize protein loss and maximize cell specific protein recovery. © 1995

ISSN:0006-3592    

DOI:10.1002/bit.260470507

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractFollowing ozone oxidation of polyester microfibers of 3.5 μm average diameter and 0.83 m2/g specific area, the fiber surface was subjected to graft polymerization of acrylic acid and subsequently immobilized with serologically active proteins includingStaphylococcus aureusprotein A, a specific antigen, and a specific antibody. The immobilization reaction was mediated by a watersoluble carbodiimide, which allowed formation of a co‐valent linkage between the ligand proteins and the grafted poly(acrylic acid)chains. The yields of the immobilized ligand proteins were of the order of 1 mg/g fiber. Their binding affinity and capacity to respective specific proteins were studied in vitro from a buffered solution and serum. It was found that the specific proteins were selectively adsorbed with dissociation constants as low as 1× 10−6M, suggesting the adsorption to take place through highly specific protein‐protein interaction. An addition of serum albumin did not significantly affect the specific binding, regardless of the ligand proteins. The binding capacity ranged from 1 × 10−13to 1× 10−11mol/cm2primarily depending on the surface density of the immobilized ligands and the number of their binding sites per molecule. © 1995 John

ISSN:0006-3592    

DOI:10.1002/bit.260470508

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractA dynamic diffusion–reaction–growth model is proposed for the study of lactic fermentation, the bioconversion of citric acid, and cell release in an immobilized cell reactor [pH‐stat continuous stirred tank‐reactor (CSTR)]. The model correctly simulates the onset of fermentation and colonization of the gel, followed by the steady state. External diffusion is nonlimiting and internal diffusion is limited by high cell densities at the periphery of the gel beads. Lactose–citrate cometabolism in the gel is related to the distribution of active included biomass within the gel and to gradients of substrates (lactose, citrate) and products (lactate, pH) in the beads. The utilization of lactose is limited by reaction, whereas that of citrate is limited by diffusion. Cell release from gel to the liquid medium occurs in the external spherical cap of the beads. In this peripheral zone viability is maintained at around 90%. © 1995 John Wile

ISSN:0006-3592    

DOI:10.1002/bit.260470509

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractThe starch‐binding domains of glucoamylase I (SBD of GA‐I) fromAspergillus awamoriand of cyclodextrin glucanotransferase (domain E of CGTase) fromBacillus maceranswere fused to the C‐terminus of β‐galactosidase (β‐gal) The majority of the fusion proteins produced inEscherichia coliwere found as inclusion bodies. Active fusion proteins were purified by partial solubilization of the inclusion bodies with 2Murea followed by affinity chromatography. Adsorption isotherms of purified fusion proteins on corn starch and cross‐linked amylose were generated. The β‐gal fusion proteins had similar affinities for cross‐linked amylose and corn starch but significantly different saturation capacities on corn starch. The adsorption and elution data from the potato starch column as well as the adsorption isotherms of p‐gal‐domain E fusion protein (BDE109) on corn starch and cross‐linked amylose demonstrated that domain E of CGTase is an independent domain, which retained its starch‐binding activity when separated from the other four (A–D) domains in CGTase

ISSN:0006-3592    

DOI:10.1002/bit.260470510

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractFor a stable and reliable operation of a BAS‐reactor a high, active biomass concentration is required with mainly biofilm‐covered carriers. The effect of reactor conditions on the formation of nitrifying biofilms in BAS‐reactors was investigated in this article. A start‐up strategy to obtain predominantly biofilm‐covered carriers, based on the balancing of detachment and a biomass production per carrier surface area, proved tp be very successful. The amount of biomass and the fraction of covered carrier were high and development of nitrification activity was fast, leading to a volumetric conversion of 5 kgN· m−3· d−1at a hydraulic retention time of 1h. A 1‐week, continuous inoculation with suspended purely nitrifying microorganisms resulted in a swift start‐up compared with batch addition of a small number of biofilms with some nitrification activity. The development of nitrifying biofilms was very similar to the formation of heterotrophic biofilms. In contrast to heterotrophic bio‐films, the diameter of nitrifying biofilms increased during start‐up. The detachment rate from nitrifying biofilms decreased with lower concentrations of bare carrier, in a fashion comparable with heterotrophic biofilms, but the nitrifying biofilms were much more robust and resistant. Standard diffusion theory combined with reaction kinetics are capable of predicting the activity and conversion of biofilms on small suspended particles. ©

ISSN:0006-3592    

DOI:10.1002/bit.260470511

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY