摘要:

AbstractThe kinetics of oxidation of elemental sulfur byThiobacillus ferrooxidansin a batch reactor was followed by measuring the concentration of adsorbed cells on the sulfur surface, the concentration of free cells in liquid medium, and the amount of sulfur oxidized. As the elemental sulfur was oxidized to sulfate, the liquid‐phase concentration of free cells continued to increase with time, whereas the surface concentration of adsorbed cells per unit weight of sulfur approached a limiting value, i.e., the maximum adsorption capacity. During sulfur oxidation, there was a close correlation between the concentrations of adsorbed and free cells, and these data were well correlated with the Langmuir isotherm. The observed rates of batch growth and sulfur oxidation were consistent with a kinetic model, assuming that the growth rate of batch growth and sulfur oxidation were consistent with a kinetic model. Assuming that the growth rate of adsorbed bacteria is proportional to the product of the concentration of adsorbed cells and the fraction of adsorption sites unoccupied by cells. The kinetic and stoichiometric parameters appearing in the model were evaluated using the experimental data and were compared with parameters determined previously for a few metal sulfides. © 1994 John Wiley&Sons, I

ISSN:0006-3592    

DOI:10.1002/bit.260440602

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

年代:1994

数据来源: WILEY

摘要:

AbstractPhase transfer studies were carried out on the solubilization of horseradish peroxidase (HRP) (E.C. 1.11.1.7) in reverse micelles formed in isooctane using the anionic surfactant, aerosol OT, at concentrations between 50 and 110mM. The selectivity of this methodology was tested, because the HRP used comprised a mixture of seven different isoenzymes with a wide range of isoelectric points. Forward and backward transfers were carried out in wellstirred vessels until equilibrium was reached. Significant protein partitioning could only be obtained by using NaCl to adjust ionic strength in pH range between 1.5 and 3.5, with a maximum at pH 3. The back transfer process was best at pH 8 with 80mMphosphate buffer and 1MKCI. A loss of 1% to 3% of the surfactant through precipitation at the interface at pH<4 was observed, which may be due to instability in this pH region, because, even without protein, a similar precipitate was noticed. Protein partitioning was approximately constant when the ionic strength was increased up to 1MNaCl at pH 3, but protein recovery in back transfer decreased accordingly. Hydrophobic interactions together with association between the protein and surfactant might be responsible for that behavior. Protein partitioning remained the same when the surfactant concentration was decreased to 50 mM, at the expense of higher variability. HPLC chromatograms showed no apparent damage to the protein after reverse micellar extraction. Protein partitioning is best when the temperature is kept at 25×C. The amount of protein and specific activity recovered strongly depends on the phase ratio used during forward transfer. Overall activity recovery varied from 87% to 136% when the phase ratio was increased from 1:1 to 30:1 in forward transfer. This behavior may be due to a change in the ratio of the three isoenzymes recovered after the backward transfer process, with the most active one being increasingly enriched at higher phase ratios. © 1994 John Wiley&Sons, In

ISSN:0006-3592    

DOI:10.1002/bit.260440603

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

年代:1994

数据来源: WILEY

摘要:

AbstractThermal deactivation of solid‐state acid phosphates (E.C. 3.1.3.2, from potato) is analyzed, both in the presence and in the absence of organic solvents. The thermal deactivation profile departs from first order kinetics and shows an unusual activity. The process is described by a phenomenological equation, whose theoretical implications are also discussed. The total amount of buffer salts in the enzyme powder dramatically affects enzyme stability in the range 70×C to 105×C. The higher salt/protein ratio increases the rate of thermal deactivation. The deactivation rate is virtually unaffected by the presence of organic solvents, independent of their hydrophilicity. © 1994 John Wiley&Sons,

ISSN:0006-3592    

DOI:10.1002/bit.260440604

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

年代:1994

数据来源: WILEY

摘要:

AbstractMicellar‐enhanced ultrafiltration (MEUF) is investigated as a large‐scale technique for separating amino acid enantiomers. Specifically,L‐5‐cholesterol glutamate, a chiral ligand‐exchange cosurfactant, is used together with a nonionic surfactant to form mixed micelles that preferentially bindD‐phenylalanine overL‐phenylalanine in the presence of copper(II). Operational selectivities as high as 4.2 are obtained. Potentiometric titrations using a water‐soluble model compound similar to the chiral cosurfactant indicate that the ternary copper complex with phenylalanine has a stereoselectivity for the D enantiomer which is significantly smaller than that observed in the MEUF system. Thus, the selectivity of the chiral legend's local solvent and structural environment. © 1994 John

ISSN:0006-3592    

DOI:10.1002/bit.260440605

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe bioconversion of octanoic acid into 2‐heptanone by spores ofPenicillium roquefortiiis performed using a fed‐batch technique with pH control by addition of the liquid substrate itself. The early stage of this process takes place with a high bioconversion rate and high yield. These values then decrease as a result of germination and growth the biocatalyst. An optimization strategy for the process would thus be to improve the characteristics of this first period, i.e., increase its duration and the reaction rate. An increase in duration is evidenced in two cases: (I) under oxygen limitation: and (ii) when the spore content in the medium is less than 107spores/mL. These conditions give insufficient overall bioconversion rates: better optimization should be achieved without oxygen limitation and with high spore content. Characterization of the first period by material and bioenergetic balances suggests that an increase in the ethanol content of the medium, which acts as an energy source and a permeabilizer, and the use of specific inhibitor of the Krebs cycle, may be a way to further improve the biocatalyst performance and stability. © 1994 John Wiley&Sons,

ISSN:0006-3592    

DOI:10.1002/bit.260440606

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

年代:1994

数据来源: WILEY

摘要:

AbstractA mathematical model has been developed that predicts the cell population dynamics and production of recombinant protein and infective extracellular virus progeny by insect cells after infection with baculovirus in batch suspension culture. Infection in the model is based on the rate of virus attachment to suspended insect cells under culture conditions. The model links the events following infection with the sequence of gene expression in the baculovirus replicative cycle. Substrate depletion is used to account for the decrease in product yield observed when infecting at high cell densities. Model parameters were determined in shaker flasks for two media: serum‐supplemented IPL‐41 medium and serum free Sf900II medium. There was good agreement between model predictions and the results from an independent series of experiments performed to validate the mode. The model predicted: (1) the optimal time of infection at high multiplicity of infection: (2) the timing and magnitude of recombinant protein production in a 2‐L bioreactor; and (3) the timing and magnitude of recombinant protein production at multiplicities of infection from 0.01 to 100 plaque‐forming units per cell. Through its ability to predict optimal infection strategies in batch suspension culture, the model has use in the design and optimization of large‐scale systems for the production of recombinant products using the baculovirus expression vector system. © 1994 John Wiley

ISSN:0006-3592    

DOI:10.1002/bit.260440607

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe incidence of apoptotic and necrotic cell death was compared in CHO, SF9 insect cells and murine plasmacytoma (J558L) and hybridoma (TB/C3) cells during in vitro cultivation in batch cultures. Acridine orange staining and fluorescence microscopy enabled the visualization of a classic morphological feature of apoptotic cell, the presence of condensed and/or fragmented chromatin. DNA gel electrophoresis was employed to show an additional characteristic of the process, the endonuclease‐mediated fragmentation of DNA into multiples of 180 base pairs. The levels of apoptosis at the end of batch cultures of plasmacytoma and hybridoma cell lines were found to be 60% and 90% of total dead cells, respectively. However, employing the above‐mentioned techniques, the biochemical and morphological features of apoptosis were not found in CHO and SF9 insect cells. Some factors affecting the induction of apoptosis during the batch culture of the hybridoma and plasmacytoma cell lines were identified. The most effective inducer was found to be glutamine limitation, followed by (in order of importance) serum limitation, glucose limitation, and ammonia toxicity. Blockage of the cell cycle of the plasmacytoma and hybridoma cells using thymidine resulted in the induction of apoptosis. This has important implications for the development of cell culture processes that minimize cell division and thereby increase specific productivity. © 1994 John Wiley&Sons,

ISSN:0006-3592    

DOI:10.1002/bit.260440608

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

年代:1994

数据来源: WILEY

摘要:

AbstractMany mammalian cell fed‐batch processes rely on maintaining the cells in a viable and productive state for extended periods of time in order to reach high final concentrations of secreted protein. In the work described herein, a nonamplified NSO cell line was transfected with a vector expressing a recombinant human anti‐HIV gp 120 monoclonal antibody (Mab) and a selectable marker, glutamine synthetase. A fed‐batch process was developed which improved product yields tenfold over the yields reached in batch culture. In this case, the clone was cultured for a period of 22 days and produced 0.85 g Mab/L. To gauge the effect of extended culture lifetime on product quality, biochemical characteristics of MAb isolated from different time points in the fed‐batch culture were determined. The apparent molecular weight of the MAb was constant throughout the course of the culture. Isoelectric focusing revealed four major charged species, with a fifth more acidic species appearing later in the culture. The antigen binding kinetics were constant for MAb isolated throughout the culture period. Glycosylation analysis, on the other hand, revealed that MAb produced later in the culture contained greater percentages of truncatedN‐acetylglucosamine and highmannoseN‐glycans. Possible contributions to this underglycosylated material from either cell lysis or synthesis from noviable cells were found to be negligible. Instead, the viable cells appeared to be secreting more truncated and high mannose MAb glycoforms as the culture progressed. © 1994 John Wil

ISSN:0006-3592    

DOI:10.1002/bit.260440609

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

年代:1994

数据来源: WILEY

摘要:

AbstractRecombinant cells ofSaccharomyces cerevisiae, expressing virus‐like particles (Ty‐VLPs), can be readily disrupted in a high pressure homogenizer and show identical disruption kinetics to the untransformed host strain. When the cells are freeze/thawed before disruption, they become about four times more resistant to homogenization. This effect increases with the number of freeze/thaw cycles, but is independent of the time the cells remain frozen. The freeze/thaw effect is observed with cells harvested during both the logarithmic and stationary phase of growth, and occurs with the untransformed host strain as well as the transformed one. Freeze/thawed cells are twice as resistant to disruption in the bead mill as fresh cells. © 1994 John Wiley&Sons,

ISSN:0006-3592    

DOI:10.1002/bit.260440610

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe use of charged peptides fused to enzymes for immobilization onto ion‐exchange membranes was explored for the enzyme ×‐galactosidase. The additional charged peptides, containing 1, 5, 11, and 16 aspartates, fused to ×‐galactosidase, for the most part didnotinterfere with the kinetic behavior for lactose hydrolysis. There was a 2‐fold decline in Vmfor the 16‐aspartate fusion, but the others were quite similar to the wild type enzyme (BGWT). BGWT and the fusions all retained approximately 50% of their activities when adsorbed onto ion‐exchange membranes. In contrast to BGWT, the enhanced binding strength of the 11 aspartate fusion provided the ability to hydrolyze whey permeate at 0.3Mionic strength without enzyme leakage, and to immobilize the enzyme directly from diluted cell extract with 83% purity. © 1994 John W

ISSN:0006-3592    

DOI:10.1002/bit.260440611

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

年代:1994

数据来源: WILEY