摘要:

AbstractSeveral researchers have demonstrated that the presence of a heterologous protein in recombinantEscherichia colielicits a response similar to the heat‐shock response, which includes enhanced protease expression. The present work detects, quantifies, and characterizes intracellular protease activity inE. colithat are “shocked” by the induction of a recombinant protein, CAT, which is an endogenous protein in someE. colistrains. A novel, sodium dodecyl sulfate gelatin poly‐acrylamide gel electrophoresis (SDS‐GPAGE) method is used to detect, quantify, and characterize the presence of these proteases. A hypothesis is proposed which links the amplified protease activity to a temporary depletion of specific amino acid pools, and a stringent‐like stress response. © 1993 John Wil

ISSN:0006-3592    

DOI:10.1002/bit.260420602

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

年代:1993

数据来源: WILEY

摘要:

AbstractIt is well recognized that metabolic fluxes are the key variables that must be determined in order to understand metabolic regulation and patterns. However, owing to difficulties in measuring the flux values, evaluation of metabolic fluxes has not been an integral part of the most metabolic studies. Flux values for metabolites of glycolysis, tricarboxylic acid (TCA) cycle, and hexose monophosphate (HMP) pathway were obtained for batch and glucose‐limited continuous cultures ofBacillus subtilisby combining the information from the stoichiometry of key biosynthetic reactions with the experimental data on concentrations of glucose and metabolic by‐products, CO2evolution, and oxygen uptake rates. The results indicate that (1) the metabolic fluxes and energetic yield as well as the extent of flux mismatch in metabolic activity of glycolysis and the TCA cycle reactions can be accurately quantified; (2) the flux through the TCA cycle in continuous culture is much in excess of cell energetic and biosynthetic demands for precursors; (3) for the range of growth rates examined the TCA cycle flux increases almost in proportion to growth rate and is significantly repressed only at very high growth rates of batch cultures; and (4) for continuous cultures the isocitrate dehydrogenase catalyzed reaction of the TCA cycle is the major source of the reduced form of nicotinamide‐adenine dinucleotide phosphate (NADPH) used in biosynthesis. © 1993 John Wiley&Son

ISSN:0006-3592    

DOI:10.1002/bit.260420603

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe current understanding of insect cell metabolism is very limited. In order to gain some insight into the growth and metabolism of insect cellsSpodoptera frugiperda(Sf9), a comprehensive characterization of culture conditions for cells grown in the IPL‐41 medium was made by measuring the amino acid composition of the growth medium and the cell extract, the macromolecular composition of the cells (DNA, RNA, and protein), medium concentrations of various metabolites and sugars, and the evolved CO2. Since in the IPL‐41‐based serum‐free medium all of the amino acids except cysteine are in great excess of what is needed by the cells for energy and protein production, a medium formulation with an osmolarity similar to the IPL‐41 but with a lower amino acid content than IPL‐41 was also developed. The new medium also lacks maltose and sucrose (contains only glucose), supported cell growth to a high cell density of 8 × 106cells/mL. The cellular and energetic yields indicated that a tight coupling between the biosynthetic and energetic reactions was attained for cells grown in the new medium. Moreover, it was found that the intermittent feeding of glucose may not be required as the cell yield and growth rate were comparable whether the same total amount of glucose was provided intermittently or was included initially in the medium. The eventual cessation of growth in the new medium is believed to be due to the amino acid limitation because concentrations of both glutamine and glutamate were very low at the end of the growth phase. Thus, further optimization, which may include higher initial glutamine in the medium or its intermittent feeding, could lead to a further increase in the cell density. Finally, a stoichiometrically based analysis of metabolic reactions confirmed the operation of the key pathways and was used to quantify the distribution of metabolites among primary metabolic reactions. The quantitative flow values were used to highlight some key aspects of insect cell metabolism. © 1993 John Wi

ISSN:0006-3592    

DOI:10.1002/bit.260420604

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

年代:1993

数据来源: WILEY

摘要:

AbstractA flow cytometric method was developed for the assay of β‐galactosidase in singleEscherichia colicells. A new fluorogenic substrate for β‐galactosidase, C12FDG, contains a lipophilic group that allows the substrate to penetrate through cell membranes under normal conditions. When the substrate is hydrolyzed by intracellular β‐galactosidase, a green fluorescent product is formed and retained inside the cell. Consequently, the stained β‐galactosidase–positive cells exhibit fluorescence, which is detected by flow cytometry. This new assay was used to analyze the segregational instability caused by a reduction in specific growth rate of the plasmid‐bearing cells in the T7 expression system. Induction results in a substantial accumulation of intracellular β‐galactosidase along with a rapid increase in the fraction of plasmid‐free cells. Once the cells lose the plasmid, they no longer produce β‐galactosidase, which is reduced by at least half every generation; thus, after staining, the fluorescent, plasmid‐bearing cells can be distinguished from the nonfluorescent, plasmid‐free cells using flow cytometry. This article describes the feasibility of the flow cytometric assay for singleE. colicells and reports the optimal assay conditions. A direct relationship between β‐galactosidase activity and green fluorescence intensity was found, and the fractions of recombinant cells in batch cultures were analyzed after various levels of inductio

ISSN:0006-3592    

DOI:10.1002/bit.260420605

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

年代:1993

数据来源: WILEY

摘要:

AbstractHighly porous biocompatible and biodegradable polymers in the form of cylindrical disks of 13.5 mm diameter were implanted in the mesentery of male syngeneic Fischer rats for a period of 35 days to study the dynamics of tissue ingrowth and the extent of tissue vascularity, and to explore their potential use as substrates for cell transplantation. The advancing fibrovascular tissue was characterized from histological sections of harvested devices by image analysis techniques. The rate of tissue ingrowth increased as the porosity and/or the pore size of the implanted devices increased. The time required for the tissue to fill the device depended on the polymer crystallinity and was smaller for amorphous polymers. The vascularity of the advancing tissue was consistent with time and independent of the biomaterial composition and morphology. Poly(L‐lactic acid) (PLLA) devices of 5 mm thickness, 24.5% crystallinity, 83% porosity, and 166 μm median pore diameter were filled by tissue after 25 days. However, the void volume of prevascularized devices (4%) was minimal and not practical for cell transplantation. In contrast, for amporphous PLLA devices of the same dimensions, and the similar porosity of 87% and median pore diameter of 179 μm, the tissue did not fill completely prevascularized devices, and an appreciable percentage (21%) of device volume was still available for cell engraftment after 25 days of implantation. These studies demonstrate the feasibility of creating vascularized templates of amorphous biodegradable polymers for the transplantation of isolated or encapsulated cell populations to regenerate metabolic organs and tissues. © 1993 John Wiley&Sons,

ISSN:0006-3592    

DOI:10.1002/bit.260420606

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

年代:1993

数据来源: WILEY

摘要:

AbstractOxygen transfer in a novel pulse bioreactor has been evaluated. The agitator consists of a series of alternately fixed and movable parallel plates mounted so that the movable plates vibrate at 30 Hz causing a pulsating fluid motion. Pure oxygen, at pressures up to 5 atm, diffuses through silicone rubber tubing that also vibrates at 30 or 60 Hz. The main feature of this bioreactor is high oxygen transfer with low shear to prevent damage to fragile animal cell membranes. We estimate that sufficient oxygen can be supplied to support over 108cells/mL of human diploid foreskin cells growing on microcarriers. © 1993 John Wiley&Sons, Inc

ISSN:0006-3592    

DOI:10.1002/bit.260420607

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

年代:1993

数据来源: WILEY

摘要:

AbstractA strain of the fission yeastSchizosaccharomyces pombewas aerobically grown in a cell‐recycle fermentor under various operating conditions, i.e., different bleeding rates and various separate feed rates of glucose and basal medium. Carbon and energy balances were analyzed during steady‐state culture regimes, allowing growth yields and maintenance coefficients to be determined under glucose‐limited and glucose‐excess environments. Special attention was given to the metabolic shift from purely oxidative to respirofermentative glucose catabolism resulting from a change in the growth‐limiting factor. No maintenance requirements for the carbon source and for energy were observed during glucose‐limited culture regimes and oxidative catabolism. Under glucose excess and respirofermentative metabolism, themGcoefficient was shown to be growth‐linked, whereas the enhancement of the apparentmecoefficient observed for increased residual glucose concentrations could be assigned to a decline in the ATP yield. © 1993 John W

ISSN:0006-3592    

DOI:10.1002/bit.260420608

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe dilution rate of an ultrafiltration membrane bioreactor in the enzymatic hydrolysis of cellulose was optimized using the kinetic model developed by Fan and Lee.4The sequence of optimal dilution rates was found to generally consist of an initial period of a minimal value (batch period), a subsequent period of maximum dilution rate, a period of a second batch, and a final period of a singular dilution rate. The effects of operating conditions, such as β‐glucosidase activity, operating time, maximum dilution rate, substrate feeding rate, and enzyme‐to‐substrate ratio on both the conversion yield and the sequence of optimal dilution rates were investigated. To evaluate the validity of kinetic model employed in this work, enzymatic hydrolysis was carried out using α‐cellulose as a substrate in the ultrafiltration membrane bioreactor. The experimental data were well consistent with the simulation results. © 1993 John Wiley

ISSN:0006-3592    

DOI:10.1002/bit.260420609

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe toxicity of homologous series of organic solvents has been investigated for the gram‐positive bacteria,Arthrobactersp. andNocardiasp., and the gram‐negative bacteria,Acinetobactersp. andPseudomonassp. The hydrophobicity of the solvent, expressed by its logPoctanol, proves to be a good measure for the toxicity of solvents in a two‐phase system. The transition from toxic to nontoxic solvents occurs between logPoctanol3 and 5 and depends on the homologous series. No correlation has been found between the hydrophobicity of the substituent on the alkyl backbone of the solvent and the location of the transition point in toxicity. The logPoctanol, above which all solvents are nontoxic, is used to express the solvent tolerance of the bacteria. In general, the solvent tolerance of gram‐negative bacteria is found to be slightly higher than that of gram‐positive bacteria, but this does not hold for all homologous series of organic solvents investigated.Because the toxicity effects of organic solvents in a two‐phase system can be ascribed to molecular as well as phase toxicity effects, molecular toxicity effects were investigated separately in a one‐phase system with subsaturating amounts of organic solvent. The solvent concentration in the aqueous phase, at which 50% of the metabolic activity of the bacteria is lost, is used to express solvent toxicity. This concentration is found to be similar for the gram‐positiveArthrobacterand the gram‐negativeAcinetobacter. Assuming the critical membrane concentration theory (G. J. Osborne et al.Enzyme Microb. Technol.1990, 12: 281–291) to be valid, it can be concluded that differences in solvent tolerance between these two bacteria, cannot be ascribed to differences in response to molecular toxicity. Prediction of the toxicity of any solvent, using the critical membrane theory, appears to be possible in the case of alkanols or alkyl acetates. However, prediction of the toxicity of ethers appears to be impossible. © 1993

ISSN:0006-3592    

DOI:10.1002/bit.260420610

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

年代:1993

数据来源: WILEY

摘要:

AbstractTriglyceride hydrolysis usingChromobacterium viscosumlipase B in a reversed micellar media was investigated in a batch‐type reactor. The dynamic evolution of the product concentration was analyzed using several mechanistic models, both from the literature and developed in this work. A kinetic model with nonlinear product inhibition was found to be the most adequate for the description of batch hydrolysis data over an extensive range of time and substrate concentration. The obtained rate equation described the time course of not only the reactions performed in this work, at different water contents (W0= 7, 24) and pH values, but also the experimental results obtained in the literature with aCandida rugosalipase. © 1993 John Wiley&Sons, I

ISSN:0006-3592    

DOI:10.1002/bit.260420611

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

年代:1993

数据来源: WILEY