摘要:

AbstractThe constitutive cytoplasmic expression inE. coliof human growth hormone (hGH) with differentN‐terminal extensions (3 or 4 amino acids) has been studied. These hGH precursors were used forin vitrocleavage 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 criticalper sein 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 inE. coli. with constitutive as well as inducible promoters we conclude that glucose fed batch processes are often superior to batch processes in the productio

ISSN:0006-3592    

DOI:10.1002/bit.260360102

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe influence of concentrated maltotetraose solutions on fungal alpha‐amylase activity and specificity has been determined. It has been found that the enzyme is not inhibited by 500 g/L substrate concentration and that transglycosylation reactions are increased with rising substrate concentration. The amount of oligosaccharides of polymerization degree higher than maltotetraose reaches 20% (w/w). Moreover, the effect of polyhydric alcohols, known as water activity depressors has been analyzed: the presence of polyols does not modify the amount of transglycosylation products, but changes the hydrolysis pattern by favoring the formation of low polymerization degree oligosaccharide

ISSN:0006-3592    

DOI:10.1002/bit.260360103

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe appearance of sustained oscillations in bioreactor variables (biomass and nutrient concentrations) in continuous cultures ofSaccharomyces cerevisiaeindicates 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, andT= 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 fermentati

ISSN:0006-3592    

DOI:10.1002/bit.260360104

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

年代:1990

数据来源: WILEY

摘要:

AbstractSustained oscillations of biomass, ethanol, and ammonium concentrations, specific growth rate, and specific uptake rates of ethanol, ammonium, and oxygen were found in continuous cultures ofSaccharomyces cerevisiaeunder controlled dissolved oxygen (DO), pH, and temperature conditions. The period of oscillations was approximately 2.5–3 h at a pH of 5.5 and 2–2.5 h at a pH of 6.5. Oscillations were observed only under conditions of low carbon (glucose below the minimum detectable level), nitrogen nutrient (ammonium concentration varied between 0.00001 and 0.0015M), and ethanol concentration (0.002–0.085 g/L) in the bioreactor.The oscillatory behavior at pH 5.5 was also characterized by partially synchronized cell growth and reproduction. Not only did the total percentage of budding cells oscillate with the same period as observed for the global biomass and nutrient concentrations, but the peaks in the individual subpopulations of initial budding, middle budding, and late budding cells appeared sequentially during the oscillation period. This provides strong evidence of the hypothesis that variations in metabolism during different periods in the cell cycle of a partially synchronized cell population are responsible for the observed oscillatory bioreactor behavior.The specific nutrient uptake rates for ammonium and oxygen as well as the net specific ethanol uptake rate oscillated with the same period as the biomass oscillations. These results show a dramatic increase in the ammonium and oxygen consumption rates prior to the initial budding of the synchronized subpopulation and a decrease in these rates during the late budding phase. At a pH of 5.5, the late budding phase is characterized by high specific ethanol productivity; however, the ethanol productivity lags the late budding phase at a pH pf 6.5. The observed time‐varying metabolism in the oscillatory operating regime appears to be the result of the metabolic changes which occur during the cell cycle. Models which can predict the oscillatory biomass concentration and nutrient levels in this regime must be capable of predicting the concentrations and metabolic rates of the subpopulations

ISSN:0006-3592    

DOI:10.1002/bit.260360105

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

年代:1990

数据来源: WILEY

摘要:

AbstractMandelonitrile lyase (EC 4.1.2.10) catalyzes the formation ofD‐mandelonitrile from HCN and benzaldehyde. Mandelonitrile lyase was immobilized by adsorption to support materials, for example, Celite. The enzyme preparations were used in diisopropyl ether for production ofD‐mandelonitrile. In order to obtain optically pureD‐mandelonitrile it was necessary to use reaction conditions which favor the enzymatic reaction and suppress the competing spontaneous reaction, which yields a racemic mixture ofD,L‐mandelonitrile. The effects of substrate concentrations, water content, and support materials on both the spontaneous and enzymatic reactions were studied. The enzymatic reaction was carried out under conditions where the importance of the spontaneous reaction was negligible and high enantiomeric purity ofD‐mandelonitrile was achieved (at least 98% enantiomeric excess). The operational stability of the enzyme preparations was studied in batch as well as in continuous systems. It was vital to control the water content in the system to maintain an active preparation. In a packed bed reactor the enzyme preparations were shown to be active and stable. The reactors were run for 50 h with only a small decrease in prod

ISSN:0006-3592    

DOI:10.1002/bit.260360106

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

年代:1990

数据来源: WILEY

摘要:

AbstractImmobilization of Saccharomyces cerevisiae ATCC 834 within alginate beads enhances microbiological conversion of benzaldehyde toL‐phenylacetyl carbinol (L‐PAC), a precursor employed for synthesis ofL‐ephedrine. Yields of 90% L‐PAC on benzaldehyde (initially 0.6% in medium) were obtained with immobilized cells, in contrast to about 10% with free cells which tend to form pellets in the presence of benzaldehyde. The predominant favorable action of immobilization appears to be a reduction in the toxic or inhibitory effects of benzaldehyde. With an initial benzaldehyde concentration of about 0.6% in the medium the optimum cell mass concentration was observed to be about 28 g cell mass (immobilized) per liter of

ISSN:0006-3592    

DOI:10.1002/bit.260360107

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe cyclic, semicontinuous production ofL‐phenylacetyl carbinol (L‐PAC) from a benzaldehyde substrate bySaccharomyces cerevisiaeATCC 834 immobilized in calcium alginate beads was substantially enhanced to about 4.5 g/L in a second cycle by reactivation in fresh medium for 24 h, following an earlier 24‐h period of production from substrate. Intermittent feeding of benzaldehyde was employed (four doses in 3 h). In subsequent similar cycles, however, the production returned to that produced in the first cycle, viz. L‐PAC concentration of 2–3 g/L in the medium. Production of L‐PAC was also increased by adaptation of the cells over 200 h of exposure to the benzaldehyde substrate (compared to wild‐type cells) and by continuous (as compared to intermittent) feeding of the substrate. A liter as great as 10 g/L was obtained with wild‐type cells by continuous feeding of benzaldehyde over 6 h. Immobilization not only protected the cells from toxic effects of substrate but also permitted them to be used during 7 cycles of semicontinuous operation over

ISSN:0006-3592    

DOI:10.1002/bit.260360108

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

年代:1990

数据来源: WILEY

摘要:

AbstractA flow cytometric kinetic study of hybidoma growth and monoclonal antibody production is presented, along with the influence of glutamine on intracellular responses such as (relative) cell size, and cell RNA and total protein content. Specific findings are: (1) RNA content remained constant throughout the growth phase, then fell drastically as the cells entered the stationary phase. Also, in stationary phase, RNA content of antibody‐producing cells was higher than for those not secreting antibody. (2) The cell size was constant and maximal throughout exponential phase, and diminished monotonically during later stages. (3) Average protein and antibody cellular content declined dramatically upon glutamine exhaustion. Thus, relative RNA levels and cell size provided quantitative determinants of both cell growth state and antibody secretion conditions. These results encourge consideration of structured kinetic studies which recognize the quality of the biophas

ISSN:0006-3592    

DOI:10.1002/bit.260360109

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe influence of glutamine (a major energy source) on both hybridoma growth and monoclonal antibody production was examined. A series of batch experiments were performed in T‐flasks containing initial glutamine levels ranging from 0.5 to 4.0 mMin RPMI 1640 with 20% v/v fetal calf serum. The maximum final cell concentration increased with initial glutamine levels in the range of 0.5–2 mM; further glutamine increases had little or no effect. Earlier studies in our laboratories demonstrated that serum component(s) strongly influence the maximum specific growth rate. Here, the present studies reveal also thestoichiometriclimitation by glutamine in the later stages of growth when its concentration is drastically reduced. For 0.5 to 1.5 mMinitial glutamine, complete substrate utilization coincided with the cessation of cell growth and the onset of the death phase. For initial glutamine concentrations higher than 2.0 mM, growth halted prior to glutamine exhaustion, presumably because serum or RPMI component(s) were exhausted. The specific antibody secretion rate was essentially non‐growth‐associatedabovea critical low glutamine concentration inboththe growth and death phases. At or below this critical value, an apparent emergence of stoichiometnc or energy limitation resulted in a dramatic drop in the secretion rate to zero. A simple unstructured model was developed that simulates these trends well. All parameters were determined using only subsets of the data. Nevertheless, these parameter values provided simulations in good agreement with all the glutamine‐limited

ISSN:0006-3592    

DOI:10.1002/bit.260360110

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

年代:1990

数据来源: WILEY

摘要:

AbstractIn fed‐batch fermentation, cells ofL. mesenteroidesimmobilized 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 simil

ISSN:0006-3592    

DOI:10.1002/bit.260360111

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

年代:1990

数据来源: WILEY