Phase behavior and rheological properties of enzymatically synthesized trehalose decanoate aqueous solutions.

Surface tension properties of an enzymatically synthesized equimolar mixture of trehalose mono- and didecanoate in aqueous solutions have been determined. At 20 degrees C a critical micellar concentration (CMC) of 50 micromol/l and a minimal surface tension of 28 mN/m have been obtained. Above the CMC, it has been shown that up to a concentration of 42 wt%, and in a 20-60 degrees C temperature range the sugar ester aqueous solutions do not form any crystalline structure, nor present any phase transition, and the trehalose decanoate molecules form an isotropic worm-like micellar phase. The rheological properties indicate however a more complicated picture in the same concentration and temperature ranges. In steady shear, the viscosity of the trehalose decanoate solutions do not exhibit any shear rate dependence from 1 to 100 s(-1) for concentrations up to 42 wt%. Below 0.8 wt%, the viscosity remains constant and close to that of water; then, between 0.8 and 23 wt%, the viscosity shows a quadratic increase with surfactant concentration. For higher concentrations, up to 42 wt%, no further significant increase in viscosity is observed. In oscillatory shear experiments, the solutions exhibit viscoelastic properties. The observed rheological behavior as a function of concentration and temperature may be due to a progressive evolution of the trehalose decanoate molecular associations: as the concentration increases, the system evolves towards an entangled and/or partially branched or cross-linked micellar network, and eventually a multiconnected network of cross-linked micelles.

Effect of acyl donor chain length and substitutions pattern on the enzymatic acylation of flavonoids.

Rutin and esculin were enzymatically acylated with different aliphatic acids as acyl donors (fatty acids, dicarboxylic acids and omega-substituted fatty acids) by an immobilized lipase from Candida antarctica. The effect of the water content and the acyl donors pattern on the flavonoid initial acylation rate and conversion yield were investigated. The obtained results indicated that the water content of the medium has a strong effect on the performance of these reactions. The best conversion yields were reached when the water content was kept lower than 200 ppm. At low water content of the medium, these syntheses are influenced by carbon chain length and substitution pattern of the acyl donors. Higher conversion yields of esculin and rutin (>70%) were obtained with aliphatic acids having high carbon chain length (>12). Moreover, it has been found that the amine and thiol groups on omega-substituted fatty acid chain were unfavourable to these reactions. The 1H NMR and 13C NMR analyses of some synthesized esters (esculin and rutin palmitate) show that only monoesters were produced and that the esterification takes place on the primary OH of glucose moiety of the esculin and on the secondary 4"'-OH of the rhamnose residue of rutin.

Characterisation of the enzyme activities involved in the valine biosynthetic pathway in a valine-producing strain of Corynebacterium glutamicum.

The enzyme activities of the valine biosynthetic pathway and their regulation have been studied in the valine-producing strain, Corynebacterium glutamicum 13032DeltailvApJC1ilvBNCD. In this micro-organism, this pathway might involve up to five enzyme activities: acetohydroxy acid synthase (AHAS), acetohydroxy acid isomeroreductase (AHAIR), dihydroxyacid dehydratase and transaminases B and C. For each enzyme, kinetic parameters (optimal temperature, optimal pH and affinity for substrates) were determined. The first enzyme of the pathway, AHAS, was shown to exhibit a weak affinity for pyruvate (K(m)=8.3 mM). It appeared that valine and leucine inhibited the three first steps of the pathway (AHAS, AHAIR and DHAD). Moreover, the AHAS activity was inhibited by isoleucine. Considering the kinetic data collected during this work, AHAS would be a key enzyme for further strain improvement intending to increase the valine production by C. glutamicum.

Flexibility of the metabolism of Corynebacterium glutamicum 2262, a glutamic acid-producing bacterium, in response to temperature upshocks.

In order to test the temperature sensitivity of glutamate production metabolism, several temperature shifts, from 33 to 37, 38, 39, 40 or 41 degrees C, were applied to the temperature-sensitive strain, Corynebacterium glutamicum 2262, cultivated in a 24-h fed-batch process. Whereas glucose was entirely dedicated to biomass synthesis when cells were grown at 33 degrees C, applying temperature upshocks, whatever their range, triggered a redistribution of the carbon utilisation between glutamate, biomass and lactate production. Although increasing the culture temperature from 33 to 37, 38, 39 or 40 degrees C resulted in final glutamate titers superior to 80 g/l, temperatures resulting in the best chanelling of the carbon flow towards glutamic acid synthesis were 39 and 40 degrees C. Moreover, this study showed that the higher the temperature, the slower the growth rate and the higher the lactate accumulation.

Exopolysaccharide production by Propionibacterium acidi-propionici on milk microfiltrate.

AIMS: The aims of this work were to evaluate growth and exopolysaccharide (EPS) production properties of Propionibacterium acidi-propionici DSM 4900 on milk permeate. METHODS AND RESULTS: Anaerobic growth on milk permeate was only possible if supplemented with yeast extract (YE). Fermentation capacities of the strain were significantly improved by further increasing the supplemented YE. At 5 g l(-1) YE, consumption of 45 g l(-1) lactose to produce 9 g l(-1) biomass, 34 g l(-1) organic acids and 0.65 g l(-1) EPS was observed. From a kinetic point of view, EPS production occurred during the bacteria growth phase. At the excreted polysaccharide level, the medium showed shear-thinning behaviour with a relatively high apparent viscosity of up to 30 mPa.s (milli.Pascal.second) at a shear rate of 17 s(-1). CONCLUSION: EPS production by P. acidi-propionici DSM 4900 on milk permeate showed promising rheological behaviour of the milk-derived medium obtained, even at a low production level. SIGNIFICANCE AND IMPACT OF THE STUDY: A kinetic study on EPS production by a food-grade bacterium that could be used in situ in alimentation was carried out.

Study of the effects of temperature, pH and yeast extract on growth and exopolysaccharides production by Propionibacterium acidi-propionici on milk microfiltrate using a response surface methodology.

AIMS: To study the effects of temperature, pH and yeast extract (YE) concentration on growth and exopolysaccharide (EPS) production by Propionibacterium acidi-propionici DSM 4900 cultivated on milk microfiltrate. METHODS AND RESULTS: A multifactorial approach using a Response Surface Methodology (RSM) was followed. The results indicated that both growth, and EPS and organic acids production, were influenced by pH, temperature and YE concentration. Biomass and organic acids production occurred in all the tested domains, whereas EPS production was only possible in a narrow pH range (5.3-6.5). The results clearly showed that the optimal conditions for EPS production were different to those for optimal growth. The effect of YE on EPS production was not only due to an increase in growth but also to a direct effect on the production of EPS. The temperature played a major role. A decrease of temperature induced a slowing down of both growth and organic acids production, making the essential factors of the medium and the precursors of EPS biosynthesis more available and hence, leading to an increase in EPS production. CONCLUSION: The effects of pH, temperature and YE were determined, allowing the definition of favourable, though non-optimal, conditions for EPS production: 23 degrees C, pH 6 and 3 g l(-1) YE concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of a multifactorial approach for investigating the effect of fermentation conditions on EPS production has been demonstrated.

Amino acids metabolism by VO 208 hybridoma cells: some aspects of the culture process and medium composition influence.

In the present study an approach has been developed in order to examine the consequence of essential and non essential amino acid supplementation on VO208 hybridoma cells behaviour. The effect of amino acid enrichment has been studied taking into account the culture process, i.e., batch or continuous culture mode and the medium composition, i.e., a home made serum-free medium or a serum containing one. A group of 4 amino acids, i.e., Ser, Pro, Gly and Arg presented atypical evolution pattern of their extracellular concentration depending on the type of the medium and on the culture mode. Some amino acids were probably involved in the limitation of the cellular proliferation. Met was one of the amino acids that appears to may have been at limiting concentration in all cases. In continuous culture mode, an enrichment of amino acids resulted in a rapid improvement of the viable cell density in both media, with or without the presence of serum. For most amino acids, supplementation during continuous culture induced an increase of the amino acid uptake rate. A comparative analysis of amino acids utilisation, depending on the culture conditions studied in the present study, has been performed in order to propose an overall picture of amino acids metabolism by VO 208 Hybridoma cell line.

Determination of growth and lysis kinetics in plant cell suspension cultures from the measurement of esterase release.

The death of Medicago sativa L. cells cultivated in a batch culture was investigated by measuring both the appearance of intact dead cells determined on the basis of the trypan blue (TB) dye exclusion, and the release of the cytoplasmic esterase activity into the culture medium upon cell death. Taking into account the strong instability of this released esterase activity, the total dead cell and lysed cell densities have been estimated. A mechanism for cell death and lysis is proposed and the specific rates of cell growth, death and lysis estimated. The specific rate of appearance of TB dead cells was low and essentially constant (0.25 day(-1)) during the first 8 days of the batch culture, and then increased above 1.5 day(-1) after 2 weeks of cultivation. Whereas no lysis occurred during the first seven days, this phenomenon occurred during the second period and accounted for about 20% of the total cell death by the end of the process. Thus, the viability determined by the trypan blue exclusion method appeared to be invalid after 7 days of culture. When lysis of viable cells is taken into consideration, the specific growth rate was significantly increased and growth was shown to continue for a further 8 days. Increased sensitivity of the cells to shear stresses and consequent cell lysis could be the result of a 35% increase in the cell size

Importance of phosphoenolpyruvate carboxylase of Corynebacterium glutamicum during the temperature triggered glutamic acid fermentation.

To give clues about the respective importance of phosphoenol-pyruvate carboxylase (PEPc) and pyruvate carboxylase (Pc) in Corynebacterium glutamicum metabolism during a temperature triggered glutamic acid fermentation, PEPc activity was genetically amplified and Pc activity was suppressed by biotin limitation in the culture medium. In absence of Pc activity, glutamate production was dramatically reduced whereas lactate excretion was strongly increased. Whereas PEPc amplification in excess of biotin (4 mg/L) only slightly modified the cell kinetics, under biotin limiting conditions this amplification strongly improved the glutamate production (4 microg/L). When Pc was absent, PEPc activity was sufficient to allow up to 70% of the maximal glutamate production rate and seemed to have an important anaplerotic role, especially at the beginning of the production phase. In contrast, Pc was predominant during the remainder of the glutamate fermentation.

Glutamate excretion as a major kinetic bottleneck for the thermally triggered production of glutamic acid by Corynebacterium glutamicum.

The study was aimed at evaluating the extent of flux control exercised by the amino acid excretion step on the glutamate production flux in C. glutamicum 2262 strain that is induced for glutamate excretion by an upward temperature shift. Cells initially induced to excrete glutamate were cultivated at different controlled temperatures between 33 and 40 degrees C, and changes in glutamate excretion flux and intracellular concentration were determined in response to increased culture temperature. The fastest growth rate of 0.45 h(-1) and the lowest glutamate excretion rate of 1 mmole/g dw x h were observed at 33 degrees C, together with a high intracellular 0.5 mmole/g dw glutamate accumulation. On the contrary, the fastest glutamate excretion rate of 6 mmole/g dw x h was obtained at 40 degrees C, when cell growth was arrested and the internal glutamate level reduced to 0.25 mmol/g dw. The observed sixfold increase in excretion flux as a result of the temperature increase clearly suggests a specific effect of temperature on the glutamate export system which appears as the major kinetic bottleneck for the glutamate production flux. This conclusion is corroborated by the high internal accumulation of glutamate which, even under the fastest excretion conditions, severely inhibits the activity of the glutamate biosynthesis pathway.

Na-butyrate increases the production and alpha2,6-sialylation of recombinant interferon-gamma expressed by alpha2,6- sialyltransferase engineered CHO cells.

A non-human like glycosylation pattern in human recombinant glycoproteins expressed by animal cells may compromise their use as therapeutic drugs. In order to correct the CHO glycosylation machinery, a CHO cell line producing recombinant human interferon- gamma (IFN) was transformed to replace the endogenous pseudogene with a functional copy of the enzyme alpha2,6-sialyltransferase (alpha2,6-ST). Both the parental and the modified CHO cell line were propagated in serum-free batch culture with or without 1 mM sodium butyrate. Although Na-butyrate inhibited cell growth, IFN concentration was increased twofold. The IFN sialylation status was determined using linkage specific sialidases and HPLC. Under non- induced conditions, IFN expressed by alpha2,6-engineered cells contained 68% of the total sialic acids in the alpha2,6- conformation and the overall molar ratio of sialic acids to IFN was 2.3. Sodium butyrate addition increased twofold the molar ratio of total sialic acids to IFN and 82% of total sialic acids on IFN were in the alpha2,6-conformation. In contrast, no effect of the sodium butyrate was noticed on the sialylation of the IFN secreted by the alpha2,6-ST deficient parental cell line. This study deals for the first time with the effect of Na-butyrate on CHO cells engineered to produce human like sialylation.

A new methodology for plant cell viability assessment using intracellular esterase activity.

A plant cell suspension culture of Alfalfa (Medicago sativa L.) was grown in a bioreactor using a batch procedure. The cytoplasmic esterase activity (EC 3.1) was extracted from the cells and measured during cultivation using fluorescein diacetate as the fluorogenic substrate. This enzymatic activity was conclusively found to be correlated to cell viability assessed with the membrane integrity test using the trypan blue dye. This new viability determination method is convenient, simple and can be reproduced because: (1) the difficult step of counting the cells when using the trypan blue exclusion method is avoided and (2) the esterase activity level per viable cell constituted of numerous enzymes depends on cell viability but is independent of cellular metabolism.

Genetic engineering of α2,6-sialyltransferase in recombinant CHO cells and its effects on the sialylation of recombinant interferon-γ.

The CHO cell line has achieved considerable commercial importance as a vehicle for the production of human therapeutic proteins, but is known to lack a functional copy of the gene coding for α2,6-sialyltransferase (EC 2.4.99.1). The cDNA for rat α2,6-ST was expressed in a recombinant CHO cell line making interferon-γ, using a novel in vitro amplification vector. The enzyme was expressed efficiently, and resulted in up to 60% of the total sialic acids on interferon-γ being linked in the α2,6-conformation. This sialic acid linkage distribution was more akin to that seen in natural human glycoproteins. In the most successful cell clones, expression of α2,6-sialyltransferase improved the overall level of sialylation by up to 56%, and had no adverse effects on cell growth, IFN-γ productivity or other aspects of IFN-γ glycosylation. These experiments demonstrate how the glycosylation machinery of rodent cells can be genetically manipulated to replicate human tissues.

Modeling of an industrial alcohol fermentation and simuiation of the plant by a process simulator.

The aim of the present study was the development of a general simulation module for fermentation within the framework of existing chemical process simulators. This module has been applied to an industrial plant which produces ethanol from beet molasses and fresh beet juice by Saccharomyces cerevisiae. An unstructured mechanistic model has been developed with kinetic laws that are based on a chemically defined reaction scheme which satisfies stoichiometric constraints. This model can be applied to different culture conditions and takes into account secondary byproducts such as higher alcohols. These byproducts are of prime importance and need to be correctly estimated because a sequence of distillation columns follow the fermentor in the plant. Important measurement campaigns have been performed on the plant to validate the model. Plant operation has been successfully simulated using the same kinetic model for both continuous and fed-batch modes of production. (c) 1995 John Wiley & Sons, Inc.

Insulin utilization and kinetic effect on hybridoma metabolism in batch and continuous cultures.

This paper discusses the insulin utilization kinetics and the effect of its concentration during batch and continuous mass cultures of the murine VO208 hybridoma cells, using a home-made serum-free medium. Our results show that insulin is utilized by the cells, with a specific rate of 1 relative units (RU) per 10(9) cells per h in batch culture. In continuous reactor running at different insulin levels, this consumption rate is observed to vary from 0.13 to 0.55 RU per 10(9) cells per h when the insulin activity increases from 0.3 to 35 RU l-1 and then to stabilize for higher insulin levels until 110 RU l-1. A low insulin amount in the medium around 0.01 RU l-1, which is near physiological levels, is found sufficient to promote the cell growth. Interestingly, we observe that too high insulin levels, above 25 RU l-1, induce a reduction of the cell density due to an inhibitory effect on the maximal specific cell growth rate. Furthermore, the specific rate of MAb production is found to be independent of the insulin amount in the medium.

Software sensors for the monitoring of perfusion cultures: evaluation of the hybridoma density and the medium composition from glucose concentration measurements.

New software sensors based on the Extended Kalman Filter technique have been developed for the monitoring of animal cell perfusion cultures. They use a kinetic model describing the growth, death and metabolism of hybridoma cells as a function of the medium composition. The model was initially validated on a batch culture and found to correctly predict the continuous perfusion culture kinetics, except for the production of ammonia and lactate. Using the measurement of a single component in the culture medium, in this case glucose, the Extended Kalman Filter provides an excellent evaluation of the time variation of the concentrations of living and dead cells, of glutamine and antibodies, during the whole perfusion culture for a retained cell density rising from 1 to 11 x 10(6) cells.ml-1 inside the reactor.

Production of a membrane-bound protein, the human gamma-glutamyl transferase, by CHO cells cultivated on microcarriers, in aggregates and in suspension.

Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h-1, the highest cell density (near 1.3 x 10(6) cells ml-1), and the highest enzyme activity around 300 mU ml-1, which corresponded to a specific cellular level of 20 mU 10(-5) cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems.

Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release.

The death of the hybridoma VO 208 in a continuous culture at pH 7 and 6.8 was investigated by measuring both the appearance of visible dead cells which do not exclude the trypan blue dye and the release of lactate dehydrogenase (LDH) in the culture medium. The intracellular LDH was found to be completely released either when live cells lysed or when they were transformed into visible dead cells. No significant lysis of blue dead cells could be observed at the two different pH. Using a LDH balance over the culture system, cell lysis was found negligible at pH 7, but accounted for 20% of the total cell death at pH 6.8. A methodology is proposed to evaluate the rate constants of hybridoma lysis and total death. For the investigated cell line in continuous culture, the calculated total cell death rate constant was found to increase from 0.002 h-1 to 0.01 h-1 when decreasing the pH from 7 to 6.8.

The use of lactate dehydrogenase (LDH) release kinetics for the evaluation of death and growth of mammalian cells in perfusion reactors.

A general methodology is proposed to estimate the actual specific growth and death rate of mammalian cells in continuous perfusion reactors from the monitoring of the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) in the culture medium. The procedure is illustrated on a perfusion culture of human tumor kidney cells growing on microcarriers and producing prourokinase (PUK). The intracellular LDH content of living attached cells is checked to be constant during the culture. However, cells detached from the microcarriers, and counted dead because of the uptake of trypan blue, have only released part of their intracellular LDH. In the culture medium, LDH is relatively stable as the loss of activity does not exceed 5% per day. The time variation of the LDH concentration in the medium is used to calculate the total amount of lysed and actually produced cells in the reactors, hence, the actual specific rates of cell growth and death. It is thus found that the stationary phase observed after 400 h of perfusion culture is the result of equal growth and death rates, with a daily renewal of living cells on the microcarriers near 10%. Moreover, for the cell line tested, the production of PUK is associated with cellular growth.

Comparison of specific rates of hybridoma growth and metabolism in batch and continuous cultures.

For the mouse hybridoma cell line VO 208, kinetics of growth, consumption of glucose and glutamine, and production of lactate, ammonia and antibodies were compared in batch and continuous cultures. At a given specific growth rate, different metabolic activities were observed: a 40% lower glucose and glutamine consumption rate, but a 70% higher antibody production rate in continuous than in batch culture. Much higher metabolic rates were also measured during the initial lag phase of the batch culture. When representing the variation of the specific antibody production rate as a function of the specific growth rate, there was a positive association between growth and antibody production in the batch culture, but a negative association during the transient phase of the continuous culture. The kinetic differences between cellular metabolism in batch and continuous cultures may be the result of modifications in the physiology and metabolism of cells which, in continuous cultures, were extensively exposed to glucose limitations.