Improvement of plasmid stability of recombinant Bacillus-subtilis cells in continuous immobilized cultures.

The immobilization of recombinant Bacillus subtilis in K-carrageenan gel beads has been performed in order to study the growth conditions inside the gel beads and to improve plasmid stability. Bacterial colonies showing high cell density were studied using scanning electron microscopy. A series of continuous cultures of free and immobilized B. subtilis MT119 (pHV1431, pIL252 and pIL252 Kpn) have been developed without selection pressure. In the free-cell systems, it was found that a loss of plasmid vectors occurred after a short period. In contrast, in the immobilized cell systems, plasmid-free segregants were not detected in any of the cases during the first 80 h of the culture.

Immobilization of L-glutamate dehydrogenase into soluble cross-linked polymers. ADP effect and electron microscopy studies.

Active soluble cross-linked L-glutamate dehydrogenase (L-glutamate: NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) albumin polymers were produced. Electron microscopic studies and kinetic properties were studied with the polymer in solution and compared with previous published data about the enzyme immobilized inside proteic films (Barbotin, J.N. and Breuil, M. (1978) Biochim. Biophys. Acta 525, 18--27). The glutaraldehyde effect on activity yield, ADP and beta-NAD+ protection, stability and pH rate profile were studied and discussed. Apparent Michaelis constants were determined with soluble polymers produced with or without ADP during the grafting process. Experiments were performed on the regulatory properties of immobilized glutamate dehydrogeanse showing the decrease of ADP activation and GTP inhibition as compared to the free form. In other respects, electron microscopy observations showed morphological differences between the two populations of soluble polymers produced in presence of ADP, obtained after gel filtration on Sepharose 6B. Linear aggregates of high molecular weight and classical soluble polymers were obtained. Similar Km values and regulatory properties were exhibited by the two forms, demonstrating the absence of interdependence between the allosteric control and the polymerization of enzyme monomers.

1H-NMR on line monitoring of water activity during lipase catalysed esterification.

1H-NMR spectroscopy is used to determine simultaneously the water activity (aw) and the time course of an esterification reaction catalysed by a lipase. Chemical shifts signals of hydroxylic hydrogens in fast exchange (i.e the average hydroxylic signal of acid, alcohol and water) varies with water activity and ester content. Calibration curves have been established from model media composed of the substrates and various ester contents, at different water activities, in order to mimic a reaction medium. One relationship is established between water activity, hydroxylic hydrogen signal chemical shift and ester content. In order to estimate the water activity evolution as a function of time, this last relationship is applied to the hydroxylic hydrogen chemical shift measured in a reaction medium where the Rhizomucor miehei lipase in a powder form is suspended in the liquid substrates. This alternative way of determining the water activity based on hydroxylic hydrogen chemical shift presents some advantages over more classical means, i.e. time saved and inaccuracies avoided by monitoring without handling the sample.

Mechanism of gluconate synthesis in Rhizobium meliloti by using in vivo NMR.

The dehydrogenation of [1-(13)C]- and [2-(13)C]glucose into gluconate was monitored by NMR spectroscopy in living cell suspensions of two Rhizobium meliloti strains. The synthesis of gluconate was accompanied, in the cellular environment, by the formation of two gluconolactones, a gamma-lactone being detected in addition to the expected delta-lactone. These lactones--as well as the gluconate--could be further metabolized by the cells. The delta-lactone was utilized faster than the gamma-lactone. The presence--in significant amounts--and the relative stability of the lactones raise the question of their possible physiological significance.

Immobilized organelles and whole cells into protein foam structures: scanning and transmission electron microscope observations.

Protein foam structures bearing cells and organelles were produced by using a co-crosslinking method with serum albumin and glutaraldehyde at sub-zero temperature. Morphological observations obtained with scanning and transmission electron microscopy indicate macroporous and homogeneous structures. The glutaraldehyde concentration was varied to reveal its effect on immobilized red cells. It was observed that the structural appearance of preatreated cells or subcellular fractions (thylakoids from lettuce ; spheroplasts and chromatophores from R. capsulata) is preserved during the immobilization process. The morphological features of foam particles are always related to the observed kinetic activities of the specimens.

A histochemical model dealing with an immobilized glucose oxidase-peroxidase system. The influence of diffusion limitations on histochemical results.

A histochemical model dealing with an immobilized bienzyme system (glucose oxidase-peroxidase) is presented. The model is an artificial proteic membrane obtained by a previously described co-cross-linking process. The kinetic properties of free and immobilized horseradish peroxidase were studied when 3,3'-diaminobenzidine is used as a hydrogen donor substrate. A new direct method was developed for immobilized enzyme activity measurements. Computer simulation based on experimental kinetic parameters was performed in order to discuss electron microscopy results. By changing diffusion limitations, various profiles of insoluble product were visualized inside the proteic film and no geometrical similarity was seen between enzyme distributions and insoluble osmiophilic product patterns.

Kinetic, computer, and electron microscopic studies dealing with an artificial enzyme membrane: theoretical and experimental evaluation of the cytochemical demonstration of acetylcholinesterase.

The cytochemical demonstration of acetylcholinesterase is theoretically and experimentally evaluated with a model system. The model is an artificial proteic membrane in which acetylcholinesterase homogeneously is immobilized chemically by a bifunctional agent, glutaraldehyde. The copper-thiocholine histochemical method is studied kinetically and the system is simulated by computer calculations based on experimental kinetic parameters and numerical analysis methods. In addition, the corresponding electron micrographs are presented. These studies lead to the conclusions that the system is ruled by diffusional constraints and that enzyme distribution and repartition of the insoluble electron dense product are not circumscribed by any specific conditions.

Stability of plasmid pHV1431 in free and immobilized cell cultures. Effect of temperature.

The segregational and structural stability of pHV1431 has been examined in Bacillus subtilis grown at 30 and 37 degrees C in continuous cultures without selection pressure. Immediately after appearance of plasmid-free cells in the reactor, a competition was observed between bacteria that favored plasmid-free cells because of the faster growth. A stronger instability was found at 30 degrees C compared to that at 37 degrees C. At 30 degrees C after 50 hours of culture, 2% of the cells carried the plasmid, whereas at 37 degrees C this percentage was reached after 130 hours. In both cases, no structural instability was observed. To improve the stability, the recombinant Bacillus subtilis (pHV1431) was immobilized in kappa-carrageenan gel beads. In comparison to free cell systems, a higher cell concentration was obtained. Moreover, the plasmid was maintained stable for longer periods; after 150 hours of culture 40% of cells in the reactor still carried the plasmid at both temperatures.

Relevance and isotopic assessment of hexose-6-phosphate recycling in micro-organisms.

Some pathways of hexose-6-phosphate recycling--those involving a breakdown of the hexose skeleton--through carbohydrate metabolism of micro-organisms were analyzed for both metabolic and isotopic effects. Two modes of recycling were proposed based on the degree of alteration of the hexose molecule through the catabolic part of the cycle. Simulated operation of most of these pathways resulted in increased synthesis of hexose-6-phosphate and NADPH, and reduced the NADH and moreover the ATP synthesis within the carbohydrate metabolism. A basic model for the quantitative assessment by means of isotopic studies of the processes of hexose-6-phosphate recycling is presented. The model was initially designed for the study of micro-organisms producing polysaccharides, but it can be extended to other situations.

Immobilized and free cell continuous cultures of a recombinant E. coli producing catechol 2,3-dioxygenase in a two-stage chemostat: improvement of plasmid stability.

The immobilization of recombinant strains of E. coli W3110/pTG205 in K-carrageenan gel beads improves the plasmid stability during continuous cultures in the absence of selection pressure. Since, xyl E gene (which encodes catechol 2,3-dioxygenase from Pseudomonas putida) transcription is controlled by the trp promoter, the effects of tryptophan (repressor) and 3 beta-indolyl acrylic acid (derepressor) on pTG 205 stability and enzyme production have been studied in both free and immobilized cell cultures. A two-stage continuous culture system running for 150 h is described. In the first stage an immobilized culture is performed in the presence of tryptophan with a significant plasmid stability. The cells released from the gel beads are continuously transferred in the second stage reactor where expression is induced by 3 beta-indolyl acrylic acid. In these conditions an efficient production of catechol 2,3-dioxygenase is observed.

Enhanced production of scopolin bySolanum aviculare cells immobilised within Ca-alginate gel beads.

Different matrices, obtained by varying calcium (0.1 to 1.5M) and alginate (1 to 1.5%) concentrations, were used to study the influence of immobilisation parameters on the behaviour ofS. aviculare. A significant modulation of cell growth, cell release, and scopolin production and excretion has been observed. Physiological and morphological characteristics ofSolanum aviculare cells immobilised within Ca-alginate beads were notably different from those of suspended cells. ImmobilisedS. aviculare have accumulated scopolin (up to 120 µg·g-1 FWB) within beads and excreted it into the culture medium (up to 8 µg·g-1 FWB). Contrary to suspended cells which have accumulated only traces of this metabolite within intracellular compartments (1 µg·g-1 FWB), no scopolin has been found into the culture medium.

Agitation rate effects on plasmid stability in immobilized and free-cell continuous cultures of recombinant E. coli.

Escherichia coli B/pTG201 recombinant cells were immobilized by entrapment in a carrageenan gel and cultivated in nonselective media to investigate the effect of agitation rate on plasmid stability, biomass concentration, and enzyme productivity. These parameters were studied in continuous cultures for free and immobilized cells, respectively. Immobilized recombinant cells exhibit an increase in the stability of the plasmid pTG201 compared to free cells, even under conditions where the tendency of plasmid stability for free cells decreased generally more rapidly under a higher agitation rate. Intensive agitation, resulting also in a strong shear stress, greatly reduced cell concentration within gel beads throughout the course of growth. Higher enzyme expression of catechol 2-3, dioxygenase was also obtained in leaked cells due to better maintenance of plasmid stability and higher plasmid copy number with regard to free cells. Enzyme productivity of leaked and free cells in minimal medium decreased with the increase in agitation rate, due to decreased plasmid stability; however, in LB medium, it increased in the presence of higher agitation rate related to important cell concentration.

Response surface analysis of chlortetracycline and tetracycline production with K-carrageenan immobilized Streptomyces aureofaciens.

A full-factorial experimental design at three levels with two independent variables, carrageenan concentration (1.0, 1.5, and 2.0%) and potassium chloride concentration (0.3, 0.7, and 1.1 M) was studied in order to analyze the effect of both factors on the antibiotic production of K-carrageenan-immobilized mycelia of Streptomyces aureofaciens. The response surfaces obtained have indicated that both carrageenan and potassium chloride concentrations have a pronounced effect on the yield of chlortetracycline (CTC) and tetracycline (TC) produced by S. aureofaciens. By exclusively varying the immobilization conditions, the tetracycline production can be enhanced more than eight times (12.3 mg g-1 biomass for immobilized cells vs. 1.5 mg g-1 biomass for free cells) in comparison with free-cell mycelial cultures.

Effects of salts on production and on O-acetylation of glucuronan excreted by the Rhizobium meliloti M5N1 CS strain.

Acetylation determined by 1H-nuclear magnetic resonance spectroscopy and production of the glucuronan excreted by the Rhizobium meliloti M5N1CS strain during cultivation in RCS medium with and without added magnesium salts have been studied. These salts induce an increase in the degree of substitution and the molar ratio of 2,3-di-O-acetyl residues. A decrease in production is observed after 75 h of fermentation as the magnesium salt concentration increases. The presence of manganese and sodium salts in the culture induces inhibition of exopolysaccharide (EPS) production. However, the structure of the EPS is similar to that of the EPS produced by standard fermentation, without modification in the degree of substitution.

Physicochemical properties of extracellular (1-->4)-beta-D-glucuronan produced by the Rhizobium meliloti M5N1CS strain during fermentation: evidence of degradation by an exoenzyme activated by Mg2+.

The mutant Rhizobium meliloti M5N1CS (NCIMB 40472) produced a partially acetylated (1-->4)-beta-D-glucuronan during fermentation. The polysaccharide (EPS) extracted from broth during fermentation by microfiltration and ultrafiltration (using a 100 kDa cut-off membrane) was characterized by size exclusion chromatography. The weight-average molecular weight (Mw) of the EPS decreased from 7.5 x 10(5) to 5 x 10(5) between 27 and 75 h of fermentation. When MgSO4.7H2O (0.8 gl-1 per day) was present in the medium during the same period, the Mw of the EPS decreased to 1.5 x 10(5). Since EPS degradation was detected after microfiltration of the fermentation medium supplemented with magnesium, the Mw decrease of the EPS extracted during fermentation may be explained by enzymic degradation of the polymer activated by Mg2+ ions.

Identification of glucuronan lyase from a mutant strain of Rhizobium meliloti.

The Rhizobium meliloti M5N1CS (NCIMB 40472) mutant strain wich induces nodule formation on alfalfa roots, produces a (1 --> 4)-beta-D-glucuronan partially acetylated. During fermentation under specific conditions, the molecular weight of the polymer decrease, the presence of polysaccharide degrading enzyme was suspected. A glucuronan lyase was identified, this new bacterial lyase produces d.p. 4 oligoglucuronans, substituents (acetates) present on the substrate reduced the enzyme activity.

Cyclic (1-->2)-beta-D-glucans excreted by the glucuronan-producing strain Rhizobium meliloti M5N1CS (NCIMB 40472) and by the succinoglycan-producing strain Rhizobium meliloti M5N1.

During fermentation, the mutant strain Rhizobium meliloti M5N1CS, which induces nodule formation on alfalfa roots, produces a partially acetylated (1-->4)-beta-D-glucuronan. In addition to this exopolysaccharide of high molecular weight, the mutant strain produces oligoglucoronates and cyclic (1-->2)-beta-D-glucans with degrees of polymerization from 17 to 30. Under the conditions applied, magnesium has no effect on cyclic glucan production by the mutant strain, but the succinoglycan production by the wild-type strain Rhizobium meliloti M5N1 increases.

alpha-Amylase production by free and immobilized Bacillus subtilis.

The effect of glucose on the alpha-amylase production by Bacillus subtilis ATCC-21556 was studied. Initial glucose concentrations up to 20 g/L were found to be directly proportional to the specific alpha-amylase production in an immobilized-cell batch system, whereas a free-cell batch system presented an inversely proportional relationship with the initial glucose concentration. This might be owing to the alpha-amylase repression by the glucose present in the culture medium. Three hundred eighty-five percent of the specific alpha-amylase production with the free-cell system was produced by the immobilized-cell batch culture.