Jean-Noël Barbotin

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.

1H NMR spectroscopic determination of poly 3-hydroxybutyrate extracted from microbial biomass

Abstract 1 H NMR analysis was performed in order to identify the nature of the poly 3-hydroxyalkanoate (PHA) accumulated by Rhizobium meliloti M5N1 and determine its concentration within the cells. Since the PHA was identified as being poly 3-hydroxybutyrate (PHB), the method of quantification was previously tested on standard PHB. The use of either methanol or benzene as the internal reference showed good accuracy. The extraction of PHB was carried out using dispersions of sodium hypochlorite and chloroform. A treatment time of 1 h with a 10% concentration of hypochlorite allowed for total recovery of PHB. After extraction of different amounts of PHB, aliquots were submitted to 1 H NMR analysis. Methanol used as the internal reference gave an overvaluation of the PHB content. However, benzene met all the requirements of an internal reference due to its chemical inertia in our experimental conditions. NMR data obtained using benzene as the internal reference are in good agreement with corresponding data obtained by gas chromatographic (GC) analysis.

A (1→4)-β-D-glucuronan excreted by a mutant of the Rhizobium meliloti M5N1 strain

Abstract A mutant of the R. meliloti M5N1 strain has been selected. This strain, R. meliloti M5N1 CS (NCIMB 40472), excretes an extracellular material composed of 2-O-Ac-β-GlcpA, 3-O-Ac-β-GlcpA, 2,3-di-O-Ac-β-GlcpA and three species of β-GlcpA residues 1→4 linked. For the culture conditions used, the weight average molecular weight of the polymer varied in the range of 6 × 104 < Mw < 4 × 105. High molecular weight glucuronate forms thermoreversible gels at 5 g L−l. In the presence of divalent cation such as Ca2+ or trivalent cations such as Cr3+ or Fe3 +, cross linking of the polymer occurs. This polysaccharide is the first exocellular (1→4)-β-D-glucuronan produced by a R. meliloti strain.

Nitrogen, carbon and light-mediated regulation studies of carotenoid biosynthesis in immobilized mycelia of Gibberella fujikuroi

Some aspects of the regulation of carotenoid biosynthesis in immobilized Gibberella fujikuroi mycelia were studied and the results compared to those obtained from freely suspended mycelia. Nitrogen limitation induced carotenoid biosynthesis in immobilized G. fujikuroi mycelia under non-limiting carbon conditions. In addition, certain water soluble polyketide like red pigment was synthetised and released into the culture medium under same conditions. It was found that carbon/nitrogen ratio plays an important role in carotenoid biosynthesis and it determines the extension of its production. Noticeable differences were also found in type and quantity of other secondary metabolites produced by both immobilized and freely suspended mycelia of G. fujikuroi. Photoinduction of carotenoid biosynthesis in immobilized mycelia was also studied. The results suggest that regulation of carotenoid biosynthesis is qualitatively similar to that observed in freely suspended mycelia and is shown to be enhanced by immobilization in natural polymers.

Synthesis of wax ester through triolein alcoholysis: Choice of the lipase and study of the mechanism

Abstract Eight microbial lipases and one animal tissue lipase were tested for their ability to support alcoholysis between triolein and stearyl alcohol to produce wax ester. The lipases from Alcaligenes sp. and Chromobacterium viscosum were shown to produce the best ester yield (about 53%); however, the reaction was catalyzed in a shorter length of time using the lipase from Alcaligenes sp. (5 h) as compared to the lipase from C. viscosum (200 h). The lipases from Mucor javanicus, Mucor miehei, and Pseudomonas fluorescens showed 35% ester yield within 150 h. The other lipases, i.e., the lipases extracted from porcine pancreas, Candida rugosa, Rhizopus javanicus, and Rhizopus niveus showed only low catalytic activity (less than 20%). The lipase from Alcaligenes sp. was used for further investigations on the mechanism of the reaction. A regioselectivity toward the sn-1,3-positions of the acylglycerols was shown during the first hours of reaction. After 5 h when ester synthesis and triolein consumption stopped, the continuous evolution of the concentration of monoolein and diolein isomer forms was ascribed to isomerization reactions.

Exopolysaccharide production by the Rhizobium meliloti M5N1 CS strain. Location and quantitation of the sites of O-acetylation

Abstract The mutant strain M5N1 CS of Rhizobium meliloti is able to produce, in a Rhizobium complete medium supplemented with fructose or sucrose (1% w/v), a partially acetylated homopolymer of glucuronic acid. During batch cultivation, the polysaccharide production is observed while the pH of the medium decreases slightly. The nature of the carbon source and the control of pH have no effect on the degree of acetylation of the polymer produced in the flasks. About 40% of glucuronic residues of the polymer are monoacetylated at C-3 or at C-2, C-3 substitution is about twice that of C-2 and remains constant during the fermentation.

Immobilization of Recombinant Bacteria: A Strategy to Improve Plasmid Stability

Recombinant DNA technology opens up important avenues in the acquisition of improved microorganisms for large-scale production of metabolites or proteins of commercial interest. The productivity of a biochemical reactor employing plasmids of genetically engineered cells is, however, largely affected by the instability of plasmids during prolonged cultivation.*,* Two kinds of instability have been observed: structural (i.e., genetic instability of the plasmid itself) and segregational (i.e., efficiency of plasmid transfer to daughter cells during cell division). All these phenomena can occur both in batch and continuous culture, but the most common problem observed in continuous culture is segregational instability. In such a case, owing to the difference of specific growth rate between the recombinant and the plasmid-free cells, the latter are thus able to rapidly overtake the overall population. Several factors have been found to influence plasmid maintenance depending on characteristics of the plasmid itself (size, copy number, expression level of the gene, etc.), the host cell (genotype), and also the growth medium and culture conditions (dilution rate, temperature, agitation, etc.). The application of selection pressure (e.g., addition of antibiotic) in the reactor to supress the growth of plasmid-free cells is one of the most commonly employed methods to overcome the drawbacks of recombinant plasmid instability. However, antibiotics are rather expensive to use in industrial-scale cultivation. Other strategies have been proposed, such as the insertion of a specific DNA sequence (active partitioning locus) that guarantees stable plasmid inheritance and high expression of the target gene.3 On the other hand, immobilization of whole living cells has also been used to enhance plasmid ~tability.~ In this paper, a short review of research on immobilized recombinant cells is presented.

NMR on-line monitoring of esterification catalyzed by cutinase.

A nuclear magnetic resonance (NMR) method has been developed to monitor on-line lipase-catalyzed esterification reactions without the need to sample the reaction medium. The technique, through (1)H NMR, measures the concentrations of alcohol, ester, hydroxylic hydrogens in the organic phase, and hydroxylic hydrogens in the aqueous phase, if any. Also, the chemical shift evolution of the two types of hydroxylic hydrogens has been followed, providing information on water content of the organic phase and on the appearance of a distinct aqueous phase. As far as (13)C NMR is concerned, it has been possible to measure, first the acid and the ester concentrations in the carbonyl region, and second, the alcohol and the ester concentrations in the methylene region. All (1)H and (13)C results are in agreement with one another. Furthermore, NMR allows for the choice of detection zone. Preliminary studies on the solid phase proved the presence of much more water in the solid phase than in the organic phase, and also gave evidence of the existence of two types of esters, one in the organic phase, mainly associated with the acid, and the other one not associated with the acid, most probably entrapped within the solid enzyme.

Lipase-catalyzed synthesis of tricaprylin in a medium solely composed of substrates. water production and elimination

The conditions for tricaprylin synthesis using Lipozyme (immobilized 1,3-specific Mucor miehei lipase) in a medium solely composed of glycerol and caprylic acid (octanoic acid) in stoichiometric amounts are 60°C with air ventilation in an open batch. The kinetics of mono-, di-, and triotanoylglycerol synthesis and caprylic acid esterification are correlated with those corresponding to water production and water activity along the esterification reaction. The technical feasibility of studying the time course of the reaction and water loss monitoring only by weight and Karl-Fisher measurements is also described.

Physicochemical properties of extracellular (1→4)-β--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.