Jean-Paul Seguin

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.

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.

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.

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.

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.

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.

Effect of Mg2+ on production and on O‐acetylation of glucuronan excreted by the Rhizobium meliloti M5N1 CS strain during fermentation

The effect of Mg2+ on glucuronan O‐acetylation and production by the Rhizobium meliloti M5N1 CS strain was studied. Magnesium ion induced the production of a more acetylated exopolysaccharide containing a greater molar ratio of 2,3‐di‐O‐acetyl residues.

13C NMR measurement of hydrogen bonding effects for hydroxylic compounds: relationships between the OH stretching frequencies, the chemical shifts of proton donor group and electron reorganization

Abstract The carbon chemical shifts induced by hydrogen bonding have been measured for complexes between phenol, methanol, benzylic alcohol and π or n bases. Linear relationships are obtained between corrected induced chemical shifts and the IR frequency shifts ΔνOH but reverse slopes result for C-1 sp2 and sp3 carbons.