Biomedical subjects

J Courtois

Publications and source records attributed to J Courtois.

At least 19 recordsLinked to original sources

PubMed2005

Purification of oligouronides using hollow-fiber membrane functionalised with L-histidine.

The pseudobioaffinity chromatography with L-histidines immobilized onto poly(ethylene-vinyl alcohol) hollow-fiber membranes was studied for a selective purification of anionic oligosaccharides. Oligoglucuronans with different degrees of polymerization and acetylation, prepared by an enzymatic degradation of bacterial polyglucuronic acid, were used as models. The adsorption and elution parameters were studied to optimise the selective adsorption. A better understanding of the physico-chemical phenomena governing this selectivity was attempted and a few hypotheses on the mechanism of selectivity are proposed.

Adsorption↗

PubMed2005

Production of glucuronan oligosaccharides using a new glucuronan lyase activity from a Trichoderma sp. strain.

Sinorhizobium meliloti M5N1CS synthesizes a homopolymer of glucuronic acids beta-(1,4) linked and variably C2 and/or C3O-acetylated. To obtain beta-Delta-(4,5)-unsaturated oligoglucuronans, various acetylated forms of this bacterial polymer were cleaved by a Trichoderma sp. GL2 glucuronan lyase. Oligomers with polymerization degrees up to 8 were then produced, purified by liquid chromatography (size exclusion and anions exchange) and characterized using 1H NMR and ESI-Q/TOF-MS. Finally, the production (in gram quantity) of pure unsaturated oligoglucuronans non-acetylated (di- and trisaccharide) was investigated thanks to the complete depolymerization of deacetylated glucuronan.

Acetylation↗

PubMed2005

Purification and characterization of a novel glucuronan lyase from Trichoderma sp. GL2.

The filamentous fungus Trichoderma sp. GL2 produces an extracellular glucuronan lyase (GL) when grown on glucuronan as the sole carbon source. In this paper, we report the purification to electrophoretical homogeneity of this polysaccharide lyase by size exclusion chromatography and anion exchange chromatography. The purified GL, classified as an endopolyglucuronate lyase, is a monomer with an apparent molecular weight of 27 kDa and an isoelectric point of 6.95. Despite an inhibition of the activity when polysaccharide substrates were substituted by acetates, the enzyme was active toward glucuronans (acetylated or not) and ulvan, leading to various (4,5)-unsaturated products as oligoglucuronans (acetylated or deacetylated), highly acetylated low-molecular-weight (LMW) glucuronans, and LMW ulvans.

Magnetic Resonance Spectroscopy↗

PubMed2005

Flax (Linum usitatissimum) seed cake: a potential source of high molecular weight arabinoxylans?

Water-soluble polysaccharides were extracted from flaxseed cake and analyzed. Two groups were separated by anion-exchange chromatography. The first one (nonretained) was the major fraction (83%) and possessed a high molecular weight (HMW) arabinoxylan (56%) with an Ara/Xyl ratio of 0.32 and an M(w) of 846 000. This polymer was accompanied by a smaller galactoglucan (44%), with an M(w) of 6.5 x 10(4). The latter group (17%), retained by the gel, was further described as a HMW pectin heterogeneous group, with, respectively, 3.1 x 10(5) and 1.3 x 10(5). Despite the presence of HMW arabinoxylans, the investigation of rheological flow sweep at the concentration of 2% (w/v) has shown a slight shear thinning behavior with a small zero-rate viscosity at 9.6 Pa.s.

Chromatography, Ion Exchange↗

PubMed2005

Methods for obtaining neutral and acid oligosaccharides from flax pectins.

Esterified acid soluble pectins from flax (Linun usitatissimum L.) were degraded either with HCl or pectin lyase. Centrifugation and 2-propanol precipitation led to the isolation of two low molecular weight polygalacturonates after acid hydrolysis of pectins. However, after pectin lyase digestion and purification by size-exclusion HPLC, (1)H NMR analyses indicated that acetylated hairy regions, large methylated and acetylated oligogalacturonides together with small unsubstituted oligogalacturonides were produced. Thus, in a few steps, a panel of substituted neutral and acidic oligosaccharides was produced from a raw plant material. Such oligosaccharides could be useful for further fractionations such as chemical saponification and enzymatic removal of neutral sugar chains from the hairy regions. The procedures used for pectin extraction, for degradation, and for the purification of fragments seem appropriate for large-scale production of biologically active oligosaccharides from flax.

Acetylation↗

PubMed2005

Structural investigations of the neutral polysaccharide of Linum usitatissimum L. seeds mucilage.

The heterogeneity of the purified water-soluble neutral fraction coming from the mucilage extract of the yellow flaxseed was investigated. After fractionation by size-exclusion chromatography, the analyse of the neutral monosaccharides composition showed a mixture of three major families of polymers. They were all identified as arabinoxylans with a constant A/X ratio of 0.24, but varying in their galactose and fucose residues in the side chains. Furthermore, the molecular weight (M(w)) analysis acquired by multi-angle laser light scattered, revealed the association of two high M(w) polymers [5.7x10(6) (11.1%) and 9.3x10(5) (42.4%) g mol(-1)] with a smaller one [3.2x10(5) g mol(-1) (45.3%)].

Adhesives↗

PubMed2004

A rapid method for determining the antimicrobial activity of novel natural molecules.

Preservatives used in the Agro-food industries may be of natural origin or obtained chemically. Because of the increasing interest of consumers in food products that contain only natural ingredients, studies on preservative molecules of natural origin, such as organic acids or peptides, have been reported in the past several years. Such studies, which require numerous assays, may be limited by the large amount of molecules required. Microscale assays provide an opportunity for testing natural components available in low quantity. This study examined a rapid method that used microplates for the evaluation of anti-microbial substances. The method was validated using five foodborne pathogens. It required a low amount of product and was convenient for the determination of correlations between the bacterial growth inhibition and concentration of the antimicrobial substance.

Anti-Bacterial Agents↗

PubMed2003

Microfiltration and ultrafiltration of polysaccharides produced by fermentation using a rotating disk dynamic filtration system.

The recovery of exopolysaccharides (EPS) produced by Sinorhizobium meliloti bacteria by dynamic microfiltration was investigated using a rotating disk device designed in our laboratory, equipped with a 0.2 microm nylon membrane. This system differs from commercially available systems by the presence of vanes on the disk which produce a very important increase in permeate flux while yielding excellent EPS transmission. For polymers produced under standard fermentation conditions (70 h at 30 degrees C), the mass flux rose to 650 g h(-1) m(-2) using a disk equipped with 2 mm vanes rotating at 2000 rpm against 380 g h(-1) m(-2) with a smooth disk at the same speed. The maximum flux observed was 1560 g h(-1) m(-2) with a 6-mm vanes disk rotating at 3000 rpm and a 36 degrees C broth. An interesting finding was that the permeate flux J(f) for various disks can be correlated by the same function of the mean shear stress at the membrane tau(wm) according to J(f) = 4.6 tau(wm) (0.717) for a 30 degrees C broth, showing that the effect of vanes is merely to increase the shear stress by raising the fluid core velocity between the membrane and the disk. With 6-mm vanes the core angular velocity was found to be 84% of disk velocity vs. 45% for a smooth disk. When the fermentation temperature was increased to 36 degrees C to produce a lower molecular weight polymer, the permeate flux rose by about 250%, much more than what could be expected from the reduction in permeate viscosity and followed the same power law with membrane shear stress as for 30 degrees C. The same device was equipped with a PES 50 kDa membrane to concentrate EPS by ultrafiltration. Permeate fluxes were of the order of 160 L h(-1) m(-2) at 2000 rpm and 30 degrees C with nearly complete EPS rejection. Finally, the net electrical power consumed by the disk was measured by subtracting the power consumed without fluid from the power during filtration at the same speed. This power increases with speed and with the presence of vanes, but since the gain provided by the vanes is very high, the specific energy per m(3) of permeate is minimal with the highest vanes tested (6 mm) and maximal for smooth disks.

Cell Division↗

PubMed2003

Polysaccharide lyases: recent developments as biotechnological tools.

Polysaccharide lyases, which are polysaccharide cleavage enzymes, act mainly on anionic polysaccharides. Produced by prokaryote and eukaryote organisms, these enzymes degrade (1,4) glycosidic bond by a beta elimination mechanism and have unsaturated oligosaccharides as major products. New polysaccharides are cleaved only by their specific polysaccharide lyases. From anionic polysaccharides controlled degradations, various biotechnological applications were investigated. This review catalogues the degradation of bacterial, plant and animal polysaccharides (neutral and anionic) by this family of carbohydrate acting enzymes.

Animals↗

PubMed2002

Cloning, sequencing and overexpression of a Sinorhizobium meliloti M5N1CS carboxymethyl-cellulase gene.

The EndS encoding sequence was isolated from Sinorhizobium meliloti M5N1CS DNA. Comparisons between the deduced amino acid sequence of the mature EndS (337 amino acids, molecular mass 36,418 Da, isoelectric point 4.92) and those of published beta-glycanases showed that this enzyme belongs to family 5 of the glycoside hydrolases. The protein was overproduced in Escherichia coli using a T7 expression system. When the purified overexpressed EndS protein was tested on cellulose-type components, the best substrate was CM-cellulose.

Base Sequence↗

PubMed2001

Structure of a polysaccharide from a Rhizobium species containing 2-deoxy-beta-D-arabino-hexuronic acid.

The structure of the extracellular polysaccharide (EPS) produced by the Rhizobium sp. B strain isolated from atypical nodules on alfalfa has been determined using a combination of chemical and physical techniques (methylation analysis, high pH-anion exchange chromatography (HPAEC), mass spectrometry and 1-D and 2-D NMR spectroscopy). As opposed to the EPS from other strains of Rhizobium, the EPS from the sp. B strain contains D-Glc together with L-Rha and 2-deoxy-D-arabino-hexuronic acid. It is a polymer of a repeating unit having the following structure: --> 4)-beta-D-Glcp-(1 --> 4)-alpha-L-Rhap -(1 --> 3)-beta-D-Glcp-(1 --> 4)-2-deoxy-beta-D-GlcpA-(1 -->. The polysaccharide also contains 0.6 O-acetyl groups per sugar which have not been located.

Acetylation↗

PubMed2001

Purification and properties of a glucuronan lyase from Sinorhizobium meliloti M5N1CS (NCIMB 40472).

A glucuronan lyase extracted from Sinorhizobium meliloti strain M5N1CS was purified to homogeneity by anion-exchange chromatography. The purified enzyme corresponds to a monomer with a molecular mass of 20 kDa and a pI of 4.9. A specific activity was found only for polyglucuronates leading to the production of 4,5-unsaturated oligoglucuronates. The enzyme activity was optimal at pH 6.5 and 50 degrees C. Zn(2+), Cu(2+), and Hg(2+) (1 mM) inhibited the enzyme activity. No homology of the enzyme N-terminal amino acid sequence was found with any of the previously published protein sequences. This enzyme purified from S. meliloti strain M5N1CS corresponding to a new lyase was classified as an endopolyglucuronate lyase.

Amino Acid Sequence↗

PubMed2000

Human plasmin enzymatic activity is inhibited by chemically modified dextrans.

Some synthetic dextran derivatives that mimic the action of heparin/heparan sulfate were shown to promote in vivo tissue repair when added alone to wounds. These biofunctional mimetics were therefore designated as "regenerating agents" in regard to their in vivo properties. In vitro, these biopolymers were able to protect various heparin-binding growth factors against proteolytic degradation as well as to inhibit the enzymatic activity of neutrophil elastase. In the present work, different dextran derivatives were tested for their capacity to inhibit the enzymatic activity of human plasmin. We show that dextran containing carboxymethyl, sulfate as well as benzylamide groups (RG1192 compound), was the most efficient inhibitor of plasmin amidolytic activity. The inhibition of plasmin by RG1192 can be classified as tight binding hyperbolic noncompetitive. One molecule of RG1192 bound 20 molecules of plasmin with a K(i) of 2.8 x 10(-8) m. Analysis with an optical biosensor confirmed the high affinity of RG1192 for plasmin and revealed that this polymer equally binds plasminogen with a similar affinity (K(d) = 3 x 10(-8) m). Competitive experiments carried out with 6-aminohexanoic acid and kringle proteolytic fragments identified the lysine-binding site domains of plasmin as the RG1192 binding sites. In addition, RG1192 blocked the generation of plasmin from Glu-plasminogen and inhibited the plasmin-mediated proteolysis of fibronectin and laminin. Data from the present in vitro investigation thus indicated that specific dextran derivatives can contribute to the regulation of plasmin activity by impeding the plasmin generation, as a result of their binding to plasminogen and also by directly affecting the catalytic activity of the enzyme.

Dextrans↗

PubMed2000

Production of a glucoglucuronan by a rhizobia strain infecting alfalfa. Structure of the repeating unit.

The Rhizobium sp. T1 strain. which induces nodule formation on alfalfa and clover roots, produces, during growth, an extracellular polysaccharide composed of D-glucose and D-glucuronic acid noted glucoglucuronan. During the bacterial growth, the pH of the medium decreases slightly. The control of pH in the growth medium slightly reduces the glucoglucuronan production. Under the conditions tested in the present work, the weight-average molecular weight of the polymers produced with or without pH control are similar: Mw approximately 2 x 10(6); the repeating unit determined by chemical and NMR analyses corresponds to the disaccharide: --> 3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1 -->.

Biopolymers↗

PubMed2000

Increase of the ATP-dependent phosphoenolpyruvate carboxykinase activity in Sinorhizobium meliloti (Rhizobium meliloti) during hypothermic environmental conditions.

Sinorhizobium meliloti growth is affected when the incubation temperature is lower than 22 degrees C. In culture media containing glucose or fructose (1%, w/v), the doubling time at 19 degrees C was about 6.25 h during the exponential growth phase, while it was 2.75 h at 30 degrees C; at 17 degrees C it was three-fold higher than at 30 degrees C. Modifications in the bacterial metabolism explain the doubling time increase when bacteria are incubated at low temperature. We determine here, the phosphoenolpyruvate carboxykinase (PEPCK) activity increases when S. meliloti cells first grown at 30 degrees C are shifted at 17 degrees C and incubated for 10 h at this low temperature; we noted the PEPCK activity was three-fold higher in cells incubated in media containing glucose and shifted from 30 to 17 degrees C than in cells maintained at 30 degrees C, while it was only 1.5-fold higher in cells grown in media containing fructose.

Cold Temperature↗

PubMed2000

The sleep data transfer between remote and central sites.

Sleep laboratory have long waiting lists due to the high prevalence of sleep disorders and the low availability of centers. One possibility for improving patient care is an ambulatory recording at home or in a distributed center. Then the data recorded must be transferred for analysis in the specialized center. Similarly, an application is needed for the transfer of raw data when the recordings are made by a health care provider sending sleep technologists to set-up the equipment in the patient's home. It is ethically preferable that the analysis be made by a physician from an independent organization from the provider to avoid any conflict of commercial interest with the provider whose main purpose is delivering therapeutic equipment (CPAP, oxygen...). The objectives were to set-up and test the transfer of sleep data files by ISDN lines between a health care provider and an expert sleep center. The application had to take into account a wide geographical dispersion of patients, general practitioners and medical experts. For that reason, it was decided to use existing Internet Web technology to facilitate portability and interoperability, reduce design time and costs and speed up deployment. The healthcare provider is the collecting site of ambulatory sleep recordings performed at the patient's home by the sleep technician of the provider. The raw data are sent to the sleep center for expert analysis. The methods used were: a PC set-up with a Gazel pro 2B PCI board, a commercially available file transfer software (DT Winstutel from ORSENNA) and an ISDN connection at 128 Kbps. The transfer security needs a set-up to maintain the confidentiality of the personal data transmitted: this is ensured through an identification sequence with several encrypted passwords. The duration of the transfer between Paris and Clamart has been tested for a 44 Moctets file: it took 66 min instead of a theoretical duration of 50 min. This difference was explained by a slower transfer rate (93 Kbps) instead of 128 Kbps. The cost of the transfer has been estimated at 5.4 Euro. This cost can be reduced by subscribing higher transfer tariffs. It amounts to 4.5% of the total cost of the ambulatory procedure at a value of 0.12 Euro per Moctet.

Computer Security↗

PubMed1999

Influence of fermentation conditions and microfiltration processes on membrane fouling during recovery of glucuronane polysaccharides from fermentation broths.

We have investigated the recovery of exopolysaccharides produced by Sinorhizobium meliloti M5N1 CS bacteria from fermentation broths using different membrane filtration processes: cross-flow filtration with a 7 mm i.d. tubular ceramic membrane of 0.5-microm pores under fixed transmembrane pressure or fixed permeate flux and dynamic filtration with a 0.2 microm nylon membrane using a 16-cm rotating disc filter. With the tubular membrane, the polysaccharide mass flux was mainly limited by polymer transmission that decayed to 10% after 90 min. The mass flux of polymer produced under standard fermentation conditions (70 h at 30 degrees C) stabilized after 70 min to 15 g/h/m(2). This mass flux rises to 36 g/h/m(2) when the mean stirring speed during fermentation is increased and to 123 g/h/m(2) when fermentation is extended to 120 h. In both cases, the mean molecular weight of polysaccharides drops from 4.0 10(5) g/mol under standard conditions to 2.7 10(5) g/mol. A similar reduction in molecular weight was observed when the fermentation temperature was raised to 36 degrees C without benefit to the mass flux. These changes in fermentation conditions have little effect on stabilized permeate flux, but raise significantly the sieving coefficient, due probably to molecular weight reduction and the filamentous aspect of the polymer as observed from SEM photographs. The polymer-mass flux was also increased by reducing transmembrane pressure (TMP) and raising the shear rate by inserting a rod in the membrane lumen. Operation under fixed permeate flux instead of constant TMP inhibited fouling during the first 4 h, resulting in higher sieving coefficients and polymer mass fluxes. The most interesting results were obtained with dynamic filtration because it allows operation at high-shear rates and low TMP. Sieving coefficients remained between 90 and 100%. With a smooth disc, the polysaccharide mass flux remained close to 180 g/h/m(2) at 1500 rpm and cell concentrations from 1 to 3 g/L. When radial rods were glued to the disc to increase wall shear stress and turbulence, the mass flux rose to 275 g/h/m(2) at the same speed and cell concentration.

Bioreactors↗

PubMed1998

In vivo 13C-NMR studies of polymer synthesis in rhizobium meliloti M5N1 strain

The use of in vivo 13C-NMR approach for the monitoring of the synthesis of various polymers within cells of Rhizobium meliloti (M5N1 strain) is reported. Significant differences in polymer biosynthesis have been shown as a function of the metabolic state of the cells and the labeled carbon source used. Consumption of carbon source and produced glycogen was complete with mid-exponential phase harvested cells. This was not the case with stationary phase harvested cells, for which polyhydroxybutyrate synthesis was higher and gluconate synthesis was lower than the former. [1-13C]fructose-grown cells produced more exopolysaccharide and polyhydroxybutyrate, but less beta-(1,2) glucan and gluconate than [1-13C]glucose-grown cells. This approach offers a suitable tool to examine the kinetics of polymer biosynthesis by Rhizobia. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗