Production of oligoglucuronans by enzymatic depolymerization of nascent glucuronan

An original bioreactor process for production of oligoglucuronans was developed using the Sinorhizobium meliloti M5N1CS strain that produces glucuronan. This anionic homopolysaccharide was composed of beta-D-(1,4)-glucopyranosyluronic residues variably O-acetylated at C-3 and/or C-2 positions according to culture conditions. It was depolymerized during its biosynthesis by addition of a fungal glucuronan lyase activity in broths. After purification by tangential ultrafiltration and low-pressure liquid chromatography, (1)H NMR and ESI-Q/TOF-MS characterized the poly- and oligoglucuronic acid fractions. This enzymatic bioreactor strategy authorized the production in gram quantity of an unsaturated and no acetylated oligoglucuronan with a degree of polymerization of 3.     

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.     

Improved isolation of glucuronan from algae and the production of glucuronic acid oligosaccharides using a glucuronan lyase

A new source for the production of bioactive glucuronic acid oligosaccharides (GlcUAOs) from the depolymerization of green seaweed Ulva lactuca glucuronan (Algal glucuronan) has been investigated. Algal glucuronan purification was optimized by the acidic precipitation method which allowed us to separate the polysaccharide mixture extracted from the cell wall of Ulva lactuca using hot water containing sodium oxalate. A series of the GlcUAOs were obtained by enzyme degradation of algal glucuronan with a glucuronan lyase (GL) isolated from Trichoderma strain. The putative bioactive GlcUAOs generated were then purified by size-exclusion chromatography in gram quantity and characterized by ¹H/¹³C NMR spectroscopy and ESI-Q/TOF-mass spectrometry.     

Production of oligoglucuronans using a monolithic enzymatic microreactor

A glucuronan lyase (EC 4.2.2.14) was immobilized on a monolithic Convective Interaction Media (CIM((R))) disk. The immobilization yield was equal to 29% of the initial activity and 35% of the initial protein amount. Degradations of three glucuronans with various O-acetylation degrees were investigated and compared with degradations using free enzyme. The immobilized glucuronan lyase was inhibited by the O-acetylation degree like the free enzyme. (1)H NMR analyses were used to study the O-acetylation degree of oligoglucuronans and demonstrated that the average degrees of polymerization were inclusive between 4 and 13 after 24h of degradation. This first immobilization of a glucuronan lyase constitutes a new tool to produce oligoglucuronans.     

Continuous production of oligoglucuronans by immobilized glucuronan lyase

A glucuronan lyase was incubated with sepharose matrices pre-activated with N-hydroxysuccinimide (NHS), cyanogen bromide (CNBr) or epoxy. The CNBr- and NHS-activated gels showed satisfactory immobilization yields whereas no enzyme could be immobilized using the epoxy coupling group. Glucuronan lyase immobilized on CNBr-gel ensured rapid conversion of several glucuronans into oligomers with an enzymatic activity identical to that of the free enzyme. As classically observed when using free enzymes, the acetylation degree of glucuronan limited enzyme activity. Nevertheless, this immobilized system made it easier to obtain accurately oligomers with different polymerization degrees, notably in modifying the glucuronans residence times in column. Thus oligoglucuronan pools with polymerization degrees between 2 and 25 could be obtained with a productivity ranging from 120 mg h^?1 to 1.2 g h^?1 using 0.9 ml of chromatographic gel with immobilized glucuronan lyase. This methodology opens the way to continuous and large oligoglucuronan productions.     

Cloning of the Trichoderma reesei cDNA encoding a glucuronan lyase belonging to a novel polysaccharide lyase family

The filamentous fungus Trichoderma reesei produces glucuronan lyase (TrGL) when it is grown on beta-(1-->4)-polyglucuronate (cellouronate) as a sole carbon source. The cDNA encoding TrGL was cloned, and the recombinant enzyme was heterologously expressed in Pichia pastoris. The cDNA of TrGL includes a 777-bp open reading frame encoding a 20-amino-acid signal peptide and the 238-amino-acid mature protein. The amino acid sequence showed no similarity to the amino acid sequences of previously described functional proteins, indicating that the enzyme should be classified in a novel polysaccharide lyase (PL) family. Recombinant TrGL catalyzed depolymerization of cellouronate endolytically by beta-elimination and was highly specific for cellouronate. The enzyme was most active at pH 6.5 and 50 degrees C, and its activity and thermostability increased in the presence of Ca2+, suggesting that its calcium dependence is similar to that of other PLs, such as pectate lyases.     

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.     

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.     

Studies on fungal polysaccharide. XVII. A new glucuronan "protuberic acid" produced by a fungus Kobayashia nipponica.

A water-soluble glucuronan "protuberic acid", [alpha] 22-D minus 83.6 degrees, was isolated and purified from Kobayashia Nipponica, and its physicochemical properties were investigated. The purified protuberic acid was homogeneous as shown by zone electrophoresis, gel filtration over Sepharose 4B, and ultracentrifugation. The sedimentation coefficient was 1.8 S and its intrinsic viscosity was 1.1 dl/g. By gel filtration the molecular weight was estimated to be about 170 000. The results of periodate oxidation, methylation analysis, and partial acid hydrolysis indicated that this acidic polysaccharide has a linear structure of mainly 1, 4-linkages and containing an acid-labile linkage. Reduced protuberic acid, [alpha] 22-D minus 44 degrees, is also described.     

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 M5N1 CS 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.     

Studies on fungal polysaccharide XVII. A new glucuronan “protuberic acid” produced by a fungus Kobayashi Nipponica

A water-soluble glucuronan “protuberic acid”, [α]_d²² −83.6° and purified from Kobayashia Nipponica, and its physicochemical properties were investigated.The purified protuberic acid was homogeneous as shown by zone electrophoresis, gel filtration over Sepharose 4B, and ultracentrifugation. The sedimentation coefficient was 1.8 S and its intrinsic viscosity was 1.1 dl/g. By gel filtration the molecular weight was estimated to be about 170 000. The results of periodate oxidation, methylation analysis, and partial acid hydrolysis indicated that this acidic polysaccharide has a linear structure of mainly 1,4-linkages and containing an acid-labile linkage. Reduced protuberic acid, [α]_d²² −44°, is also described.     

Efficient purification of small unsaturated oligoglucuronides by reversed-phase chromatography

Ion-exchange chromatography has been applied to purification of unsaturated oligoglucuronans. After an isocratic elution on a strong anion-exchange column, the collected fractions were desalted by low pressure size exclusion chromatography. However, this efficient separation was limited by the time required to desalt. So, we developed a reversed-phase chromatography method using back ionization of oligomers. Two C18 columns were tested with trifluoroacetic acid (TFA 0.7%) as eluent. Different selectivities and column stabilities were observed in this acidic condition. The scale up for semi-preparative applications enabled us to recover pure unsaturated oligoglucuronans without desalting step.     

Enzymatic degradation of amylouronate (α-(1 → 4)-linked glucuronan) by α-glucuronidase from Paenibacillus sp. TH501b

Enzymatic degradation of amylouronate (α-(1 → 4)-linked polyglucuronic acid sodium salt, α-(1 → 4)-linked glucuronan), which was prepared from water-soluble starch by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation, was investigated. A bacterial strain TH501b capable of degrading amylouronate was isolated from soil samples collected in the natural environment. Molecular analysis of the 16S rRNA gene showed that TH501b belongs to the genus Paenibacillus. A hydrolytic enzyme responsible for the degradation of amylouronate, amylouronate hydrolase-I (AUH-I), was detected in the cell-free extract of TH501b. AUH-I was purified by four steps of column chromatography and some properties were characterized. The molecular mass of the native AUH-I was estimated to be approximately 115 kDa by size exclusion chromatography (SEC), whereas sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) showed two major bands at 80 kDa and 46 kDa, respectively. The enzyme was most active at pH 6.0–7.0 and 30 °C. The SEC analysis of reaction products revealed that AUH-I liberated glucuronate as a sole product from amylouronate, indicating that AUH-I hydrolyzed amylouronate exolytically, and thus, was classified as α-glucuronidase.     

Chemical composition and 13C NMR spectroscopic characterisation of ulvans from Ulva (Ulvales, Chlorophyta)

The chemical composition and structures of several ulvan extracts isolated from various Ulva species were studied. They were all composed mainly of rhamnose, glucuronic acid, xylose, glucose and sulphate with smaller amounts of iduronic acid and traces of galactose. Proteins were also present, most likely as contaminants. Precise quantification of the uronic acid content by chemical-enzymatic hydrolysis coupled to HPAEC-PAD analysis and by colorimetry was not achieved, most likely due to the incomplete hydrolysis of glucuronan segments, inadequate HPAEC-pulsed-amperometric response factor for iduronic acid and to a possible differential colorimetric response of the two uronic acids. ¹³C NMR spectroscopic investigation of different ulvans demonstrated that they were all based on ulvanobiuronic acid 3-sulphate A and B repeating units [β-D-Glc pA-(1->4)-α-L-Rhap3S and α-L-IdopA-(1->4)-α-L-Rha p3S, respectively] as well as contiguous β 1->4 linked D-glucuronic acids possibly occurring either in ulvan or as a separate glucuronan. Marked variations in the content of the repeating structures were seen among the different samples. However, due to the limited number of samples studied, no conclusion was reached concerning the effects of species and ecophysiological conditions on the chemistry of ulvan. This revised version was published online in June 2006 with corrections to the Cover Date.     

Studies of low molecular weight samples of glucuronans with various acetylation degree

Partially acetylated, high molecular weight glucuronans were produced by a Sinorhizobium meliloti mutant strain. Two native glucuronan samples with various degrees of acetylation were sonicated to obtain lower molecular weight samples and with low viscosity suitable for chemical modification and (13)C NMR experiments. The average degree of substitution (DS) of the polymer was estimated by Fourier transform infrared (FTIR) and NMR. (13)C NMR spectra were obtained and used to suggest a complete assignment of the signals. The nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear multi-bond coherence (HMBC) experiments were used to elucidate connectivities between the various residues and deduce the linkage of these residues within the polysaccharide.     

Controlled sulfatation of natural anionic bacterial polysaccharides can yield agents with specific regenerating activity in vivo

The regenerating activities of chemically modified anionic bacterial polysaccharides by O-sulfonation were investigated using a in vivo model of rat injured muscle regeneration. Glucuronan (GA), a linear homopolysaccharide of -->4)-beta-D-GlcpA-(1--> residues partially acetylated at the C-3 and/or the C-2 position, and glucoglucuronan (GGA), a linear heteropolysaccharide of -->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1--> residues were sulfated. SO3-DMF sulfatation complex provided polysaccharides with different sulfur contents, however, a depolymerization occurred because we did not use large excess of pyridine to obtain pure modified polysaccharides. A regenerating activity on injured extensor digitorum longus (EDL) muscles on rats was obtained with these two sulfated anionic polymers. The position of sulfate groups on glucoglucuronan (primary or secondary alcohol) seems to have no influence on the biological activity by opposition to the degree of sulfatation both for the glucuronans and the glucoglucuronans. The yield of acetate groups in the glucuronan polymer modulated the specific activity.     

Cellouronate (β-1,4-linked polyglucuronate) lyase from Brevundimonas sp. SH203: Purification and characterization

Biodegradation of cellouronate (β-1,4-linked polyglucuronic acid sodium salt, β-1,4-linked glucuronan), which was prepared from regenerated cellulose by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) mediated oxidation, was investigated. A bacterial strain with the ability to degrade cellouronate was isolated from soil collected in a natural environment, and identified as Brevundimonas sp. SH203 by comparing the nucleotide sequences of its 16S rDNA with those registered in the GenBank database. Cellouronate lyase-I (CUL-I), being responsible for the depolymerization of cellouronate, was purified to homogeneity from cell-free extracts. CUL-I was a monomeric protein with the molecular mass of 39 kDa by SDS–PAGE and 37 KDa by size exclusion chromatography (SEC). The enzyme activity was optimum at pH 7.5 and was inhibited by some divalent metal ions such as Mg²⁺, Fe²⁺ and Mn²⁺. The enzymatic reaction products were analyzed by SEC, TLC and ¹³C NMR. The results indicated that CUL-I catalyzed to depolymerize cellouronate endolytically to oligocellouronates and monomeric uronate.     

Characterisation of sorbed water molecules on neutral and ionic polysaccharides

Ionic and neutral polysaccharides with well-defined structures were chosen to investigate the mechanism of water sorption at different relative humidities. From an experimental point of view, the freezing water was determined by DSC when the total sorbed water was obtained from thermogravimetry. The isotherms of sorption and enthalpies of interaction were determined using the combination of a microbalance and a microcalorimeter. It is shown that freezing water appears for P/P₀ > 0.85 especially with the neutral polymers. The differential molar enthalpy of interaction is higher for P/P₀ < 0.85 corresponding to the fixation of two water molecules forming double H-bonds; this result is confirmed by molecular modelling; saturation is obtained experimentally for 4 water molecules interacting per glucose unit. On ionic polymers, the water retention increases especially over P/P₀ 0.8 and the enthalpy of interaction is higher for the first water molecules sorbed. For P/P₀ 0.8, the numbers of bound water molecules found are 2 per glucopyranosyl unit for neutral polysaccharides, 5 for glucuronan and 9–10 for carboxymethylcellulose (CMC) of D = 2 and hyaluronan (HA)     

Conformational and configurational features of acidic polysaccharides and their interactions with calcium ions: a molecular modeling investigation.

Modeling simulations have been performed on the four regular glycuronans: alpha-D-(1--->4) polygalacturonic, alpha-L-(1--->4) polyguluronic, beta-D-(1--->4) polymannuronic, and beta-D-(1--->4) polyglucuronic acids. The goal of this study was to characterize the similarities and differences in conformational and configurational behavior as well as in calcium binding in order to progress in the understanding of the physicochemical properties of the parent polysaccharides of industrial interest, namely pectin, alginate and glucuronan. This required the evaluation of the accessible conformational space for the disaccharide subunits of the four homopolymers, using the flexible residue protocol of the MM3 molecular mechanics procedure. The results were used to access the configurational statistics of representative polysaccharide chains, as well as for the determination of the regular polysaccharide helices and their conformational transitions. The surfaces of all regular helices likely to occur for each polyuronide were explored for cation binding using the GRID procedure. Both alpha-D-(1--->4) polygalacturonate and alpha-L-(1--->4) polyguluronate chains exhibit a high specificity for calcium binding, and have well-defined chelation sites. In contrast, beta-D-(1--->4) polymannuronate and beta-D-(1--->4) polyglucuronate chains do not display any stereospecificity for calcium binding. The results gathered from molecular modeling lead to a clear understanding of the different structural features that are displayed by the four ionic polymers.     

Ceramide-1-phosphate binds group IVA cytosolic phospholipase a2 via a novel site in the C2 domain

Previously, ceramide-1-phosphate (C1P) was demonstrated to be a potent and specific activator of group IV cytosolic phospholipase A(2)alpha (cPLA(2)alpha) via interaction with the C2 domain. In this study, we hypothesized that the specific interaction site for C1P was localized to the cationic beta-groove (Arg(57), Lys(58), Arg(59)) of the C2 domain of cPLA(2)alpha. In this regard, mutants of this region of cPLA(2)alpha were generated (R57A/K58A/R59A, R57A/R59A, K58A/R59A, R57A/K58A, R57A, K58A, and R59A) and examined for C1P affinity by surface plasmon resonance. The triple mutants (R57A/K58A/R59A), the double mutants (R57A/R59A, K58A/R59A, and R57A/K58A), and the single mutant (R59A) demonstrated significantly reduced affinity for C1P-containing vesicles as compared with wild-type cPLA(2)alpha. Examining these mutants for enzymatic activity demonstrated that these five mutants of cPLA(2)alpha also showed a significant reduction in the ability of C1P to: 1) increase the V(max) of the reaction; and 2) significantly decrease the dissociation constant (K (A)(s)) of the reaction as compared with the wild-type enzyme. The mutational effect was specific for C1P as all of the cationic mutants of cPLA(2)alpha demonstrated normal basal activity as well as normal affinities for phosphatidylcholine and phosphatidylinositol-4,5-bisphosphate as compared with wild-type cPLA(2)alpha. This study, for the first time, demonstrates a novel C1P interaction site mapped to the cationic beta-groove of the C2 domain of cPLA(2)alpha.