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.     

Purification of heparinase and heparitinase by affinity chromatography on glycosaminoglycan-bound ah-sepha-rose 4b

Heparinase and heparitinase were separated from an extract of Flavobacterium heparinum, induced with heparin by using column chromatography on hydroxylapatite. As the heparinase preparation contained chondroitinases B and C, chondroitinase B was removed by rechromatography on a hydroxylapatite column. Chondroitinase C was then eliminated by column chromatography on O-phosphono(“phospho”)-cellulose. The heparinase preparation thus obtained was free from sulfoamidase for 2-deoxy-2-sulfoamino-D-glucose (GlcN-2S), sulfatase for 2-amino-2-deoxy-6-O-sulfo D-glucose (GlcN-6S), as well as (δ_4,5glycosiduronase for the unsaturated disaccharides obtained from heparin. The remaining sulfatase for 4-deoxy-α-L-thero-hex-4-enopyranosyluronic acid 2-sulfate (δUA-2S) in the heparinase preparation was removed by affinity chromatography with dermatan sulfate-bound AH-Sepharose 4B coated with dermatan sulfate. The heparitinase preparation separated by column chromatography on hydroxyla patite was purified by affinity chromatography with heparin-bound AH-Sepharose 4B coated with heparin. Sulfatase for 2-amino-2-deoxy-6-O-sulfo-D-glucose (GlcN-6S) and δ_4,5glycosiduronase for the unsaturated disaccharides obtained from heparin were removed by this chromatography. Sulfatase for 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid 2-sulfate (δUA-2S) remaining in the heparitinase preparation was finally removed by column chromatography on hydroxylapatite. The recoveries of the purified preparations of heparinase and heparitinase were estimated to be 39 and 50%, respectively, from the crude enzyme fractions obtained by the first column chromatography on hydroxyl- patite. The purified heparinase and heparitinase were free from all enzymes that could degrade the sulfated unsaturated disaccharides produced from heparin with heparinase.     

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.     

Preparation of unsaturated disaccharides by eliminative cleavage of heparin and heparan sulfate with heparitinases.

1. Six kinds of unsaturated disaccharides were prepared by enzymatic digestion of heparin and heparan sulfate with heparitinases I0 and IV, and subsequent column chromatography. They were identified by HPLC showing good separation from each other. 2. The content of each unsaturated disaccharide fraction was determined colorimetrically, and found to range from 130.7 to 722.3 mumol. 3. Molecular extinction coefficient of each unsaturated disaccharide was calculated from absorbance at a wavelength of around 230 nm where a peak appeared on the ultraviolet spectrum of each disaccharide solution at pH 2. The values varied from 6000 to 6600.     

Structural parameters and natural occurrence of 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid

2-Deoxy-2,3-didehydro-N-glycoloylneuraminic acid has been found to occur in porcine, bovine and equine submandibular glands as well as in the urine of pig, horse and rat. This novel, unsaturated sialic acid was isolated by gel filtration and ion-exchange chromatography. Final purification was achieved by column chromatography or by preparative thin-layer chromatography on cellulose. The structural analysis was performed by combined capillary gas-liquid chromatography/mass spectrometry. The various data were compared with those from synthetic 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid. Besides of the unsaturated N-glycoloylated sialic acid, also the corresponding N-acetylated derivative was present in the materials analyzed. The inhibitory effect of 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid on Vibrio cholerae sialidase using N-acetylneuraminyl-(alpha 2----3)-lactose as substrate is slightly higher (50% inhibition at 10 microM) when compared with 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (50% inhibition at 15 microM).     

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.     

High-performance liquid chromatographic separation and on-line mass spectrometric detection of saturated and unsaturated oligogalacturonic acids

A method for the simultaneous determination of saturated and unsaturated oligogalacturonic acids up to degree of polymerization (dp) of 7 by high-performance liquid chromatography (HPLC) is presented. For this purpose, a Cyclobond I 2000 column and a volatile mobile phase consisting of ammonium formate and methanol were used, allowing direct coupling of HPLC to a mass spectrometer via an electrospray interface (ESI-MS) without additional desalting. The analytical system was used for the characterization of digests obtained by incubation of polygalacturonic acid with commercial enzyme preparations.     

Synthesis of tetrazole analogs of gamma- and delta-amino acids.

N-benzyloxycarbonyl-protected alpha- or beta-amino alcohols, easily prepared from alpha- and beta-amino acids, were converted into aldehydes and directly reacted with (triphenyl phosphoranylidene) acetonitrile, leading to unsaturated nitriles. Treatment of nitriles with NaN(3) and ZnBr(2) produced unsaturated gamma- and delta-amino tetrazoles, which were deprotected and converted to the corresponding saturated compounds by catalytic hydrogenation. For the case of delta-amino tetrazole, the methylation of the acidic moiety occurred after treatment with CH(2)N(2), leading to the N(1)- and N(2)-methylated constitutional isomers, which were separated by column chromatography and hydrogenated.     

Analysis of unsaturated disaccharides from glycosaminoglycuronan by high-performance liquid chromatography

A rapid and simple analytical method for unsaturated disaccharide isomers formed by enzymatic digestion from hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin by high-performance liquid chromatography using an amine-bound silica column with a linear gradient of sodium dihydrogen phosphate was developed. The analyses were performed on isomers of two groups belonging to the chondroitin sulfate family and the heparin sulfate family. In both families, disaccharide isomers eluted in the order non-, mono-, di-, and trisulfated disaccharides by elevating salt concentrations. The method was applied to the analysis of constituent disaccharides of representative sulfated glycosaminoglycans, which proved that most constituents could be quantified separately. This method is advantageous in that enzymatic digests can be applied directly on a column without any pretreatment and good resolution of several disaccharides can be obtained by one chromatography.     

High performance liquid chromatography of unsaturated disaccharides produced from chondroitin sulfates by chondroitinase.

High performance liquid chromatography was performed by the ion pair method on unsaturated disaccharides produced from chondroitin sulfates by the action of chondroitinase. Completely separated peaks corresponding to deltaDi-0S, deltaDi-4S, and deltaDi-6S were obtained on a column of mu-bondapak-C18 with 0.035 M tetra-butylammonium phosphate (pH 7.54) as a mobile phase. There was a linear relationship between the ratio of the peak area and the amount of each standard tested.     

Effects of Phosphatidylcholine or Ergosteryl Oleate on Physiological Properties of Saccharomyces sake

Chromatographically homogeneous phosphatidylcholine identified by thin-layer chromatography on silica gel G was isolated from mycelia of Aspergillus oryzae or egg yolk by a combination of alumina and silicic acid column chromatography. Although the 2-position in both Asp. oryzae- and egg yolk-phosphatidylcholine was occupied nearly exclusively by unsaturated fatty acids, significant proportions of unsaturated fatty acids were also found in the 1 -position of Asp. oryzae-phosphatidylcholine. In nitrogen gas-sparging anaerobic culture of Saccharomvces sake Kyokai No. 7, supplementing the basal synthetic medium with phosphatidylcholine enhanced the yeast growth and fermentative activity, whereas adding ergosteryl oleate enhanced alcohol-endurability. Supplementation with both phosphatidylcholine and ergosteryl oleate promoted the yeast growth, fermentative activity and alcohol-endurability of cells.     

Protein kinase C of a human megakaryoblastic leukemic cell line (MEG-01). Analysis of subspecies and activation by diacylglycerol and free fatty acids

Protein kinase C (PKC) from a human megakaryoblastic leukemic cell line (MEG-01) was resolved into two fractions by hydroxyapatite column chromatography, which are indistinguishable from the brain type II (beta I/beta II) and type III (alpha) subspecies, by biochemical and immunoblot analysis. In the presence of both phosphatidylserine and diacylglycerol, several free unsaturated fatty acids (FFA's), such as arachidonic, oleic, linoleic and linolenic acids, further enhanced the enzyme activation, and allowed the enzyme to exhibit almost full activity at nearly basal levels of Ca2+ concentration. The concentration of unsaturated FFA's giving rise to the maximum enzyme activation was around 2 x 10(-5) M. Palmitic and stearic acids were inactive. The result implies that, in addition to diacylglycerol, the receptor-mediated release of unsaturated FFA's from membrane phospholipids may also take part in the activation of PKC.     

Unsaturated disaccharides in the enzymatic digests of heparan sulfates

High performance liquid chromatography was performed by an ion-pair reversed-phase method of six standard unsaturated disaccharides derived from heparan sulfate and heparin. Separation of delta Di-GlcNAc, delta Di-GlcN(2S), delta Di-GlcNAc(6S), delta Di-GlcN(2,6- or 2,2'-diS) and delta Di-GlcN(2,6,2'-triS) was achieved on a column of Jasco SC-02 with 10 mM tetrabutylammonium phosphate (pH 7.0) containing 30 or 47% methanol as a mobile phase. delta Di-GlcN(2,6-diS) and delta Di-GlcN(2,2'-diS) were separated on the same column with 35 mM triethylamine phosphate (pH 5.3). Four preparations (BL-1.0-1, BL-1.0-2, BL-1.0-3, and BL-1.25-1) separated from crude bovine lung heparan sulfate, a standard bovine lung heparan sulfate (BL-ST), bovine kidney heparan sulfate 1.0 M Fr and 1.25 M Fr (BK-1.0 and BK-1.25), and porcine kidney heparan sulfate 1.0 M Fr (PK-1.0) were digested with a mixture of heparinase, and heparitinases 1 and 2. The resulting foregoing unsaturated disaccharides in the digests were analyzed by the above HPLC procedures. The proportions of the unsaturated disaccharides in the digests of BL-1.25-1 and BL-ST were similar, but those of the others differed from each other. It is noteworthy that delta Di-GlcNAc plus delta Di-GlcNAc(6S) in the digest of BL-1.0-1 was approximately 95% of the total unsaturated disaccharides. Small amounts of delta Di-GlcN (2,6,2'-triS) were found in all the samples. It was found that delta Di-GlcN(2,2'-diS) was a prominent component in the disulfated unsaturated disaccharides from BL-1.25-1 and BK-1.25.     

High-performance liquid chromatography of pyridylamino derivatives of unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases

A sensitive method was developed for the separation and quantitation of four unsaturated disaccharides (delta Di-0S, delta Di-4S, delta Di-6S, and delta Di-diS) by high performance liquid chromatography. The unsaturated disaccharides were coupled with a fluorescent compound, 2-aminopyridine. Complete separation of the resulting pyridylamino derivatives was achieved on a column of muBondapak-C18 with 8 mM KH2PO4-Na2HPO4 (pH 6.0)/methanol (30/l, by volume) as a mobile phase. There was a linear relationship between the fluorescence emission (peak height), and the amount of each authentic disaccharide used for the coupling reaction. This method was applied to analyze commercially available chondroitin sulfates A and C, dermatan sulfate, and urinary glycosaminoglycans obtained from patients with mucopolysaccharidosis after digestion with chondroitinases. The data indicated that the present method is useful for the separation and quantitation of nmol-pmol levels of the unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases and can be used for their structural characterization.     

Abnormal urinary excretion of unsaturated dicarboxylic acids in patients with medium-chain acyl-CoA dehydrogenase deficiency

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently described metabolic disorder of fatty acid oxidation in humans. Acute episodes are usually characterized biochemically by the appearance of nonketotic dicarboxylic aciduria. In addition, other abnormal metabolites, such as suberylglycine, n-hexanoylglycine, 3-phenylpropionylglycine, and octanoylcarnitine, are excreted in the urine. Urinary organic acids were determined using dual capillary column gas-liquid chromatography and gas-liquid chromatography/mass spectrometry. In three cases of MCAD deficiency we observed a disproportionate increase in the excretion of unsaturated dicarboxylic acids compared to either fasting control children with expected ketotic dicarboxylic aciduria or patients with nonketotic dicarboxylic aciduria not associated with MCAD deficiency. The most significant increase was in the urinary excretion of cis-4-decendioic acid. Additionally, the urinary excretions of cis-3-octenedioic and cis-5-decenedioic acids were slightly decreased whereas the excretion of cis-5-dodecenedioic acid was increased. These data are consistent with the notion that as a result of MCAD deficiency the metabolic oxidation of unsaturated fatty acids such as linoleate and oleate is inhibited more than saturated fatty acids.     

A method for fractionation of cerebrosides into classes with different fatty acid compositions

A method is described for the separation of beef brain cerebrosides into three fractions containing different classes of fatty acids: nonhydroxy (I), unsaturated nonhydroxy (II), and hydroxy fatty acid cerebrosides (III). The procedure consists of benzoylation of either crude or purified cerebrosides, followed by column chromatographic separation of benzoylated derivatives containing nonhydroxy acids from those containing hydroxy fatty acids. The benzoyl groups are removed by sodium methoxide-catalyzed transesterification; from the reaction mixtures, fractions I and III precipitate. The fraction II present in mother liquor of I was shown to contain mainly short-chain and unsaturated nonhydroxy fatty acid cerebrosides. The fatty acid composition of each fraction was obtained by gas-liquid chromatography.     

Isolation and Identification of Cryptic Bioactive Regions in Bovine Achilles Tendon Collagen

Several proteins are known to host specific regions within their sequence, that when exposed or excised out proteolytically can display a range of physiological activities quite different from that of the parent protein. Collagen, a class of structural biopolymers and an important component of the extracellular matrix, is now known to harbor several such bioactive peptides which can act as physiological regulators. This study was undertaken to identify such cryptic sites from bovine Achilles tendon collagen and an antioxidative assay was used to screen for bioactivity. Bacterial crude protease was used to hydrolyze collagen and the hydrolysate was subjected to separation through ion-exchange column chromatography. Fractions were screened using conventional antioxidative assays and further purified by gel permeation chromatography. Two biologically active cryptic peptides were obtained displaying high antioxidative properties, E1 and F3. At low concentrations, both peptides displayed higher chelating ability than EDTA and were able to reduce the auto-oxidation of unsaturated fatty acid. The molecular weights of the peptides were found out through column chromatography and Tricine SDS PAGE; both displayed molecular mass below 4?kDa. Overall E1 displayed a comparatively better antioxidative ability than the others and was further characterized by circular dichroism studies and sequencing. A BLAST search of the active peptide sequence revealed that an almost similar peptide also resides in human collagen Type I.     

A simplified procedure for synthesis of di-[14C]acyl-labeled lecithins.

A simplified procedure for synthesis of 1,2-di-[1'-14C]oleoyl-, 1,2-di-[1'-14C]linoleoyl-, and 1,2-di-[1'-14C]eicosatrienoyl-sn-glycero-3-phosphorylcholine is described. The method involves acylation of the CdCl2 complex of glycerophosphorylcholine with a 14C-labeled fatty acid in the presence of trifluoracetic anhydride and pyridine. The 14C-labeled lecithin is isolated in pure form by preparative thin-layer chromatography and alumina column chromatography in an overall yield of 12-24%. No isomerization or peroxidation of the unsaturated acids was detected.     

Endo-polygalacturonate lyase of Cytophaga johnsonii

Summary An extracellular endopolygalacturonate lyase of Cytophaga johnsonii was purified from the culture filtrate. It appeared to be homogeneous as judged by polyacrylamide gel electrophoresis at pH 8.6 as well as pH 4.3. The purified enzyme had a pH optimum around 9.0 and required Ca⁺⁺ ions for its maximum activity. The apparent K _mfor polygalacturonic acid was found to be 0.22%. Both paper and column chromatography indicated formation and accumulation of an unsaturated monomer along with unsaturated di-, tri-, tetra- and pentamers from polygalacturonic acid by the enzyme action, indicating that the enzyme cleaved the substrate randomly in a non-hydrolytic manner. The glycosidic linkage next to the non-reducing end of polygalacturonic acid was not resistant to attack by this enzyme unlike in other known polygalacturonate lyases.Abbreviations PG lyase Polygalacturonate lyase - Tris Tris (hydroxymethyl) aminomethane