Biosynthesis of the storage polysaccharide from the snail Biomphalaria glabrata,identification and specificity of a branching β1→6 galactosyltransferase

Adult snails synthesize in their albumen glands a storage polysaccharide called galactan which is utilized by the developing embryos. With [6-³H]-uridine 5diphosphogalactose the incorporation of labelled d-galactose into the polysaccharide can be traeed in freshly removed glands maintained in a bathing buffer. After centrifugation of homogenized glands, galactosyltrasferase activity is only found in the insoluble fraction. Chaps extracts of this material retain almost all of their activity and can be used for comparison of the incorporation rates into different native galactans or in various oligosaccharides. A highly efficient -(16) galactosyltransferase was detected when methyl 3-O-(-d-galactopyranosyl)--d-galactopyranoside was offered as acceptor. The substitution at the penultimate residue resulted in a branched trisaccharide as demonstrated by ¹H-NMR-spectroscopy and permethylation analysis of the reaction product. Comparable results were obtained with various oligosaccharides containing an internal galactose unit glycosidically linked 13. Attempts to separate and purify the various enzymes involved resulted in the isolation of a fraction which is able to transfer d-Gal exclusively to native galactan, but not to oligosaccharides. A further fraction was obtained from a different resin with activity for native galactan and 6-O-(-d-galactopyranosyl)-d-galactopyranose. but without any for methyl-3-O-(-d-galactopyranosyl)--d-galactopyranose. It is thus concluded that at least three different enzymes are involved in the biosynthesis of this snail galactan.Abbreviation Gal galactose - glc gas-liquid chromatography - Gro glycerol - tlc thin layer chromatography     

Differential binding characteristics and applications of dGalβ1→3dGalNAc specific lectins

Summary The binding properties of Arachis hypogaea (PNA), Bauhinia vurpurea alba (BPL), Maclura pomifera (MPL) and Sophora japonica (SJL) lectins were studied by quantitative precipitin and precipitin inhibition assays, demonstrating them to be most specific for dGal13dGalNAc residues. Additionally, each lectin had its own binding characteristic such as different binding abilities to dGal14dGlcNAc or dGal13dGlcNAc1linked oligosaccharides, and/or dGalNAc1linked to the Ser or Thr of the protein moiety. These differential binding characteristics can be used for investigating fine differences of the carbohydrate structure of the glycoconjugates, especially those having dGal13dGalNAc residues as terminal non-reducing ends.Abbreviations dGal d-galactopyranose - dMan d-mannopyranose - dGalNAc 2-acetamido-2-deoxy-d-galacto-pyranose - dGlcNAc 2-acetamino-2-deoxy-d-glucopyranose - LFuc L-fucose - NeuNAc N-acetylneuraminic acid - Ser serine - Thr Threonine - RCA Ricinus communis agglutinin - SBA Soy bean agglutinin (Glycine max) - HPA Helix pomatia agglutinin - DBL Dolichos biflorus lectin - GCL Geodia cydonium lectin     

Purification and properties of a novel β-galactosidase or exo-(1 → 4)-β-d-galactanase from the cotyledons of germinated Lupinus angustifolius L. seeds

The main polysaccharide component of the thickened cell walls in the storage parenchyma of Lupinus angustifolius L. cotyledons is a linear (1 4)--linked d-galactan, which is mobilised after germination (L.A. Crawshaw and J.S.G Reid, 1984, Planta 160, 449–454). The isolation from the germinated cotyledons of a -d-galactosidase or exo-(1 4)--d-galactanase with a high specificity for the lupin galactan is described. The enzyme, purified using diethylaminoethyl-cellulose, carboxymethyl-cellulose and affinity chromatography on lactose-agarose, gave two bands (major 60 kDa, minor 45 kDa) on sodium dodecyl sulphate-gel electrophoresis, and two similar bands on isoelectric focusing (major, pI 7.0, minor pI 6.7, both apparently possessing enzyme activity). The minor component cross-reacted with an antiserum raised against, and affinity-purified on, the major band. Both components had a common N-terminal sequence. The minor component was probably a degradation product of the major one. The enzyme had limited -galactosidase action, catalysing the hydrolysis of p-nitrophenyl--d-galactopyranoside and (1 4)- and (1 6)--linked galactobioses. Lactose [-d-galactopyranosyl-(1 4)-d-glucose] was hydrolysed only very slowly and methyl--d-galactopyranoside not at all. Lupin galactan was hydrolysed rapidly and extensively to galactose, whereas other cell-wall polysaccharides (xyloglucan and arabinogalactan) with terminal non-reducing -d-galactopyranosyl residues were not substrates. A linear (1 4)--linked galactopentaose was hydrolysed efficiently to the tetraose plus galactose, but further sequential removals of galactose to give the tetraose and lower homologues occurred more slowly. Galactose, -galactonolactone and Cu⁺² were inhibitory. No endo--d-galactanase activity was detected in lupin cotyledonary extracts, whereas exo-galactanase activity varied pari passu with galactan mobilisation. Exo-galactanase protein was detected, by Western immunoblotting of cotyledon extracts, just before the activity could be assayed and then increased and decreased in step with the enzyme activity. The exo-galactanase is clearly a key enzyme in galactan mobilisation and may be the sole activity involved in depolymerising the dominant (1 4)--galactan component of the cell wall.Abbreviations CM carboxymethyl - DEAE diethylaminoethyl - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TLC thin-layer chromatography We wish to thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for the award of a studentship to M.S. Buckeridge, and the Government of São Paulo State, Brazil for granting him leave of absence. We are grateful to Dr. Amanda Heyller (Unilever Research Laboratory, Colworth House, Bedford, UK) for N-terminal sequence determinations, to Dr. Stuart Wilson (Stirling) for preparing gelatin SDS-gels and to Cristina Fanutti (Stirling) for purifying the xyloglucan oligosaccharide.     

Negative-ion fast atom bombardment tandem mass spectrometry for characterization of sulfated unsaturated disaccharides from heparin and heparan sulfate

Negative-ion fast atom bombardment tandem mass spectrometry has been used in the characterization of non-, mono-, di- and trisulfated disaccharides from heparin and heparan sulfate. The positional isomers of the sulfate group of monosulfated disaccharides were distinguished from each other by negative-ion fast atom bombardment tandem mass spectra, which provide an easy way of identifying the positional isomers. This fast atom bombardment collision induced dissociation mass spectrometry/mass spectrometry technique was also applied successfully to the characterization of di- and trisulfated disaccharides.Abbreviations FABMS fast atom bombardment mass spectrometry - CID collision induced dissociation - MIKE mass analysed ion kinetic energy - MS/MS mass spectrometry/mass spectrometry - HPLC high performance liquid chromatography - UA d-gluco-4-enepyranosyluronic acid - CS chondroitin sulfate - DS dermatan sulfate - HA hyaluronan - Hep heparin - HS heparan sulfate - UA(14) GlcNAc 2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose - UA(14)GlcNAc6S 2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcNAc 2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcNAc6S 2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(14)GlcN6S 2-amino-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcN 2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcN6S 2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(14)GlcNS 2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose - UA(14)GlcNS6S 2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcNS 2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcNS6S 2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(13)GalNAc 2-acetamido-2-deoxy-3-O-(-d-Gluco-4-enepyranosyluronic acid)-d-galatose - UA(13)GalNAc4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA(13)GalNAc6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA2S(13)GalNAc 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-galactose - UA2S(13)GalNAc4S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA2S(13)GalNAc6S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA(13)GalNAcDiS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose - UA(13)GlcNAc 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose.     

Production of β-fructofuranosidase byAspergillus japonicus

A newly isolated strain, MU-2, which produces very high -fructofuranosidase activity, was identified asAspergillus japonicus. For enzyme production by the strain, sucrose at 20% (w/v) was the best carbon source and yeast extract at 1.5 to 3% (w/v) the best nitrogen source. Total enzymatic activity and cell growth were at maximum after 48 h, at 1.57×10⁴ U/flask and 0.81 g dry cells/flask, respectively. The optimum pH value of the enzymatic reaction was between 5.0 and 5.5 and the optimum temperature 60 to 65°C. The enzyme produced 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose by fructosyl-transferring activity. The strain was found to be very useful for industrial production of -fructofuranosidase.     

β-Glucanases in developing cotton (Gossypium hirsutum L.) fibres

Cotton fibres possess several -glucanase activities which appear to be associated with the cell wall, but which can be partially solubilised in buffers. The main activity detected was that of an exo-(13)--d-glucanase (EC 3.2.1.58) but which also had the characteristics of a -glucosidase (EC 3.2.1.21). Endo-(13)--d-glucanase activity (EC 3.2.1.39) and much lower levels of (14)--d-glucanase activity were also detected. The exo-(13)--glucanase showed a maximum late on (40 days post-anthesis) in the development of the fibres, whereas the endo-(13)--glucanase activity remained constant throughout fibre development. The -glucanase complex associated with the cotton-fibre cell wall also functions as a transglucosylase introducing, inter alia, (16)--glucosyl linkages into the disaccharide cellobiose to give the trisaccharide 4-O--gentiobiosylglucose.Abbreviations CMC carboxymethylcellulose - ONPG o-nitrophenyl--d-glucopyranoside - TLC thin-layer chromatography Presented at the Third Cell Wall Meeting held in Fribourg in 1984     

Curdlan and other bacterial (1→3)-β-d-glucans

Three structural classes of (13)--d-glucans are encountered in some important soil-dwelling, plant-associated or human pathogenic bacteria. Linear (13)--glucans and side-chain-branched (13,12)--glucans are major constituents of capsular materials, with roles in bacterial aggregation, virulence and carbohydrate storage. Cyclic (13,16)--glucans are predominantly periplasmic, serving in osmotic adaptation. Curdlan, the linear (13)--glucan from Agrobacterium, has unique rheological and thermal gelling properties, with applications in the food industry and other sectors. This review includes information on the structure, properties and molecular genetics of the bacterial (13)--glucans, together with an overview of the physiology and biotechnology of curdlan production and applications of this biopolymer and its derivatives.     

Purification and properties ofβ-fructofuranosidase from Aspergillus japonicus

-Fructofuranosidase fromAspergillus japonicus, which produces 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose, was purified to homogeneity by fractionation with calcium acetate and ammonium sulphate and chromatography with DEAE-Cellulofine and Sephadex G-200. Its molecular size was estimated to be about 304,000 Da by gel filtration. The enzyme was a glycoprotein which contained about 20% (w/w) carbohydrate. Optimum pH for the enzymatic reaction was 5.5 to 6. The enzyme was stable over a wide pH range, from pH 4 to 9. Optimum reaction temperature for the enzyme was 60 to 65°C and it was stable below 60°C. The K_m value for sucrose was 0.21m. The enzyme was inhibited by metal ions, such as those of silver, lead and iron, and also byp-chloromercuribenzoate.     

Synthesis of FimH receptor-active manno-oligosaccharides by reverse hydrolysis using α-mannosidases from Penicillium citrinum,Aspergillus phoenicis and almond

Recombinant Penicillium citrinum -1,2-mannosidase, expressed in Aspergillus oryzae, was employed to carry out regioselective synthesis of -d-mannopyranosyl-(12)-d-mannose. Yields (w/w) of 16.68% disaccharide, 3.07% trisaccharide and 0.48% tetrasaccharide were obtained, with 12 linkages present at 98.5% of the total linkages formed. Non-specific -mannosidase from almond was highly efficient in reverse hydrolysis and oligosaccharide yields of 45–50% were achieved. The products of the almond mannosidase were a mixture of disaccharides (30.75%, w/w), trisaccharides (12.26%, w/w) and tetrasaccharides (1.89%, w/w) with 12, 13 and 16 isomers. -1,2-linkage specific mannosidase from P. citrinum and -1,6-linkage-specific mannosidase from Aspergillus phoenicis were used in combination to hydrolyse the respective linkages from the mixture of isomers, resulting in -d-mannopyranosyl-(13)-d-mannose in 86.4% purity. The synthesised oligosaccharides can potentially inhibit the adhesion of pathogens by acting as "decoys" of receptors of type-1 fimbriae carried by enterobacteria.     

Observations on the cell-wall compositions of the bracket fungi Laetiporus sulphureus and Piptoporus betulinus

Homogenized tissues and their alkali-soluble and alkali-insoluble fractions of fruiting bodies of the basidiomycetes Laetiporus sulphureus and Piptoporus betulinus were investigated using X-ray diffraction, infrared spectrometry and chemical methods. The presence of (13)--d-glucan, (13)--d-glucan and chitin was established. The relative amounts of these polysaccharides were different in the two species and differences were also found between context and trama. The proportion of (13)--d-glucan was exceptionally high in the context of L. sulphureus (about 78%). In addition, the trama of both species contained a substance resembling a cyclic wax by its X-ray pattern and solubility properties. The substances identified are considered to belong to the hyphal wall     

Studies of the action of ceramide-like substances (d- andl-PDMP) on sphingolipid glycosyltransferases and purified lactosylceramide synthase

We have studied the effects ofD-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and itsL-enantiomer on glycosphingolipids in cultured normal human kidney proximal tubular cells. We found thatD-PDMP exerted a concentration-dependent reduction in the metabolic labelling and cellular levels of glucosylceramide (GlcCer), lactosylceramide (LacCer), and the globo-series glycosphingolipids, GbOse₃Cer and GbOse₄Cer. It also directly inhibited the activity of UDP-glucose:ceramide 1 4-glucosyltransferase (GlcT-1) and UDP-galactose: GlcCer 1 4 galactosyltransferase (GalT-2). In contrast,L-PDMP had opposite effects on the metabolic labelling of GlcCer, LacCer, and GbOse₃Cer. The levels of GlcCer and LacCer were increased, while the labelling and level of GbOse₄Cer were strongly reduced. Purified GalT-2 from human kidney was inhibited byD-PDMP and stimulated byL-PDMP. It appears likely that the different glycosphingolipid glycosyltransferases possess similar binding sites for the ceramide moiety, which are blocked by binding toD-PDMP and, in the case of GbOse₄Cer synthase, byL-PDMP as well. The stimulatory effects ofL-PDMP on GlcCer and LacCer synthases may be the result of binding to a modulatory site on the glycosyltransferases; in intact cells, the enzyme-analog complex may afford protection against the normal catabolic inactivation of the enzymes.Abbreviations GalT-2 UDP-galactose:GlcCer -galactosyltransferase - GbOse₃Cer Gal1 4Gal1 GlcCer - GbOse₄Cer GalNAc1 3Gal1 4Gal1 GlcCer - GlcCer glucosylceramide - GlcT-1 UDP-glucose:ceramide -glucosyltransferase - GSLs glycosphingolipids - LacCer lactosylceramide - PDMP threo-1-phenyl-2-decanolyamino-3-morpholino-1-propanol     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Synthesis of Gal-β-(1→4)-GlcNAc-β-(1→6)-[Gal-β- (1→3)]-GalNAc-α-OBn oligosaccharides bearing O-methyl or O-sulfo groups at C-3 of the Gal residue: specific acceptors for Gal: 3-O-sulfotransferases

Our recent studies have revealed the existence of two distinct Gal: 3-O-sulfotransferases capable of acting on the C-3 position of galactose in a Core 2 branched structure, e.g., Gal14GlcNAc16(Gal13)GalNac1OBenzyl as acceptor to give 3-O-sulfoGal14GlcNAc13(Gal13)GalNAc1OB 20 and Gal14GlcNAc16(3-O-sulfoGal13)GalNAc1OB 23. We herein report the synthesis of these two compounds and also that of other modified analogs that are highly specific acceptors for the two sulfotransferases. Appropriately protected 1-thio-glycosides 7, 8, and 10 were employed as glycosyl donors for the synthesis of our target compounds.     

Synthesis of N-linked pentasaccharides with isomeric glycosidic linkage

As part of a program to explore the structural requirement of N-glycans in the carbohydrate-mediated biological interactions, N-linked pentasaccharide core structure was stereochemically modified in terms of glycosidic linkage. Three isomers, -D-Man-(13)-[-D-Man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, -D-Man-(13)-[-D-Man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, and -D-Man-(13)-[-D-man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, were synthesized. Synthesis of the pentasaccharide with natural linkage is also described.     

Subcellular localization and topology of β(1→4)galactosyltransferase that elongates β(1→4)galactan side chains in rhamnogalacturonan I in potato

The subcellular localization and topology of rhamnogalacturonan I (RG-I) (14)galactosyltransferase(s) ([14]GalTs) from potato (Solanum tuberosum L.) were investigated. Using two-step discontinuous sucrose step gradients, galactosyltransferase (GalT) activity that synthesized 70%-methanol-insoluble products from UDP-[¹⁴C]Gal was detected in both the 0.5 M sucrose fraction and the 0.25/1.1 M sucrose interface. The former fraction contained mainly soluble proteins and the latter was enriched in Golgi vesicles that contained most of the UDPase activity, a Golgi marker. By gel-filtration analysis, products of 180–2,000 Da were found in the soluble fraction, whereas in the Golgi-enriched fraction the products were larger than 80 kDa and could be digested with rhamnogalacturonan lyase and (1,4)endogalactanase to yield smaller rhamnogalacturonan oligomers, galactobiose and galactose. The endogalactanase requires (14)galactans with at least three galactosyl residues for cleavage, indicating that the enzyme(s) present in the 0.25/1.1 M Suc interface transferred one or more galactosyl residues to pre-existing (14)galactans producing RG-I side chains in total longer than a trimer. Thus, the (14)GalT activity that elongates (14)-linked galactan on RG-I was located in the Golgi apparatus. This (14)GalT activity was not reduced after treatment of the Golgi vesicles with proteinase, but approximately 75% of the activity was lost after treatment with proteinase in the presence of Triton X-100. In addition, the (14)GalT activity was recovered in the detergent phase after treatment of Golgi vesicles with Triton X-114. Taken together, these observations supported the view that the RG-I (14)GalT that elongates (14)galactan was mainly located in the Golgi apparatus and integrated into the membrane with its catalytic site facing the lumen.Abbreviations GalT Galactosyltransferase - (14)GalT (14)-Galactosyltransferase - H ⁺ -ATPase Proton ATPase - HG Homogalacturonan - HSP70 ER resident Bip - mMDH Mitochondrial malate dehydrogenase - RG-I Rhamnogalacturonan I - RG-II Rhamnogalacturonan II - RGP Reversibly glycosylated polypeptide - RG-Lyase Rhamnogalacturonan lyase - Suc Sucrose - UDPase Uridine-5-diphosphatase     

Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

d-Xylose as inducer of the xylan-degrading enzyme system in the yeast Pullularia pullulans

Summary The specificity of induction of wooddegrading enzymes from Pullularia pullulans was investigated using series of mono-, di- and (14)--trisaccharides or glycanes. A strain of P. pullulans (1740), unable to grow on Avicel or carboxymethyl-cellulose (CMC), uses xylan and steamexploded wood as carbon sources. This strain, thus grown, was evaluated for various enzyme activities. d-Xylose was the nutritional inducer of -xylosidase and -xylanase. d-Glucuronic acid induced activity on CMC and -glucosidase activity was observed regardless of carbon source used. (14)--Xylobiose was not an inducer of -xylanase production, but high levels of this enzyme were obtained with either structural isomers (12) or (13)-. Since synthesis of this enzyme was stimulated by increasing xylose concentration yp to 40 g/l, it is suggested that xylose enters the cells by passive transport and is unable to induce a permease system.Affiliated to the Scientific, Technological and Medical University of Grenoble     

Hydrolysis of oligosaccharides of the β-(1→4)-linked d-xylose series by an endo(1→4)-β-d-xylanase from the anaerobic rumen fungus Neocallimastix frontalis

An endo-(14)--d-xylanase from Neocallimastix frontalis was purified by anion-exchange chromatography. The enzyme had an apparent molecular mass of 30 kDa on SDS-PAGE and exhibited maximum activity at 50°C and at pH values between 6.0 and 6.6. Kinetic studies on the hydrolysis of xylo-oligosaccharides, ranging from xylobiose to xylodecaose, showed that xylohexaose and xyloheptaose were the preferred substrates for the enzyme and that xylobiose, xylotriose and xylotetraose were not hydrolysed. Xylose was not a product of the hydrolysis of any of the xylo-oligosaccharide substrates tested. The enzyme appeared to have a strong preference for the hydrolysis of the internal glycosidic bonds of the oligosaccharides, which is typical of endo-(14)--d-xylanase activity, but it differed from other fungal endo-(14)--d-xylanases in that it had uniform action on the various internal linkages in the xylo-oligosaccharides.V. Garcia-Campayo, S.I. McCrae and T.M. Wood are with The Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB2 9SB, UK     

Structures of the N-linked sugar chains in the PAS-6 glycoprotein from the bovine milk fat globule membrane

The structures of the N-linked sugar chains in the PAS-6 glycoprotein (PAS-6) from the bovine milk fat globule membrane were determined. The sugar chains were liberated from PAS-6 by hydrazinolysis, and the pyridylaminated sugar chains were separated into a neutral (6N) and two acidic chains (6M and 6D), the acidic sugar chains then being converted to neutral sugar chains (6MN and 6DN). 6N was separated into two neutral fractions (6N13 and 6N5.5), while 6MN and 6DN each gave a single fraction (6MN13 and 6DN13). The structure of 6N5.5, which was the major sugar chain in PAS-6, is proposed to be Man16 (Man13) Man14GlcNAc14GlcNAc-PA; 6N13, 6MN13 and 6DN13 are proposed to be Gal13Gal14GlcNAc12Man16 (Gal13Gal14GlcNAc12Man13) Man14GlcNAc14 (Fuc16)GlcNAc-PA;6M and 6D had 1 or 2 additional NeuAc residues at the non-reducing ends of 6MN13 and 6DN13, respectively. © 1998 Rapid Science Ltd     

Organization of genes required for gellan polysaccharide biosynthesis in<Emphasis Type="Italic"> Sphingomonas elodea</Emphasis> ATCC 31461

Sphingomonas elodea ATCC 31461 produces gellan, a capsular polysaccharide that is useful as a gelling agent for food and microbiological media. Complementation of nonmucoid S. elodea mutants with a gene library resulted in identification of genes essential for gellan biosynthesis. A cluster of 18 genes spanning 21 kb was isolated. These 18 genes are homologous to genes for synthesis of sphingan polysaccharide S-88 from Sphingomonas sp. ATCC 31554, with predicted amino acid identities varying from 61% to 98%. Both polysaccharides have the same tetrasaccharide repeat unit, comprised of [4)--l-rhamnose-(13)--d-glucose-(14)--d-glucuronic acid-(14)--d-glucose-(1]. Polysaccharide S-88, however, has mannose or rhamnose in the fourth position and has a rhamnosyl side chain, while gellan has no sugar side chain but is modified by glyceryl and acetyl substituents. Genes for synthesis of the precursor dTDP-l-rhamnose were highly conserved. The least conserved genes in this cluster encode putative glycosyl transferases III and IV and a gene of unknown function, gelF. Three genes (gelI, gelM, and gelN) affected the amount and rheology of gellan produced. Four additional genes present in the S-88 sphingan biosynthetic gene cluster did not have homologs in the gene cluster for gellan biosynthesis. Three of these gene homologs, gelR, gelS, and gelG, were found in an operon unlinked to the main gellan biosynthetic gene cluster. In a third region, a gene possibly involved in positive regulation of gellan biosynthesis was identified.