Substrate specificities and kinetic properties of two (1→3), (1→4)-β-d-glucan endo-hydrolases from germinating barley (Hordeum vulgare)

Two β-d-glucan endo-hydrolases purified from germinating barley (Hordeum vulgare) hydrolyse (1→4)-β linkages in (1→3),(1→4)-β-d-glucans where the d-glucosyl residue is substituted at O-3, but will not hydrolyse (1→3)-β-d-glucans or (1→4)-β-d-glucans. Methylation analysis of hydrolytic products released from barley (1→3),(1→4)-β-d-glucan indicates that 3-O-β-cellobiosyl-d-glucose and 3-O-β-cellotriosyl-d-glucose are the major oligomers formed. The enzymes exhibit characteristic endo-hydrolase action-patterns on this substrate. Both enzyme can therefore be classified as (1→3),(1→4)-β-d-glucan 4-glucanohydrolases (EC 3.2.1.73). The reduced, pneumococcal polysaccharide RS III, which consists of alternating (1→3)- and (1→4)-linked β-d-glucosyl residues, is hydrolysed by the enzymes to release laminaribiose as a major oligomeric product. Although the kinetic parameters of the two enzymes are similar, one hydrolyses barley (1→3),(1→4)-β-d-glucan at a significantly higher rate than the other and is more stable at elevated temperatures.     

Synthesis of analogs of a metabolite of sodium 2-pyridinethiolate N-oxide (pyrithione)

Antitumor activities of two (1 → 6)-branched (1 → 3)-β-d-glucans, isolated from the fruiting body of Auricularia auricula-judae (“kikurage”, an edible mushroom), and other branched polysaccharides containing a backbone chain of (1 → 3)-α-d-glucosidic or (1 → 3)-α-d-mannosidic linkages [and their corresponding (1 → 3)-d-glycans, derived by mild, Smith degradation] were compared. Among these polysaccharides, a water-soluble, branched (1 → 3)-β-d-glucan (glucan I) of A. auriculajudae exhibited potent, inhibitory activity against implanted Sarcoma 180 solid tumor in mice. The alkali-insoluble, branched (1 → 3)-β-d-glucan (glucan II), a major constituent of the fruiting body, showed essentially no inhibitory activity. When the latter glucan, having numerous branches attached, was modified by controlled, periodate oxidation, borohydride reduction, and mild, acid hydrolysis, the resulting, water-soluble, degraded glucan, having covalently linked polyhydroxy groups attached at O-6 of the (1 → 3)-linked d-glucosyl residues, exhibited potent antitumor activity. Further investigations using the glucan-polyalcohol indicated that the attachment of the polyhydroxy groups to the (1 → 3)-β-d-glucan backbone may enhance the antitumor potency of the glucan. On the other hand, partial introduction     

The molecular structures of some glucans from the cell walls of Schizosaccharomyces pombe

Extraction of the cell walls of Schizosaccharomyces pombe with dilute alkali at 4° yields a mixture of polysaccharides including galactomannan, (1→3)-α-d-glucan, and a branched (1→3)-β-d-glucan. The alkali-insoluble residue contains a lightly branched (1→3)-β-d-glucan, together with smaller amounts of an extremely highly branched (1→6)-β-d-glucan. The properties of the three distinct β-d-glucans are compared with those isolated from other yeasts.     

Structure of an l-arabino-d-xylan from the bark of Cinnamomum zeylanicum

An arabinoxylan isolated from the bark of Cinnamomum zeylanicum was composed of l-arabinose and d-xylose in the molar ratio 1.6:1.0. Partial hydrolysis furnished oligosaccharides which were characterised as α-d-Xylp-(1→3)-d-Ara, β-dXylp-(1→4)-d-Xyl, β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-d-Xyl, β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-β-d-Xylp-Xylp-(1→4)-d-Xyl, xylopentaose, and xylohexaose. Mild acid hydrolysis of the arabinoxylan gave a degraded polysaccharide consisting of l-arabinose (8%) and d-xyolse (92%). Methylation analysis indicated the degraded polysaccharide to be a linear (1→4)-linked d-xlan in which some xylopyranosyl residues were substituted at O-2 or O-3 with l-arabinofuranosyl groups. These data together with the results of methylation analysis and periodate oxidation of the arabinoxylan suggested that it contained a (1→4)-linked β-d-xylan backbone in which each xylopyranosyl residue was substituted both at O-2 and O-3 with l-arabinofuranosyl, 3-O-α-d-xylopyranosyl-l-arabinofuranosyl, and 3-O-l-arabinofuranosyl-l-arabinofuranosyl groups.     

The structure of the low molecular-weight glucans isolated from the siphonous green alga caulerpa simpliciuscula

A β-d-glucan of low molecular weight isolated from the marine alga Caulerpa simpliciuscula has been shown to contain 30 glucose residues. At least 27 of these are β-d-(1→3) linked. There are 1-2β-(1→6) branches per molecule, with a maximum of 4 d-glucose residues per side chain. As normally isolated, this glucan is associated with a soluble (1→4)-α-d-glucan (soluble starch) of the same molecular weight, in the ratio of 3 molecules of β-d-glucan per molecule of α-d-linked glucan.     

Fine structure of (1→3),(1→4)-β-d-glucan from Zea shoot cell-walls

The insoluble material that remains after extraction of Zea shoots with cold buffer was treated successively with 3m LiCl and hot water. The polysaccharides solubilized by these treatments were mostly (1→3),(1→4)-β-d-glucans. The β-d-glucan from the hot-water-soluble fraction was hydrolyzed by Bacillus subtilis (1→3),(1→4)-β-d-glucan 4-glucanohydrolase. The oligosaccharides were characterized by methylation analysis of the enzymic fragments and by methylation analysis of secondary fragments generated by treatment of the isolated oligosaccharides with Streptomyces QM B814 cellulase. The results demonstrate that the native polysaccharide consists mainly of cellotriosyl and cellotetraosyl residues joined by single (1→3) linkages. Evidence is presented to show that certain other glucosyl sequences are also present in the native polysaccharide including (a) two, three, or four contiguous (1→3)-linkages; (b) blocks of more than four (1→4)-linked glucose residues; (c) regions having alternating (1→3)- and (1→4)-linkages.     

The structure of a dextran produced by Leuconostoc mesenteroides NRRL B-1397: the linkages and length of the branches

The structure of a dextran produced by Leuconostoc mesenteroides NRRL B-1397 has been investigated in relation to its immunological properties. The methylated dextran yielded on acid hydrolysis 2,3,4,6-tetra-, 2,3,4-tri-, 3,4,-di-, and 2,4-di-O-methyl-d-glucose, in the molar ratio of 1.0:3.1:0.7:0.2, together with a trace of 2,4,6-tri-O-methyl-dglucose, indicating that the branches occur mainly at O-2 and the remainder at O-3. A carboxyl-dextran, obtained by catalytic oxidation of the dextran to convert the terminal, non-reducing d-glucose residues d-glucuronic acid residues, was partially hydrolyzed with acid. Fractionation gave 2-O-(α-d-glucopyranosyluronic acid) d-glucose (major), 6-O-(α-d-glucopyranosyluronic acid)-d-glucose, and mixtures of aldotri-, aldotetra-, and aldopentaouronic acid that contain both (1 → 6)- and (1 → 2)-d-glucosidic linkages. It is concluded that the branches at O-2 are mainly single d-glucose units, whereas those occurring at O-3 may be longer than two glucose units, forming a highly branched structure having an average repeating- unit of 5 sugar residues.     

Synergism between enzymes of Sclerotium rolfsii involved in the solubilization of crystalline cellulose

Synergism between (1→4)-β-d-glucan cellobiohydrolase, endo-(1→4)-β-d-glucanases, and β-d-glucosidases of Sclerotium rolfsii for solubilization of native and amorphous celluloses is discussed. Besides synergism between cellobiohydrolase and endo-β-glucanases of S. rolfsii, a synergistic effect between endo-β-glucanases and β-glucosidases [which behaved rather as (1→4)-β-d-glucan glucohydrolases] was observed for solubilization of crystalline and amorphous celluloses. It seems that a cellobiohydrolase initiates the attack on crystalline cellulose and an endo-β-d-glucanase the attack on amorphous cellulose.     

Enzymic degradation of chemically modified extracellular polysaccharides from Rhizobia

Extracellular polysaccharides from Rhizobium trifolii, U226, Coryn and Bart A; Rhizobium phaseoli, U453; Rhizobium leguminosarum, U331; and Rhizobium meliloti, U27, after chemical modification, become substrates for certain β-d-glucan hydrolases. The Streptomyces (1 → 4)-β-d-glucan endohydrolase (EC 3.2.1.4) hydrolyses reduced and deacetylated rhizobial polysaccharides, both before and after removal of carboxyethylidene substituents, to produce a series of oligosaccharides. The Rhizopus arrhizus (1 → 3)-β-d-glucan endohydrolase (EC 3.2.1.6) hydrolyses only fully modified polysaccharides to yield, in the case of R. meliloti U27, laminarabiose, and, in all other instances, a disaccharide identified β-d-Gal-(1 → 3)-D-Glc. The same disaccharides are released by the Rhizopus enzyme from oligosaccharides produced by the action of the Streptomyces enzyme on fully modified polysaccharides. The results are discussed in relation to the available data for the structure of the polysaccharides and the specificity of the enzymes.     

Synthesis of two isomeric methyl β-D-xylotriosides containing a (1→2)-β-linkage

Partial hydrolysis with acid, methylation analysis (including uronic acid degradation), Smith degradation, and p.m.r. spectroscopy have been used to determine the primary structure of the capsular polysaccharide of Klebsiella serotype k64. The hexasaccharide repeating-unit, which also contains one O-acetyl substituent, comprises a 4)-α-d-GlcpA-(1 → 3)-α-d-Manp-(1 → 3)-β-d-Glcp-(1 → 4)-α-d-Manp-(1 → chain with a 4,6-O-(l-carboxyethylidene)-β-d-glucopyranosyl and an l-rhamnosyl group attached to the 4-linked d-mannosyl residue at O-2 and O-3, respectively.     

Structural features of two amyloids from the hemi-cellulosic fraction of field-bean (Dolichos lablab) hulls

Two amyloid-type fractions were isolated from field-bean (Dolichos lablab) hulls by 10% alkali extraction followed by acetylation and solvent fractionation. The major, chloroform-insoluble fraction and a minor, chloroform-soluble fraction were found to be homogeneous in sedimentation analysis and molecular-sieve chromatography. The polysaccharides contained xylose and glucose in various proportions. Methylation analysis, periodate oxidation, Smith degradation, oxidation by chromium trioxide, and oligosaccharide studies indicated a new type of structure for the major fraction (glucose:xylose ratio of 1.9:1) in that it had a backbone of (1→4)-linked β-d-glucose residues interspersed with single or multiple residues of (1→4)-linked β-d-xylose, and to which some single d-xylosyl groups are attached through O-6 of d-glucose. In contrast, the minor fraction (glucose:xylose ratio of 1:3.7) had a backbone of (1→4)-linked β-d-xylose interspersed with (1→4)-β-d-glucose and having a side chain of d-xylose, attached through O-6 of d-glucose. The third fraction was found to be a mixture of linear (1→4)-d-glucan and (1→4)-d-xylan.     

Pyruvic acid-containing mono- and oligo-saccharides from rhizobium trifolii bart A

After partial, acid hydrolysis of the extracellular, acid polysaccharide from Rh. trifolii Bart A, the following products were isolated and characterized: 3,4-O-(1-carboxyethylidene)-d-galactose, 4,6-O-(1-carboxyethylidene)-d-galactose, 3-O-[3,4-O-(1-carboxyethylidene)-β-d)-galactopyranosyl]-d-glucose, 3-O-[4,6-O-(1-carboxyethylidene)-β-d-galactopyranosyl]-d-glucose, O-[3,4-O-(1-carboxyethylidene)-β-d-galactopyranosyl ]-(1→3)-O-d-glucopyranosyl-(1→4)-d-glucose, and O-[4,6-O-(1- carboxyethylidene)-β-d-galactopyranosyl]-(1→3)-O-β-d-glucopyranosyl-(1→4)-d-glucose. The presence of pyruvic acid linked either to O-3 and O-4 or to O-4 and O-6 of the d-galactopyranosyl group of these saccharides indicates that both structures may be present in the original polysaccharide.     

Further study of the structure of lentinan,an anti-tumor polysaccharide from Lentinus edodes

The structure of lentinan, an anti-tumor polysaccharide from Lentinus edodes, has been further investigated. Periodate oxidation, Smith degradation, methylation analysis, and bioassay were the principal methods used. These studies showed that a branched molecule having a backbone of (1→3)-β-d-glucan and side chains of both β-d-(1→3)- and β-d-(1→6)-linked d-glucose residues, together with a few internal β-d-(1→6)-linkages, is present.     

Fine structure of (1→3)-β-d-glucans: curdlan and paramylon

Paramylon, a natural (1→3)-β-d-glucan found in Euglena gracilis, and curdlan, a “regenerated” (1→3)-β-d-glucan found in Alcaligenes faecalis, have been studied. Differences in chemical and physical properties are compared to each other and it is concluded that this system is a “native-regenerated” pair just as the well known “native cellulose-regenerated cellulose” system. X-Ray diffraction and density measurements indicate for paramylon a very high level of crystallinity, approaching 90%, whereas curdlan powder is only 30% crystalline. The effect of hydrolytic treatment on the crystallinity of the different samples shows the same trends as for cellulosic materials. From the negative birefringence of annealed fibers and positive birefringence of the paramylon granules, a tangential disposition of the chains in the granules may be concluded. Microfibril formation from curdlan precipitated from solution is in line with a proposed triple-helical structure for the crystalline form of this polysaccharide.     

Purification of some glycoside hydrolases by affinity chromatography

Two glycoproteins have been isolated from the cell walls of baker's yeast. One is a glucan-protein complex which has been partially characterised as having a branched carbohydrate structure composed of chains of (1→3)-linked β-d-glucosyl residues, some of which are attached by (1→6)-linkages to the main chain. Immobilization of this glycoprotein was achieved by covalent attachment to Sepharose, and the product was used to isolate a number of (1→3)-β-d-glucan hydrolases from Helix pomatia, malted barley, and Basidiomycete QM806. The second glycoprotein, a mannan-protein complex, after immobilization, has been used in the purification of an α-d-mannosidase from jack-bean meal.     

Water-soluble (1→3), (1→4)-β-d-glucans from barley (Hordeum vulgare) endosperm. I. Physicochemical properties

Physicochemical methods have been used to define molecular weight, molecular weight distribution, solution behaviour and shape of (1→3), (1→4)-β-d-glucans purified from the 40°C water-extract of barley endosperm by precipitation with 30% saturated ammonium sulphate. The molecular weight and solution properties of a (1→3), (1→4)-β-d-glucan from Australian grown barley (cv. Clipper) are compared with a commercially available preparation. Weight and number average molecular weights are 290 000 and 210 000 respectively for the Clipper (1→3), (1→4)-β-d-glucan and 160 000 and 150 000 respectively for the commercial preparation. The degree of polydispersity is small, but this probably results from the selection of a specific population of (1→3), (1→4)-β-d-glucan molecules during isolation. The higher molecular weight of the Clipper (1→3), (1→4)-β-d-glucan is reflected in higher sedimentation coefficient and intrinsic viscosity values. Viscosity and sedimentation data indicate that the molecules are highly asymmetric, with axial ratios of approximately 100 and 80 for the Clipper (1→3), (1→4)-β-d-glucan and the commercial preparation, respectively. Both polysaccharides appear to exist in solution as extended, worm-like chains.     

Strucrural investigation of Klebsiella serotype K7 polysaccharide

Methylation analysis of and partial hydrolysis studies on the Klebsiella K7 capsular polysaccharide and its carboxyl-reduced derivative indicated the recurrence of D-glucopyranuronic acid, D-mannopyranose, and D-glucopyranose residues, linearly linked in a specific manner, in the molecular structure. D-Galactopyranose and pyruvic acid residues are linked to the main chain on the D-mannose residues (at O-3) and the D-glucose residues (at O-4 and O-6), respectively; the simplest interpretation of this evidence is that nine sugar residues and pyruvic acid constitute a repeating unit. The sequence →3)-β-D-GlcAp-(1→2)-α-D-Manp-(1→2)-α-D-Manp-(1→3)-D-Glcp→ was demonstrated by the isolation from the polysaccharide of an aldotetraouronic acid of this structure.     

The structure of the O-glycosylically-linked oligosaccharide chains of LPG-I,a glycoprotein present in articular lubricating fraction of bovine synovial fluid

Periodate oxidation of LPG-1 established that N-acetylneuraminic acid residues are linked preponderantly α-(2→3) to D-galactose residues. The resistance of 2-acetamido-2-deoxyD-galactose residues to periodate oxidation suggests that they are linked at either O-3 or O-4 to D-galactose residues. After treatment of LPG-I with alkaline sulfite, ≈80% of 2-acetamido-2-deoxygalactose was recovered as the sulfonic acid derivative. The Gal→GalNAc disaccharide released from sialic-acid-free LPG-I by digestion with endo-2-acetamido-2-deoxy-α-D-galactosidase (which suggests an α-D-GalNAc→-L-Ser or -L-Thr linkage) gave a high color-yield in the Morgan—Elson reaction, indicating that 2-acetamido-2-deoxy-D-galactose residues are linked at C-3 to D-galactose residues. The migration of the released Gal-GalNAc disaccharide was the same as that of a standard sample of O-β-D-galactosyl-(1→3)-2-acetamido-2-deoxy-D-galactose. Treatment of sialic acid-free LPG-I with Streptococcus pneumoniae β-D-galactosidase, which hydrolyzes only galactosides linked β-D-(1→4) gave no free D-galactose, whereas treatment of LPG-I with bovine testes β-D-galactosidase released > 90% of D-galactose. These results provide evidence for β-D-Galp-(1→3)-α-D-GalNAcp-(1→3)-L-Ser or -L-Thr and α-NeuAc-(2→3)-β-D-Galp-(1→3)-α-D- GalNAcp-(1→3)-L-Ser or -L-Thr structures. The sensitivity of the methods used and the recovery of constituents following treatment of LPG-I do not rule out the occurrence of small amounts of other tri- or tetra-saccharide chains.     

Chemical structure of an acidic polysaccharide produced by serratia piscatorum

The acidic polysaccharide of Serratia piscatorum consists of L-rhamnopyranosyl, D-galactopyranosyl, and D-galactopyranosyluronic acid residues in the molar ratio of 2:1:1. Some of the D-galactopyranosyluronic acid residues are acetylated at O-2 or O-3, or both. Smith degradation and methylation analysis indicated that the L-rhamnopyranosyl, D-galactopyranosyl, and D-galactopyranosyluronic acid residues are substituted with glycosidic linkages at O-3, O-3, and O-4, respectively. Partial acid hydrolysis of the native polysaccharide gave four acidic oligosaccharides, each of which was isolated and characterized, suggesting the following tetrasaccharide repeating unit: →3)-L-Rhap-(1→4)-D-GalAp-(1→3)-L-Rhap-(1→3)-D-Galp-(1→.