Modes of action of β-mannanase enzymes of diverse origin on legume seed galactomannans

β-Mannanase activities in the commercial enzyme preparations Driselase and Cellulase, in culture solutions of Bacillus subtilis (TX1), in commercial snail gut (Helix pomatia) preparations and in germinated seeds of lucerne, Leucaena leucocephala and honey locust, have been purified by substrate affinity chromatography on glucomannan-AH-Sepharose. On isoelectric focusing, multiple protein bands were found, all of which had β-mannanase activity. Each preparation appeared as a single major band on SDS-polyacrylamide gel electrophoresis. The enzymes varied in their final specific activities, Km values, optimal pH, isoelectric points and pH and temperature stabilities but had similar MWs. The enzymes have different abilities to hydrolyse galactomannans which are highly substituted with galactose. The preparations Driselase and Cellulase contain β-mannanases which can attack highly substituted galactomannans at points of single unsubstituted d-mannosyl residues if the d-galactose residues in the vicinity of the bond to be hydrolysed are all on only one side of the main chain.     

Affinity interactions between agarose and β-1,4-glycans: a model for polysaccharide associations in algal cell walls

This paper reviews the extensive and previously unpublished work on the interactions between agarose and 1,4-linked β-d-glycans carried out at Unilever Research, Colworth Laboratory, UK. The effect of the following variables is discussed: (i) galactose content of galactomannans; (ii) substitution patterns in the agarose molecule; (iii) structural variations in the 1,4-β-d-glycan main chain; and (iv) molecular size of the 1,4-β-d-glycans.Double helices of agarose, a non-substituted regular polysaccharide comprising 1,3-linked β-d-galactose and 1,4-linked 3,6-anhydro-α-l-galactose, bind in an ordered cooperative fashion to an extended ribbon ordered conformation of sequences of 1,4-linked β-d-mannopyranose residues in plant galactomannans to give mixed gelling systems. This interaction survives, in a modified form, substitution along the agarose molecule by O-methyl ether and O-sulphate esters at O6 of the d-galactose and O2 of the 3,6-anhydro-l-galactose, and 4,6-linked pyruvic acid ketal groups on the d-galactose. The higher the level of substitution on the agarose, the weaker the interaction with galactomannan.In general, the higher the level of galactose substitution in the galactomannan the lower the extent of interaction with agarose. Evidence is presented, however, which indicates that the fine structural distribution of galactose along the galactomannan molecule is also an important determinant for the co-gelling interaction. Substituted 1,4-linked β-d-glucomannans, β-d-glucans and β-d-xylans which can form closely similar extended ribbon order conformations to the galactomannans also participate in co-gelling interactions with agarose. These β-d-glycans are similar in structure to important skeletal polysaccharides such as hemicelluloses and cellulose. This suggests that the binding between agars and β-d-glycans might mimic biological cohesion between skeleton and gel phases in natural red seaweed cell walls. The sensitivity of the interactions studied to fine details of agar and β-d-glycan structure is what might be expected on biological grounds, since the wide and subtle variations of natural polysaccharide structure are presumed to represent a mechanism for control of their intermolecular interactions.     

13C-n.m.r.-spectral study of galactosyltransferase specificity with regard to the carbohydrate side-chain of native ovalbumin

As a prelude to studies using bovine N-acetylglucosaminide-β-(1→4)-galactosyltransferase to label membrane-surface glycoproteins with isotopically enriched d-galactose, the structural specificity of the enzymic reaction with water-soluble, hen ovalbumin has been examined. The enzyme-catalyzed transfer of d-galactose from UDP-d-galactose requires a (nonreducing) terminal 2-acetamido-2-deoxy-d-glucosyl group and exhibits selectivity towards saccharide chains containing d-mannose. This study considers the structural specificity of the enzyme with regard to the anomeric linkage between 2-acetamido-2-deoxy-d-glucose and d-mannose in the carbohydrate chains of hen ovalbumin. Uniformly ¹³C-enriched d-galactose was enzymically attached to the ovalbumin carbohydrate chain (which exhibits microheterogeneity in its structure), the protein was hydrolyzed, and separate glycopeptide fractions were chromatographically isolated. The ¹³C-n.m.r. spectra (60.5 MHz) of the fractions revealed two peaks for the anomeric carbon atom of d-galactose. The two peaks, at 104.20 and 104.39 p.p.m., were ascribed to d-galactosyl groups attached to 2-acetamido-2-deoxy-d-glucose respectively linked β-(1→4) and β-(1→2), to d-mannose in the glycopeptide chains. Quantifying of the spectral data revealed no specificity of d-galactosyltransferase towards the linkage from the terminal 2-acetamido-2-deoxy-d-glucosyl group to the penultimate d-mannosyl residue.     

Mammalian-lung galactan

Exopolysaccharides of Agrobacterium tumefaciens and Rhizobium meliloti, containing d-glucose, d-galactose, pyruvic acid, and O-acetyl groups in the approximate proportions 6:1:1:1.5, were analysed by methylation. They were found to contain the following main structural units (all β-glycosidic): chain residues of (1→3)-linked d-glucose (24%), (1→3)-linked d-galactose (15%), (1→4)-linked d-glucose (20%), and (1→6)-linked d-glucose (18%); (1→4,1→6)-linked branching residues of d-glucose (12%), and terminal d-glucose residues substituted at positions 4 and 6 by pyruvate (11%). Uronic acid-containing exopolysaccharides of Rhizobium leguminosarum, R. phaseoli, and R. trifolii contained d-glucose, d-glucuronic acid, d-galactose, pyruvic acid, and O-acetyl groups in the approximate proportions 5:2:1:2:3. Methylation gave identical patterns of methylated sugar components, from which the following structural elements were deduced: chain residues of (1→3)-linked d-glucose substituted at positions 4 and 6 by pyruvate (13%), (1→4)-linked d-glucose (32%), and (1→4)-linked d-glucuronic acid (20%); (1→4,1→6)-linked branching residues of d-galactose and/or d-glucose (13%), and terminal d-glucose and/or d-galactose residues substituted at positions 4 and 6 by pyruvate (13%).     

Isolation and characterization of β-d-glucan,heteropolysaccharide, and trehalose components of the basidiomycetous lichen Cora pavonia

Cora pavonia, a lichen having a basidiomycetous mycobiont, is rich in protein (36%), of which 10% is tyrosine. α,α-Trehalose is present and was isolated in 4.4% yield. The lichen was found to contain polysaccharides typical of basidiomycetes and different from those of ascomycetes and ascomycetous lichens. Isolated and characterized were a β-d-glucan and a heteropolysaccharide containing l-rhamnose, l-fucose, d-xylose, d-mannose, d-glucose, and d-galactose. The β-d-glucan was highly branched with 21% of nonreducing end-groups, contained 3-O-, 6-O-, and 3,6-di-O-substituted β-d-glucopyranosyl units, and had a main chain consisting of (1→3)- and (1→6)-links. The heteropolysaccharide component contained mainly mannose and xylose, having a mannose-containing nucleus and a main chain with preponderant (1→3)-linked α-d-mannopyranosyl residues. These were unsubstituted (10%), and 4-O- (10%) and 2,4-di-O-substituted (10%) with residues of β-d-xylopyranose. On methylation analysis of the heteropolysaccharide, a capillary column of DB-210 proved to be particularly useful for gas-liquid chromatographic resolution of partially O-methylated alditol acetates.     

Interaction properties of d-galactose-depleted guar galactomannan samples

The interaction of galactose-depleted guar galactomannan with agarose has been studied by optical rotation, and with xanthan using an Instron Materials Tester and a Rheometrics Mechanical Spectrometer. Samples were prepared by treatment of guar galactomannan with highly purified α-d-galactosidase. Modified guar galactomannans with a d-galactose content of 19–25%, in admixture with agarose, showed similar optical rotation changes on heating and cooling as did mixtures of agarose and carob galactomannan (23% d-galactose content). Modified samples with 13–16% d-galactose, in the presence of agarose, showed more pronounced optical rotation changes on heating and cooling, but samples with less than 13% d-galactose were only sparingly soluble even on autoclaving. The degree of interaction of galactose-depleted guar galactomannan samples with xanthan, as measured rheologically, increased as the d-galactose content decreased, paralleling the optical rotation changes with galactomannan/agarose mixtures. In the presence of xanthan, samples with a d-galactose content of 25% or less formed firm rubbery gels.     

Structural analysis of galactomannans by NMR spectroscopy

The structure of naturally occurring galactomannans was characterized by high resolution NMR spectroscopy involving two-dimensional (2D) NMR measurements of the field gradient DQF-COSY, HMQC, HMBC, and ROESY experiments. Four galactomannans with different proportions of galactose (G) and mannose (M), from fenugreek gum (FG), guar gum (GG), tara gum (TG), and locust bean gum (LG), were investigated. Because these galactomannans had very high molecular weights, hydrolysis by dilute H₂SO₄ was carried out to give the corresponding low molecular weight galactomannans, the structural identities of which were established by comparison of the specific rotations, shape of the GPC profiles, and NMR spectra with those of higher molecular weight galactomannans. The correlation signals GH1-GC4, -GC5, and -MC6 in HMBC and GH1-GH6 in ROESY spectra of FG showed that more than two galactopyranose units with the 1 → 4 linkage were connected at C6 of the mannopyranose main chain. The coupling constant (J_H1,2) of galactose was 3.4 Hz, indicating that galactose has an α-linkage. The main chain mannose was found to connect through the 1 → 4 linkage, because of the appearance of the correlation signals MH1-MC4, and MC1-MH4 in the HMBC spectrum due to the long-range correlation signals between two neighboring mannopyranose residues through the M4-O-M1 bond. Although the main chain mannose J_H1,2 was not observed, probably because of the high molecular weight, the specific rotation of LG with a higher proportion of mannose was low, [α]_D²⁵ = +10.8°, compared with that of FG with a lower proportion of mannose, [α]_D²⁵ = +90.5°, suggesting that the mannose in the main chain had a α-linkage. These results suggest that the galactomannans comprise a (1 → 4)-β-mannopyranosidic main chain connected with more than two (1 → 4)-α-galactopyranosidic side chains, in addition to the single galactopyranose side chain, at C6 of the mannopyranose main chain.     

The carbohydrates of the green seaweeds urospora wormskioldii and codiolum pusillum

Urospora wormskioldii and Codiolum pusillum are different life forms of this arctic alga. They both metabolise d-glucose, d-fructose, sucrose, myo-inositol, glyceric acid, and malto-oligosaccharides. In Codiolum, 1,3-linked d-glucose and l-rhamnose oligosaccharides were also present. The major polysaccharide extracted by water from both forms is a polydisperse, sulphated glucuronoxylorhamnan. Polysaccharides containing 1,3-, 1,4-, and triply linked d-glucose residues were also isolated from the aqueous extracts. Pure amylopectin-type polysaccharides were isolated from acid extracts of both forms of the weed. The major difference between the two forms was the presence in Codiolum of a sulphated (1→4)-linked β-d-mannan branched at C-6 and sulphated at C-2. The similarities and differences of the carbohydrates with those of Urospora penicilliformis and other green seaweeds are discussed.     

Structure and biodegradation of the polysaccharide components of egg masses isolated from snails of an Ampullarius species

A polysaccharide preparation, isolated from egg masses deposited by snails of an Ampullarius species, was purified via precipitation with Cetavlon in the presence of sodium borate, and found to contain d-galactose and a smaller proportion of d-glucose, and to have two components with sedimentation coefficients of 10S and 40S. A polysaccharide, isolated from freshly laid egg masses, was highly branched and was shown to contain nonreducing α-d-glucopyranosyl and β-d-galactopyranosyl end-groups, and 3,6-di-O-substituted β-d-galactopyranosyl residues. One or more of the polysaccharide components was a d-glucopyrano-d-galactopyranan with non-reducing α-d-glucopyranosyl end-groups (1→4)-linked to β-d-galactopyranosyl residues. The polysaccharide preparations, obtained from freshly laid egg masses and from those that were left for 10 and 15 days after being laid, were structurally different from each other. With the passage of time, progressive diminution of the 10S component and the proportion of d-glucose in the polysaccharide took place, suggesting that each constituent was consumed preferentially by the snail embryos as an energy source.     

Structural studies of plant gum from sap of the lac tree, Rhus vernicifera

The plant gum isolated from sap of the lac tree, Rhus vernicifera (China), was separated into two fractions having mol. wt. 84,000 and 27,700 by aqueous-phase gel-permeation chromatography. The fractions contain d-galactose (65 mol%), 4-O-methyl-d-glucuronic acid (24 mol%), d-glucuronic acid (3 mol%), l-arabinose (4 mol%), and l-rhamnose (3 mol%). Smith degradation of the carboxyl-reduced polysaccharides gives products of halved molecular weight, and these consist of a β-(1→3)-linked galactopyranan main chain and side chains made up of galactopyranose residues. Peripheral groups, such as α-d-Galp-, α-d-Galp-(1→6)-β-d-Galp-, 4-O-methyl-β-d-GlcpA-, and 4-O-methyl-β-d-GlcpA-(1→6)-β-d-Galp-, are attached to this interior core through β-(1→3)- or β-(1→6)-linkages.     

Diverse patterns of cell wall mannan/galactomannan occurrence in seeds of the Leguminosae

Endosperms from seeds of different subfamilies of Leguminosae were submitted to sequential aqueous and alkaline aqueous extractions. The extractions from species belonging to the Mimosoideae and Faboideae subfamilies yielded galactomannans with constant Man:Gal ratios, whereas the extractions from Caesalpinioideae seeds gave rise to galactomannans with increasing values of the Man:Gal ratio. The presence of a family of galactomannans within the same species may be a trait found only in Caesalpinioideae subfamily. The final insoluble residues that were obtained after the removal of galactomannans from the Caesalpinioideae and Faboideae subfamilies are composed of pure mannans and do not contain cellulose, while those from the Mimosoideae subfamily are composed of cellulose. A mannan was isolated from the unripe endosperm of Caesalpinia pulcherrima, suggesting no developmental relationship between galactomannan and mannan. These results are consistent with the presence of a distinctive cell wall pattern in the endosperms of Leguminosae species.     

The mucilage of Opuntia ficus-indica

The mucilage extracted from the cladodes (modified stems) of Opuntia ficus-indica contains residues of d-galactose, d-xylose, l-arabinose, l-rhamnose, and d-galacturonic acid. Seasonal variation in the sugar composition of the mucilage has been investigated. Fractionation studies indicate that the mucilage is essentially homogeneous. The rate of release of the constituent sugars and the change in optical rotation on mild hydrolysis coupled with the results of chromic acid oxidation suggest that the mucilage contains α-arabinofuranosyl, β-xylopyranosyl, β-rhamnopyranosyl, β-galactopyranosyl, and α-galactopyranosyluronic acid residues. The results also suggest a core containing galacturonic acid, rhamnose, and galactose, to which xylose and arabinose are attached in peripheral positions.     

The mannan from Schizolobium parahybae endosperm is not a reserve polysaccharide

In higher plants, mannans are found as dominant reserve material in the endosperm of Arecaceae seeds and also in some species from Apiaceae, Rubiaceae and Asteraceae. A linear β(1 → 4)-d-mannan was now isolated from the endosperm of Schizolobium parahybae, family Caesalpiniaceae, a native of Southern Brazil. Its seeds were germinated and the consumption of polysaccharides from the endosperm, namely galactomannans and β(1 → 4)-d-mannan, was analysed at differents stages of germination. At the 6th day after germination no residual 3:1 Man:Gal galactomannan was found, indicating that complete degradation of galactomannan had been reached. However, after 12 days of germination, the mannan was recovered from the remaining endosperm. Its presence in the endosperm after germination demonstrated that it is not a reserve material as described for seeds of other species.     

The preparation of a crystalline guanosine trialcohol and its sodium salt

A polysaccharide has been extracted from Cassia corymbosa seeds with cold, acidulated water, and purified to give a water-soluble product containing d-galactose and d-mannose in 4:7 molar ratio. Acid-catalyzed fragmentation, periodate oxidation, methylation, and enzymic hydrolysis showed that the seed gum has a branched structure consisting of a linear chain of β-d-(1→4)-linked mannopyranosyl units, some of which are substituted at O-6 by two α-d-(1→6) galactopyranosyl units mutually linked glycosidically. Methylation analysis of the galactomannan afforded 2,3,4-tri- and 2,3,4,6-tetra-O-methylgalactose, along with 2,3-di- and 2,3,6-tri-O-methylmannose, in the molar ratios of 2:2:2:5. Both the methylation and the periodate-oxidation studies showed ～36.4% of end groups. The significance of these results, together with the findings of partial hydrolysis with acid, are discussed, in relation to ascertaining the structure of the repeating unit of the polysaccharide.     

Distribution of D-galactosyl groups in guaran and locust-bean gum

Distribution of α-d-galactopyranosyl side-chain groups in two galactomannans, guaran and locust-bean gum, was determined by measurement of the O-acetyl-O-methyl-d-mannitol derivatives obtained from the corresponding primary C-p-tolylsulfonyl polysaccharide derivatives. The O-acetyl-O-methyl-d-mannitol derivatives were produced by β-elimination and methylation, with sodium (methylsulfinyl)methide and methyl iodide, of the primary C-p-toluenesulfinylated galactomannans, followed by sequential acid hydrolysis, reduction, and acetylation of the partially degraded p-tolyl sulfones. The results indicated that side-chain units of guaran are alternately disposed along the d-mannan backbone, whereas those of locust-bean gum are disposed in uniform blocks along the backbone.     

Isolation of a collagen fraction from the body-wall glycoproteins of the leech (Hirudo medicinalis), and characterization of its carbohydrate-amino acid portion

Fractionation of the leech (Hirudo medicinalis) body-wall glycoproteins yielded a collagen fraction containing only d-glucose and d-galactose as its carbohydrate constituents. Digestion of the collagen with trypsin and pronase, and alkaline degradation of the resulting glycopeptides, gave a product that contained a disaccharide linked to hydroxylysine. Mild, acid hydrolysis of the N-acetylated glycopeptides yielded a disaccharide consisting of a d-glucose and a d-galactose residue. Various chemical and enzymic reactions of the disaccharide, the glycosyloxylysine, and the glycopeptide fraction indicated that the disaccharide is 2-O-α-d-glucopyranosyl-d-galactose, and that this is β-glycosidically linked to O-5 of the hydroxylysine residue in the collagen.     

STRUCTURAL STUDIES ON THE GALACTOMANNANS OF LICHENS OF THE GENUSCLADONIA

Galactomannans were isolated fromCladonia signata,C. furcata,C. imperialis, andC. clathratavia successive alkaline extraction and precipitation with Fehling solution and Cetavlon. They were investigated using¹³C-NMR spectroscopy, methylation analysis, and Smith degradation, and their specific rotations and monosaccharide compositions determined. As with galactomannans of otherCladoniaspecies, they contained (1→6)-linked main chains of α-mannopyranose, which were non-substituted (structure4in Fig. 2), mono-substituted at O-2 with α-mannopyranose (structure6) or α-galactopyranose (structure1), O-4 with β-galactopyranose (structure2), and disubstituted at O-2 and O-4 with α-mannopyranosyl and β-galactopyranosyl units, respectively (structure5). Disubstitution was present to a greater extent in the galactomannans ofC. clathrataandC. imperialisthan in those ofC. signataandC. furcata. In the case of the galactomannans ofC. furcata,C. clathrata, andC. imperialis, substitution also occurred at O-2 withO-β-galactofuranosyl-(1→6)-O-α-mannopyranosyl units (structure7). As observed in previous investigations, the C-1 portion of the¹³C-NMR of mannose-containing polysaccharides is typical of the lichen species. However, those of galactomannans ofC. imperialisandC. clathrataare almost identical and, although other chemical data showed many structures in common, some differences were evident.     

The structure of the neutral polysaccharide gum secreted by Rhizobium strain cb744

A previous investigation of the structure of the extracellular polysaccharide gum from the nitrogen-fixing Rhizobium strain cb744 (a member of the slow-growing Cowpea group) indicated that there were two β-(1→4)-linked d-glucopyranosyl residues for each α-(1→4)-linked d-mannopyranosyl residue, and that each mannose was substituted at O-6 by a β-d-galactopyranosyl residue having 71% of the galactose present as 4-O-methylgalactose. The present study shows that, although the gum appeared to have a simple tetrasaccharide repeating unit, it is composed of two closely associated components. One is a (1→4)-linked α-d-mannan substituted at each O-6 by a β-d-galactopyranosyl residue (71% 4-O-methylated). The second component is a (1→4)-linked β-d-glucan. The existence of the two polysaccharides was established by separation of the β-d-galactosidase-treated gum on a column of concanavalin A-Sepharose 4B. The d configurations were determined and the anomeric attribution of the linkages confirmed by the use of enzymes. The interaction between the two gum components is discussed.     

Evaluation of the cold-active Pseudoalteromonas haloplanktis β-galactosidase enzyme for lactose hydrolysis in whey permeate as primary step of d-tagatose production

d-Tagatose is an innovative natural low-calorie bulk sweetener with a broad potential for low-calorie and low-glycaemic foods and drinks. Production of this healthy sweetener is realized through enzymatic d-galactose isomerization. d-Galactose needs to be produced in situ due to its limited availability. Whey permeate contains a substantial amount of lactose, which is an interesting source for d-galactose production through enzymatic lactose hydrolysis. In this context, the cold-active β-galactosidase from the psychrophile Pseudoalteromonas haloplanktis was studied. Optimal parameters for efficient lactose hydrolysis in whey permeate have been deduced, viz. optimal incubation temperature, pH and lactose concentration. Hydrolysis efficiencies above 96.0% were realized within 24 h at 23 °C and pH 7.0 in whey permeate with a maximum dry matter content of 10.0% (w/w). In addition, the effect of the presence of d-glucose and d-galactose was investigated up to concentrations of 100 g l⁻¹. d-Glucose inhibited lactose hydrolysis more strongly compared to d-galactose. Also, the operational stability of the cold-active β-galactosidase was studied. Hydrolysis efficiencies above 90.0% were maintained during 7 subsequent hydrolysis cycles.     

An arabinogalactan from the seeds of Pseudium guava

Hemicelluloses of seeds of Pseudium guava containing d-galactose (59.6), d-arabinose (35.9), and a uronic acid (4.5%) were analyzed by methylation analysis and Smith-degradation analysis, and the following structural elements were deduced; chain residues of (1→4)-linked d-galactose, (1→5)-linked d-arabinose, and terminal d-arabinose residues. The following structure was assigned to the polysaccharide. →5)-d-Araf-(1→4)-d-Galf-(1→5)-d-Araf-(1→