α-Galactosidase (Aspergillus niger)

Galactomannan has been extracted from the endosperm of seeds of Gleditsia triacanthos (honey locust) at different stages of development, when the seed was accumulating storage material. Properties of the different samples have been studied. The molecular size distribution became more disperse as galactomannan accumulated and the galactose: mannose ratio decreased slightly. Some possible reasons for these changes are discussed.

A range of galactomannans varying widely in the contents of D-galactose have been compared for self-association and their interaction properties with agarose and xanthan. Whereas, in general, the most interactive galactomannans are those in which the (1→4)-β-D-mannan chain is least substituted by α-D-galactosyl stubs, evidence is presented which indicates that the distribution of D-galactosyl groups along the backbone (fine structure) can have a significant effect on the interaction properties. For galactomannans containing <30% of D-galactose, those which contain a higher frequency of unsubstituted blocks of intermediate length in the β-D-mannan chain are most interactive. For galactomannans containing >40% of D-galactose, those which contain a higher frequency of exactly alternating regions in the β-D-mannan chain are most interactive. This selectivity, on the basis of galactomannan fine-structure, in mixed polysaccharide interactions in vitro could mimic the selectivity of binding of branched plant-cell-wall polysaccharides in biological systems.

A range of galactomannans varying widely in the content of D-galactose have been compared for self-association, and their interaction properties with agarose and xanthan. The results presented indicate that in general the most interactive galactomannans are those in which the D-mannan main chain bears fewest D-galactose stubs, and confirm that the distribution of D-galactose groups along the main chain can have a significant effect on the interactive properties of the galactomannans. It has been shown that freeze — thaw precipitation of galactomannans requires regions of totally unsubstituted D-mannose residues along the main chain, and that a threshold for significant freeze — thaw precipitation occurs at a weight-average length of totally unsubstituted residues of approximately six. For galactomannans having structures above this threshold their interactive properties with other polysaccharides are controlled by structural features associated with totally unsubstituted regions of the D-mannan backbone. In contrast, for galactomannans below this threshold, their interactive properties are controlled by structural features associated with unsubstituted sides of D-mannan backbone.

The distribution of D-galactosyl groups along the D-mannan backbone (fine structure) of carob and guar galactomannans has been studied by a computer analysis of the amounts and structures of oligosaccharides released on hydrolysis of the polymers with two highly purified β-D-mannanases isolated from germinated guar seed and from Aspergillus niger cultures. Computer programmes were developed which accounted for the specific subsite-binding requirements of the β-D-mannanases and which simulated the synthesis of galactomannan by processes in which the D-galactosyl groups were transferred to the growing D-mannan chain in either a statistically random manner or as influenced by nearest-neighbour/second-nearest-neighbour substitution. Such a model was chosen as it is consistent with the known pattern of synthesis of similar polysaccharides, for example, xyloglucan; also, addition to a preformed mannan chain would be unlikely, due to the insoluble nature of such polymers. The D-galactose distribution in carob galactomannan and in the hot- and cold-water-soluble fractions of carob galactomannan has been shown to be non-regular, with a high proportion of substituted couplets, lesser amounts of triplets, and an absence of blocks of substitution. The probability of sequences in which alternate D-mannosyl residues are substituted is low. The probability distribution of block sizes for unsubstituted D-mannosyl residues indicates that there is a higher proportion of blocks of intermediate size than would be present in a galactomannan with a statistically random D-galactose distribution. Based on the almost identical patterns of amounts of oligosaccharides produced on hydrolysis with β-D-mannanase, it appears that galactomannans from seed of a wide range of carob varities have the same fine-structure. The D-galactose distribution in guar-seed galactomannan also appears to be non-regular, and galactomannans from different guar-seed varieties appear to have the same fine-structure.

β-D-Mannosidase (β-D-mannoside mannohydrolase EC 3.2.1.25) was purified 160-fold from crude gut-solution of Helix pomatia by three chromatographic steps and then gave a single protein band (mol. wt. 94,000) on SDS-gel electrophoresis, and three protein bands (of almost identical isoelectric points) on thin-layer iso-electric focusing. Each of these protein bands had enzyme activity. The specific activity of the purified enzyme on p-nitrophenyl β-D-mannopyranoside was 1694 nkat/mg at 40° and it was devoid of α-D-mannosidase, β-D-galactosidase, 2-acet-amido-2-deoxy-D-glucosidase, (1→4)-β-D-mannanase, and (1→4)-β-D-glucanase activities, almost devoid of α-D-galactosidase activity, and contaminated with <0.02% of β-D-glucosidase activity. The purified enzyme had the same K_m for borohydride-reduced β-D-manno-oligosaccharides of d.p. 3-5 (12.5mM). The initial rate of hydrolysis of (1→4)-linked β-D-manno-oligosaccharides of d.p. 2-5 and of reduced β-D-manno-oligosaccharides of d.p. 3-5 was the same, and o-nitrophenyl, methylumbelliferyl, and naphthyl β-D-mannopyranosides were readily hydrolysed. β-D-Mannobiose was hydrolysed at a rate ~25 times that of 6¹-α-D-galactosyl-β-D-mannobiose and 6³-α-D-galactosyl-β-D-mannotetraose, and at ~90 times the rate for β-D-mannobi-itol.

Hydrolysis of galactomannan in endosperms of germinating guar is due to the combined action of three enzymes, α-galactosidase, β-mannanase and exo-β-mannanase. α-Galactosidase and exo-β-mannanase activities occur both in endosperm and cotyledon tissue but β-mannanase occurs only in endosperms. On seed germination, β-mannanase and endospermic α-galactosidase are synthesized and activity changes parallel galactomannan degradation. Galactomannan degradation and synthesis of these two enzymes are inhibited by cycloheximide. In contrast, endospermic exo-β-mannanase is not synthesized on seed germination, but rather is already present throughout endosperm tissue. It has no action on native galactomannan. α-Galactosidase, β-mannanase and exo-β-mannanase have been purified to homogeneity and their separate and combined action in the hydrolysis of galactomannan and effect on the rate of uptake of carbohydrate by cotyledons, studied. Results obtained indicated that these three activities are sufficient to account for galactomannan degradation in vivo and, further, that all three are required. Cotyledons contain an active exo-β-mannanase and sugar-uptake experiments have shown that cotyledons can absorb mannobiose intact, indicating that this enzyme is involved in the complete degradation of galactomannan on seed germination.

Treatment of hot-water-soluble carob galactomannan with β-D-mannanases from A. niger or lucerne seed affords an array of D-galactose-containing β-D-mannosaccharides as well as β-D-manno-biose, -triose, and -tetraose (lucerne-seed enzyme only). The D-galactose-containing β-D-mannosaccharides of d.p. 3–9 produced by A. niger β-D-mannanase have been characterised, using enzymic, n.m.r., and chemical techniques, as 6¹-α-D-galactosyl-β-D-mannobiose, 6¹-α-D-galactosyl-β-D-mannotriose, 6³,6⁴-di-α-D-galactosyl-β-D-mannopentaose (the only heptasaccharide), and 6³,6⁴-di-α-D-galactosyl-β-D-mannohexaose, 6⁴,6⁵-di-α-D-galactosyl-β-D-mannohexaose, and 6¹, 6³,6⁴-tri-α-D-galactosyl-β-D-mannopentaose (the only octasaccharides). Four nonasaccharides have also been characterised. Penta- and hexa-saccharides were absent. Lucerne-seed β-D-mannanase produced the same branched tri-, tetra- and hepta-saccharides, and also penta- and hexa-saccharides that were characterised as 6¹-α-D-galactosyl-β-D-mannotetraose, 6³-α-D-galactosyl-β-D-mannotetraose, 6¹,6³-di-α-D-galactosyl-β-D-mannotetraose, 6³-α-D-galactosyl-β-D-mannopentaose, and 6⁴-α-D-galactosyl-β-D-mannopentaose. None of the oligosaccharides contained a D-galactose stub on the terminal D-mannosyl group nor were they substituted on the second D-mannosyl residue from the reducing terminal.

Purified (1→4)-β-D-mannanase from Aspergillus niger and lucerne seeds has been incubated with mannosaccharides and end-reduced (1→4)-β-D-mannosaccharides and, from the products of hydrolysis, a cyclic reaction-sequence has been proposed. From the heterosaccharides released by hydrolysis of the hot-water-soluble fraction of carob galactomannan by A. niger β-D-mannanase, a pattern of binding between the β-D-mannan chain and the enzyme has been deduced. The products of hydrolysis with the β-D-mannanases from Irpex lacteus, Helix pomatia, Bacillus subtilis, and lucerne and guar seeds have also been determined, and the differences from the action of A. niger β-D-mannanase related to minor differences in substrate binding. The products of hydrolysis of glucomannan are consistent with those expected from the binding pattern proposed from the hydrolysis of galactomannan.

A β-D-mannoside mannohydrolase enzyme has been purified to homogeneity from germinated guar-seeds. Difficulties associated with the extraction and purification appeared to be due to an interaction of the enzyme with other protein material. The purified enzyme hydrolysed various natural and synthetic substrates, including β-D-manno-oligosaccharides and reduced β-D-manno-oligosaccharides of degree of polymerisation 2 to 6, as well as p-nitrophenyl, naphthyl, and methylumbelliferyl β-D-mannopyranosides. The preferred, natural substrate was β-D-mannopentaose, which was hydrolysed at twice the rate of β-D-mannotetraose and five times the rate of β-D-mannotriose. This result, together with the observation that α-D-mannose is released on hydrolysis, indicates that the enzyme is an exo-β-D-mannanase.

N.m.r., enzymic, and chemical techniques have been used to characterise the D-galactose-containing tri- and tetra-saccharides produced on hydrolysis of carob and L. leucocephala D-galacto-D-mannans by Driselase β-D-mannanase. These oligosaccharides were shown to be exclusively 6¹-α-D-galactosyl-β-D-mannobiose and 6¹-α-D-galactosyl-β-D-mannotriose. Furthermore, these were the only D-galactose-containing tri- and tetra-saccharides produced on hydrolysis of carob D-galacto-D-mannan by β-D-mannanases from other sources, including Bacillus subtilis, Aspergillus niger, Helix pomatia gut solution, and germinated legumes. Acid hydrolysis of lucerne galactomannan yielded 6¹-α-D-galactosyl-β-D-mannobiose and 6²-α-D-galactosyl-β-D-mannobiose.

An enzymic technique has been developed for the rapid and accurate quantitation of the galactomannan content of guar seeds and milling fractions. The technique involves the measurement of the galactose component of galactomannans using galactose dehydrogenase. The galactomannans are converted to galactose and manno-oligosaccharides using partially purified enzymes from a commercial preparation and from germinated guar seeds. Simple procedures have been devised for the preparation of these enzymes. Application of the technique to a number of guar varieties gave values for the galactomannan content ranging from 22.7 to 30.8% of seed weight.

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.

A series of galactomannans with varying degrees of galactose substitution have been extracted from the endosperms of legume seeds with water and alkali and the amount of substitution required for water solubility has been determined. Some were heterogeneous with respect to the degree of galactose substitution. The structural requirements for hydrolysis by plant β-mannanase have been studied using the relative rates and extents of hydrolysis of these galactomannans. A more detailed examination of the products of hydrolysis of carob galactomannan has been made. At least two contiguous anhydromannose units appear to be needed for scission. This is similar to the requirement for hydrolysis by microbial enzymes. Judas tree (Cercis siliquastrum) endosperm contained a polysaccharide with a unique composition for a legume seed reserve. Gel chromatography and electrophoresis on cellulose acetate indicated homogeneity. Hydrolysis with a mixture of β-mannanase and α-galactosidase gave a glucose-mannose disaccharide and acetolysis gave a galactose-mannose. These results, as well as the pattern of hydrolysis by β-mannanase were consistent with a galactoglucomannan structure.

Structural changes in galactomannan on germination of lucerne, carob, honey locust, guar and soybean seeds, as measured by viscosity, elution volumes on gel filtration and ultra-centrifugation were slight consistent with a rapid and complete hydrolysis of a molecule once hydrolysis of the mannan chain starts. β-Mannanase activity increased and then decreased, paralleling galactomannan depletion. Multiple forms of β-mannanase were isolated and these were located in the endosperm. β-Mannanase had limited ability to hydrolyse galactomannans with high galactose contents. Seeds containing these galactomannans had very active α-galactosidases. β-Mannosidases were present in both endosperm and cotyledon-embryo and could be separated chromatographically. The level of activity was just sufficient to account for mannose production from manno-oligosaccharides.

Germinating seeds of lucerne, guar, carob and soybean initially depleted raffinose series oligosaccharides and then galactomannan. This depletion was accompanied by a rapid increase and then a decrease in α-galactosidase levels. Lucerne and guar contained two α-galactosidase activities, carob three and soybean four. One of these in each plant, from its location in the endosperm, time of appearance and kinetic behaviour, appeared to be primarily involved in galactomannan hydrolysis. This enzyme in lucerne had MW of 23 000 and could not be separated from β-mannanase by (NH₄)₂SO₄ fractionation, DEAE, CM or SE-cellulose chromatography or gel filtration, but only by polyacrylamide gel electrophoresis. In guar, carob and soybean, it could be separated by ion-exchange chromatography and gel filtration. In lucerne, carob and guar most of the total increase in activity was due to this enzyme. The other α-galactosidases had MWs of about 35 000 and could be separated from β-mannanase by dissection, ion exchange cellulose chromatography and gel filtration. They were located in the cotyledon-embryo and appeared to be primarily involved in galactosylsucrose oligosaccharide hydrolysis.

Background and Aims: Although the plant economic spectrum seeks to explain resource allocation strategies, carbohydrate storage is often omitted. Belowground storage organs are the centre of herb perennation, yet little is known about the role of their turnover, anatomy, and carbohydrate storage in relation to the aboveground economic spectrum. Methods: We collected aboveground traits associated with the economic spectrum, storage organ turnover traits, storage organ inner structure traits, and storage carbohydrate concentrations for approximately eighty temperate meadow species. Key Results: The suites of belowground traits were largely independent from one another, but there was significant correlation between the aboveground traits with both inner structure and storage carbohydrates. Anatomical traits diverged according to leaf nitrogen concentration on one side and vessel area and dry matter content on the other; carbohydrates separated along leaf nitrogen and plant height. Conclusions: Contrary to our expectations, aboveground traits and not storage organ turnover were correlated with anatomy and storage carbohydrates. Belowground traits associated with the aboveground economic spectrum also did not fall clearly within the fast-slow economic continuum, thus indicating the presence of a more complicated economic space. Our study implies that the generally over-looked role of storage within the plant economic spectrum represents an important dimension of plant strategy.

Fructans are important in the survival of plants and also valuable for humans as potentially health promoting food ingredients. In this study fructan content and composition were determined in grains of 20 barley breeding lines and cultivars with a wide variation in chemical composition, morphology and country of origin, grown at one site in Chile. There was significant genotypic variation in grain fructan content ranging from 0.9% to 4.2% of grain dry weight. Fructan degree of polymerisation (DP) was analysed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Changes in the distribution of different chain lengths and the pattern of structures of fructan were detected with increasing amount of fructan in the different barleys. A positive correlation was found between fructan content and the relative amount of long chain fructan (DP > 9) (r = 0.54, p = 0.021). Our results provide a basis for selecting promising barley lines and cultivars for further research on fructan in barley breeding with the aim to produce healthy food products.

Dietary inulin can affect the composition and metabolic activity of the gastrointestinal microbiota in piglets. To investigate whether the chain length of inulin may influence its stability in the gut and the bacterial community, 18 weaned piglets were housed 2 per cage, with 1 female and 1 castrated male animal each. The piglets received a control diet without or with 4% inulin, defined by an average degree of polymerisation (DP) of 31 (short-chain, I31) or 57 (long-chain, I57), with 6 piglets/diet. After a short feeding period of 6 d, fructan concentrations, selected bacterial groups, lactic acid, short-chain fatty acid concentrations, and the pH were determined in the digesta of different segments of the gastrointestinal tract. The results indicated that differences in the microbial degradation of inulin were depending on the DP. Compared to the short-chain inulin, the concentrations of the long-chain inulin were numerically greater in the small intestine and caecum, and greater in the digesta of the ascending colon. Differences were also observed in the bacterial composition of the digesta, showing greater cell numbers of enterococci (P=0.029), bifidobacteria (P=0.029), and Lactobacillus mucosae (P=0.028) in the ileum in group I57 compared to group I31. However, most bacteria tended to be numerically reduced in the ileum in group I31 compared to both control and I57 groups. Minor effects were observed in the ascending colon: L. reuteri and L. amylovorus were decreased in group I57 compared to the control group (P=0.031 and 0.034, respectively), and L. mucosae was decreased in group I31 compared to the control animals (P=0.029). The concentrations of bacterial metabolites were distinctively changed in the large intestine of the piglets fed inulin. The pH was lower in the rectum contents in group I57 compared to the control piglets (P=0.026), but lactic acid and total short-chain fatty acid concentrations were not affected. The molar ratios of propionic acid increased in the caecal contents (P=0.040) and in both, the ascending and descending colonic digesta (P=0.017 and 0.013, respectively) in group I57 compared to the control group, while acetic acid decreased (P<0.001) and n-valeric acid increased (PP=0.011, respectively) in the digesta of the ascending and descending colon in group I57. In conclusion, the microbial degradation of inulin was dependent on its chain length. Long-chain inulin affected the microbial fermentation more pronounced compared to short-chain inulin. The effects were already observed after 6 d, a relatively short application period, indicating that inulin may be used specifically during the sensitive post-weaning period for piglets.

An improved method for the measurement of fructans in wheat grains is presented. A mild acid treatment is used for fructan hydrolysis, followed by analysis of the released glucose and fructose with high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Not only the amount of fructose set free from fructans but also the released glucose can be quantified accurately, allowing determination of the average degree of polymerization of fructans (DP_av). Application of the mild acid treatment to different grain samples demonstrated that a correction should be made for the presence of sucrose and raffinose, but not for stachyose or higher raffinose oligosaccharides. The fructan content and DP_av of spelt flour, wheat flour, and whole wheat flour were 0.6%, 1.2%, and 1.8% of the total weight and 4, 5, and 6, respectively. Validation experiments demonstrate that the proposed quantification method is accurate and repeatable and that also the DP_av determination is precise.

Extractable dietary fiber (DF) plays an important role in nutrition. This study on porridge making with whole grain rye investigated the effect of rest time of flour slurries at room temperature before cooking and amount of flour and salt in the recipe on the content of DF components and molecular weight distribution of extractable fructan, mixed linkage (1→3)(1→4)-β-D-glucan (β-glucan) and arabinoxylan (AX) in the porridge. The content of total DF was increased (from about 20% to 23% of dry matter) during porridge making due to formation of insoluble resistant starch. A small but significant increase in the extractability of β-glucan (P = 0.016) and AX (P = 0.002) due to rest time was also noted. The molecular weight of extractable fructan and AX remained stable during porridge making. However, incubation of the rye flour slurries at increased temperature resulted in a significant decrease in extractable AX molecular weight. The molecular weight of extractable β-glucan decreased greatly during a rest time before cooking, most likely by the action of endogenous enzymes. The amount of salt and flour used in the recipe had small but significant effects on the molecular weight of β-glucan. These results show that whole grain rye porridge made without a rest time before cooking contains extractable DF components maintaining high molecular weights. High molecular weight is most likely of nutritional importance.