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α-Galactosidase (Guar)

alpha-Galactosidase Guar E-AGLGU
Product code: E-AGLGU
€168.00

1,000 Units

Prices exclude VAT

Available for shipping

Content: 1,000 Units
Shipping Temperature: Ambient
Storage Temperature: 2-8oC
Formulation: In 3.2 M ammonium sulphate (stabilised with BSA)
Physical Form: Suspension
Stability: > 4 years at 4oC
Enzyme Activity: α-Galactosidase
EC Number: 3.2.1.22
CAZy Family: GH27
CAS Number: 9025-35-8
Synonyms: alpha-galactosidase; alpha-D-galactoside galactohydrolase
Source: Guar
Molecular Weight: 44,500
Concentration: Supplied at ~ 500 U/mL
Expression: Purified from Guar seed
Specificity: Hydrolysis of terminal, non-reducing α-D-galactose residues in α-D-galactosides, including galactose oligosaccharides, galactomannans and galactolipids.
Specific Activity: ~ 50 U/mg (40oC, pH 4.5 on p-nitrophenyl-α-D-galactopyranoside, before addition of BSA)
Unit Definition: One Unit of activity is the amount of enzyme required to release one µmole of p-nitrophenol (pNP) per minute from p-nitrophenyl-α-D-galactopyranoside per min at pH 4.5 and 40oC.
Temperature Optima: 40oC
pH Optima: 4.5
Application examples: Applications in carbohydrate and glycobiology research.

High purity α-Galactosidase (Guar) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.

Documents
Certificate of Analysis
Safety Data Sheet
FAQs Booklet
Publications
Megazyme publication
Enzymic analysis of the fine structure of galactomannans.

McCleary, B. V. (1994). “Methods in Carbohydrate Chemistry”, Vol. X, (J. N. BeMiller, D. J. Manners and R. J. Sturgeon, Eds.), John Wiley & Sons Inc., pp. 175-182.

A number of methods have been described for the analysis of the fine structure of galactomannans, i.e., the distribution of D-galactosyl units along the D-mannan backbone (1). Such studies include the analysis of x-ray diffraction data of stretched fibers of galactomannans (2,3), 1H- and 13C-nmr (nuclear magnetic resonance) of native and partially depolymerized galacto¬mannans (4) and a range of chemical procedures (5-7), including those employing a detailed theoretical analysis of the kinetics of reaction (8). An alternative approach involves the characterization and quantification of the oligosaccharides produced on hydrolysis of galactomannans by highly purified and well-characterized β-mannanases (EC 3.2.1.78) (9,10). The β-mannanases employed were purified to homogeneity by affinity chromatography on gIucornannan-AH-Sepharose 4B. They were characterized by a range of physicochemicai procedures by determining the kinetics of their action on β-mannooligosaccharides, and by characterizing the structures of oligosaccharides produced on hydrolysis of galactomannans and glucomannans (11). From these studies, a basic model describing the subsite binding requirements of all the β-mannanases examined was proposed (Fig. 1). This model was then modified to account for the slight differences noted in the types of oligosaccharides produced by β-mannanases from different sources. The β-mannanases which differ most significantly in their action patterns on galactomannans are those from Aspergillus niger culture filtrates and from germinated guar seed.

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Megazyme publication
α-D-Galactosidase from lucerne and guar seed.

McCleary, B. V. (1988). “Methods in Enzymology”, Volume 160, (H. Gilbert, Ed.), Elsevier Inc., pp. 627-632.

α-Galactosidase has been shown to occur in a wide range of plants and animals and to be synthesized by microorganisms. This enzyme has been purified from several sources using conventional chromatographic procedures and a range of affinity supports. Of the affinity procedures, that employing N-ɛ-aminocaproyl-α-D-galactopyranosylamine coupled to Sepharose 4B as described by Harpaz et al. is effective and reliable and can be used to purify α-galactosidase from a wide range of biological materials. The affinity technique described by Harpaz et al. was employed to purify α-galactosidase from green coffee beans and from soybean seed. However, neither of these materials is a good source of this activity. This chapter describes the large-scale purification of α-galactosidases with high activity on galactomannan from germinated seeds of lucerne and guar employing the affinity matrix.

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Megazyme publication
Galactomannan changes in developing Gleditsia Triacanthos Seeds.

Mallett, I., McCleary, B. V. & Matheson, N. K. (1987). Phytochemistry, 26(7), 1889-1894.

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.

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Megazyme publication
Effect of galactose-substitution-patterns on the interaction properties of galactomannas.

Dea, I. C. M., Clark, A. H. & McCleary, B. V. (1986). Carbohydrate Research, 147(2), 275-294.

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.

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Megazyme publication

Effect of the molecular fine structure of galactomannans on their interaction properties - the role of unsubstituted sides.

Dea, I. C. M., Clark, A. H. & McCleary, B. V. (1986). Food Hydrocolloids, 1(2), 129-140.

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.

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Megazyme publication
The fine structures of carob and guar galactomannans.

McCleary, B. V., Clark, A. H., Dea, I. C. M. & Rees, D. A. (1985). Carbohydrate Research, 139, 237-260.

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.

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Megazyme publication

Enzymes metabolizing polysaccharides and their application to the analysis of structure and function of glycans.

Matheson, N. K. & McCleary, B. V. (1985). “The Polysaccharides”, Volume 3, (G. O. Aspinall, Ed.), Academic Press Inc., pp. 1-105.

Enzymes metabolizing polysaccharides were used in such processes as baking and brewing for countless centuries before the relationship between the chemical structure of the polysaccharides and their modification by enzymes was known. In the past two centuries, studies of the structures and functions of polysaccharides involved in the storage of chemical energy, in the structural parts of tissues, and as information carriers, as well as the enzymes that metabolize them, have been carried out.

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Megazyme publication
β-D-mannosidase from Helix pomatia.

McCleary, B. V. (1983). Carbohydrate Research, 111(2), 297-310.

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 Km 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.

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Megazyme publication
Enzymic interactions in the hydrolysis of galactomannan in germinating guar: The role of exo-β-mannanase.

McCleary, B. V. (1983). Phytochemistry, 22(3), 649-658.

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.

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Megazyme publication
Characterisation of the oligosaccharides produced on hydrolysis of galactomannan with β-D-mannase.

McCleary, B. V., Nurthen, E., Taravel, F. R. & Joseleau, J. P. (1983). Carbohydrate Research, 118, 91-109.

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.

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Megazyme publication
Action patterns and substrate-binding requirements of β-D-mannanase with mannosaccharides and mannan-type polysaccharides.

McCleary, B. V. & Matheson, N. K. (1983). Carbohydrate Research, 119, 191-219.

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.

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Megazyme publication
Purification and properties of a β-D-mannoside mannohydrolase from guar.

McCleary, B. V. (1982), Carbohydrate Research, 101(1), 75-92.

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.

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Megazyme publication
Preparative–scale isolation and characterisation of 61-α-D-galactosyl-(1→4)-β-D-mannobiose and 62-α-D-galactosyl-(1→4)-β-D-mannobiose.

McCleary, B. V., Taravel, F. R. & Cheetham, N. W. H. (1982). Carbohydrate Research, 104(2), 285-297.

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.

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Megazyme publication
Effect of galactose content on the solution and interaction properties of guar and carob galactomannans.

McCleary, B. V., Amado, R., Waibel, R. & Neukom, H. (1981). Carbohydrate Research, 92(2), 269-285.

Guar galactomannan has been modified by treatment with an α-D-galactosidase A preparation from lucerne seeds. This enzyme was purified by affinity chromatography on N-ϵ-aminocaproyl-α-D-galactopyranosylamine linked to Sepharose 4B, had a high activity towards galactomannans, and was completely devoid of β-D-mannanase. On incubation for 2 h, this enzyme removed > 75% of the galactose from guar galactomannan with no concurrent decrease in viscosity. Eventual decrease in viscosity was associated with the formation of insoluble, mannan-type precipitates. This phenomenon, although directly related to the galactose content of the galactomannan, was also time-dependent. The limiting viscosity numbers calculated for the “mannan backbones” of α-D-galactosidase-treated, guar galactomannan having galactose-mannose ratios of 38:62 to 15:85 were the same. Modified, guar galactomannan (at 0.4% w/v) having a galactose-mannose ratio of 20:80, or less, forms a gel on storage at 4° over several weeks. Also, gel particles form when solutions of these galactomannans are passed through a freeze-thaw cycle. Samples containing < 10% of galactose rapidly precipitate from solution even at 30°. The interaction of guar galactomannan with xanthan is greatly increased by removal of galactose residues. Samples having galactose-mannose ratios of ~19:81 interact with xanthan to essentially the same degree as carob galactomannan (Gal/Man = 23:77).

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Megazyme publication

An enzymic technique for the quantitation of galactomannan in guar Seeds.

McCleary, B. V. (1981). Lebensmittel-Wissenschaft & Technologie, 14, 56-59.

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.

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Megazyme publication
Hydrolysis of legume seed D-galacto-D-mannans by α-D-galactosidases and β-D-mannanases.

McCleary, B. V. (1980). “Mechanisms of Saccharide Polymerization and Depolymerization”, (J. John. Marshall, Ed.), Academic Press Inc., pp. 285-300.

D-Galacto-D-mannans occur in the endosperms of a wide range of leguminous seeds in amounts varying from 0.1% (soybean) to 45% (Cassia brewsterii) of seed weight (1). The polysaccharides from different species have different proportions of D-galactose and D-mannose, but essentially always consist of a β-1,4-linked mannan backbone with single D-galactose branches lined α-1,6 (2).

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

McCleary, B. V. (1979). Phytochemistry, 18(5), 757-763.

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.

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Megazyme publication
Galactomannans and a galactoglucomannan in legume seed endosperms: Structural requirements for β-mannanase hydrolysis.

McCleary, B. V., Matheson, N. K. & Small, D. B. (1976). Phytochemistry, 15(7), 1111-1117.

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.

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Megazyme publication
Galactomannan structure and β-mannanase and β-mannosidase activity in germinating legume seeds.

McCleary, B. V. & Matheson, N. K. (1975). Phytochemistry, 14(5-6), 1187-1194.

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.

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Megazyme publication
α-D-galactosidase activity and galactomannan and galactosylsucrose oligosaccharide depletion in germinating legume seeds.

McCleary, B. V. & Matheson, N. K. (1974). Phytochemistry, 13(9), 1747-1757.

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.

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Publication
Enzyme-modified guar gum/xanthan gelation: An analysis based on cascade model.

Mao, C. F., Zeng, Y. C. & Chen, C. H. (2012). Food Hydrocolloids, 27(1), 50-59.

The gel properties of a mixture of enzymatically-modified guar gum (EMG) and xanthan (XG) were investigated. The guar gum sample treated with α-galactosidase to remove galactose residues had a mannose/galactose ratio of 3.02, and was capable of forming a synergistic gel with xanthan. The concentration-dependent and temperature-dependent modulus data for the EMG/XG gel were analyzed by the two-component cascade model which assumed a heterotypic association between segments of EMG and XG. The optimal functionalities (number of cross-linking sites per chain), fEMG and fXG, were found to be 300 and 30, respectively. The former is equal to the number of segment size with more than six unsubstituted mannose residues in a galactomannan backbone, while the latter corresponds to a cross-link density of one per 37 repeating units in a xanthan molecule. The cascade analysis of the composition dependence of critical gelling concentration was performed by introducing the effect of the homotypic association of XG or EMG, which became significant in the gel point measurement. The enthalpy change per cross-link for the heterotypic association was found to be two times higher than that for the homotypic association.

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Publication
A novel enzymatic technique for limiting drug mobility in a hydrogel matrix.

Burke, M. D., Park, J. O., Srinivasarao, M. & Khan, S. A. (2005). Journal of Controlled Release, 104(1), 141-153.

An oral colon specific drug delivery platform has been developed to facilitate targetted release of therapeutic proteins as well as small molecule drugs. A simple enzymatic procedure is used to modify the molecular architecture of a lightly chemically crosslinked galactomannan hydrogel as well as a model drug–galactomannan oligomer conjugate, fluoroisocynate (FITC) tagged guar oligomer, to entrap the model drug. The enzyme-modified hydrogel retains the drug until it reaches the colonic environment where bacteria secrete enzymes (namely β-mannanase) to degrade the gel and release the drug molecule. Laser scanning confocal microscopy combined with fluorescence recovery after photobleaching is used to quantify the diffusion of the drug conjugate. The diffusion coefficient of solutes in the lightly crosslinked galactomannan hydrogel is approximately equal to the diffusion coefficient in the guar solution for simple diffusional drug loading. After drug loading, α-galactosidase treatment generates additional physical crosslinks in the hydrogel matrix as well as between the drug–oligomer conjugate and the hydrogel, which reduces diffusion of the drug–oligomer conjugate significantly. Degradation of the hydrogel by β-mannanase results in a slow and controlled rate of FITC–guar oligomer diffusion, which generates an extended release profile for the model drug.

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Publication
Does the branching degree of galactomannans influence their effect on whey protein gelation?

Tavares, C., Monteiro, S. R., Moreno, N. & Lopes da Silva, J. A. (2005). Colloids and Surfaces A: Physicochemical and Engineering Aspects, 270, 213-219.

The influence of the degree of branching of galactomannans on the heat-induced gelation of whey proteins was investigated, using oscillatory rheological measurements at low strain amplitude and microstructural analysis by confocal laser scanning microscopy. Galactomannans were from different origins and/or enzymatically modified, with mannose-to-galactose ratios ranging from 1.5 to 3.7. Whey protein gels were formed at 13% protein, pH 7 and low ionic strength. Galactomannan concentration ranged from 0 to 0.6%. Within the range of concentrations used, the presence of the galactomannan decreased the gelling temperature and had a positive effect on the gel strength of the whey protein gel. These effects are more pronounced as the degree of branching decreases. The effect of the original guar sample was quite different from all the other samples, either in terms of rheology or microstructure, particularly for the higher galactomannan concentration. The mixed gels appeared as biphasic systems, with the polysaccharide enriched phase dispersed on the protein matrix at low polysaccharide concentrations, but progressing to a phase inversion at higher polysaccharide concentrations, especially for the lower branched samples. The linear viscoelasticity seems to be insensitive to some of the microstructural changes observed within the mixed gels. The branching degree of the galactomannan does have an effect on microstructure and viscoelasticity of the WPI gels, but this effect is limited to a short range of mannose-to-galactose ratios, above which this effect is insignificant.

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Publication
Enzymatic Modification of Guar Solutions.

Tayal, A., Pai, V., Kelly, R. M. & Khan, S. A. (2002). Water Soluble Polymers, (pp. 41-49), Springer US.

Structurally modified guar galactomannans find application in food and petroleum industries as rheology modifiers. Enzymes provide a powerful and convenient method to modify guar structure. In this study, the kinetics of enzymatic degradation of guar solutions were investigated using SEC and rheology. Molecular information from SEC reveals the degradation reaction to be zeroth order in guar concentration. Further, the rate constant was proportional to enzyme concentration, demonstrating that the enzyme acts as a true catalyst. The zero shear viscosity was very sensitive to degradation, with several orders of magnitude change being observed over the course of polymer chain scission. A unique correlation was developed between degradation time, guar molecular weight and viscosity. This enables superposition of the viscosity-time profiles for different enzyme concentrations to a master curve; providing for a priori prediction of guar solution viscosity as a function of degradation time and enzyme concentration.

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Evolution of microstructure and rheology in mixed polysaccharide systems.

Pai, V., Srinivasarao, M. & Khan, S. A. (2002). Macromolecules, 35(5), 1699-1707.

Synergistic biopolymer blends composed of xanthan and enzymatically modified guar galactomannan are investigated in terms of their time-dependent properties. In particular, a side-chain cleaving enzyme, α-galactosidase, is used to cleave off galactose sugar units from guar to produce modified galactomannans with varying galactose contents of 25.2 and 16.2%. Laser scanning confocal microscopy and dynamic rheology are used to monitor the properties of each of these two modified guar gum in solution as well as in blends with xanthan as they are allowed to age over a period of 3 weeks. Our results indicate that solutions of guar with a higher galactose (25.2%) content undergo no rheological change over the period of observation and show a constant gel elastic modulus (G‘) in blends with xanthan. Confocal images of the solutions and the blends also indicate that the systems are stable over a period of 3 weeks. In contrast, guar gum with a lower galactose content (16.2%) forms interchain associations in solution, developing aggregates that convert it from a macromolecular solution to a gel. This is reflected in its dynamic moduli which increase significantly with time and show a transition from frequency-dependent behavior with G‘ ‘ (viscous modulus) > G‘ (elastic modulus) to a frequency-independent character with G‘ > G‘ ‘. This process of association and phase separation is directly observed in confocal images of the modified guar as well as in its blend, though not to the same extent in the latter. The presence of a second component thus seems to retard the association process. Interestingly, the blend moduli remain unchanged in magnitude and show gellike features even though the mode of association and concomitant microstructure changes.

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Gelation and rheology of xanthan/enzyme-modified guar blends.

Pai, V. B. & Khan, S. A. (2002). Carbohydrate Polymers, 49(2), 207-216.

The rheological behavior and synergistic character of mixed polysaccharide systems are examined for blends of xanthan with enzymatically-modified guar. In particular, the enzyme α-galactosidase is used to selectively cleave off the galactose side chains of guar in order to obtain galactomannans with tailored molecular architecture: EMG1 with a relatively high galactose content of 33.6% and a mannose (M) to galactose (G) ratio of 1.85, and EMG2 with a lower galactose content (25.2%) and an M/G ratio of 2.86. Blends of xanthan with enzymatically-modified guar gum samples are examined in terms of their dynamic rheological properties and compared to those of xanthan - locust bean gum blends. The extent of synergism, illustrated by the gel elastic modulus G′ and yield stress τc, is found to increase with increasing extent of enzymatic modification. At constant ionic strength, the EMG2 and locust bean blends behave similarly, with increasing extent of synergy as the temperature of mixing is increased. Additionally, at a fixed mixing temperature, the blends made in water have a higher elastic modulus than those made in salt. In contrast, the EMG1 blends are weaker and the dynamic moduli are unaffected by changes in the mixing temperature or ionic strength. These results are consistent with those of other researchers and are directly related to both the level of disorder in the xanthan molecule as well as the galactose content and fine structure of the galactomannan.

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Determination of locust bean gum and guar gum by polymerase chain reaction and restriction fragment length polymorphism analysis.

Meyer, K., Rosa, C., Hischenhuber, C. & Meyer, R. (2001). Journal of AOAC International, 84(1), 89-99.

A polymerase chain reaction (PCR) was developed to differentiate the thickening agents locust bean gum (LBG) and the cheaper guar gum in finished food products. Universal primers for amplification of the intergenic spacer region between trnL 3’ (UAA) exon and trnF (GAA) gene in the chloroplast (cp) genome and subsequent restriction analysis were applied to differentiate guar gum and LBG. The presence of <5% (w/w) guar gum powder added to LBG powder was detectable. Based on data obtained from sequencing this intergenic spacer region, a second PCR method for the specific detection of guar gum DNA was also developed. This assay detected guar gum powder in LBG in amounts as low as 1% (w/w). Both methods successfully detected guar gum and/or LBG in ice cream stabilizers and in foodstuffs, such as dairy products, ice cream, dry seasoning mixes, a finished roasting sauce, and a fruit jelly product, but not in products with highly degraded DNA, such as tomato ketchup and sterilized chocolate cream. Both methods detected guar gum and LBG in ice cream and fresh cheese at levels <0.1%.

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Diffusion of macromolecules in polymer solutions and gels: a laser scanning confocal microscopy study.

Burke, M. D., Park, J. O., Srinivasarao, M. & Khan, S. A. (2000). Macromolecules, 33(20), 7500-7507.

Laser scanning confocal microscopy combined with fluorescence recovery after photobleaching is an effective tool to measure the diffusion coefficients of macromolecules in cross-linked hydrogels and polymer solutions. In this study, the effects of enzyme treatment on the diffusion of macromolecules (FITC-dextran) in guar solutions and titanium-guar hydrogels are examined. Enzyme treatment with β-mannanase, a polymer backbone cleaving enzyme, quickly increases the diffusion coefficient of the probe molecules in both solutions and hydrogels to that in water. Enzyme treatment of guar solutions and hydrogels with α-galactosidase, a side chain cleaving enzyme, displays a unique behavior due to changes in the fine structure of guar. The removal of galactose branches from the mannan backbone of guar creates additional hyperentanglements (i.e., cross-links), which reduce the water holding capacity of guar and induce syneresis. If the depth at which the diffusion coefficient is measured remains constant, a minimum is observed in the diffusion coefficient as α-galactosidase enzyme treatment time increases. At the site of measurement, the sample changes from a homogeneous guar system to a phase-separated polymer-rich hydrogel and finally to a dilute polymer phase as the polymer-rich hydrogel phase precipitates below the site of measurement. The diffusion coefficient in the dilute polymer phase increases to that in water, while the diffusion coefficient in the hydrogel phase continues to decrease to a value of approximately 6 × 10-8 cm2/s.

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Rheology and microstructural changes during enzymatic degradation of a guar-borax hydrogel.

Tayal, A., Pai, V. B. & Khan, S. A. (1999). Macromolecules, 32(17), 5567-5574.

Hydrogels composed of borax cross-linked guar galactomannans are enzymatically degraded using endo-β-mannanase, an enzyme which cleaves the polymer chain backbone. Dynamic rheological measurements show the elastic (G‘) and viscous (G‘ ‘) moduli to be sensitive to gel structure and to reduce significantly during the enzymatic hydrolysis process. The reduction in rheological properties shows three distinct regimes:  an initial large decrease, a slower reduction rate at intermediate times, and an accelerated reduction at longer degradation times. In contrast, the polymer chain molecular weight, obtained from gel permeation chromatography, reduces rapidly at short times and at a slower rate subsequently. We therefore find the kinetics of moduli reduction to be dictated by the relationship between gel structure and rheological properties, rather than purely the rates of chain scission. At short times, the large decrease in moduli is analogous to changes in molecular weight and can directly be attributed to chain scission. At long times, corresponding to when the product of polymer concentration and intrinsic viscosity, c[η], reaches a critical value (≤2.5), the chains are too short to overlap and the long range network breaks down rapidly, leading to accelerated moduli reduction. Additionally, a synergistic increase in the degradation rate is observed on using a combination of backbone-cleaving β-mannanase enzyme and a side-chain-cleaving α-galactosidase enzyme, as compared to using only β-mannanase. This can be attributed to an enhancement of mannanase activity due to removal of the sterically hindering galactose side chains. Finally, a comparison of gel and solution degradation reveals very similar behavior in molecular weight changes for both but contrasting trends in rheology.

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Glycosidases—a great synthetic tool.

Scigelova, M., Singh, S. & Crout, D. H. G. (1999). Journal of Molecular Catalysis B: Enzymatic, 6(5), 483-494.

Glycosidases were used to prepare oligosaccharide structures of physiological and medicinal relevance. The study included an extensive screening of crude enzymatic preparations for α- and β-galactosidase, α- and β-mannosidase, β- N-acetylglucosaminidase, β- N-acetylgalactosaminidase and α-L-fucosidase activities. The enzymes were assessed with respect to regioselectivity of glycosyl transfer on to carbohydrate acceptors. The purification procedures for individual biocatalysts are described in detail.

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