endo-1,4 β-Mannanase (Cellvibrio japonicus)

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.

We propose an injectable nanocomposite hydrogel that is photo-curable via light-induced thiol-ene addition between methacrylate modified O-acetyl-galactoglucomannan (GGMMA) and thiolated cellulose nanocrystal (CNC-SH). Compared to free-radical chain polymerization, the orthogonal step-growth of thiol-ene addition allows a less heterogeneous hydrogel network and more rapid crosslinking kinetics. CNC-SH reinforced the GGMMA hydrogel as both a nanofiller and a crosslinker to GGMMA resulting in an interpenetrating network via thiol-ene addition. Importantly, the mechanical stiffness of the GGMMA/CNC-SH hydrogel is mainly determined by the stoichiometric ratio between the thiol groups on CNC-SH and the methacrylate groups in GGMMA. Meanwhile, the bioactive glass nanoparticle (BaGNP)-laden hydrogels of GGMMA/CNC-SH showed a sustained release of therapeutic ions in simulated body fluid in vitro, which extended the bioactive function of hydrogel matrix. Furthermore, the suitability of the GGMMA/CNC-SH formulation as biomaterial resin to fabricate digitally designed hydrogel constructs via digital light processing (DLP) lithography printing was evaluated.

We describe a method for containing insoluble particulates for use as substrates in either bacterial growth or enzyme assays. This method was designed for high-throughput screening of environmental or engineered bacteria. Benchmarking this method with several model bacteria uncovered phenotypes not observable with the particulate substrates alone.

The galactomannan extracted from Delonix regia (DRGM) seeds was hydrolyzed at different enzyme dosages and reaction times. A linear relation between inverse Molecular weight (Mn) and hydrolysis time was obtained, and the Mn reduction was controlled from 4.86 × 10⁵ to 1.95 × 10⁵ Da. The hydrolysis does not affect galactomannan structure as observed by FT-IR and DRX, maintaining an amorphous structure with only a slight increase in galactose:mannose relationship as observed by ¹H NMR. A rheological behavior shift was observed below Mn = 4.15 × 10⁵ Da when viscoelastic behavior changes from a weak gel to a macromolecular solution in a 3% (w/v) galactomannan hydrolysate. Shear viscosity of 3% (w/v) galactomannan dispersion in water follows closely a power law equation where the consistency index decreased, and the flow behavior index increased with increasing hydrolysis degree. These results indicate the conditions for obtaining specific viscoelastic properties of DRGM dispersions for specific applications by a careful control of the enzymatic hydrolysis process.

The development of a bio-based economy requires the utilization of lignocellulosic biomass in a cost-effective way. The economic viability of lignocellulosic biomass-based industries is hindered by our imperfect understanding of biomass structure and suboptimal industrial processes. To achieve such goals requires direct and rapid monitoring of lignocellulosic polymers as they are physically, chemically, and/or enzymatically treated. In this study, the recently reported fluorescent protein tagged carbohydrate binding modules method (FTCM) was used to specifically track mechanical, chemical and enzymatic-induced variations of hemicelluloses at the surface of different wood fibers. Our results showed that susceptibility to hydrolysis in kraft pulp was higher for xylan, while mannan was more vulnerable in mechanical pulps. Furthermore, FTCM rapidly and efficiently detected enzymatic inactivation and the apparent complementarity (additive and/or synergistic effect) between cellulase and other enzymes (xylanase and mannanase), significantly bolstering cellulose and hemicelluloses hydrolysis. Subsequent addition of xylanase and mannanase enzymes directly proved that xylan was acting as a physical shield which was covering mannan in bleached kraft pulp. This study suggests that mannan was closely associated with cellulose or was deeply embedded in the cell wall organization of such fibers. FTCM provided direct support for previous models on fiber structure that were based on time-consuming and complicated approaches (i.e.chromatography, spectroscopy and microscopy). FTCM allowed for the monitoring of layers of polymers as they were exposed after treatments, providing key information regarding hydrolysis optimization and the specific susceptibility of xylan and mannan to biomass treatments. We believe that by applying this simple and rapid method on site, biomass industries could substantially improve cost-effectiveness of production of biofuels and other lignocellulosic biomass-based products.

The effectiveness of enzymatic hydrolysis of palm-kernel expeller (PKE) is dependent on various factors that influence the stability and functionality of the enzymes. In the present study, parameters influencing the enzymatic treatment of PKE were optimised employing response surface methodology. In addition, the effectiveness of enzymatic hydrolysed PKE in increasing inclusion rates in broiler diets was evaluated. Results showed that temperature, enzyme concentration and duration of hydrolysis had significant (<i>P</i> &lt; 0.01) effects on the enzymatic hydrolysis of PKE. Using the crude enzyme produced by <i>Aspergillus terreus</i> K1 isolated in our laboratory, maximum reduction of crude fibre (40%) was achieved by fermenting the PKE at 60% initial moisture with 9.0 U/g PKE mannanase at 51&deg;C for 18 h, with the production of 9.9% (w/w) of monosaccharides and oligosaccharides. Results of the growth-performance study indicated that inclusion rate of PKE with or without enzyme treatment in broiler diet is 5% for starter period and 20% for the finisher diet, without any detrimental effect on animal performance. Although the inclusion rate of enzyme-treated PKE can be increased to 30% without affecting average daily gain, feed conversion ratio of the birds will be compromised.

Enzymatic hydrolysis was proposed for the first time to purify the sub-fraction of water-extracted cassia polysaccharide (CP-40), and a homogeneous polysaccharide CP-40-M was obtained with relatively high yield. The structural features of CP-40-M were characterized using size-exclusion chromatography equipped with multiple detectors (SEC-MALLS), high-performance anion-exchange chromatography (HPAEC), methylation and gas chromatography-mass (GC–MS), as well as nuclear magnetic resonance (NMR) spectra. The weight-average molecular weight for CP-40-M was determined to be 0.29 × 10⁵ Da, and the molar ratio of xylose to glucuronic acid was 4.62. The structure of CP-40-M was elucidated to be glucuronoxylan, with glucopyranosyluronic acid group terminally attached to O-2 of the →4)-β-Xylp-(1→ backbone. It was the first time to obtain this type of xylan from the water extract of Cassia obtusifolia seeds. The structure elucidation of CP-40-M was meaningful for better understanding the natural characteristics of cassia polysaccharide and important for their potential use in food industry and folk medicine.