Galactomannan Degradation Research Articles

High efficient preparation of low molecular weight galactomannan from Leucaena leucocephala galactomannan through the combination of hydrogen peroxide and oxalic acid

Researchers have always been interested in polysaccharide degradation because of the increased biological activity and usability following degradation. In this work, low molecular weight galactomannan (LMW-GM) was produced through the degradation of galactomannan by H2O2 and oxalic acid (OA). The optimal reaction conditions were found by conducting the response surface optimization experiment based on single-factor experiment and kinetics analysis. Under these conditions, the LMW-GM yield was 69.48 ± 1.02 %. Ultimately, an analysis of the degradation process revealed that OA attacked GM indiscriminately, and H2O2 has a stronger effect on the removal of branched chains while degrading GM. Hence, the degradation steps were rearranged as H2O2 was added 20 min before OA at a constant total time. The LMW-GM yield was successfully increased to 76.49 ± 1.27 %. The goal of this work is hopefully to give a theoretical foundation for the low-cost preparation and industrial production of the degradation of galactomannan.

Defective copper-cobalt binuclear Prussian blue analogue nanozymes with high specificity as lytic polysaccharide monooxygenase-mimic via axial ligation of histidine

Degradation of polysaccharides based on lytic polysaccharide monooxygenases (LPMOs) has received considerably interest in the environment and energy fields since 2010. With the rapid development of nanozymes in various fields, it is highly desirable but challenging to develop LPMO-like nanozymes with high specificity and satisfied activity. Here, a defective copper-cobalt binuclear Prussian blue analogue (CuCoPBA) nanozyme was developed via a facile and ingenious methodology based on single histidine (His). For the first time, His-CuCoPBA nanozyme was found to exhibit LPMO-like activity with H2O2 as a cosubstrate at room temperature and neutral pH, which can efficiently catalyze the degradation of galactomannans selectively. Significantly, the high degradation activity at pH 10 expands the application of Fenton-like nanozymes in alkaline condition. Singlet oxygen (1O2), as a main reactive intermediate, plays a crucial role in the galactomannan degradation catalyzed by His-CuCoPBA nanozyme. Both control experimental and density functional theory (DFT) results indicate Cu-NxHis contributes to the efficiently and selectively catalytic activity of His-CuCoPBA nanozymes by emulating the binding and catalytic sites of LPMOs. The present work not only represents a fundamental breakthrough toward degradation of polysaccharide based on nanozyme, but also contributes to understanding the catalytic mechanism of natural Cu-dependent LPMOs.

Biochemical analyses of a novel acidophilic GH5 β-mannanase from Trichoderma asperellum ND-1 and its application in mannooligosaccharides production from galactomannans.

In this study, an acidophilic GH5 β-mannanase (TaMan5) from Trichoderma asperellum ND-1 was efficiently expressed in Pichia pastoris (a 2.0-fold increase, 67.5 ± 1.95 U/mL). TaMan5 displayed the highest specificity toward locust bean gum (Km = 1.34 mg/mL, Vmax = 749.14 μmol/min/mg) at pH 4.0 and 65°C. Furthermore, TaMan5 displayed remarkable tolerance to acidic environments, retaining over 80% of its original activity at pH 3.0-5.0. The activity of TaMan5 was remarkably decreased by Cu2+, Mn2+, and SDS, while Fe2+/Fe3+ improved the enzyme activity. A thin-layer chromatography (TLC) analysis of the action model showed that TaMan5 could rapidly degrade mannan/MOS into mannobiose without mannose via hydrolysis action as well as transglycosylation. Site-directed mutagenesis results suggested that Glu205, Glu313, and Asp357 of TaMan5 are crucial catalytic residues, with Asp152 playing an auxiliary function. Additionally, TaMan5 and commercial α-galactosidase displayed a remarkable synergistic effect on the degradation of galactomannans. This study provided a novel β-mannanase with ideal characteristics and can be considered a potential candidate for the production of bioactive polysaccharide mannobiose.

Guar gum fortified white breads for prospective postprandial glycaemic control – Effects on bread quality and galactomannan molecular weight

Guar galactomannans effectively reduce post-prandial glycaemic responses and can be used to improve the health impact of white bread. Here we compare the impact of four guars containing galactomannans with varying weight-average molecular weight (Mw) at three different wheat flour substitution levels (5, 10 and 15%) on bread quality. The negative impact of galactomannan incorporation on bread quality becomes more pronounced at higher substitution levels and with higher Mw. However, Mw is likely to be an important parameter for the efficacy of such breads. But we observed a degradation of galactomannan during baking, which was likely caused by enzymatic activity in the dough. A decreased contact time between wheat flour and guar gum, along with pre-hydration of the guar, improved sensory attributes such as texture, markedly decreased the presence of large air holes in the crumb and reduced the degree of depolymerisation of galactomannan during baking. The results from this study give an important starting point for future work of optimizing the baking procedure of guar fortified breads keeping in mind not only bread quality but also a retained Mw of the active ingredient.

Characterization of a Novel Thermophilic Mannanase and Synergistic Hydrolysis of Galactomannan Combined with Swollenin

Aspergillus fumigatus HBFH5 is a thermophilic fungus which can efficiently degrade lignocellulose and which produces a variety of glycoside hydrolase. In the present study, a novel β-mannanase gene (AfMan5A) was expressed in Pichia pastoris and characterized. AfMan5A is composed of 373 amino acids residues, and has a calculated molecular weight of 40 kDa. It has been observed that the amino acid sequence of AfMan5A showed 74.4% homology with the ManBK from Aspergillus niger. In addition, the recombined AfMan5A exhibited optimal hydrolytic activity at 60 °C and pH 6.0. It had no activity loss after incubation for 1h at 60 °C, while 65% of the initial activity was observed after 1 h at 70 °C. Additionally, it maintained about 80% of its activity in the pH range from 3.0 to 9.0. When carob bean gum was used as the substrate, the Km and Vmax values of AfMan5A were 0.21 ± 0.05 mg·mL−1 and 15.22 ± 0.33 U mg−1·min−1, respectively. AfMan5A and AfSwol showed a strong synergistic interaction on galactomannan degradation, increasing the reduction of the sugars by up to 31%. Therefore, these findings contribute to new strategies for improving the hydrolysis of galactomannan using the enzyme cocktail.

Characterization of digestate microbial community structure following thermophilic anaerobic digestion with varying levels of green and food wastes.

The properties of digestates generated through anaerobic digestion are influenced by interactions between the digester microbial communities, feedstock properties and digester operating conditions. This study investigated the effect of varying initial feedstock carbon to nitrogen (C/N) ratios on digestate microbiota and predicted abundance of genes encoding lignocellulolytic activity. The C/N ratio had a significant impact on the digestate microbiome. Feedstocks with intermediate C/N ratio (20-27) (where higher biomethane potential was observed) showed higher relative abundance of archaea compared to feedstocks with C/N ratios at 17 and 34. Within microbial networks, four microbial clusters and eight connector microorganisms changed significantly with the C/N ratio (P < 0.05). Feedstocks with C/N < 23 were richer in organisms from the family Thermotogaceae and genus Caldicoprobacter and enhanced potential for degradation of maltose, galactomannans, melobiose and lactose. This study provides new insights into how anaerobic digestion conditions relate to the structure and functional potential of digester microbial communities, which may be relevant to both digester performance and subsequent utilization of digestates for composting or amending soil.

A facile quantitative characterization method of incomplete degradation products of galactomannan by ethanol fractional precipitation

A novel method for the quantitative characterization of incomplete degradation products of galactomannan (IDPG) was developed by ethanol fractional precipitation. In this work, IDPG in the enzymatic hydrolyzate of Sesbania cannabina galactomannan was fractionated into several fractions with high recovery (>96 %) by ethanol fractional precipitation. The average molecular weights of obtained fractions were in descending order with varied corresponding contents of galactomannan degradation products. Thus, the quantitative characterization of IDPG can be achieved. Moreover, the results of statistical validation and method robustness showed that the method has good reproducibility. Surprisingly, when this method was applied to the guar gum or locust bean gum enzymatic hydrolyzates, it was found that the lower the mannose to galactose ratio of un-hydrolyzed galactomannan, the higher the molecular weight of their degradation product obtained under same ethanol concentration. This method is effectively applied to the quantitative characterization of IDPG from other sources.

What happens when the rain is back? A hypothetical model on how germination and post-germination occur in a species from transient seed banks.

We hypothesize that by simulating the natural priming in seeds of a species that forms transient seed banks it is possible to clarify molecular aspects of germination that lead to the recruitment of seedlings when the next rainy season begins. We used seeds of Solanum lycocarpum as a biological model. Our findings support the idea that the increment of seed germination kinetics when the rainy season returns is mainly based on the metabolism and embryonic growth, and that the hydropriming, at the end of seed dispersion, increases the germination window time of these seeds by mainly increasing the degradation of galactomannan of the cell wall. This can improve the energy supply (based on carbon metabolism) for seedling growth in post-germination, which improves the seedling’s survival chances. From these findings, we promote a hypothetical model about how the priming at the end of the rainy season acts on mRNA synthesis in the germination of seeds from transient banks and the consequence of this priming at the beginning of the following rainy season. This model predicts that besides the Gibberellin and Abscisic Acid balance (content and sensitivity), Auxin would be a key component for the seed-seedling transition in Neotropical areas.Seed collection was performed under authorization number SISGEN AB0EB45.

Backbone 1H, 13C, and 15N resonance assignments of BoMan26A, a \u03b2-mannanase of the glycoside hydrolase family 26 from the human gut bacterium Bacteroides ovatus

Bacteroides ovatus is a member of the human gut microbiota. The importance of this microbial consortium involves the degradation of complex dietary glycans mainly conferred by glycoside hydrolases. In this study we focus on one such catabolic glycoside hydrolase from B. ovatus. The enzyme, termed BoMan26A, is a β-mannanase that takes part in the hydrolytic degradation of galactomannans. The crystal structure of BoMan26A has previously been determined to reveal a TIM-barrel like fold, but the relation between the protein structure and the mode of substrate processing has not yet been studied. Here we report residue-specific assignments for 95% of the 344 backbone amides of BoMan26A. The assignments form the basis for future studies of the relationship between substrate interactions and protein dynamics. In particular, the potential role of loops adjacent to glycan binding sites is of interest for such studies.

Galactomannan degradation by thermophilic enzymes: a hot topic for biotechnological applications.

Extremophilic microorganisms are valuable sources of enzymes for various industrial applications. In fact, given their optimal catalytic activity and operational stability under harsh physical and chemical conditions, they represent a suitable alternative to their mesophilic counterparts. For instance, extremophilic enzymes are important to foster the switch from fossil-based to lignocellulose-based industrial processes. Indeed, more stable enzymes are needed, because the conversion of the lignocellulosic biomass to a wide palette of value-added products requires extreme chemo-physical pre-treatments. Galactomannans are part of the hemicellulose fraction in lignocellulosic biomass. They are heteropolymers constituted by a β-1,4-linked mannan backbone substituted with side chains of α-1,6-linked galactose residues. Therefore, the joint action of different hydrolytic enzymes (i.e. β-mannanase, β-mannosidase and α-galactosidase) is needed to accomplish their complete hydrolysis. So far, numerous galactomannan-degrading enzymes have been isolated and characterized from extremophilic microorganisms. Besides applications in biorefinery, these biocatalysts are also useful to improve the quality (i.e. digestibility and prebiotic properties) of food and feed as well as in paper industries to aid the pulp bleaching process. In this review, an overview about the structure, function and applications of galactomannans is provided. Moreover, a survey of (hyper)-thermophilic galactomannans-degrading enzymes, mainly characterized in the last decade, has been carried out. These extremozymes are described in the light of their biotechnological application in industrial processes requiring harsh conditions.

Galactomannan Degrading Enzymes from the Mannan Utilization Gene Cluster of Alkaliphilic Bacillus sp. N16-5 and Their Synergy on Galactomannan Degradation.

Two glycoside hydrolases encoded by the mannan utilization gene cluster of alkaliphilic Bacillus sp. N16-5 were studied. The recombinant Gal27A (rGal27A) hydrolyzed both galactomannans and oligo-galactomannans to release galactose, while the recombinant Man113A (rMan113A) showed poor activity toward galactomannans, but it hydrolyzed manno-oligosaccharides to release mannose and mannobiose. rGal27A showed synergistic interactions with rMan113A and recombinant β-mannanase ManA (rManA), which is also from Bacillus sp. N16-5, in galactomannan degradation. The synergy degree of rGal27A and rManA on hydrolysis of locust bean gum and guar gum was 1.13 and 2.21, respectively, and that of rGal27A and rMan113A reached 2.00 and 2.68. The main products of galactomannan hydrolyzed by rGal27A and rManA simultaneously were galactose, mannose, mannobiose, and mannotriose, while those of galactomannan hydrolyzed by rGal27A and rMan113A were galactose and mannose. The yields of mannose, mannobiose, and mannotriose dramatically increased compared with the hydrolysis in the presence of rManA or rMan113A alone.

Three novel mutations in α-galactosidase gene involving in galactomannan degradation in endosperm of curd coconut

The deficiency of α-galactosidase activity in coconut endosperm has been reported to cause a disability to hydrolyze oligogalactomannan in endosperm resulting in curd coconut phenotype. However, neither the α-galactosidase encoding gene in coconut nor the mutation type has been identified and characterized in normal and curd coconuts. In this study, cDNA and genomic DNA encoding α-galactosidase gene alleles from a normal and two curd coconuts were successfully cloned and characterized. The deduced amino acid of wild type α-galactosidase contains 398 amino acid residues with a 17 N-terminal amino acids signal peptide sequence. Three mutant alleles, the first 19-amino acids from 67 to 85 (ADALVSTGLARLGYQYVNL) deletion with S137R and the second R216T, were identified from curd coconut plant no.1 while the third P250R was identified from curd coconut plant no. 10. All mutations of α-galactosidase gene were confirmed by the analysis of parental genomic DNA from normal and curd coconuts. Heterologous expression in Komagataella phaffii (Pichia pastoris) indicated that recombinant P250R, R216T and 19-amino acids deletion-S137R mutant proteins showed no α-galactosidase activity. Only the recombinant wild-type protein was able to detect for α-galactosidase activity. These results are in accordance with the no detection of α-galactosidase activity in developing curd coconut endosperms by tissue staining. While, the accumulation of enzyme activity was present in the solid endosperm of normal coconut. The full-length cDNA and parental genomic DNA sequences encoding α-galactosidase in normal coconut as well as identified curd coconut mutant alleles are reported in Genbank accession no. KJ957156 and KM001681-3. Transcription level of the α-galactosidase gene in mature curd coconut endosperm was at least 20 times higher than normal. In conclusion, absence of α-galactosidase activity caused by gene mutations associates with an accumulation of oligogalactomannan in endosperms, resulting in curd coconut phenotype.

Insights into the substrate specificity and synergy with mannanase of family 27 α-galactosidases from Neosartorya fischeri P1.

Thermophilic Neosartorya fischeri P1 is an excellent carbohydrate-active enzyme (CAZyme) producer. Two α-galactosidases of GH (glycoside hydrolase ) family 27 with a very low sequence identity (28.7%), Gal27A and Gal27B, were identified in strain P1 and functionally expressed in Pichia pastoris. In comparison to other characterized GH27 fungal counterparts, rGal27B has a higher temperature optimum (75 °C) and better thermostability (>50% activity at 70 °C for 15 min), and rGal27A shows stability over the broadest pH range (pH 2.0-12.0). Moreover, great distinctions lie in the two enzymes. When using pNPG as the substrate, rGal27B had a higher turnover number (1621.4 vs. 368.3 s(-1)) but lower affinity (2.84 vs. 0.8 mM) and catalytic efficiency (460.8 vs. 580.3 s(-1) mM(-1)) than rGal27A. rGal27B acted on galacto-oligosaccharides, whereas rGal27A was active on polymeric substrates. Although both enzymes showed synergy in galactomannan degradation when combined with a β-mannanase of the same strain, enzyme combinations including rGal27A released more reducing sugars (up to 11.67-fold). Homology modeling predicts different loops in N. fischeri α-galactosidases, highlighting the larger tunnel structure in Gal27A to accommodate/bind branched galactomannan with high galactose contents. Phylogenetic analysis reveals the far relationship of Gal27A and Gal27B that they may evolve in different action modes, and their coexistence widens the substrate spectrum for nutrient utilization. This study illustrates the substrate profiles and synergistic mechanism of GH27 α-galactosidases of different structures.

Carbohydrate mobilisation in germinating seed of Enterolobium contortisiliquum and Peltophorum dubium (Fabaceae), two tropical trees used for restoration

Enterolobium contortisiliquum (Vell.) Morong. and Peltophorum dubium (Spreng.) are two leguminous species native to Brazil that are frequently used to restore degraded areas. Seed of E. contortisiliquum are exalbuminous, whereas seed of P. dubium have a mucilaginous endosperm and both are orthodox, dormant and have a water-impermeable seed coat. There is little information about the dynamics of their germination and understanding this process is important for propagation, conservation and satisfactory practices for restoration of degraded areas. Thus, in this study we evaluated and compared the carbohydrate mobilisation of the seed of both species during germination and early seedling development. Data obtained showed differences in the composition and in the mobilisation of the storage carbohydrates in the studied species. Whereas the main storage of the E. contortisiliquum embryo is starch, the main reserve found in P. dubium is the galactomannan stored in the endosperm. The carbohydrates first hydrolysed in both species are raffinose family oligosaccharides that are used in the embryo development during germination. Starch found in cotyledons of E. contortisiliquum or accumulated after galactomannan degradation in the embryo of P. dubium is not used during germination and early seedling growth in either species.

ManR, a novel Zn(II)2Cys6 transcriptional activator, controls the β-mannan utilization system in Aspergillus oryzae

Fungal endo-β-mannanases (β-mannanases) are widely used as industrial enzymes; however, no transcriptional regulator of β-mannanases has been identified in fungi or other eukaryotic cells to date. To identify a transcriptional regulator of β-mannanases in Aspergillus oryzae, a gene-disruptant library of transcriptional regulators was screened for mutants exhibiting reduced β-mannanase activity by using konjac glucomannan as the substrate, and ManR, a Zn(II)2Cys6 type DNA binding protein was identified. Moreover, a manR-overexpressing strain showed significantly increased β-mannanase activity. DNA microarray analysis of the manR-disruptant strain further indicated that when konjac glucomannan is used as the carbon source, ManR positively regulates the gene expression of not only β-mannanase, but also the enzymes involved in the degradation of galactomannans and glucomannans such as α-galactosidase, β-mannosidase, acetylmannan esterase, and β-glucosidase. Furthermore, we demonstrated that the presence of 1,4-β-d-mannobiose increased the expression of the endo-β-mannanase gene (manG, AO090010000122), and that ManR plays a key role in the inducible expression of manG in A. oryzae. Therefore, we conclude that ManR is a positive regulator of the β-mannan utilization system in A. oryzae. This is the first study to identify a transcriptional regulator of this system in eukaryotic cells.

A Galactomannan-Driven Enhancement of the In Vitro Multiplication Rate for the Marubakaido Apple Rootstock (Malus prunifolia (Willd.) Borkh) is Not Related to the Degradation of the Exogenous Galactomannan

Agar is a complex mixture of gel-forming polysaccharides. Gelling agents are very often used to provide proper support for plants grown in semisolid culture media. And agar is the most frequently used gelling agent in plant tissue culture media. Galactomannans, another group of gel-forming polysaccharides, consists of a (1 → 4)-linked β-D: -mannopyranosyl backbone partially substituted at O-6 with D: -galactopyranosyl side groups. In this work, we demonstrate that a statistically significant 2.7-fold increase on the multiplication rate (MR) for in vitro-grown Marubakaido (Malus prunifolia) shoots was associated with a 12.5% replacement of agar in the semi-solid culture media for a galactomannan obtained from seeds of Schizolobium paraybae. This increase on MR was due mainly to a 1.9-fold increase in the number of main branches and an 8.6-fold increase in the number of primary lateral branches. Gas liquid chromatography and thin layer chromatography analyzes demonstrated that the galactomannan-driven enhancement of the in vitro multiplication rate of the Marubakaido apple rootstock was not related to the galactomannan degradation. To the best of our knowledge, this is the first report on the successful use of partial replacement of high quality agar by a galactomannan from S. paraybae in a micropropagation system for a tree species.

A celluloytic complex from Clostridium cellulovorans consisting of mannanase B and endoglucanase E has synergistic effects on galactomannan degradation

In our previous study using a fluorescently labeled cohesin biomarker, we detected and identified a putative cellulosomal mannanase belonging to the glycosyl hydrolase family 26 from Clostridium cellulovorans in xylan-containing cultures. In this study, a mannanase gene, manB from C. cellulovorans, was expressed in Escherichia coli. The optimal pH of a purified enzyme was around pH 7.0 and the optimal temperature was 40 °C. The purified mannanase B (ManB) showed high hydrolytic activity toward galactomannan. An assembly of ManB with mini-CbpA, which contains a carbohydrate-binding module that provides proximity to insoluble substrates, increased the activity toward galactomannan [locust bean gum (LBG) and guar gum] 1.7- and 2.0-fold over those without mini-CbpA. We tested the synergistic effects on galactomannan (LBG and guar gum) degradation using cellulosomal mannanase ManB with cellulosomal endoglucanase E, which was predicted to have mannanase activity in C. cellulovorans as a cellulolytic complex. When assembled with the mini-CbpA, the mixture of endoglucanase E (EngE) and ManB at a molar ratio of 1:2 showed the highest synergistic effect (2.4-fold) on LBG. The mixture at a ratio of 1:3 showed the highest synergistic effect (2.8-fold) on guar gum. These synergistic actions indicated that ManB assembled with mini-CbpA hydrolyzed insoluble galactomannan, which in turn promoted soluble galactomannan degradation by EngE.

Effects of abscisic acid, ethylene and sugars on the mobilization of storage proteins and carbohydrates in seeds of the tropical tree Sesbania virgata (Leguminosae)

Endospermic legumes are abundant in tropical forests and their establishment is closely related to the mobilization of cell-wall storage polysaccharides. Endosperm cells also store large numbers of protein bodies that play an important role as a nitrogen reserve in this seed. In this work, a systems approach was adopted to evaluate some of the changes in carbohydrates and hormones during the development of seedlings of the rain forest tree Sesbania virgata during the period of establishment. Seeds imbibed abscisic acid (ABA), glucose and sucrose in an atmosphere of ethylene, and the effects of these compounds on the protein contents, α-galactosidase activity and endogenous production of ABA and ethylene by the seeds were observed. The presence of exogenous ABA retarded the degradation of storage protein in the endosperm and decreased α-galactosidase activity in the same tissue during galactomannan degradation, suggesting that ABA represses enzyme action. On the other hand, exogenous ethylene increased α-galactosidase activity in both the endosperm and testa during galactomannan degradation, suggesting an inducing effect of this hormone on the hydrolytic enzymes. Furthermore, the detection of endogenous ABA and ethylene production during the period of storage mobilization and the changes observed in the production of these endogenous hormones in the presence of glucose and sucrose, suggested a correlation between the signalling pathway of these hormones and the sugars. These findings suggest that ABA, ethylene and sugars play a role in the control of the hydrolytic enzyme activities in seeds of S. virgata, controlling the process of storage degradation. This is thought to ensure a balanced flow of the carbon and nitrogen for seedling development.

Storage proteins and cell wall mobilisation in seeds of Sesbania virgata (Cav.) Pers. (Leguminosae)

The endosperm of seeds of Sesbania virgata (Cav.) Pers. accumulates galactomannan as a cell wall storage polysaccharide. It is hydrolysed by three enzymes, one of them being α-galactosidase. A great amount of protein bodies is found in the cytoplasm of endospermic cells, which are thought to play the major role as a nitrogen reserve in this seed. The present work aimed at understanding how the production of enzymes that degrade storage compounds is controlled. We performed experiments with addition of inhibitors of transcription (actinomycin-d and α-amanitin) and translation (cycloheximide) during and after germination. In order to follow the performance of storage mobilisation, we measured fresh mass, protein contents and α-galactosidase activity. All the inhibitors tested had little effect on seed germination and seedling development. Actinomycin-d and cycloheximide provoked a slight inhibition of the storage protein degradation and concomitantly lead to an elevation of the α-galactosidase activity. Although α-amanitin showed some effect on seedling development at latter stages, it presented the former effect and did not change galactomannan degradation performance. Our data suggest that some of the proteases may be synthesised de novo, whereas α-galactosidase seems to be present in the endosperm cells probably as an inactive polypeptide in the protein bodies, being probably activated by proteolysis when the latter organelle is disassembled. These evidences suggest the existence of a connection between storage proteins and carbohydrates mobilisation in seeds of S. virgata, which would play a role by assuring a balanced afflux of the carbon and nitrogen to the seedling development.

Determination of galactose and mannose residues in natural galactomannans using a fast and efficient high-performance liquid chromatography/UV detection

The present work describes the validation of an easy, fast and efficient precolumn derivatization method for the quantification of oligosides, mannose and galactose obtained by degradation of galactomannans. This work combines an acid hydrolysis and an enzymatic degradation of natural galactomannans with the quantification of released residues by reversed-phase HPLC–UV, the most usual HPLC system in laboratories. In case of enzymatic degradation, mannotetraose has been detected and quantified for the first time, and an application to the evaluation of the galactosyl distribution in galactomannans is proposed. After an acidic hydrolysis, this method also allowed to obtain the mannose/galactose (M/G) ratio.