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|Ivory nut seeds
|Mannose = 98
|Main Chain Glycosidic Linkage:
|Substrate For (Enzyme):
High purity Mannan (Ivory Nut) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
Treated with sodium borohydride to lower reducing sugar levels. Traces of arabinose and xylose.
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Novel bi-modular GH19 chitinase with broad pH stability from a fibrolytic intestinal symbiont of Eisenia fetida, Cellulosimicrobium funkei HY-13.
Bai, L., Kim, J., Son, K. H., Chung, C. W., Shin, D. H., Ku, B. H., Kim, D. Y. & Park, H. Y. (2021). Biomolecules, 11(11), 1735.
Endo-type chitinase is the principal enzyme involved in the breakdown of N-acetyl-d-glucosamine-based oligomeric and polymeric materials through hydrolysis. The gene (966-bp) encoding a novel endo-type chitinase (ChiJ), which is comprised of an N-terminal chitin-binding domain type 3 and a C-terminal catalytic glycoside hydrolase family 19 domain, was identified from a fibrolytic intestinal symbiont of the earthworm Eisenia fetida, Cellulosimicrobium funkei HY-13. The highest endochitinase activity of the recombinant enzyme (rChiJ: 30.0 kDa) toward colloidal shrimp shell chitin was found at pH 5.5 and 55 °C and was considerably stable in a wide pH range (3.5–11.0). The enzyme exhibited the highest biocatalytic activity (338.8 U/mg) toward ethylene glycol chitin, preferentially degrading chitin polymers in the following order: ethylene glycol chitin > colloidal shrimp shell chitin > colloidal crab shell chitin. The enzymatic hydrolysis of N-acetyl-β-d-chitooligosaccharides with a degree of polymerization from two to six and colloidal shrimp shell chitin yielded primarily N,N′-diacetyl-β-d-chitobiose together with a small amount of N-acetyl-d-glucosamine. The high chitin-degrading ability of inverting rChiJ with broad pH stability suggests that it can be exploited as a suitable biocatalyst for the preparation of N,N′-diacetyl-β-d-chitobiose, which has been shown to alleviate metabolic dysfunction associated with type 2 diabetes.Hide Abstract
Development and evaluation of an agar capture system (ACS) for high-throughput screening of insoluble particulate substrates with bacterial growth and enzyme activity assays.
Garcia, C. A. & Gardner, J. G. (2021). Journal of Microbiological Methods, 106337.
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.Hide Abstract
A novel AA10 from Paenibacillus curdlanolyticus and its synergistic action on crystalline and complex polysaccharides.
Limsakul, P., Phitsuwan, P., Waeonukul, R., Pason, P., Tachaapaikoon, C., Poomputsa, K., Kosugi, A., Sakka, M., Sakka, K. & Ratanakhanokchai, K. (2020). Applied Microbiology and Biotechnology, 104, 1-18.
Lytic polysaccharide monooxygenases (LPMOs) play an important role in the degradation of complex polysaccharides in lignocellulosic biomass. In the present study, we characterized a modular LPMO (PcAA10A), consisting of a family 10 auxiliary activity of LPMO (AA10) catalytic domain, and non-catalytic domains including a family 5 carbohydrate-binding module, two fibronectin type-3 domains, and a family 3 carbohydrate-binding module from Paenibacillus curdlanolyticus B-6, which was expressed in a recombinant Escherichia coli. Comparison of activities between full-length PcAA10A and the catalytic domain polypeptide (PcAA10A_CD) indicates that the non-catalytic domains are important for the deconstruction of crystalline cellulose and complex polysaccharides contained in untreated lignocellulosic biomass. Interestingly, PcAA10A_CD acted not only on cellulose and chitin, but also on xylan, mannan, and xylan and cellulose contained in lignocellulosic biomass, which has not been reported for the AA10 family. Mutation of the key residues, Trp51 located at subsite − 2 and Phe171 located at subsite +2, in the substrate-binding site of PcAA10A_CD revealed that these residues are substantially involved in broad substrate specificity toward cellulose, xylan, and mannan, albeit with a low effect toward chitin. Furthermore, PcAA10A had a boosting effect on untreated corn hull degradation by P. curdlanolyticus B-6 endo-xylanase Xyn10D and Clostridium thermocellum endo-glucanase Cel9A. These results suggest that PcAA10A is a unique LPMO capable of cleaving and enhancing lignocellulosic biomass degradation, making it a good candidate for biotechnological applications.Hide Abstract
Structural and biochemical characterization of the Cutibacterium acnes exo-β-1,4-mannosidase that targets the N-glycan core of host glycoproteins.
Reichenbach, T., Kalyani, D., Gandini, R., Svartström, O., Aspeborg, H. & Divne, C. (2018). PloS One, 13(9), e0204703.
Commensal and pathogenic bacteria have evolved efficient enzymatic pathways to feed on host carbohydrates, including protein-linked glycans. Most proteins of the human innate and adaptive immune system are glycoproteins where the glycan is critical for structural and functional integrity. Besides enabling nutrition, the degradation of host N-glycans serves as a means for bacteria to modulate the host’s immune system by for instance removing N-glycans on immunoglobulin G. The commensal bacterium Cutibacterium acnes is a gram-positive natural bacterial species of the human skin microbiota. Under certain circumstances, C. acnes can cause pathogenic conditions, acne vulgaris, which typically affects 80% of adolescents, and can become critical for immunosuppressed transplant patients. Others have shown that C. acnes can degrade certain host O-glycans, however, no degradation pathway for host N-glycans has been proposed. To investigate this, we scanned the C. acnes genome and were able to identify a set of gene candidates consistent with a cytoplasmic N-glycan-degradation pathway of the canonical eukaryotic N-glycan core. We also found additional gene sequences containing secretion signals that are possible candidates for initial trimming on the extracellular side. Furthermore, one of the identified gene products of the cytoplasmic pathway, AEE72695, was produced and characterized, and found to be a functional, dimeric exo-β-1,4-mannosidase with activity on the β-1,4 glycosidic bond between the second N-acetylglucosamine and the first mannose residue in the canonical eukaryotic N-glycan core. These findings corroborate our model of the cytoplasmic part of a C. acnes N-glycan degradation pathway.Hide Abstract
Zhang, Q. & Mischnick, P. (2018). Macromolecular Chemistry and Physics, In Press.
Konjak glucomannan (KGM) is methylated with NaOH/MeI in water in the presence of borate and as a reference without this additive. With increasing equiv. of borate increasing suppression of 2- and 3-O-methylation of mannosyl residues (M) is observed, while glucosyl units (G) are mainly affected at O-6, but to much lower extent. Raising the temperature and/or addition of acetone as a co-solvent enhance reactivity, but at the cost of M/G regioselectivity. O-Methyl konjac glucomannans (M-KGM) with an average degree of substitution (DS) up to 0.8 and a DS ratio for G and M up to 2 are obtained. From liquid chromatrography–electrospray ionization–mass spectrometry (LC-ESI-MS) of the oligosaccharides obtained from M-KGM, it is concluded that borate-mediated transient protection might also depend on the location of M within KGM. Comparison with random and block models supports random distribution of M and G in KGM. Thermogravimetric analysis shows higher decomposition temperature of M-KGMs with increasing DS.Hide Abstract
Salmeán, A. A., Guillouzo, A., Duffieux, D., Jam, M., Matard-Mann, M., Larocque, R., Pedersen, H. L., Michel, G., Czjzek, M., Willats, W. G. T. & Hervé, C. (2018). Scientific Reports, 8(1), 2500.
Marine algae are one of the largest sources of carbon on the planet. The microbial degradation of algal polysaccharides to their constitutive sugars is a cornerstone in the global carbon cycle in oceans. Marine polysaccharides are highly complex and heterogeneous, and poorly understood. This is also true for marine microbial proteins that specifically degrade these substrates and when characterized, they are frequently ascribed to new protein families. Marine (meta)genomic datasets contain large numbers of genes with functions putatively assigned to carbohydrate processing, but for which empirical biochemical activity is lacking. There is a paucity of knowledge on both sides of this protein/carbohydrate relationship. Addressing this ‘double blind’ problem requires high throughput strategies that allow large scale screening of protein activities, and polysaccharide occurrence. Glycan microarrays, in particular the Comprehensive Microarray Polymer Profiling (CoMPP) method, are powerful in screening large collections of glycans and we described the integration of this technology to a medium throughput protein expression system focused on marine genes. This methodology (Double Blind CoMPP or DB-CoMPP) enables us to characterize novel polysaccharide-binding proteins and to relate their ligands to algal clades. This data further indicate the potential of the DB-CoMPP technique to accommodate samples of all biological sources.Hide Abstract
Morrill, J., Månberger, A., Rosengren, A., Naidjonoka, P., von Freiesleben, P., Krogh, K. B., Nylander, T., Karlsson, E. N., Adlercreutz, P. & Stålbrand, H. (2018). Applied Microbiology and Biotechnology, 102, 5149-5163.
β-Mannanases catalyze the conversion and modification of β-mannans and may, in addition to hydrolysis, also be capable of transglycosylation which can result in enzymatic synthesis of novel glycoconjugates. Using alcohols as glycosyl acceptors (alcoholysis), β-mannanases can potentially be used to synthesize alkyl glycosides, biodegradable surfactants, from renewable β-mannans. In this paper, we investigate the synthesis of alkyl mannooligosides using glycoside hydrolase family 5 β-mannanases from the fungi Trichoderma reesei (TrMan5A and TrMan5A-R171K) and Aspergillus nidulans (AnMan5C). To evaluate β-mannanase alcoholysis capacity, a novel mass spectrometry-based method was developed that allows for relative comparison of the formation of alcoholysis products using different enzymes or reaction conditions. Differences in alcoholysis capacity and potential secondary hydrolysis of alkyl mannooligosides were observed when comparing alcoholysis catalyzed by the three β-mannanases using methanol or 1-hexanol as acceptor. Among the three β-mannanases studied, TrMan5A was the most efficient in producing hexyl mannooligosides with 1-hexanol as acceptor. Hexyl mannooligosides were synthesized using TrMan5A and purified using high-performance liquid chromatography. The data suggests a high selectivity of TrMan5A for 1- hexanol as acceptor over water. The synthesized hexyl mannooligosides were structurally characterized using nuclear magnetic resonance, with results in agreement with their predicted β-conformation. The surfactant properties of the synthesized hexyl mannooligosides were evaluated using tensiometry, showing that they have similar micelle-forming properties as commercially available hexyl glucosides. The present paper demonstrates the possibility of using β-mannanases for alkyl glycoside synthesis and increases the potential utilization of renewable β-mannans.Hide Abstract
Zhang, Q. & Mischnick, P. (2017). Macromolecular Chemistry and Physics, 218(17), 1700119.
Methylation in water with NaOH/MeI is applied to study the influence of the stereochemistry on relative reactivities of D-mannosyl (M) compared to D-glucosyl (G) units in konjac glucomannan (KGM). The pH is kept constant at 13.6 over the course of the reaction and aliquots are removed after various time intervals. Methyl distribution in G and M residues is determined after perethylation, hydrolysis, and conversion to O-ethyl-O-methyl-alditol acetates. The order of relative rate constants determined for the O-methyl Konjac glucomannans (M-KGMs) in degree of substitution (DS) range 0.3–0.8 is G-k6 > M-k6 > G-k2 ≈ M-k2 > M-k3 > G-k3. Oligosaccharides obtained by partial hydrolysis after full protection of M-KGM with MeI-d3 are labeled with m-amino-benzoic acid and measured by liquid chromatography–electrospray ionization–mass spectrometry. DS/DP profiles are in full agreement with random distribution of methyl groups. Thermal properties of M-KGMs are analyzed by differential scanning calorimetry and thermogravimetric analysis. Decomposition temperature increases with DS, while the temperature of an endothermic change decreases.Hide Abstract
Kim, D. Y., Lee, S. H., Lee, M. J., Cho, H. Y., Lee, J. S., Rhee, Y. H., Shin, D. H., Son K. H. & Park, H. Y. (2017). International Journal of Biological Macromolecules, 106, 620-628.
The gene (1488-bp) encoding a novel GH10 endo-β-1,4-xylanase (XylM) consisting of an N-terminal catalytic GH10 domain and a C-terminal ricin- type β-trefoil lectin domain-like (RICIN) domain was identified from Luteimicrobium xylanilyticum HY-24. The GH10 domain of XylM was 72% identical to that of Micromonospora lupine endo-β-1,4-xylanase and the RICIN domain was 67% identical to that of Actinospica robiniae hypothetical protein. The recombinant enzyme (rXylM: 49 kDa) exhibited maximum activity toward beechwood xylan at 65°C and pH 6.0, while the optimum temperature and pH of its C-terminal truncated mutant (rXylM△RICIN: 35 kDa) were 45°C and 5.0, respectively. After pre- incubation of 1 h at 60°C, rXylM retained over 80% of its initial activity, but the thermostability of rXylM△RICIN was sharply decreased at temperatures exceeding 40°C. The specific activity (254.1 U mg-1) of rXylM toward oat spelts xylan was 3.4-fold higher than that (74.8 U mg-1) of rXylM△RICIN when the same substrate was used. rXylM displayed superior binding capacities to lignin and insoluble polysaccharides compared to rXylM△RICIN. Enzymatic hydrolysis of β-1,4-D-xylooligosaccharides (X3-X6) and birchwood xylan yielded X3 as the major product. The results suggest that the RICIN domain in XylM might play an important role in substrate-binding and biocatalysis.Hide Abstract
Gruben, B. S., Mäkelä, M. R., Kowalczyk, J. E., Zhou, M., Benoit-Gelber, I. & De Vries, R. P. (2017). BMC genomics, 18(1), 900.
Background: The Aspergillus niger genome contains a large repertoire of genes encoding carbohydrate active enzymes (CAZymes) that are targeted to plant polysaccharide degradation enabling A. niger to grow on a wide range of plant biomass substrates. Which genes need to be activated in certain environmental conditions depends on the composition of the available substrate. Previous studies have demonstrated the involvement of a number of transcriptional regulators in plant biomass degradation and have identified sets of target genes for each regulator. In this study, a broad transcriptional analysis was performed of the A. niger genes encoding (putative) plant polysaccharide degrading enzymes. Microarray data focusing on the initial response of A. niger to the presence of plant biomass related carbon sources were analyzed of a wild-type strain N402 that was grown on a large range of carbon sources and of the regulatory mutant strains δxlnR, δaraR, δamyR, δrhaR and δgalX that were grown on their specific inducing compounds. Results: The cluster analysis of the expression data revealed several groups of co-regulated genes, which goes beyond the traditionally described co-regulated gene sets. Additional putative target genes of the selected regulators were identified, based on their expression profile. Notably, in several cases the expression profile puts questions on the function assignment of uncharacterized genes that was based on homology searches, highlighting the need for more extensive biochemical studies into the substrate specificity of enzymes encoded by these non-characterized genes. The data also revealed sets of genes that were upregulated in the regulatory mutants, suggesting interaction between the regulatory systems and a therefore even more complex overall regulatory network than has been reported so far. Conclusions: Expression profiling on a large number of substrates provides better insight in the complex regulatory systems that drive the conversion of plant biomass by fungi. In addition, the data provides additional evidence in favor of and against the similarity-based functions assigned to uncharacterized genes.Hide Abstract