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|Stability:||> 10 years under recommended storage conditions|
|Substrate For (Enzyme):||endo-1,3-β-Glucanase|
High purity Laminaripentaose for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.
Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.
Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.Hide Abstract
β-Glucosidase BGL1 from Coprinopsis cinerea Exhibits a Distinctive Hydrolysis and Transglycosylation Activity for Application in the Production of 3-O-β-D-Gentiobiosyl-D-laminarioligosaccharides.
Kang, L., Zhang, X., Wang, R., Liu, C., Yi, L., Liu, Z., Zhang, Z. & Yuan, S. (2019). Journal of Agricultural and Food Chemistry, 67(38), 10744-10755.
We previously reported that β-glucosidase BGL1 at low concentration (15 µg mL-1) from Coprinopsis cinereal exhibited hydrolytic activity only toward laminarioligosaccharides but not toward cellooligosaccharides and gentiobiose. This study shows that BGL1 at high concentration (200 µg mL-1) also hydrolyzed cellobiose and gentiobiose, which accounted for only 0.83 and 2.05% of its activity toward laminaribiose, respectively. Interestingly, BGL1 at low concentration (1.5 µg mL-1) showed transglycosylation but BGL1 at high concentration (200 µg mL-1) did not. BGL1 utilizes only laminarioligosaccharides but not glucose, gentiobiose, and cellobiose to synthesize the higher oligosaccharides. BGL1 transferred one glucosyl residue from substrate laminarioligosaccharide to another laminarioligosaccharide as an acceptor in a β(1 → 3) or β(1 → 6) fashion to produce higher laminarioligosaccharides or 3-O-β-D-gentiobiosyl-D-laminarioligosaccharides. The BGL1-digested laminaritriose exhibited approximately 90% enhancement in the anti-oxidant activity compared to that of untreated laminaritriose, implying a potential application of BGL1-based transglycosylation for the production of high value-added rare oligosaccharides.Hide Abstract
Mechanisms of redundancy and specificity of the Aspergillus fumigatus Crh transglycosylases.
Fang, W., Sanz, A. B., Bartual, S. G., Wang, B., Ferenbach, A. T., Farkaš, V., Hurtado-Guerrero, R., Arroya, J. & Van Aalten, D. M. (2019). Nature Communications, 10(1), 1-10.
Fungal cell wall synthesis is achieved by a balance of glycosyltransferase, hydrolase and transglycosylase activities. Transglycosylases strengthen the cell wall by forming a rigid network of crosslinks through mechanisms that remain to be explored. Here we study the function of the Aspergillus fumigatus family of five Crh transglycosylases. Although crh genes are dispensable for cell viability, simultaneous deletion of all genes renders cells sensitive to cell wall interfering compounds. In vitro biochemical assays and localisation studies demonstrate that this family of enzymes functions redundantly as transglycosylases for both chitin-glucan and chitin-chitin cell wall crosslinks. To understand the molecular basis of this acceptor promiscuity, we solved the crystal structure of A. fumigatus Crh5 (AfCrh5) in complex with a chitooligosaccharide at the resolution of 2.8 Å, revealing an extensive elongated binding cleft for the donor (−4 to −1) substrate and a short acceptor (+1 to +2) binding site. Together with mutagenesis, the structure suggests a “hydrolysis product assisted” molecular mechanism favouring transglycosylation over hydrolysis.Hide Abstract
HPAEC-PAD and Q-TOF-MS/MS analysis reveal a novel mode of action of endo-β-1,3(4)-D-glucanase Eng16A from coprinopsis cinerea on barley β-glucan.
Xiong, Y., Wang, Y., Li, M., Kang, L., Zhou, J., Liu, C., Liu, Z., Zhang, Z. & Yuan, S. (2019). Food Chemistry, 287, 160-166.
We previously reported that an endo-β-1,3(4)-d-glucanase, Eng16A, from C. cinerea shows a higher degradation activity toward barley β-glucan than laminarin. HPAEC-PAD and Q-TOF-MS/MS analyses show that Eng16A-digestion products of barley β-glucan not only contain some oligosaccharides with (1 → 3)-β-linkage adjacent to the reducing end, which is consistent with β-1,3(4)-glucanase-digestion products, but also include some oligosaccharides containing (1 → 4)-β-linkage adjacent to the reducing end which is consistent with cellulase-digestion products. Thus, Eng16A possesses both cellulase and β-1,3(4)-glucanase activities. Because Eng16A does not degrade cellulose, we propose that the insertion of a (1 → 3)-β-linkage among the groups of (1 → 4)-β-linkages may make these (1 → 4)-β-linkages prone to cleavage by Eng16A. Furthermore, Eng16A also possesses transglycosylation activity which leads to some products containing one or a few consecutive (1 → 3)-β-linkages adjacent to the non-reducing end. Therefore, HPAEC-PAD and Q-TOF-MS/MS analyses provide an efficient approach to reveal complicated modes of action of some endo-β-1,3(4)-d-glucanases on barley β-glucan.Hide Abstract
Molecular recognition of the beta‐glucans laminarin and pustulan by a SusD‐like glycan‐binding protein of a marine Bacteroidetes.
Mystkowska, A. A., Robb, C., Vidal‐Melgosa, S., Vanni, C., Fernandez‐Guerra, A., Höhne, M. & Hehemann, J. H. (2018). The FEBS journal, 285(23), 4465-4481.
Marine bacteria catabolize carbohydrate polymers of algae, which synthesize these structurally diverse molecules in ocean surface waters. Although algal glycans are an abundant carbon and energy source in the ocean, the molecular details that enable specific recognition between algal glycans and bacterial degraders remain largely unknown. Here we characterized a surface protein, GMSusD from the planktonic Bacteroidetes‐Gramella sp. MAR_2010_102 that thrives during algal blooms. Our biochemical and structural analyses show that GMSusD binds glucose polysaccharides such as branched laminarin and linear pustulan. The 1.8 Å crystal structure of GMSusD indicates that three tryptophan residues form the putative glycan‐binding site. Mutagenesis studies confirmed that these residues are crucial for laminarin recognition. We queried metagenomes of global surface water datasets for the occurrence of SusD‐like proteins and found sequences with the three structurally conserved residues in different locations in the ocean. The molecular selectivity of GMSusD underscores that specific interactions are required for laminarin recognition. In conclusion, our findings provide insight into the molecular details of β‐glucan binding by GMSusD and our bioinformatic analysis reveals that this molecular interaction may contribute to glucan cycling in the surface ocean.Hide Abstract
Wang, D., Kim, D. H., Yoon, J. J. & Kim, K. H. (2017). Process Biochemistry, 52, 233-237.
We report the first hydrothermal hydrolysis of curdlan, a water insoluble β-1,3-glucan, to produce β-1,3-glucooligosaccharides, which are high-value materials with health-benefiting activities. In this study, hydrothermal hydrolysis was tested for the liquefaction and saccharification of curdlan. The optimal hydrothermal hydrolysis conditions were 180°C and 60 min, respectively, resulting in a high degree of liquefaction (98.4%) and low byproduct formation. Under the optimal conditions, 17.47 g/L of β-1,3-glucooligosaccharides was produced from 20 g/L of curdlan, representing a conversion yield of 87.4% (w/w). Using this process, β-1,3-glucooligosaccharides were conveniently produced in a one-step reaction without any chemicals or enzymes. This hydrothermal hydrolysis for curdlan exhibited the best performance among various hydrolysis processes reported to date. This method can be applied to large-scale production of β-1,3-glucooligosaccharides for the functional food and biopharmaceutical industries.Hide Abstract
Mechelke, M., Herlet, J., Benz, J. P., Schwarz, W. H., Zverlov, V. V., Liebl, W. & Kornberger, P. (2017). Analytical and Bioanalytical Chemistry, 1-13.
The rising importance of accurately detecting oligosaccharides in biomass hydrolyzates or as ingredients in food, such as in beverages and infant milk products, demands for the availability of tools to sensitively analyze the broad range of available oligosaccharides. Over the last decades, HPAEC-PAD has been developed into one of the major technologies for this task and represents a popular alternative to state-of-the-art LC-MS oligosaccharide analysis. This work presents the first comprehensive study which gives an overview of the separation of 38 analytes as well as enzymatic hydrolyzates of six different polysaccharides focusing on oligosaccharides. The high sensitivity of the PAD comes at cost of its stability due to recession of the gold electrode. By an in-depth analysis of the sensitivity drop over time for 35 analytes, including xylo- (XOS), arabinoxylo- (AXOS), laminari- (LOS), manno- (MOS), glucomanno- (GMOS), and cellooligosaccharides (COS), we developed an analyte-specific one-phase decay model for this effect over time. Using this model resulted in significantly improved data normalization when using an internal standard. Our results thereby allow a quantification approach which takes the inevitable and analyte-specific PAD response drop into account.Hide Abstract
Deng, Y., Li, M., Chen, L. X., Chen, X. Q., Lu, J. H., Zhao, J. & Li, S. P. (2017). Carbohydrate Polymers, In Press.
The chain conformation, chemical characters and immunomodulatory activity of polysaccharide from Dendrobium devonianum (DDP) were investigated.Results showed that molecular weights, polydispersity index, radius of gyrations of DDP were 3.99 × 105 Da, 1.27, 74.1 nm, respectively. By applying the polymer solution theory, the exponent (v) values of <S2>z 1/2 = kMwv was calculated as 0.38, which revealed that DDP existed as a globular shape in aqueous solution, and further confirmed by AFM analysis. Furthermore, the main monosaccharide compositions were Man and Glc with the ratio of 29.61:1.00. Indeed, the main glycosidic linkages were β-1,4-Manp, and substituted with acetyl groups at O-2 and O-3 position. Notably, DDP could promote the immune functions of macrophages including NO release and phagocytosis. Thus, DDP could be explored as a natural immune-stimulating agent in the health and functional food area as well as pharmaceutical industries.Hide Abstract
Lee, K. C., Arai, T., Ibrahim, D., Kosugi, A., Prawitwong, P., Lan, D., Murata, Y. & Mori, Y. (2014). BioResources, 9(1), 1072-1084.
A laminarinase (endo-β-1,3-glucanase) was purified to homogeneity from Penicillium rolfsii c3-2(1) IBRL, which was originally produced in liquid culture containing 1% xylan from birchwood, via anion-exchange chromatography, gel filtration on Sephacryl S-100, and hydrophobic interaction chromatography. A single protein band with a molecular weight of 75 kDa was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which had an optimum catalytic activity at pH 4.0 to 5.0 and 70°C. This purified enzyme was most stable in the pH range 4 to 7, while it was thermostable up to 55°C and retained up to 90% of its activity after 4 h pre-incubation. A substrate laminarin kinetic study yielded estimated Km and Vmax values of 0.0817 mg/mL and 372.2 µmol/min/mg, respectively. Laminari-oligosaccharide degradation, which was analyzed by thin layer chromatography, yielded the major hydrolysis products laminaribiose and glucose.Hide Abstract
Sack, E. L. W., van der Wielen, P. W. J. J. & van der Kooij, D. (2011). Applied and Environmental Microbiology, 77(19), 6931-6938.
Biopolymers are important substrates for heterotrophic bacteria in oligotrophic freshwater environments, but information on bacterial growth kinetics with biopolymers is scarce. The objective of this study was to characterize bacterial biopolymer utilization in these environments by assessing the growth kinetics of Flavobacterium johnsoniae strain A3, which is specialized in utilizing biopolymers at µg liter-1 levels. Growth of strain A3 with amylopectin, xyloglucan, gelatin, maltose, or fructose at 0 to 200 µg C liter-1 in tap water followed Monod or Teissier kinetics, whereas growth with laminarin followed Teissier kinetics. Classification of the specific affinity of strain A3 for the tested substrates resulted in the following affinity order: laminarin (7.9 × 10-2 liter·µg-1 of C·h-1) >> maltose > amylopectin ≈ gelatin ≈ xyloglucan > fructose (0.69 × 10-2 liter·µg-1 of C·h-1). No specific affinity could be determined for proline, but it appeared to be high. Extracellular degradation controlled growth with amylopectin, xyloglucan, or gelatin but not with laminarin, which could explain the higher affinity for laminarin. The main degradation products were oligosaccharides or oligopeptides, because only some individual monosaccharides and amino acids promoted growth. A higher yield and a lower ATP cell-1 level was achieved at ≤10 µg C liter-1 than at >10 µg C liter-1 with every substrate except gelatin. The high specific affinities of strain A3 for different biopolymers confirm that some representatives of the classes Cytophagia-Flavobacteria are highly adapted to growth with these compounds at µg liter-1 levels and support the hypothesis that Cytophagia-Flavobacteria play an important role in biopolymer degradation in (ultra)oligotrophic freshwater environments.Hide Abstract