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|Stability:||> 10 years under recommended storage conditions|
|Substrate For (Enzyme):||endo-1,3-β-Glucanase|
High purity Laminaritetraose 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
Taxogenomic assessment and genomic characterisation of Weissella cibaria strain 92 able to metabolise oligosaccharides derived from dietary fibres.
Anna, M., Phebe, V., Guðmundsdóttir, E. E., Santesson, S., Nilsson, A., Óli, H. G., Linares-Pasten, J. A. & Nordberg, K. E. (2020). Scientific Reports, 10(1), 5853.
The importance of the gut microbiota in human health has led to an increased interest to study probiotic bacteria. Fermented food is a source of already established probiotics, but it also offers an opportunity to discover new taxa. Four strains of Weissella sp. isolated from Indian fermented food have been genome sequenced and classified into the species W. cibaria based on whole-genome phylogeny. The genome of W. cibaria strain 92, known to utilise xylooligosaccharides and produce lactate and acetate, was analysed to identify genes for oligosaccharide utilisation. Clusters including genes involved in transportation, hydrolysis and metabolism of xylooligosaccharides, arabinooligosaccharides and β-glucosides were identified. Growth on arabinobiose and laminaribiose was detected. A 6-phospho-β-glucosidase clustered with a phosphotransferase system was found upregulated during growth on laminaribiose, indicating a mechanism for laminaribiose utilisation. The genome of W. cibaria strain 92 harbours genes for utilising the phosphoketolase pathway for the production of both acetate and lactate from pentose and hexose sugars but lacks two genes necessary for utilising the pentose phosphate pathway. The ability of W. cibaria strain 92 to utilise several types of oligosaccharides derived from dietary fibres, and produce lactate and acetate makes it interesting as a probiotic candidate for further evaluation.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
Comparative analysis and biochemical characterization of two endo-β-1, 3-glucanases from the thermophilic bacterium Fervidobacterium sp.
Burkhardt, C., Schäfers, C., Claren, J., Schirrmacher, G. & Antranikian, G. (2019). Catalysts, 9(10), 830.
Laminarinases exhibit potential in a wide range of industrial applications including the production of biofuels and pharmaceuticals. In this study, we present the genetic and biochemical characteristics of FLamA and FLamB, two laminarinases derived from a metagenomic sample from a hot spring in the Azores. Sequence comparison revealed that both genes had high similarities to genes from Fervidobacterium nodosum Rt17-B1. The two proteins showed sequence similarities of 62% to each other and belong to the glycoside hydrolase (GH) family 16. For biochemical characterization, both laminarinases were heterologously produced in Escherichia coli and purified to homogeneity. FLamA and FLamB exhibited similar properties and both showed highest activity towards laminarin at 90° C and pH 6.5. The two enzymes were thermostable but differed in their half-life at 80° C with 5 h and 1 h for FLamA and FLamB, respectively. In contrast to other laminarinases, both enzymes prefer β-1,3-glucans and mixed-linked glucans as substrates. However, FLamA and FLamB differ in their catalytic efficiency towards laminarin. Structure predictions were made and showed minor differences particularly in a kink adjacent to the active site cleft. The high specific activities and resistance to elevated temperatures and various additives make both enzymes suitable candidates for application in biomass conversion.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
A novel thermostable GH3 β-glucosidase from Talaromyce leycettanus with broad substrate specificity and significant soybean isoflavone glycosides-hydrolyzing capability.
Reichenbach, T., Li, X., Xia, W., Bai, Y., Ma, R., Yang, H., Luo, H. & Shi, P. (2018). BioMed Research International, 2018, 4794690.
A novel β-glucosidase gene (Bgl3B) of glycoside hydrolase (GH) family 3 was cloned from the thermophilic fungus Talaromyce leycettanus JM12802 and successfully expressed in Pichia pastoris. The deduced Bgl3B contains 860 amino acid residues with a calculated molecular mass of 91.2 kDa. The purified recombinant Bgl3B exhibited maximum activities at pH 4.5 and 65°C and remained stable at temperatures up to 60°C and pH 3.0-9.0, respectively. The enzyme exhibited broad substrate specificities, showing β-glucosidase, glucanase, cellobiase, xylanase, and isoflavone glycoside hydrolase activities, and its activities were stimulated by short-chain alcohols. The catalytic efficiencies of Bgl3B were 693 and 104/mM/s towards pNPG and cellobiose, respectively. Moreover, Bgl3B was highly effective in converting isoflavone glycosides to aglycones at 37°C within 10 min, with the hydrolysis rates of 95.1%, 76.0%, and 75.3% for daidzin, genistin, and glycitin, respectively. These superior properties make Bgl3B potential for applications in the food, animal feed, and biofuel industries.Hide Abstract
Cao, H., Zhang, Y., Shi, P., Ma, R., Yang, H., Xia, W., Cui, Y., Luo, H., Bai, Y. & Yao, B. (2018). Journal of Industrial Microbiology & Biotechnology, 197, 1-10.
In the feed industry, β-glucosidase has been widely used in the conversion of inactive and bounded soybean isoflavones into active aglycones. However, the conversion is frequently inhibited by the high concentration of intestinal glucose in monogastric animals. In this study, a GH1 β-glucosidase (AsBG1) with high specific activity, thermostability and glucose tolerance (IC50 = 800 mM) was identified. It showed great glucose tolerance against substrates with hydrophobic aryl ligands (such as pNPG and soy isoflavones). Using soybean meal as the substrate, AsBG1 exhibited higher hydrolysis efficiency than the GH3 counterpart Bgl3A with or without the presence of glucose in the reaction system. Furthermore, it is the first time to find that the endogenous β-glucosidase of soybean meal, mostly belonging to GH3, plays a role in the hydrolysis of soybean isoflavones and is highly sensitive to glucose. These findings lead to a conclusion that the GH1 rather than GH3 β-glucosidase has prosperous application advantages in the conversion of soybean isoflavones in the feed industry.Hide Abstract
Mitsuya, D., Sugiyama, T., Zhang, S., Takeuchi, Y., Okai, M., Urano, N. & Ishida, M. (2018). Journal of Bioscience and Bioengineering, 126(2), 169-175.
We isolated a laminarin-degrading cold-adapted bacterium strain LA from coastal seawater in Sagami Bay, Japan and identified it as a Pseudoalteromonas species. We named the extracellular laminarinase LA-Lam, and purified and characterized it. LA-Lam showed high degradation activity for Laminaria digitata laminarin in the ranges of 15-50°C and pH 5.0-9.0. The major terminal products degraded from L. digitata laminarin with LA-Lam were glucose, laminaribiose, and laminaritriose. The degradation profile of laminarioligosaccharides with LA-Lam suggested that the enzyme has a high substrate binding ability toward tetrameric or larger saccharides. Our results of the gene sequence and the SDS-PAGE analyses revealed that the major part of mature LA-Lam is a catalytic domain that belongs to the GH16 family, although its precursor is composed of a signal peptide, the catalytic domain, and three-repeated unknown regions.Hide Abstract
Li, K., Chen, W., Wang, W., Tan, H., Li, S. & Yin, H. (2018). Carbohydrate Polymers, 201, 122-130.
Curdlan is a water-insoluble microbial exo-polysaccharide that is hardly degraded. The gene CcGluE encoding an endo-β-1 →3-glucanase consisting of 412 amino acids (44 kDa) from Cellulosimicrobium cellulans E4-5 was cloned and expressed in Escherichia coli. The recombinant CcGluE hydrolysed curdlan powder effectively. CcGluE shows high endo-β-1 →3 glucanase activity and low β-1,4 and β-1,6 glucanase activities with broad substrate specificity for glucan, including curdlan, laminarin and β-1 →3/1 →6-glucan, and the highest catalytic activity for curdlan. Moreover, the hydrolytic products of curdlan were glucan oligosaccharides with degrees of polymerisation of 2-13, and the main products were glucobiose and glucotriose. Degradation mode analysis indicated that CcGluE is more likely to hydrolyse glucopentaose and revealed that CcGluE was an endo-glucanase. Furthermore, upon combination with a homogenising pre-treatment method with curdlan, the degradation efficiency of CcGluE for curdlan powder was greatly improved 7.1-fold, which laid a good foundation for the utilisation of curdlan.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
Wu, D. T., Cheong, K. L., Deng, Y., Lin, P. C., Wei, F., Lv, X. J., Long, Z. R., Zhoa, J., Ma, S. C. & Li, S. P. (2015). Carbohydrate polymers, 134, 12-19.
Water-soluble polysaccharides from 51 batches of fruits of L. barbarum (wolfberry) in China were investigated and compared using saccharide mapping, partial acid hydrolysis, single and composite enzymatic digestion, followed by polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) analysis and high performance thin layer chromatography (HPTLC) analysis, respectively. Results showed that multiple PACE and HPTLC fingerprints of partial acid and enzymatic hydrolysates of polysaccharides from L. barbarum in China were similar, respectively. In addition, results indicated that β-1,3-glucosidic, α-1,4-galactosiduronic and α-1,5-arabinosidic linkages existed in polysaccharides from L. barbarum collected in China, and the similarity of polysaccharides in L. barbarum collected from different regions of China was pretty high, which are helpful for the improvement of the performance of polysaccharides from L. barbarum in functional/health foods area. Furthermore, polysaccharides from Panax notoginseng, Angelica sinensis, and Astragalus membranaceus var. mongholicus were successfully distinguished from those of L. barbarum based on their PACE fingerprints. These results were beneficial to improve the quality control of polysaccharides from L. barabrum and their products, which suggested that saccharide mapping based on PACE and HPTLC analysis could be a routine approach for quality control of polysaccharides.Hide Abstract
Wu, D. T., Cheong, K. L., Wang, L. Y., Lv, G. P., Ju, Y. J., Feng, K., Zhao, J. & Li, S. P. (2014). Carbohydrate Polymers, 103, 100-109.
Polysaccharides from seven species of natural and cultured Cordyceps were firstly investigated and compared using saccharide mapping, partially acidic/enzymatic (α-amylase, β-glucanase and pectinase) digestion followed with polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) and high performance thin layer chromatography (HPTLC) analysis, respectively, to obtain the comprehensive profiles of hydrolysates of the polysaccharides and their characters. The results showed that 1,4-α-D-glucosidic, 1,4-β-D-glucosidic and 1,4-α-D-galactosidic linkages were existed in natural and cultured Cordyceps sinensis, cultured Cordyceps militaris, natural Cordyceps gracilis and Cordyceps ciecadae. The similarity of polysaccharides from cultured C. militaris to natural C. sinensis was relatively high, which might contribute to the rational use of C. militaris. Moreover, different species of natural and cultured Cordyceps can be differentiated based on the saccharide mapping, which is helpful to well understand the structural characters of polysaccharides from different species of Cordyceps and to improve the quality control of polysaccharides in natural and cultured Cordyceps.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