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.
Characterization and discrimination of polysaccharides from different species of Cordyceps using saccharide mapping based on PACE and HPTLC.
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.
Purification and Characterization of a Thermostable Laminarinase from Penicillium rolfsii c3-2 (1) IBRL.
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.
Production of high-value β-1,3-glucooligosaccharides by microwave-assisted hydrothermal hydrolysis of curdlan.
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.
Characterization and comparison of polysaccharides from Lycium barbarum in China using saccharide mapping based on PACE and HPTLC.
Wu, D. T., Cheong, K. L., Deng, Y., Lin, P. C., Wei, F., Lv, X. J., Long, Z. R., Zhao, 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. barbarum and their products, which suggested that saccharide mapping based on PACE and HPTLC analysis could be a routine approach for quality control of polysaccharides.
HPAEC-PAD for oligosaccharide analysis—novel insights into analyte sensitivity and response stability.
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.
Chemical characterization and immunomodulatory activity of acetylated polysaccharides from Dendrobium devonianum.
Deng, Y., Li, M., Chen, L. X., Chen, X. Q., Lu, J. H., Zhao, J. & Li, S. P. (2017). Carbohydrate Polymers, 180, 238-245.
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
2>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.
Enzymatic properties and the gene structure of a cold-adapted laminarinase from Pseudoalteromonas species LA.
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.
Effective degradation of curdlan powder by a novel endo-β-1→ 3-glucanase.
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.
A highly glucose-tolerant GH1 β-glucosidase with greater conversion rate of soybean isoflavones in monogastric animals.
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.