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Zhang H.,Jiangnan University | Zhu L.,Jiangsu Rayguang Biotech Co. | Zhan X.,Jiangnan University
Gaodeng Xuexiao Huaxue Xuebao/Chemical Journal of Chinese Universities | Year: 2014

Carbohydrate microarray has become a powerful tool to explore the structure-function relationship between protein-carbohydrate, but it depends on enough reducing terminal oligosaccharides with aldehyde group. Nostoc oligosaccharides are a kind of oligo (1→2)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside. To acquire a full series of α-1,2-gluco-oligosaccharides from oligo (1→2)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside, the terminal fructose of four Nostoc oligosaccharides were removed using acid hydrolysis methods under the condition of 0.5 mol/L trifluoroacetic acid (TFA) treated at 95 ℃ for 9 min, respectively. Following hydrolyzed products were separated and purified using high performance liquid chromatography (HPLC), and then electrospray ionization-collision-induce dissociation tandem mass spectrometry (ESI-CID-MS/MS) and matrix assisted laser desorption ionization (MALDI)-MS were used to identify the removing of terminal fructose and sequenced, α-1,2-Glc5, Glc7, Glc8 and Glc9 were acquired. The mixture of Nostoc-Octa and Nostoc-Hexa treated with 0.5 mol/L TFA at 95 ℃ for 45 min were used to acquire low degree of polymerization (DP) of α-1,2-gluco-oligosaccharides, which were separated with P2 column, ESI-MS and MALDI-MS were used to analysis these oligosaccharides, α-1,2-gluco-oligosaccharides with the degree of polymerization 2, 3, 4 and 6 were obtained. Finally, full series of α-1-2-gluco-oligosaccharides from 2 mer to 9 mer were acquired successfully. ©, 2014, Higher Education Press. All right reserved. Source

Zhang H.,Jiangnan University | Zhu L.,Jiangsu Rayguang Biotech Co. | Zhang S.,Jiangnan University | Zhan X.,Jiangnan University
Gaodeng Xuexiao Huaxue Xuebao/Chemical Journal of Chinese Universities | Year: 2014

To acquire the mechanism of lentinan hydrolysis and full series of gluco-oligosaccharides from hydrolyzed lentinan, the hydrolysis time of lentinan treated with 0.02 mol/L trifluoroacetic acid (TFA) at 100℃ was optimized through analysis the distribution of different degree of polymerization (DP) oligosaccharides at 2, 4, and 6 h. And then lentinan hydrolysis was repeated another three times until no precipitation can be observed under optimal hydrolysis time. The mass spectrometry character of 7-mer oligosaccharides from all the four batch hydrolysis products was analyzed using ESI-CID-MS/MS based on the mass spectrometry character of 7-mer β-1, 3-and β-1, 6-gluco-oligosaccharides, and acquiring a hydrolysis mechanism. Finally, all the four batch hydrolysis products were combined and separated with P4 column and full series of gluco-oligosacchaides from 2-mer to 13-mer were acquired. The optimal time for lentinan hydrolysis was 4 h, and β-1, 3/1, 6-gluco-oligosaccharides in hydrolysis lentinan was observed at the first batch, and then the amount of β-1, 3/1, 6-gluco-oligosaccharides reduced with the increasing of hydrolysis times, and there only observed β-1, 3-gluco-oligosaccharides at the fourth hydrolysis products. This results also suggested that the triple helix structure zone containing β-1, 3/1, 6-linkage of lentinan is loose, but the only containing β-1, 3-linkage triple helix structure zone in lentinan is tight. The theory acquired in this work will supply useful information for preparation gluco-oligosaccharides in large-scale and it also expand the gluco-oligosaccharides library for exploring protein-carbohydrate interaction using carbohydrate microarray. ©, 2014, Higher Education Press. All right reserved. Source

Zhang H.-T.,Jiangnan University | Zhu L.,Jiangsu Rayguang Biotech Co. | Zhang S.,Jiangnan University | Zhan X.-B.,Jiangnan University | Lin C.-C.,Jiangnan University
Carbohydrate Research | Year: 2014

An efficient, highly sensitive, and ultramicroscale analytical method for the identification of fructose removed from fructofuranosyl-containing gluco-oligosaccharides, including malto-oligosyl fructofuranosides and oligomeric (1→2)-α-d-glucopyranosyl-(1→2)-β-d-fructofuranosides by ESI-CID-MS/MS has been developed with proven applications far superior to the existing method using NMR. With the established principle of diagnostic fragmentation by ESI-CID-MS/MS, the terminal saccharide (either glucose or fructose) can be readily and unambiguously determined at high sensitivity without a tedious derivatization process. Detection of the A-type fragmentation 0,4A-h type ion, and 0,2A type ion are useful as a diagnostic fragmentation tool to identify whether fructose terminal is removed from oligosaccharides. It will facilitate the efficient production of suitable oligosaccharide microarrays crucial for studies on carbohydrate-protein interaction in seeking functional carbohydrates. © 2014 Elsevier B.V. All rights reserved. Source

Li J.,Jiangnan University | Zhu L.,Jiangsu Rayguang Biotech Co. | Zhan X.-B.,Jiangnan University | Zhan X.-B.,Jiangsu Rayguang Biotech Co. | And 4 more authors.
Food Science and Biotechnology | Year: 2014

Endo-β-1,3-glucanase (Endo23) was purified from a Trichoderma reesei GIMCC 3.498 fermentation broth using anion exchange and 2-stage size exclusion chromatography. Purification of 44.5× and a 12% recovery yield of enzyme activity were achieved. The Mw and isoelectric point were estimated to be 24 kDa and 3.85 using SDS-PAGE and IEF, respectively. The highest substrate specificity was observed for water-insoluble curdlan. The optimal conditions for hydrolyzing curdlan were pH 5.0 and 50°C. The main hydrolytic products were glucobiose and glucotriose. Minor amounts of glucose and glucotetraose were detected. Hg2+, Fe2+, Fe3+, and Sn2+ inhibited the hydrolysis activity of Endo23 at 5 and 50 mM. K+ slightly promoted Endo23 activity. Endo23 belongs to the category EC3.2.1.39. The peptide sequences of Endo23 showed identity with conserved sequences that typically exist in β-1,3-glucanases of the glycoside hydrolase family. The Endo23 sequence was partially similar to a hypothetical lignocellulase from Penicillium oxalicum 114-2. © 2014 The Korean Society of Food Science and Technology and Springer Science+Business Media Dordrecht. Source

Li J.,Jiangnan University | Zhu L.,Jiangsu Rayguang Biotech Co. | Lu G.,Jiangnan University | Zhan X.-B.,Jiangnan University | And 3 more authors.
PLoS ONE | Year: 2014

Activation of the innate immune system before the invasion of pathogens is a promising way to improve the resistance of plant against infection while reducing the use of agricultural chemicals. Although several elicitors were used to induce the resistance of potato plant to microbial pathogen infection, the role of curdlan oligosaccharide (CurdO) has not been established. In the current study, the defense responses were investigated at biochemical and proteomic levels to elucidate the elicitation effect of CurdOs in foliar tissues of potato (Solanum tuberosum L. cv. McCain G1). The results indicate that the CurdOs exhibit activation effect on the early- and late-defense responses in potato leaves. In addition, glucopentaose was proved to be the shortest active curdlan molecule based on the accumulation of H2O2 and salicylic acid and the activities of phenylalanine amino-lyase, β-1,3-glucanase and chitinase. The 2D-PAGE analysis reveals that CurdOs activate the integrated response reactions in potato cells, as a number of proteins with various functions are up-regulated including disease/defense, metabolism, transcription, and cell structure. The pathogenesis assay shows that the ratio of lesion area of potato leaf decreased from 15.82%±5.44% to 7.79%±3.03% when the plants were treated with CurdOs 1 day before the infection of Phytophthora infestans. Furthermore, the results on potato yield and induction reactions indicate that the defense responses induced by CurdOs lasted for short period of time but disappeared gradually. © 2014 Li et al. Source

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