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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 (0,4)A-h type ion, and (0,2)A 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. Copyright © 2014 Elsevier Ltd. All rights reserved.


Zheng Z.-Y.,Jiangnan University | Zheng Z.-Y.,Jiangsu Rayguang Biotech Co. | Wang S.-Z.,Jiangnan University | Li G.-S.,Jiangnan University | And 6 more authors.
Applied Microbiology and Biotechnology | Year: 2013

To determine the factors influencing the resulting molecular weight of polysialic acid (PSA), batch fermentations by using Escherichia coli were conducted. It was found that temperature and pH were significant factors affecting the PSA production and its resulting molecular weight. When pH was set at 6.4, temperature of 37 C was suitable for cell growth and PSA production while 33 C facilitated production of higher molecular weight of PSA. pH 6.4 was favorable for PSA production while pH 7.4 was good for higher molecular weight of PSA at 37 C. Intramolecular self-cleavage of PSA might lead to relatively low molecular weight under mild acidic condition. Our data suggest that the PSA molecular weight is significantly affected by the pH condition rather than the temperature. It is concluded that the resulting PSA molecular weight not only depends on fermentation conditions but also relates to cell growth rate and PSA production rate. Higher PSA molecular weight was made when its production rate was faster than degradation rate. A novel two-stage pH control fermentation process for production of high molecular weight PSA was developed. At the first stage, pH was set at 6.4 to encourage cell growth and PSA production, whereas pH was set at 7.4 at the second stage to promote the formation of higher molecular weight PSA. PSA yield up to 5.65 g/L and its resulting molecular weight of 260 kDa was attained, the highest level ever reported. © 2012 Springer-Verlag Berlin Heidelberg.


Li J.,Jiangnan University | Zhu L.,Jiangsu Rayguang Biotech Company | 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.


Li J.,Jiangnan University | Zhu L.,Jiangsu Rayguang Biotech Co. | Zheng Z.-Y.,Jiangnan University | Zhan X.-B.,Jiangnan University | And 4 more authors.
Applied Microbiology and Biotechnology | Year: 2013

Biologically active β-1,3-oligosaccharides with rapidly growing biomedical applications are produced from hydrolysis of curdlan polysaccharide. The water-insoluble curdlan impedes its hydrolysis efficiency which is enhanced by our newly developed alkali-neutralization treatment process to increase the stability of curdlan suspension to more than 20 days, while the untreated control settled within 5 min. A putative double-layer structure model comprising of a compact core and a hydrated outer layer was proposed to describe the treated curdlan particles based on sedimentation and scanning electron microscopy observation. This model was verified by single- and two-step acid hydrolysis, indicative of the reduced susceptibility to hydrolysis when close to the compact core. Electrospray ionization-mass spectrometry, thin-layer chromatography analyses, and effective HPLC procedure led to the development of improved process to produce purified individual β-1,3-oligosaccharides with degrees of polymerization from 2 to 10 and potential for biomedical applications from curdlan hydrolyzate. Our new curdlan oligosaccharide production process offers an even better alternative to the previously published processes. © 2013 Springer-Verlag Berlin Heidelberg.


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.


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.


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.


Zhang H.-T.,Jiangnan University | Zhu L.,Jiangsu Rayguang Biotech Co. | Liu D.,Jiangnan University | Zhan X.-B.,Jiangnan University | And 3 more authors.
Biochemical Engineering Journal | Year: 2015

Dissolved oxygen tension (DOT) is an important factor influencing curdlan fermentative production by Agrobacterium sp. ATCC 31749. Therefore, optimization of dissolved oxygen profile is crucial to improve curdlan production. In this study, an unstructured mathematical models that consider nitrogen source as growth-limiting substrates were developed to estimate the kinetic parameters of cell growth, curdlan formation, and glucose consumption during Agrobacterium sp. ATCC 31749 fermentation at various DOTs (5-75%). The estimated kinetic parameters were used to establish models with Origin 8.0. All models of kinetic parameters were coupled with the kinetic model derived from Agrobacterium sp. ATCC 31749 fermentation to determine the effect of DOT on curdlan fermentation. The combined kinetic models satisfactorily fit the experimental data. The optimal dissolved oxygen profile was obtained using genetic algorithms coupled with iterative dynamic programming algorithms in MATLAB 10b. The optimal dissolved oxygen during curdlan production (20-120. h) was approximately 52% DOT, and the maximum curdlan concentration of 48.5. g/L was obtained at this DOT profile. To our knowledge, this study is the first to optimize DOT for curdlan fermentation under nitrogen-limited conditions. This report significantly contributes to the improvement of curdlan production on an industrial scale. © 2015 Elsevier B.V.


PubMed | Jiangsu Rayguang Biotech Co. and Jiangnan University
Type: | Journal: Carbohydrate polymers | Year: 2016

A coupled fermentation system of Agrobacterium sp. ATCC 31749 and Trichoderma harzianum GIM 3.442 (AT-CFS) with wheat bran as the optimal nitrogen source was established for producing low-molecular-weight curdlan with high production, which can potentially reduce the cost of low-molecular-weight curdlan biosynthesis. The initial inoculate ratio, pH and the fermentation time were optimized. Compared with the curdlan from the single fermentation system of Agrobacterium sp. ATCC 31749 (A-SFS), the molecular weight (Mw) of the curdlan produced from AT-CFS decreased by 34.01% (from 110.85kDa to 73.15kDa), and the curdlan production (47.9g/L) and conversion rate of glucose to curdlan (0.60gg


PubMed | Jiangsu Rayguang Biotech Co and Jiangnan University
Type: | Journal: 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 (12)--D-glucopyranosyl-(12)--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 (0,4)A-h type ion, and (0,2)A 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.

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