•A new sialidase gene ccsia from C. cellulans sp. 21 was cloned for the first time.•The recombinant sialidase CcSia was characterized.•High cell density fermentation was introduced to improve the production of CcSia.•Enzyme CcSia was applied for high yield preparation of ganglioside GM1.Cellulosimicrobium cellulans employs extracellular sialidase to selectively convert polysialogangliosides to ganglioside GM1. We cloned this novel sialidase gene (ccsia) from C. cellulans sp. 21, and overexpressed recombinant sialidase (CcSia) protein in E. coli BL21 (DE3) by high cell density fermentation. The presence of an N-terminal hexa-His tag allowed for purification using nickel affinity chromatography (2.3-fold, specific activity 41.5 U/mg). As determined by gel electrophoresis and gel filtration chromatography, the molecular weight of CcSia was found to be about 75 kDa, consistent with sequence analysis (75,271 Da). CcSia transformed polysialogangliosides GD1a, GD1b and GT1b into GM1. For this reaction, the response surface approach showed that optimal conditions in a 1-L system were 2 h incubation at 32.5 °C and pH 5.2, with substrate concentrations of 10 g/L and crude enzyme concentration 1 g/L, respectively. Under above conditions, 10 g/L of ganglioside was completely converted to the product GM1 with a yield of 52%. Our studies demonstrate CcSia could be used for industrial preparation of ganglioside GM1 by the pharmaceutical industry.Download high-res image (85KB)Download full-size image

•Rg2 has protective effects on Aβ25-35-induced neurotoxicity in PC12 cells.•Rg2 protection against Aβ25-35-induced toxicity is concentration-dependent.•Rg2 pretreatment decreased Aβ25-35-induced apoptosis.•The protective effect is dependent on Rg2-induced enhanced Akt phosphorylation.•Protective effect of Rg2 is associated with PI3K/Akt signaling pathway enhancement.Alzheimer's disease (AD) is one of the most debilitating neurodegenerative diseases in an aging population. Excessive accumulation of β-amyloid (Aβ) has been proposed as a pivotal event in the pathogenesis of AD. Ginsenoside Rg2 has been reported to exert neuroprotective effects. However, the underlying mechanism for its neuroprotection is not well-understood. In this study, we investigated the protective effects of ginsenoside Rg2 on Aβ25-35-induced neurotoxicity in PC12 cells and identified a potential molecular signaling pathway involved. The results showed that pretreatment of PC12 cells with ginsenoside Rg2 followed by Aβ25-35 increased cell viability in a concentration-dependent manner compared to cells that were not pretreated. In addition, ginsenoside Rg2 pretreatment attenuated Aβ25-35-induced increases in the release of lactate dehydrogenase, the intracellular calcium concentration, and levels of reactive oxygen species. Pretreatment with ginsenoside Rg2 increased the Bcl-2/Bax ratio. Moreover, ginsenoside Rg2 attenuated the cleavage of caspase-3 induced by Aβ25-35 thereby improving cell survival. Ginsenoside Rg2 significantly enhanced the phosphorylation of Akt in PC12 cells. Additionally, pretreatment with the phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, completely abolished the protective effects of ginsenoside Rg2 against Aβ25-35-induced neuronal cell apoptosis. These findings unambiguously suggested that the protective effect of ginsenoside Rg2 against Aβ25-35-induced apoptosis in PC12 cells was associated with enhancement of the PI3K/Akt signaling pathway.

•The properties of pectinate, alginate and chitosan nanofiber mats were compared.•The three mats had comparable mechanical strength and vapour permeability.•The pectinate nanofiber mat could absorb more exudates faster.•The pectinate nanofiber mat could suppress bacteria’s growth more effectively.Polysaccharides including pectin, alginate and chitosan are fabricated into dressings of micrometer-scaled architecture (micro- fiber or particle) and widely applied in wound treatments clinically. This work characterized and compared the properties of electrospun nanofibrous dressings of these polysaccharides. We found that although the three polysaccharide nanofiber mats had comparable mechanical strength and vapour permeability, the pectinate nanofiber mat could absorb 1.2 times and 3.6 times more exudates than the alginate and chitosan nanofiber mats, respectively, within less time. Moreover, the pectinate nanofiber mat showed much higher antibacterial activity (73.1%) than the alginate and chitosan nanofiber mats (11.8% and 17.1%, respectively). Further examinations demonstrated that the superior absorbency and antibacterial activity of the pectinate nanofiber mat were associated with the moderate extent of swelling of pectinate nanofibers under hydrated conditions. All these results suggest that the pectinate nanofiber mat might be a superior wound dressing to the alginate and chitosan nanofiber mats.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a potent cancer cell-specific apoptosis-inducing cytokine with little toxicity to most normal cells. However, acquired resistance of cancer cells to TRAIL is a roadblock. Agents that can either potentiate the effect of TRAIL or overcome resistance to TRAIL are urgently needed. This article reports that ginsenoside compound K (CK) potentiates TRAIL-induced apoptosis in HCT116 colon cancer cells and sensitizes TRAIL-resistant colon cancer HT-29 cells to TRAIL. On a cellular mechanistic level, CK downregulated cell survival proteins including Mcl-1, Bcl-2, surviving, X-linked inhibitor of apoptosis protein and Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein, upregulated cell pro-apoptotic proteins including Bax, tBid and cytochrome c, and induced the cell surface expression of TRAIL death receptor DR5. Reduction of DR5 levels by siRNAs significantly decreases CK- and TRAIL-mediated apoptosis. Importantly, our results indicate, for the first time, that DR5 upregulation is mediated by autophagy, as blockade of CK-induced autophagy by 3-MA, LY294002 or Atg7 siRNAs substantially decreases DR5 upregulation and reduces the synergistic effect. Furthermore, CK-stimulated autophagy is mediated by the reactive oxygen species–c-Jun NH2-terminal kinase pathway. Moreover, we found that p53 and the C/EBP homologous (CHOP) protein is also required for DR5 upregulation but not related with autophagy. Our findings contribute significantly to the understanding of the mechanism accounted for the synergistic anticancer activity of CK and TRAIL, and showed a novel mechanism related with DR5 upregulation.

•A new method is developed using cation exchange resin to methyl-esterify pectin.•The method can product high DE of HG and RG-I pectin without destroying structures.•Pectins with varying DE can be prepared by controlling the reaction conditions.This study developed a new method to methyl-esterify pectin using a cation exchange resin. Homogalacturonan (HG)-type pectin (WGPA-3-HG) and rhamnogalacturonan (RG)-I-type pectin (AHP-RG) obtained from the roots of Panax ginseng and sunflower heads, respectively, were used as models. Compared to commonly used methyl-esterification methods that use either methyl iodide or acidified methanol, the developed method can methyl-esterify both HG- and RG-I-type pectins without degrading their structures via β-elimination or acid hydrolysis. In addition, by modifying reaction conditions, including the mass ratio of resin to pectin, reaction time, and temperature, the degree of esterification can be controlled. Moreover, the resin and methanol can be recycled to conserve resources, lower costs, and reduce environmental pollution. This new methodology will be highly useful for industrial esterification of pectin.

Tumor-associated carbohydrate antigens (TACAs), which are aberrantly expressed on the surface of tumor cells, are important targets for anticancer vaccine development. Herein, several N-acyl modified Tn analogues were synthesized and conjugated with carrier protein CRM197. The immunological results of these glycoconjugates indicated that 6–CRM197 elicited higher titers of antibodies which cross-reacted with native Tn antigen than the unmodified 2–CRM197 did. The IFN-γ-producing frequency of lymphocytes in mice treated with 6–CRM197 was obviously increased, compared to that of mice vaccinated with 2–CRM197 (p = 0.016), which was typically associated with the Th1 response. Moreover, the elicited antisera against antigen 6–CRM197 reacted strongly with the Tn-positive tumor cells, implying the potential of this glycoconjugate as an anticancer vaccine.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a potent cancer cell-specific apoptosis-inducing cytokine with little toxicity to most normal cells. Here, we report that gefitinib and TRAIL in combination produce a potent synergistic effect on TRAIL-sensitive human colon cancer HCT116 cells and an additive effect on TRAIL-resistant HT-29 cells. Interestingly, gefitinib increases the expression of cell surface receptors DR4 and DR5, possibly explaining the synergistic effect. Knockdown of DR4 and DR5 by siRNA significantly decreases gefitinib- and TRAIL-mediated cell apoptosis, supporting this idea. Because the inhibition of gefitinib-induced autophagy by 3-MA significantly decreases DR4 and DR5 upregulation, as well as reduces gefitinib- and TRAIL-induced apoptosis, we conclude that death receptor upregulation is autophagy mediated. Furthermore, our results indicate that death receptor expression may also be regulated by JNK activation, because pre-treatment of cells with JNK inhibitor SP600125 significantly decreases gefitinib-induced death receptor upregulation. Interestingly, SP600125 also inhibits the expression CHOP, yet CHOP has no impact on death receptor expressions. We also find here that phosphorylation of Akt and ERK might also be required for TRAIL sensitization. In summary, our results indicate that gefitinib effectively enhances TRAIL-induced apoptosis, likely via autophagy and JNK- mediated death receptor expression and phosphorylation of Akt and ERK.

Neutral (WGPN) and acidic (WGPA) polysaccharides were fractionated from ginseng polysaccharide. WGPN and WGPA decreased fasting blood glucose by different manners of administration. Intra-gastric administration of WGPA showed a marked hypoglycemic effect, which may be related to its anti-oxidative activity. The results indicated that WGPA may have anti-diabetic potential.

In previous study, an unbranched (1 → 6)-β-d-glucan with Mw 2.6 kDa was isolated from fruit bodies of Bulgaria inquinans (Fries). In present paper, three branched (1 → 6)-β-d-glucans were obtained from the water-extracted residues by a sequential KOH-extraction, namely BIK2, BIK10 and BIK30. Their molecular weights were determined to be 37.5 kDa (BIK2), 288.9 kDa (BIK10) and 175.5 kDa (BIK30). Structural analysis indicated that their backbones were substituted by single glucosyls at C-3 positions, the branching ratios were 0.01 (BIK2), 0.17 (BIK10), 0.25 (BIK30). Immunological tests showed that all the four β-d-glucans could significantly increase the ConA or LPS-induced lymphocytes proliferation in vivo. Moreover, branched (1 → 6)-β-d-glucans have more significantly lymphocytes proliferation activities than unbranched (1 → 6)-β-d-glucan, and the effect of (1 → 6)-β-d-glucans on lymphocytes proliferation increases along with molecular weights. The present results well enrich the structure–activity relationships of (1 → 6)-β-d-glucan, and indicate (1 → 6)-β-d-glucans from B. inquinans (Fries) are potential immunostimulating agents.Highlights► Three branched (1 → 6)-β-d-glucans were isolated from B. inquinans (Fries). ► All the (1 → 6)-β-d-glucans could significantly increase lymphocytes proliferation in vivo. ► Branched (1 → 6)-β-d-glucans had more significantly activities than unbranched one. ► The activity of branched (1 → 6)-β-d-glucans increased along with molecular weight.

Pathogenic fungus Penicillium oxalicum sp. 68 was screened from soil and identified by ITS sequencing. The strain was found to be able to transform protopanaxadiol-type ginsenosides to produce a series of bioactive metabolites. Glycosidase from the culture of P. oxalicum sp. 68 was partially purified with a simple two-step procedure consisting of DEAE-cellulose chromatography and ammonium sulfate precipitation. Bioactive ginsenoside Compound K was prepared selectively and efficiently by biotransformation of ginsenosides Rb1, Rb2, Rc and Rd using the partially purified glycosidase. The optimal conditions for transforming Rb1 into Compound K were pH 4.0, 55 °C and 0.5 mg mL−1 Rb1. The sole product is Compound K and the maximum yield reached 87.7 % (molar ratio). The transformation pathways of Rb1, Rb2, Rc and Rd are Rb1→Rd→F2→Compound K, Rb2→CO→CY→Compound K, Rc→Mb→Mc→Compound K and Rd→F2→Compound K, respectively. This biotransformation method showed great potential for preparing minor bioactive ginsenosides, especially Compound K, in the pharmaceutical industry because of its high specificity and favorable environmental compatibility.

A water-soluble polysaccharide (HA) was extracted from the Herba Asari root. HA was separated into a starch-like glucan fraction (HA1) and a pectin fraction (HA2) using DEAE-cellulose. HA2 was further fractionated into three pectic polysaccharides, HA2-a, HA2-b and HA2-c, using ion-exchange chromatography. NMR and sugar composition analyses demonstrated that HA2-a is an arabinogalactan (AG) and HA2-b and HA2-c are xylogalacturonans (XGA) with AG domains. Lymphocyte proliferation assays showed that both the neutral polysaccharide and acidic polysaccharide were potent B and T cell stimulators that may have two different modes of action.Highlights► The polysaccharide isolated from the Herba Asari root was totally fractionated. ► A glucan, an arabinogalactan and two xylogalacturonan fractions were obtained. ► The glucan and the pectin have the stimulation activity on both T- and B-cell. ► Neutral and acidic polysaccharides probably have different stimulatory mechanisms.

In this paper, we further analysed the structure of a type I rhamnogalacturonan (RG-I) pectin (WGPA-2-RG) fractionated from ginseng polysaccharides. Methylation and periodate oxidation analyses showed that WGPA-2-RG has a backbone consisting of alternating rhamnose (Rha) and galacturonic acid (GalA) residues and side chains consisting of type II arabinogalactan (AG-II). Partial acidic hydrolysis for 6 h completely removed arabinose (Ara), partial galactose (Gal), but little GalA and Rha. During partial hydrolysis, the molecular weight of WGPA-2-RG decreased smoothly, suggesting that the Ara and cleavable Gal residues exist on the surface of the molecule, while GalA and Rha residues exist in the core of the molecule. The bioactivity assay showed that the arabinogalactan side chains of WGPA-2-RG are essential structures for stimulating NO secretion and lymphocyte proliferation. However, removal of the Ara and Gal residues through hydrolysis did not appreciably affect the ability of WGPA-2-RG to enhance macrophage phagocytosis.Highlights► The ginseng pectic fraction WGPA-2-RG was identified to have RG-I structure. ► WGPA-2-RG has a small backbone and long AG-II side chains. ► Most of the Gal are close to molecular core and all Ara on molecule surface. ► Ara residues are important to lymphocyte proliferation and NO production.

Two novel N-acetylneuraminic acid derivatives, luciferyl N-acetylneuraminic acid (1) and luciferyl 4,7-di-O-methyl-N-acetylneuraminic acid (2), were designed and synthesized as substrates for the rapid detection of influenza virus neuraminidase. The sensitivity and specificity of the assays with compound 1 or 2 as the substrate for detection of neuraminidases from influenza virus (H1N1 and H5N1) and bacteria (A. ureafaciens and C. perfringens) were evaluated. Compound 1 was sensitive to neuraminidases from both influenza virus and bacteria. Bioluminescent assays with this compound with H1N1 and H5N1 neuraminidases were approximately 20- and 16-fold more sensitive, respectively, than the fluorescent method with the commercial substrate 4-MUNANA. In contrast, compound 2 was only sensitive to the neuraminidases from influenza virus, showing approximately 10- and 8-fold greater sensitivity than 4-MUNANA for the detection of H1N1 and H5N1 neuraminidases, respectively. The data showed that compound 2 could be used in assays for detection of an influenza viral neuraminidase.

The pH-modified citrus pectin (MCP) has been demonstrated to inhibit galectin-3 in cancer progression. The components and structures of MCP related to this inhibition remained unknown. In this paper, we fractionated MCP on DEAE-cellulose column into a homogenous neutral fraction MCP-N (about 20 kDa) and a pectin mixture fraction MCP-A (wide molecular distribution on Sepharose CL-6B chromatography). Both MCP-N and MCP-A inhibited hemagglutination mediated by galectin-3 with minimum inhibition concentration (MIC) 625 and 0.5 μg/ml, respectively. MCP-N was identified to be a type I arabinogalactan (AG-I) with a main chain of β-1→4-galactan. MCP-N was digested by α-L-arabinofuranosidase to give its main chain structure fraction (M-galactan, around 18 kDa), which was more active than the original molecule, MIC 50 μg/ml. The acidic degradation of M-galactan increased the inhibitory activity, MIC about 5 times lower than M-galactan. These results above showed that the functional motif of the β-1→4-galactan fragment might lie in the terminal residues rather than in the internal region of the chain. Therefore, MCP-N and its degraded products might be developed to new potential galectin-3 inhibitors. This is the first report concerning the fractionation of MCP and its components on galectin-3 inhibition. The information provided in this paper is valuable for screening more active galectin-3 inhibitors from natural polysaccharides.

A ginseng pathogen, Cylindrocarpon destructans, and five nonpathogens were tested for their sensitivity to a total ginsenoside fraction (T-GF), a protopanaxadiol-type ginsenoside fraction (PPD-GF) and a protopanaxatriol-type ginsenoside fraction (PPT-GF) from the roots of Panax ginseng C.A. Meyer. The results showed that T-GF inhibited growth of the five ginseng nonpathogens, while it promoted growth of the ginseng pathogen C. destructans. PPT-GF and PPD-GF both inhibited the growth of the five ginseng nonpathogens, although the activity of PPT-GF was higher than that of PPD-GF. PPT-GF and PPD-GF exhibited different activities on C. destructans: PPT-GF inhibited its growth, whereas PPD-GF significantly enhanced its growth. The subsequent analysis of enzymatic degradation of ginsenosides by the test fungi showed that C. destructans can consecutively hydrolyze the terminal monosaccharide units from the sugar chains attached at C3 and C20 in PPD-type ginsenosides by extracellular glycosidase activity to yield four major products, gypenoside XVII (G-XVII), compound O, compound Mb and the ginsenoside F2. By contrast, the ginseng nonpathogens Aspergillus nidulans and Cladosporium fulvum have no extracellular glycosidase activity toward sugar chains attached to C3 in PPD-type ginsenosides. These results indicated that ginsenosides might act as host chemical defenses, while the ginseng root pathogenic fungi might counter their toxicity by converting PPD-type ginsenosides into growth or host recognition factors. The ability of ginseng root pathogens to deglycosylate PPD-type ginsenosides may be a pathogenicity factor.Graphical abstractPPT-type ginsenosides from Chinese ginseng inhibited growth of all six test fungi. PPD-type ginsenosides promoted growth of the ginseng pathogen Cylindrocarpon destructans, while inhibiting that of nonpathogens. C. destructans can consecutively hydrolyze sugar chains attached at C3 and C20 in PPD-type ginsenosides by an extracellular glycosidase.Highlights► PPT-type ginsenosides from Chinese ginseng inhibited growth of all six test fungi. ► PPD-type ginsenosides promoted growth of the ginseng pathogen Cylindrocarpon destructans. ► C. destructans can enzymatically degrade PPD-type ginsenosides.

Panax ginseng C. A. Meyer is a well-known plant medicine in the world. Ginseng polysaccharides mainly contain starch-like glucan and pectin. In this paper, a novel glucan WGPA-UH-N1 was purified from ginseng pectin by the treatment of de-esterification and endo-polygalacturonase, followed by the chromatographies on DEAE-Sepharose Fast Flow and Sephadex G-50 column. WGPA-UH-N1 has molecular weight about 17 kDa. WGPA-UH-N1 was determined to be a linear α-(1 → 6)-D-glucan without side chains by FT-IR, 13 C-NMR, 1 H-NMR, HMQC and HMBC spectra. It is the first time to isolate a linear α-(1 → 6)-D-glucan from Panax ginseng C. A. Meyer. Immunological activity assays showed that WGPA-UH-N1, although not effective on the phagocytosis of macrophage, could significantly induce lymphocyte proliferation without mitogenic stimuli at 1.0 mg/mL or with LPS at 0.5 mg/mL, also significantly increase NO production at the range of 0.1–1.0 mg/mL in a dose-dependent manner. The immunological activities of WGPA-UH-N1 are different from those of the β-(1 → 6)-D-glucan (BIWP2) isolated from the fruit bodies of Bulgaria Inquinans (Fries).

In this paper, ginseng pectin (WGPA) was fractionated and two new fractions were characterised. Nine fractions, including seven characterised in our previous paper, were tested for their effects on cell migration. The HG-domain rich pectins caused significant inhibition on cell migration. The order of the inhibitory effects is WGPA-1-HG < -2-HG < -3-HG < -4-HG. At 0.015 mg/ml, WGPA-3-HG significantly inhibited L-929 cell migration. WGPA-3-RG and -4-RG, containing both HG- and RG-I-domains, showed a slightly stronger inhibition than the HG-domain rich fractions. WGPA-N, WGPA-1-RG and -2-RG, containing no, trace or minor HG and RG-I domains, showed less effects on cell migration. The inhibitory effect of ginseng pectic polysaccharides was related to GalA content (HG domain) and Rha content (RG-I domain). The effect is probably mediated through decreasing cell adhesion and cell spreading.

Five rhamnogalacturonan I (RG-I) domains RG-I-1, RG-I-2, RG-I-3A, RG-I-3B and RG-I-4 were isolated from ginseng pectin by endo-polygalacturonase hydrolysis and a combination of ion-exchange and gel-permeation chromatography. The five domains all contain galacturonic acid, rhamnose, galactose and arabinose as main components and their rhamnose/galacturonic acid is from 0.26 to 0.64, among the range of RG-I. The molecular weights of RG-I-1 (5 kDa), RG-I-2 (4 kDa) and RG-I-3B (6 kDa) are smaller than those of RG-I-3A (45 kDa) and RG-I-4 (60 kDa). 13C NMR spectra of all domains showed RG-I features. RG-I-2 and RG-I-3B contained RG-I domains linked with highly methyl-esterified and acetylated homogalacturonan domains and the side chains probably belonging to type I and type II arabinogalactans, while RG-I-3A and RG-I-4 may have the side chains of type I arabinogalactans. 4-O-methyl-β-d-glucuronic acid residues were present at non-reducing terminals of RG-I-2.

The phytotoxic effect of four glycoalkaloids and two 6-O-sulfated glycoalkaloid derivatives were evaluated by testing their inhibition of cucumber root growth. The bioassays were performed using both compounds singly and in equimolar mixtures, respectively. Cucumber root growth was reduced by chaconine (C), solanine (S), solamargine (SM) and solasonine (SS) with IC50 values of 260 (C), 380 (S), 530 (SM), and 610 μM (SS). The inhibitory effect was concentration-dependent. 6-O-sulfated chaconine and 6-O-sulfated solamargine had no inhibitory effects, which indicated that the carbohydrate moieties play an important role in inhibiting cucumber root growth. The equimolar mixtures of paired glycoalkaloids, both chaconine/solanine and solamargine/solasonine, produced synergistic effects on inhibition of cucumber root growth. By contrast, mixtures of unpaired glycoalkaloids from different plants had no obviously synergistic effects. The growth inhibited plant roots lacked hairs, which implied that inhibition was perhaps at the level of root hair growth.The phytotoxic effects of four natural glycoalkaloids, chaconine, solanine, solamargine, solasonine and two glycoalkaloid derivatives 6-O-sulfated chaconine and 6-O-sulfated solamargine, were investigated by inhibiting cucumber root growth individually and in 1:1 combination.

Water-soluble polysaccharides isolated from the roots of Panax ginseng C. A. Meyer were completely fractionated into two neutral fractions (WGPN and WGPA-N) and six acidic fractions (WGPA-1-RG, WGPA-2-RG, WGPA-1-HG, WGPA-2-HG, WGPA-3-HG and WGPA-4-HG) by a combination of ethanol precipitation, ion-exchange and gel permeation chromatographies. The analytical results showed that WGPN was a starch-like glucan; WGPA-N was a mixture of starch-like glucan and arabinogalactan; WGPA-1-RG and WGPA-2-RG were composed of major neutral sugars and minor acidic sugars that belong to the type-I rhamnogalacturonan (RG-I)-rich pectins, while fractions WGPA-1-HG to WGPA-4-HG were mainly composed of galacturonic acid (GalA, 62.4–92.1%) and have been identified to be homogalacturonan (HG)-rich pectins with different degrees of methyl-esterification, ranging from 0% to 30%. High performance gel permeation chromatography (HPGPC) showed that the six acidic fractions were homogenous, with molecular weights approximately ranging from 3.5 × 103 to 1.1 × 105. Lymphocyte proliferation assays showed that both the neutral polysaccharides and acidic polysaccharides were potent B and T cell stimulators.

A low molecular-weight polysaccharide named BIWP2 was purified from the fruit bodies of Bulgaria Inquinans (Fries) via hot-water extraction, followed by freeze-thawing and gel filtration chromatography on Sephadex G-75. Monosaccharide composition analysis revealed that BIWP2 contained exclusively glucose. High performance size exclusion chromatography (HPSEC) showed that it was a homogeneous polysaccharide fraction. Its molecular weight was estimated to be 2.6 KD and the polydispersity index (Mw/Mn) was calculated to be 1.4. Periodate oxidation, methylation, and NMR analyses indicated that BIWP2 was a linear β-(1→6)-d-glucan without side chains. This is the first time to report a linear β-(1→6)-d-glucan with low molecular weight in non-lichenized ascomycete.

A water-soluble glucan (RCP-1) was prepared from the roots of Rubus crataegifolius Bge. by extraction with hot-water, deproteination by Sevag reagent, α-amylase treatment and ultrafiltration. RCP-1 consisted of only glucose, and its molecular weight was determined to be ∼7 KD by high performance gel permeation chromatography (HPGPC). Fourier transform infra-red spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (NMR), methylation and periodate oxidation analyses indicated that RCP-1 was an α-d-glucan. Its main chains were composed of (1→4)- and (1→6)-linked α-glucopyranosyls, and side chains were single α-glucopyranosyl residues attached to the O-6 of glucosyls in the main chains. RCP-1 could increase both cytotoxic activity against B16 melanoma cells and the production of nitric oxide (NO) of macrophages in vitro. Furthermore, in vivo bioassay tests indicated that RCP-1 could remarkably enhance T and B lymphocyte proliferations, augment the phagocytosis of macrophages and increase the tumour necrosis factor-alpha (TNF-α) levels in serum.

Fourteen phytopathogenic fungi were tested for their ability to transform the major ginsenosides to the active minor ginsenoside Rd. The transformation products were identified by TLC and HPLC, and their structures were assigned by NMR analysis. Cladosporium fulvum, a tomato pathogen, was found to transform major ginsenoside Rb1 to Rd as the sole product. The following optimum conditions for transforming Rd by C. fulvum were determined: the time of substrate addition, 24 h; substrate concentration, 0.25 mg ml−1; temperature, 37°C; pH 5.0; and biotransformation period, 8 days. At these optimum conditions, the maximum yield was 86% (molar ratio). Further, a preparative scale transformation with C. fulvum was performed at a dose of 100 mg of Rb1 by a yield of 80%. This fungus has potential to be applied on the preparation for Rd in pharmaceutical industry.

Eight novel toxoflavin glycosides, which are potential prodrugs in antibody directed enzyme prodrug therapy (ADEPT), were synthesized. The structures of all toxoflavin glycosides were characterized by 13C NMR spectroscopy, elemental analysis, and MS. Their enzymatic hydrolysis activities were tested against β-glucosidase (EC.3.2.1.21).

•A new GH3 gene ccxyl3a was cloned from C. cellulans sp. 21 and heterologously expressed in E. coli.•Enzyme CcXyl3A was a multifunctional enzyme with β-xylosidase/α-l-arabinofuranosidase/β-glucosidase activities.•CcXyl3A synergistically acted with Thermomyces lanuginosus xylanase in beechwood xylan degradation.A multifunctional β-xylosidase/α-l-arabinofuranosidase/β-glucosidase gene (ccxyl3a) belonging to glycoside hydrolase family 3 (GH3) was cloned from Cellulosimicrobium cellulans sp. 21 and expressed in Escherichia coli BL21 (DE3). The molecular mass of recombinant CcXyl3A was estimated to be approximately 95 kDa. With p-nitrophenyl-β-d-xyloside (pNPβXyl) as a substrate, the purified protein presented an optimal pH of 8.5 and an optimal temperature of 45 °C. Moreover, CcXyl3A was activated in the presence of the metals K+ and Na+. Purified CcXyl3A demonstrated multifunctional activities on pNPβXyl, p-nitrophenyl-β-d-glucoside (pNPβGlc), and p-nitrophenyl-α-l-arabinofuranoside (pNPαAraf). The greatest catalytic activity were found on pNPβXyl followed by pNPαAraf and pNPβGlc, respectively. Using xylooligosaccharides as substrate, CcXyl3A completely hydrolyzed xylobiose, xylotriose, xylotetraose and xylohexaose, xylose was the sole product. In addition, CcXyl3A synergistically acted with Thermomyces lanuginosus xylanase in the degradation of beechwood xylan, released xyloses from intermediate xylooligosaccharides produced by T. lanuginosus xylanase. To date, this is the first report to demonstrate the cloning and characterization of a multifunctional GH3 enzyme in C. cellulans that may have applications in hemicellulose degradation.Download full-size image

•A new GH1 gene ccbgl1a from C. cellulans sp. 21 was cloned and expressed in E. coli.•Enzyme CcBgl1A was specific to β-1,2-glycosidic bond at C-3 position of ginsenosides.•CcBgl1A was applied for one-pot preparation of ginsenoside GXVII, CO, Mb and F2.A novel β-glucosidase gene (ccbgl1a) was cloned from the ginsenosides-transforming strain Cellulosimicrobium cellulans sp. 21. This enzyme was overexpressed in Escherichia coli, the recombinant β-glucosidase (CcBgl1A) containing N-terminal His-tag was sufficiently purified by nickel metal affinity chromatography with purification factor of 1.9-fold and specific activity of 31.5 U/mg. The molecular mass of recombinant CcBgl1A was estimated to be approximately 46 kDa. CcBgl1A exhibited optimal activity at 35 °C and pH 5.5. However, above 40 °C, the enzyme stability significantly decreased. The enzyme showed high bioconversion ability on protopanaxadiol-type ginsenosides mixture (PPDGM), which could hydrolyze the outer C-3 glucose moieties of ginsenosides Rb1, Rb2, Rc and Rd into the rare ginsenosides Gypenoside XVII (Gyp XVII), compound O, ginsenoside Mb and ginsenoside F2. Scaled-up production using 1 g of the PPDGM resulted in 292 mg Gyp XVII, 134 mg CO, 184 mg Mb, and 62 mg F2, with chromatographic purities. These results suggest that CcBgl1A would be potentially useful in the preparation of pharmacologically active minor ginsenosides Gyp XVII, CO, Mb and F2.Download full-size image

•Electrospun nanofibers of three representative pectins were crosslinked.•Three feasible crosslinking methods were established.•Pectin structure affected properties of Ca2+-crosslinked nanofibers remarkably.•ADH crosslinking enhanced mechanical strength and inhibited degradation.•ADH crosslinking and high DM facilitated cell adhesion and proliferation.We reported crosslinking of electrospun nanofibers of three representative pectins (high-methoxylated, low-methoxylated, low-methoxylated and amidated pectin) and characterization of the crosslinked nanofibers. One mono-crosslinking strategy and two dual-crosslinking strategies were developed. Mono-crosslinking is achieved using calcium ions (Ca2+) to crosslink carboxylate ions in galacturonic acid residues. Dual-crosslinking is achieved using covalent crosslinking reagents glutaraldehyde (GLU) or adipic acid dihydrazide (ADH) to further crosslink hydroxyl groups or carboxylate ions after Ca2+ crosslinking. Mechanical tests and degradation experiments indicated pectin structure affected mechanical and degradation properties of Ca2+-crosslinked nanofibers remarkably. Subsequent GLU crosslinking improved their mechanical strength moderately but did not inhibit their degradation, while subsequent ADH crosslinking improved their mechanical strength and slowed down their degradation dramatically. Cell studies demonstrated that most crosslinked pectin nanofibers were of no obvious cytotoxicity, and both ADH crosslinking and high degree of methoxylation facilitated cell adhesion and proliferation on pectin nanofiber mats.