Q-Sepharose Fast Flow Research Articles

Screening of a readily selenium-enriched Spirulina polysaccharide and characterization of its structure and bioactivity

The potential of selenium-containing polysaccharides as organic selenium nutritional supplements has attracted considerable interest; however, they also encounter obstacles, including the presence of impurities and a low selenium binding efficiency. In this study, a novel method combining Q-Sepharose Fast-Flow (QFF) column chromatography with the HNO3–Na2SeO3 method was proposed. Through the application of this methodology, the selenium binding efficiency of polysaccharides was enhanced by a factor of 3.41. The selenium binding capacities of rhamnose and glucose were the highest among the monosaccharides, reaching 770.7 mg/L and 955.3 mg/L, respectively. Spirulina polysaccharide-3 (SPS-3) was selected for further investigation because of its elevated total rhamnose and glucose contents and optimal selenium binding efficiency. Employing common assays, a comparative analysis of selenium-containing Spirulina polysaccharide-3 (SeCSPS) and SPS-3 is presented. Upon selenium binding, the surface morphology of the monosaccharides changed, whereas the main chain structure remained unaltered. The antihyperglycemic activity of SeCSPS was enhanced by selenium binding. Furthermore, both SeCSPS and SPS-3 can be degraded by the simulated digestive system. In conclusion, SeCSPS can be employed as an organic selenium supplement in food or nutraceuticals while also resulting in a high selenium content. The findings of this study will provide a viable avenue for the high-value utilization of Spirulina, which will help resolve the selenium deficiency issue faced by populations with low selenium intake globally.

Purification and characterization of amylases from three freshwater fish species providing new insight application as enzyme molecular markers for zymography.

Purification of amylases from digestive tracts of three freshwater fish species with Q-Sepharose Fast Flow and Sephacryl S-200 columns displayed two isoforms of amylases from Osteochilus hasselti (O1, O2) and three isoforms of those from both Hampala dispar (UB, H1, H2) and Puntioplites proctozystron (P1, P2, P3). The optimum pH values displayed at 7.0 and 8.0, while the optimum temperatures revealed at 40 and 50°C. Almost isoenzyme activities were activated by NaCl and CaCl2, whereas EDTA and SDS strongly inhibited all enzymatic activities. Verification with an atomic absorption spectrophotometry exhibited the presence of Ca2+ ions in the range of 0.02-13.53ppm per mg protein indicating that amylases are Ca2+ dependent. Molecular weight analysis revealed 12 to 147kDa. The UB, O1, and H2 amylases with appropriate molecular masses of 64, 49, and 25kDa validated with LC-MS/MS were selected. Three certain enzymes revealed high stability in a sample buffer after five cycles of freeze-thawing process upon storage at - 20°C for 12weeks. No protein degradation was observed on polyacrylamide gel, and the enzymes still displayed sharp and clear bands on zymograms. The result suggested that the purified fish amylases, which expressed high activities and stabilities, were potentially used as enzyme molecular weight markers for zymography.

Effects of beauvericin on the blood cells of Bombyx mori

In this study, silkworms were treated by injection of the bioactive depsipeptide beauvericin (BEA) to explore its effect on the cellular immunity of larvae of the silkworm Bombyx mori. The results showed that: The LC50 of BEA for silkworms on the 3rd day of the 4th instar was 362.36 µM. The total count of circulating hemocytes in the silkworms decreased at 12 h after injection with 350 µM BEA, and reached the minimum value at 72 h post-treatment; at 48 h post-treatment, a large number of nodules formed by the aggregation of blood cells of the silkworms were observed under the light microscope. The survival rate of hemocytes in the larvae treated with BEA was significantly reduced in a dose-dependent manner in vivo and in vitro. The encapsulation of Q-Sepharose Fast Flow (QFF) gel particles by hemocytes in the treatment group was significantly higher than that in the control group at 1.5 h and 3 h post-treatment (P < 0.05). Moreover, the melanization ratio of QFF gel particles kept increasing with treatment time. The melanization rate at 24 h after treatment was significantly higher than that at other times (P < 0.05), reaching 55.33 %. Under the scanning electron microscope, BEA-treated larvae showed protrusions on the surface of their blood cells in vivo. Under the transmission electron microscope, it was observed that silkworm hemocytes were vacuolated. This study demonstrated that BEA had an effect on the blood cells of silkworms, and has thrown some light on the inhibitory effect and mechanism of BEA on insect cellular immunity.

Biochemical characterization of an alkaline and detergent-stable Lipase from Fusarium annulatum Bugnicourt strain CBS associated with olive tree dieback.

This work describes a novel extracellular lipolytic carboxylester hydrolase named FAL, with lipase and phospholipase A1 (PLA1) activity, from a newly isolated filamentous fungus Ascomycota CBS strain, identified as Fusarium annulatum Bunigcourt. FAL was purified to about 62-fold using ammonium sulphate precipitation, Superdex® 200 Increase gel filtration and Q-Sepharose Fast Flow columns, with a total yield of 21%. The specific activity of FAL was found to be 3500 U/mg at pH 9 and 40°C and 5000 U/mg at pH 11 and 45°C, on emulsions of triocanoin and egg yolk phosphatidylcholine, respectively. SDS-PAGE and zymography analysis estimated the molecular weight of FAL to be 33 kDa. FAL was shown to be a PLA1 with a regioselectivity to the sn-1 position of surface-coated phospholipids esterified with α-eleostearic acid. FAL is a serine enzyme since its activity on triglycerides and phospholipids was completely inhibited by the lipase inhibitor Orlistat (40 μM). Interestingly, compared to Fusarium graminearum lipase (GZEL) and the Thermomyces lanuginosus lipase (Lipolase®), this novel fungal (phospho)lipase showed extreme tolerance to the presence of non-polar organic solvents, non-ionic and anionic surfactants, and oxidants, in addition to significant compatibility and stability with some available laundry detergents. The analysis of washing performance showed that it has the capability to efficiently eliminate oil-stains. Overall, FAL could be an ideal choice for application in detergents.

Purification, Biochemical and Kinetic Characterization of a Novel Alkaline sn-1,3-Regioselective Triacylglycerol Lipase from Penicilliumcrustosum Thom Strain P22 Isolated from Moroccan Olive Mill Wastewater.

A novel extracellular lipase from a filamentous fungus Ascomycota strain, P22, was isolated from olive mill wastewater, then purified and characterized. This strain was identified as Penicillium crustosum Thom based on sequencing analyses. Penicillium crustosum Thom strain P22 lipase (PCrL) was purified 63-fold to homogeneity using ammonium sulfate precipitation and chromatography on a Q-Sepharose Fast Flow column, with a total yield of 34%. The purified PCrL had a molecular mass of 28 kDa, estimated by SDS-PAGE. The 20 NH2-terminal amino-acid residues showed a high degree of homology with those of other Penicillium lipases. The specific activity of PCrL at pH 9 and 37 °C were found to be 5000 and 10,000 U/mg on olive oil and trioctanoin emulsions, respectively. PCrL exhibited clear regioselectivity toward the sn-1 position of the surface-coated triglycerides which were esterified with α-eleostearic acid at the sn-1/3 position. PCrL was completely inhibited by 53 µM of Orlistat, 5 mM of phenylmethylsulfonylfluoride, and 2 mM of diiodopropyl fluorophosphate, suggesting that it belonged to the serine lipase family. PCrL showed high activity and stability in the presence of water-immiscible organic solvents, surfactant, and oxidizing agents, and showed considerable compatibility with commercial laundry detergents. Washing performance analysis revealed that it could effectively remove oil stains. Hence, PCrL has several attractive properties that make it a promising potential candidate for detergent formulations.

Structure and In Vitro Fermentation Characteristics of Polysaccharides Sequentially Extracted from Goji Berry (Lycium barbarum) Leaves.

Herein, the chelating agent-soluble fraction (CA), sodium carbonate-soluble fraction (SC), and sodium hydroxide-soluble fraction (SH) were sequentially extracted from the cell wall of goji berry (Lycium barbarum) leaves. Furthermore, SC was purified with Q-Sepharose fast flow resin to obtain the neutral sugar fraction (SC-I) and acid sugar fraction (SC-II). Physicochemical properties of polysaccharides were characterized by high-performance anion-exchange chromatography with pulsed amperometry detection, size exclusion chromatography-multi-angle laser light scattering, Fourier transform infrared spectroscopy, nuclear magnetic resonance, and atomic force microscopy analysis. Additionally, the impact of polysaccharides on modulating human gut microbiota was investigated by in vitro fermentation. A high amount of galacturonic acid (GalA) in CA showed that it was an aggregation of linear homogalacturonan. SC was the main pectic polysaccharide fraction and rich in neutral sugars. SC-I was the neutral sugar fraction with an extremely high molecular weight (2.055 × 106 Da), while SC-II was the acid sugar fraction with a low molecular weight (1.766 × 105 Da). SH seemed like a mixture of pectin and hemicellulose. All the five polysaccharides significantly (P < 0.05) increased the abundance of Bacteroides, Bifidobacteria, and Lactobacilli. To the best of our knowledge, this is the first report on the structure and fermentation characteristics of goji berry leaf polysaccharides, which is meaningful to provide a structural basis for further bioactivity research.

Effects of non-starch polysaccharides from pure wheat malt beer on beer quality, in vitro antioxidant, prebiotics, hypoglycemic and hypolipidemic properties

Pure wheat malt beer (PWMB) with potentially functional properties may attract consumer interest. To explore the functional properties of non-starch polysaccharides (NSPs) in PWMB, NSPs were extracted from PWMB and separated into PWMB non-starch polysaccharides-1 (PNSP-1) and PWMB non-starch polysaccharides-2 (PNSP-2) by Q-Sepharose Fast Flow column. PNSP-1 exhibited more effects on increasing the viscosity of beers, which had higher purity of 92.58% containing 88.53% arabinoxylan with arabinose/xylose (A/X) 0.60 and 2.78% β-glucan but no glucomannan and mannan; while PNSP-2 showed greater effects on promoting beer haze and foam retention, which had more diverse NSPs including 46.03% arabinoxylan with A/X 1.13, 41.47% arabinogalactan, 10.26% glucomannan, and 2.70% mannan but no β-glucan. Diversity of sugar composition resulted in a better antioxidant capacity of PNSP-2. NSPs proliferated Lactobacillus casei LC6117 and LC0331, but did not promote L. plantarum LP-28 and L. fermentium LF-2H. PNSP-2 was also utilized by L. rhamnosus SRG. PNSP-2 had better effects on prebiotics activities. PNSP, PNSP-1 and PNSP-2 all exhibited mild inhibitory effects on α-glucosidase and lipase but not on α-amylase, and showed a binding capacity to fat and cholesterol. This work is of great reference significance for improving the viscosity, haze, foam retention and health value of wheat beer.

A jacalin-related lectin domain-containing lipase from chestnut (Castanea crenata): Purification, characterization, and protein identification

A novel lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was discovered from Korean chestnut (Castanea crenata). The lipase was isolated and purified by ammonium sulfate precipitation and a fast protein liquid chromatography system equipped with HiTrap DEAE-Sepharose Fast Flow, HiTrap Q-Sepharose Fast Flow, and HiPrep Sephacryl S-100 Hi-Resolution columns. The purified C. crenata lipase showed a 15.8% yield, purification fold number of 465.8, and specific activity against triolein of 88.5 mU/mg. The enzyme exhibited hydrolytic activity toward tributyrin, trilaurin, and triolein, and was maximally active at pH 8.0 and 35 °C, with triolein used as the substrate. The activation energy (Ea) and deactivation energy (Ed) of triolein hydrolysis were 38.41 and 83.35 kJ/mol, respectively. In the enzyme kinetic study, Vmax, Km, and kcat were 110.58 mU/mg, 0.11 mM, and 0.221 min−1, respectively. The relatively low Km value indicated that the lipase has high affinity for its substrate. Moreover, Mg2+ and Ca2+ increased the lipase activity to 115.4% and 108.3%, respectively. The results of peptide fingerprinting revealed that the C. crenata lipase with a molecular weight of 33.3 kDa was structurally similar to the mannose-binding lectin of the jacalin-related lectin domain superfamily, implying that it has potential as a therapeutic agent for use in the biomedical industry.

Isolation and Characterization of a Novel Sialoglycopeptide Promoting Osteogenesis from Gadus morhua Eggs.

Gadus morhua eggs contain several nutrients, including polyunsaturated fatty acids, lecithin and glycoproteins. A novel sialoglycopeptide from the eggs of G. morhua (Gm-SGPP) was extracted with 90% phenol and purified by Q Sepharose Fast Flow (QFF) ion exchange chromatography, followed by S-300 gel filtration chromatography. Gm-SGPP contained 63.7% carbohydrate, 16.2% protein and 18.6% N-acetylneuraminic acid. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that Gm-SGPP is a 7000-Da pure sialoglycopeptide. β-elimination reaction suggested that Gm-SGPP contained N-glycan units. Amino acid N-terminal sequence analysis indicated the presence of Ala-Ser-Asn-Gly-Thr-Gln-Ala-Pro amino acid sequence. Moreover, N-glycan was connected at the third Asn location of the peptide chain through GlcNAc. Gm-SGPP was composed of D-mannose, D-glucuronic acid and D-galactose. Fourier transform-infrared spectroscopy (FT-IR), 1H-nuclear magnetic resonance spectroscopy (1H-NMR) and methylation analysis were performed to reveal the structure profile of Gm-SGPP. In vitro results showed that the proliferation activity of MC3T3-E1 cells was significantly promoted by Gm-SGPP. In vivo data revealed that Gm-SGPP increased the calcium and phosphorus content of tibias and promoted longitudinal bone growth in adolescent rats.

Purification of Dickeya solanil-Asparaginase and Study of the Influence of TiO2 and ZnO Nanoparticles on Its Enzymatic Activity

l-asparaginase catalyzes the hydrolysis of l-asparagine to l-aspartate and ammonia. In the present study, we describe the production, purification, and preliminary characterization of Dickeya solanil-asparaginase. The purification of l-asparaginase was done using ion-exchange chromatography on HiTrap Q-Sepharose Fast Flow followed by FPLC-gel filtration chromatography on Superdex 75 pg. The presence of the enzyme was confirmed by enzymatic activity measurements along with SDS-PAGE analysis. The purified l-asparaginase was shown to exhibit specificity towards l-asparagine, while being inactive against l-glutamine. This data allows us to suggest that the l-asparaginase from Dickeya solani might be clinically more relevant than, e.g., the l-asparaginase isolated from E. coli which hydrolyzed also l-glutamine and produces l-glutamate, a neurotoxic agent. We also demonstrated that the enzymatic activity was enhanced in the presence of TiO2 and ZnO nanoparticles, making them good candidates to improve l-asparaginase activity. Indeed, the results obtained show that the TiO2 nanoparticles increased the activity of l-asparaginase by a factor of 6.0 while the ZnO nanoparticles increased it twice.

Purification, structural characterization, and immunomodulatory activity of the polysaccharides from Ganoderma lucidum

A preliminary relationship was illustrated between the structural characteristics and corresponding immunomodulatory activities of G. lucidum polysaccharides. Two polysaccharides (GLP-1 and GLP-2) were purified from Ganoderma lucidum extracts by gradient ethanol precipitation and a Q-Sepharose Fast Flow (QFF) strong anion-exchange column. The monosaccharide composition, high-performance gel permeation chromatography–multi-angle laser light scattering–refractive index (HPGPC-MALLS-RI), Fourier-transform infrared spectroscopy (FT-IR), atomic force microscopy (AFM), methylation analysis, and nuclear magnetic resonance (NMR) were used to characterize these polysaccharides. The GLP-1 polysaccharide was elucidated as d-galactoglucan with a flexible random linear conformation that mainly composed of →6)-β-d-Glcp-(1→, →6)-α-d-Galp-(1→, and →3)-β-d-Glcp-(1→ residues. GLP-2 was found to be a relatively homogeneous β-d-glucan that possessing →6)-β-d-Glcp-(1→ and →3)-β-d-Glcp-(1→ residues packaged into a spherical conformation. Immunomodulatory activities in vivo demonstrated that GLP-1 produced better protection of the spleen and thymus and was more effective for promoting hematopoiesis and improving IgA levels in serum. Our results suggest that the immunomodulatory activities of G. lucidum polysaccharides are highly corresponded to their structural characteristics such as carbohydrate composition, molecular weight and advanced conformation. This study provides a preliminary basis for studying the relationship between polysaccharide structure characterization and pharmacological activities.

Alkaline Extraction, Structural Characterization, and Bioactivities of (1→6)-β-d-Glucan from Lentinus edodes.

The purpose of this study is to develop a robust approach to obtain β glucans from Lentinus edodes and to characterize their structural and biological properties for sustainable utilization. The alkali extraction was optimized with an orthogonal experimental design, and a concise process for obtaining specific targeting polysaccharides from Lentinus edodes was developed in this study. After purification with a Q-Sepharose Fast Flow strong anion-exchange column, the monosaccharide composition, a methylation analysis, and NMR spectroscopy were employed for their structural characterizations. LeP-N2 was found to be composed of (1→6)-β-d-glucans with minor β-(1→3) glucosidic side chains. Atomic force microscopy (AFM) and high-performance gel permeation chromatography–refractive index–multi-angle laser light scattering (HPGPC-RI-MALLS) also revealed LeP-N2 exhibiting a compact unit in aqueous solution. This (1→6)-β-d-glucan was tested for antioxidant activities with IC50 at 157 μg/mL. Moreover, RAW 264.7 macrophage activation indicated that the release of nitric oxide (NO) and reactive oxygen species (ROS) were markedly increased with no cytotoxicity at a dose of 100 μg/mL. These findings suggest that the (1→6)-β-d-glucans obtained from Lentinus edodes could serve as potential agents in the fields of functional foods or medicine.

Heterologous expression, purification and function of the extracellular domain of human RANK

BackgroundReceptor activator of NF-κB ligand (RANKL)/RANK signaling essentially functions within the skeletal system, particularly participating in osteoclastogenesis and bone resorption. In addition, this signaling pathway has also been shown to influence tumor progression as well as the development and function of the immune system. Therefore, blocking the interaction between RANKL and RANK is a new therapeutic approach to prevent bone-related diseases and cancer.ResultsThe coding sequence encoding the extracellular domain of human RANK (RANK-N) was codon optimized for Pichia pastoris and cloned into the pPIC9K vector, and the recombinant plasmid was then transformed into P. pastoris. The expression of RANK-N protein was confirmed using SDS-PAGE with Coomassie Brilliant Blue stain and western blotting. Recombinant RANK-N protein was purified by a multistep process including ultrafiltration (UF), Sephadex G-50 size-exclusion chromatography and Q-Sepharose Fast Flow ion exchange chromatography, which resulted in a purity >95%. We found that the RANK-N protein can block RANKL-RANK signaling both in vitro and in vivo. Furthermore, using a patient-derived xenograft of human colon cancer, we found that the recombinant RANK-N protein can inhibit the growth of colorectal cancer.ConclusionsThe results show that a simple system to express and purify functional RANK-N protein has been developed. This work has thus laid a foundation for further research and clinical applications of RANK-N protein in treating bone-related diseases or even colorectal cancer.

Expression of adenylate kinase fused MEK1R4F in Escherichia coli and its application in ERK phosphorylation.

To construct a highly expressed and active MEK1R4F (a constitutively active form of mitogen-activated protein kinase kinase 1) by fusion of soluble adenylate kinase (Adk) tag, resulting in Adk-MEK1R4F protein suitable for preparation of phosphorylated ERK. We fused the Adk to the N-terminus of MEK1R4F through overlapping PCR. The expression of Adk-MEK1R4F fusion protein increased ~10-fold in Escherichia coli, and was purified to 95% via two purification steps including Ni-NTA and Q Sepharose fast flow (QFF) chromatography. The purified Adk-MEK1R4F protein was functional for ERK phosphorylation and could use ADP in addition to ATP. The Adk-MEK1R4F had higher catalytic activity than regular MEK1R4F both in vitro and in cell-free extracts system. Adenylate kinase was used as a soluble tag to facilitate MEK1R4F protein expression and its application in large-scale phosphorylated ERK1/2 preparation and purification.

Aspartate aminotransferase is potently inhibited by copper complexes: Exploring copper complex-binding proteome

Recent researches indicated that a copper complex-binding proteome that potently interacted with copper complexes and then influenced cellular metabolism might exist in organism. In order to explore the copper complex-binding proteome, a copper chelating ion-immobilized affinity chromatography (Cu-IMAC) column and mass spectrometry were used to separate and identify putative Cu-binding proteins in primary rat hepatocytes. A total of 97 putative Cu-binding proteins were isolated and identified. Five higher abundance proteins, aspartate aminotransferase (AST), malate dehydrogenase (MDH), catalase (CAT), calreticulin (CRT) and albumin (Alb) were further purified using a SP-, and (or) Q-Sepharose Fast Flow column. The interaction between the purified proteins and selected 11 copper complexes and CuCl2 was investigated. The enzymes inhibition tests demonstrated that AST was potently inhibited by copper complexes while MDH and CAT were weakly inhibited. Schiff-based copper complexes 6 and 7 potently inhibited AST with the IC50 value of 3.6 and 7.2μM, respectively and exhibited better selectivity over MDH and CAT. Fluorescence titration results showed the two complexes tightly bound to AST with binding constant of 3.89×106 and 3.73×106M−1, respectively and a stoichiometry ratio of 1:1. Copper complex 6 was able to enter into HepG2 cells and further inhibit intracellular AST activity.

Extraction and purification of an endo-1,4-β-xylanase from wheat malt

Wheat and wheat malt are known to be high quality raw materials in the beer brewing process. The size and structure of wheat arabinoxylans (AXs) and their enzymatic degradation products have a profound impact on beer properties, such as viscosity, turbidity, filtration rate and foaming properties. AXs are of great benefit to human health due to their anti-tumor properties and potential roles in the immune system. Endo-1,4-β-xylanase is a key enzyme for degradation of AXs and production of small molecule AXs, thereby altering the properties of AXs, such as solubility, viscosity, and turbidity. In this study, extraction and purification of an endo-1,4-β-xylanase from wheat malt was described. The results showed that, after 40%–60% ammonium sulfate precipitation, Q-Sepharose Fast Flow anion exchange chromatography, Phenyl-Sepharose 6 Fast Flow Hydrophobic chromatography, and twice sephacryl S-100 HR gel filtration, a final endo-1,4-β-xylanase was obtained. The purified wheat malt endo-1,4-β-xylanase showed a single band on SDS-PAGE with a molecular weight of 27.8 kDa, which had a purification fold of 12.08 and a specific activity of 4.47 μ/mg.

Purification and characterization of a high salt-tolerant alginate lyase from Cobetia sp. WG-007.

An alginate lyase producing bacterial strain, Cobetia sp. WG-007, was isolated and identified from rotting seaweed. The alginate lyase, Aly-W02, was purified by procedures of ultrafiltration, Q-Sepharose Fast Flow, Phenyl Sepharose 6 Fast Flow, and Superdex-G100 with specific activity of 21,285.5 U/mg. Aly-W02 had an apparent molecular mass of 35 kDa. It exhibited maximum activity at 45°C in 50 mM sodium phosphate buffer (pH 8.5). This alginate lyase was stable in the pH range of 6.0-8.5. Among the tested metal ions, the addition of K+ , Na+ , and Mg2+ ions can enhance the enzyme activities, while Ba2+ , Ni+ , Cu2+ , Mn2+ , Zn2+ , Ag+ , and ethylenediaminetetraacetic acid decreased the activities. It displayed high salt-tolerant ability; 0.8 M NaCl or 1.5 M KCl significantly enhanced the enzyme activity. Furthermore, Aly-W02 mainly released disaccharide, trisaccharide, and tetrasaccharid from alginate. It showed potential in producing low molecular weight alginate oligosaccharides.

Purification and characterization of chondroitinase ABC from Acinetobacter sp. C26

An extracellular chondroitinase ABC (ChSase ABC, EC 4.2.2.4) produced by cultivating Acinetobacter sp. C26, was purified to homogeneity from the supernatant by ammonium sulfate fractionation, Q-Sepharose Fast Flow and Sephadex G-100 chromatography. The 76kDa enzyme was purified 48.09-fold to homogeneity with specific activity of 348.64U/mg, Using the chondroitin sulfate A (CS-A) as substrate, the maximal reaction rate (Vmax) and Michaelis-Menten constant (Km) of ChSase ABC were found to be 10.471μmol/min/ml and 0.105mg/ml, respectively. The enzyme showed the highest activity at the optimal conditions of pH 6.0 and 42 ∘C, respectively. This enzyme was stable at pH 5–10, 5–9 and 5–7 at 4°C, 37°C and 42°C, respectively. Investigation about thermal stability of ChSase ABC displayed that it was stable at 37°C. ChSase ABC activity was increased in presence of Na+, K+, Mn2+, 1,10-phenanthrolin and strongly inhibited by Cu2+, Hg2+, Al3+and SDS. These properties suggested that ChSase ABC from Acinetobacter sp. C26 bring promising prospects in medical and industry applications.

Structure and antitumor activity of the extracellular polysaccharides from Aspergillus aculeatus via apoptosis and cell cycle arrest.

Two extracellular polysaccharides, designated as WPA and WPB, were isolated from the fungus Aspergillus aculeatus using Q-Sepharose fast flow and Sephacryl S-300 column chromatography. WPA composed of mannose and galactose in a molar ratio of 3.9:1.0, and WPB mainly contained mannose. The molecular weight of WPA and WPB was about 28.1kDa and 21.0kDa, respectively. On the basis of methylation and NMR analysis, the possible main chain of WPA was [→5)-β-D-Galf-(1→2,6)-α-D-Manp(1→], and WPB was mainly [→2,6)-α-D-Manp(1→], both with [α-D-Manp(1→2)-α-D-Manp(1→2)-α-D-Manp(1→] substituted at C-2 of [→2,6)-α-D-Manp(1→]. Meanwhile, WPA displayed a stronger anti-proliferative effect than WPB on HeLa, MCF-7 and MGC-803 cells in vitro. WPA and WPB could arrest HeLa cells in G2/M phase and induce HeLa cells apoptosis. Thus, our study provides evidence that WPA and WPB may be taken as potential candidates for treating cervical carcinoma.

Chemical Structure and Immunomodulating Activities of an α-Glucan Purified from Lobelia chinensis Lour.

A neutral α-glucan, named BP1, with a molecular mass of approximately 9.45 kDa, was isolated from Lobelia chinensis by hot-water extraction, a Q-Sepharose Fast Flow column and Superdex-75 column chromatography. Its chemical structure was characterized by monosaccharide analysis, methylation analysis and analysis of its FT-IR, high performance gel permeation chromatography (HPGPC) and 1D/2D-NMR spectra data. The backbone of BP1 consists of →6α-d-Glcp1→6,3α-d-Glcp1→(6α-d-Glcp1)x-6,3α-d-Glcp1-(6α-d-Glcp1)y→. The side chains were terminal α-d-Glcp1→ and α-d-Glcp1→ (6α-d-Glcp1)z→4α-d-Glcp1→3α-d-Glcp1→4α-d-Glcp1→ (x + y + z = 5), which are attached to the backbone at O-3 of 3,6α-d-Glcp1. The results of the effect of BP1 on mouse macrophage cell line RAW 264.7 indicate that BP1 enhances the cell proliferation, phagocytosis, nitric oxide production and cytokine secretion in a dose-dependent manner. Because the inhibitor of Toll-like receptor 4 blocks the BP1-induced secretion of TNF-α and IL-6, we hypothesize that α-glucan BP1 activates TLR4, which mediates the above-mentioned immunomodulating effects.