Sepharose CL-6B Research Articles

Mechanical characterisation of agarose-based chromatography resins for biopharmaceutical manufacture

Mechanical characterisation of agarose-based resins is an important factor in ensuring robust chromatographic performance in the manufacture of biopharmaceuticals. Pressure-flow profiles are most commonly used to characterise these properties. There are a number of drawbacks with this method, including the potential need for several re-packs to achieve the desired packing quality, the impact of wall effects on experimental set up and the quantities of chromatography media and buffers required. To address these issues, we have developed a dynamic mechanical analysis (DMA) technique that characterises the mechanical properties of resins based on the viscoelasticity of a 1ml sample of slurry. This technique was conducted on seven resins with varying degrees of mechanical robustness and the results were compared to pressure-flow test results on the same resins. Results show a strong correlation between the two techniques. The most mechanically robust resin (Capto Q) had a critical velocity 3.3 times higher than the weakest (Sepharose CL-4B), whilst the DMA technique showed Capto Q to have a slurry deformation rate 8.3 times lower than Sepharose CL-4B. To ascertain whether polymer structure is indicative of mechanical strength, scanning electron microscopy images were also used to study the structural properties of each resin. Results indicate that DMA can be used as a small volume, complementary technique for the mechanical characterisation of chromatography media.

Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies

Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules.

Saccharide binding by intelectins

This communication probes ligand binding by human Intelectin-1 with several saccharides. Human Intelectin-1 was previously reported to bind to microbial glycans via ribofuranoside or galactofuranoside residues, whereas subsequently, a crystal structure of ligand bound hITLN1 indicated that hITLN1 does not bind to ribofuranoside but distinguishes between microbial and human glycans through a glycan motif – a terminal, acyclic 1,2-diol, which is present on galactofuranose and other microbial saccharides. Here, we demonstrate that besides glycerol and glycerol derivatives (which have an acyclic 1,2-diol), and 2-deoxy-d-galactose, d-ribose and 2-deoxy-d-ribose, which have been previously reported as human Intelectin-1 ligands, 2-C-hydroxymethyl-d-ribose, d-talose, d-idose, d-altrose and sorbitol also elute human Intelectin-1 from Sepharose CL-6B. Interestingly, Sepharose, 2-deoxy-d-galactose (in its pyranose form), 2-C-hydroxymethyl-d-ribose, d-ribose and 2-deoxy d-ribose lack a terminal, acyclic 1,2-diol. We discuss the implications of these observations and rationalize the discrepancies in the apparent affinity of saccharide ligands for hITLN1 with different assay formats. We also report the distinct saccharide binding profiles of the hITLN1 homologues, HaloITLN and XL35ITLN, and demonstrate that hITLN1 binding to a saccharide ligand may modulate binding to its protein ligand, lactoferrin and vice versa.

Effects of bed compression on protein separation on gel filtration chromatography at bench and pilot scale.

BACKGROUNDPoorly packed chromatography columns are known to reduce drastically the column efficiency and produce broader peaks. Controlled bed compression has been suggested to be a useful approach for solving this problem. Here the relationship between column efficiency and resolution of protein separation are examined when preparative chromatography media were compressed using mechanical and hydrodynamic methods. Sepharose CL‐6B, an agarose based size exclusion media was examined at bench and pilot scale. The asymmetry and height equivalent of a theoretical plate (HETP) was determined by using 2% v/v acetone, whereas the void volume and intraparticle porosity (ϵ p) were estimated by using blue dextran. A protein mixture of ovalbumin (chicken), bovine serum albumin (BSA) and γ'‐ globulin (bovine) with molecular weights of 44, 67 and 158 kDa, respectively, were used as a ‘model’ separation challenge.RESULTSMechanical compression achieved a reduction in plate height for the column with a concomitant improvement in asymmetry. Furthermore, the theoretical plate height decreased significantly with mechanical compression resulting in a 40% improvement in purity compared with uncompressed columns at the most extreme conditions of compression used.CONCLUSIONThe results suggest that the mechanical bed compression of Sepharose CL‐6B can be used to improve the resolution of protein separation. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Extracción purificación y caracterización de inhibidores de tripsina provenientes de semillas andinas

El presente trabajo determinó las mejores condiciones para inmovilizar covalentemente tripsina en una matriz de sepharosa, con el fin de aplicarla en la purificación de inhibidores de la misma proteasa. Los mayores valores de retención de actividad funcional y de actividad enzimática inmovilizada se obtuvieron en una matriz de Sepharosa 6B-CL a temperatura ambiente, a un pH de 10,5, con una carga enzimática de 25 mg/mL de matriz, en un tiempo mínimo de inmovilización de 12 h, para alcanzar un preparado inmovilizado estable. Posteriormente, se seleccionaron los inhibidores de tripsina más activos a través de la comparación de extractos provenientes de semillas de amaranto (Amaranthus caudatus L.), arveja (Pisum sativum), lupino o “chocho” (Lupinus mutabilis), fréjol (Phaseolus vulgaris) y sangorache (Amaranthus hybridus L.) que fueron purificados parcialmente mediante ultrafiltración centrífuga, tratamiento calórico y precipitación con TCA. El permeado y retenido de sangorache fueron los extractos inhibidores más activos y se purificaron selectivamente, por separado, mediante cromatografía de afinidad en la matriz de tripsina-glioxil-sepharosa 6B-CL. Su caracterización cinética determinó la presencia de dos inhibidores; el inhibidor I que correspondió a una inhibición competitiva y el inhibidor II, a una de tipo mixta.

Studying the Effect of Partially Purified ArthrobacterB1.Polysaccharide on Nucleosomal Distribution of HepG2 Tumor Cell Line

This study was designed to investigate the effect of partially purified polysaccharide extracted from ArthrobacterB1 on the DNA banding pattern of HepG2 tumor cell line invitro. The polysaccharide was extracted from Arthrobacter B1using phenol sulfuric acid methodwith a recovery yield of 181mg. Partial purification of polysaccharide was performed by using sepharose Cl-6B gel filtration chromatography and two peaks were obtained. Chemical analysis involving the estimation of carbohydrate and protein contents which showed that the large first peak contained the higher carbohydrate contents (30.1%) as compared with the crude polysaccharide 15.9% with low protein constituents (5.7%).The effect of Arthrobacter B1polysaccharide on HepG2 tumor cells was examined by studying whether bacterial polysaccharide participating in affecting the structure of tumor cell’s DNA or not. Results indicated that only intact nucleosomal DNA bands were separated on the gel for the untreated cells as compared with the banding pattern of the colchicine treated DNA, in which besides to intact nucleosomal DNA, mononucleosomes, dinucleosomes and oligonucleosomes were separated on the gel following electrophoresis process. Such pattern does not affected by the gradual increase in MNase concentration. On the other hand, the DNA banding pattern for the cells treated with Arthrobacter B1 polysaccharide showed significant effect by the separation of nucleosomal DNA, in which mononucleosomes, dinucleosomes and oligonucleosomes were clearly separated from intact nucleosomal DNA, and such effect was markedly appeared by increasing the concentration of MNase as compared with the cells treated colchicine.

Proteins Binding to Immobilized Rusticyanin Detected by Affinity Chromatography

Recombinant rusticyanin was produced in Pichia pastoris, then purified and immobilized on Sepharose CL-4B with periodate activation. Cellular lysate of acidophilic Acidithiobacillus ferrooxidans was applied to an affinity column with immobilized rusticyanin. Rusticyanin-binding proteins, separated using 1D PAGE and identified by mass spectrometry, included anticipated interacting partners, such as cytochromes Cyc1 and Cyc2, which are involved in the downhill electron pathway from ferrous iron to oxygen. However, the results indicate that rusticyanin’s functional protein-protein interaction (PPI) network could be more complex than expected, including various proteins involved in different cellular processes. Although affinity purification coupled to mass spectrometry should mostly detect proteins that bind stably, and thus are likely participants in functional in vivo PPIs, further verification is needed to exclude non-functional interactants. Nevertheless, our preliminary PPI data confirm some previous experimental findings and indicate potentially fruitful directions for probing additional roles of rusticyanin in sulfur metabolism, copper resistance, anaerobic iron reduction, iron transport, and oxidative stress in extreme acidophiles.

Plasmid purification by using a new naphthalene tripodal support

Abstract The aim of this work was to employ a new naphthalene tripodal support for the isolation of supercoiled (sc) isoform of plasmid (pDNA) from a native sample. This support is for the first time synthesized and used in pDNA purification. The naphthalene tripodal ligand was synthesized and characterized to assess its purity and subsequently immobilized onto an epoxy-activated Sepharose CL-6B, using mild conditions and resulting in a ligand density of 0.32 mmol naphthalene tripodal/g derivatized Sepharose CL-6B. The complete characterization of naphthalene tripodal Sepharose CL-6B support was performed by High Resolution Magic Angle Spinning (HR-MAS) NMR spectroscopy, scanning electron microscopy (SEM) and elemental analysis. The affinity was measured by SPR biosensor between naphthalene tripodal ligand immobilized on the surface and sc pVAX1- LacZ and the K D was 8.65 × 10 −8 ± 1.0 × 10 −8 M in 10 mM Tris-HCl pH 8.0, at T = 25 °C, indicating a high affinity. For comparison reasons, the affinity ligand 3,8-diamino-6-phenylphenanthridine (DAPP) was also immobilized on the chip surface and the K D for sc pVAX1- LacZ is lower than with naphthalene tripodal. Saturation transfer difference-nuclear magnetic resonance spectroscopy (STD-NMR) experiments showed that the interactions between the naphthalene tripodal–Sepharose CL-6B and DAPP-Sepharose supports and the 5′-mononucleotides are mainly hydrophobic and π-π stacking. The isolation of sc pDNA isoform was achieved with low salt concentrations, using 95 mM NaCl in binding step and 550 mM NaCl in elution step at T = 4 °C and pH 8, thus reducing the economic and environmental impact.

Hemocytes and hemocytic responses in the mole crab Emerita emeritus (Linnaeus 1767).

The mole crab, Emerita emeritus, collected from the sandy shores of a Chennai beach, was investigated for cellular immune responses based on the morphology and defensive reactions of the circulating haemocytes. Three haemocyte morphotypes were identified using light and electron microscopy, and separated in a discontinuous percoll gradient. A phagocytosis study using human B erythrocyte as a target cell under phase-contrast optics showed that granular and semi-granular haemocytes were phagocytic, and this response was enhanced by using serum (opsonin)-coated human B erythrocyte in unfractionated and fractionated haemocytes. Observation of TEM image of phagocytosis revealed that the initial recognition and binding of the target cell was restricted to granular and semigranular haemocytes, which were lacking with hyaline cells. However, the encapsulation of DEAE Sepharose CL 6B beads, either untreated or coated with serum (opsonin), was restricted to hyaline cells. This suggests the occurrence of two cell lines in haemocytes, based on the differences observed in the response of haemocytes to bind target cells for phagocytosis or encapsulation. The present study also differentiated the activation of PO in the plasma, serum, and haemocyte lysate supernatant (HLS).

Purification, characterization and partial primary structure analysis of rutin-degrading enzyme in tartary buckwheat seeds

Rutin-degrading enzymes (RDE) can degrade rutin into poorly water soluble compound, quercetin, and cause the bitter taste in tartary buckwheat. In the present study RDE from Yu 6-21 tartary buckwheat seeds was purified by ammonium sulphate precipitation, followed by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B, ion exchange chromatography on CM-Cellulose and gel filtration chromatography on Sephadex G-150. Purified RDE showed single band with molecular weight of 66 kDa on SDS-PAGE. The optimum pH and temperature of RDE were 5.0 and 50 ℃ respectively. The Km was 0.27 mmol/L, and the Vmax was 39.68 U/mg. The RDE activity could be inhibited by Cu²⁺, Zn²⁺, Mn²⁺ and EDTA, and showed tolerance to 50% methanol (V/V). The N terminal sequence (TVSRSSFPDGFLFGL) was obtained by Edman degradation method and 15 internal peptide sequences were determined by MALDI-TOF-MS (matrix-assisted laser desorption ionization time of flight mass spectrometry). These results established the foundations for identification of the candidate gene of RDE via transcriptome data and further studying RDE biological function.

Growth inhibition and antibiofilm potential of Ag nanoparticles coated with lectin, an arthropod immune molecule

Lectins from the haemolymph of arthropods, including crustaceans, are molecules potentially involved in the immune recognition and phagocytosis. Here, lectin was purified from the haemolymph of blue swimmer crab Portunus pelagicus, using mannose-coupled sepharose CL-4B affinity column chromatography. In SDS-PAGE analysis, lectin showed a molecular mass of approximately 155kDa. The synthesis of lectin-coated silver nanoparticles (lectin-coated AgNPs) was confirmed by UV–Vis spectroscopy, XRD, FTIR, SEM, TEM, SAED, and EDX analysis. TEM analysis revealed that lectin-coated AgNPs were spherical in shape with size of 30–57nm. Their antibacterial activity against human pathogenic Gram negative and Gram-positive bacteria was determined by agar well diffusion method. Lectin-coated AgNPs showed significant antimicrobial activity when compared to lectin and silver nitrate tested alone. The antibiofilm properties of lectin-coated AgNPs were also investigated on human pathogenic Gram-negative Proteus vulgaris, Pseudomonas aeruginosa and Gram-positive Enterococcus faecalis and Bacillus pumilus. Lectin-coated AgNPs showed antibiofilm activity on the bacteria as well as on Candida albicans. Lectin-coated AgNPs reduced the biofilm architecture interfering with cell adhesion and polysaccharide matrix. This was additionally confirmed by exopolysaccharide (EPS) quantification index revealing the trouble in the structural reliability of biofilm by decrease in EPS and bacterial adhesion to hydrocarbons.

Purification, characterization and amino acid content of cholesterol oxidase produced by Streptomyces aegyptia NEAE 102

BackgroundThere is an increasing demand on cholesterol oxidase for its various industrial and clinical applications. The current research was focused on extracellular cholesterol oxidase production under submerged fermentation by a local isolate previously identified as Streptomyces aegyptia NEAE 102. The crude enzyme extract was purified by two purification steps, protein precipitation using ammonium sulfate followed by ion exchange chromatography using DEAE Sepharose CL-6B. The kinetic parameters of purified cholesterol oxidase from Streptomyces aegyptia NEAE 102 were studied.ResultsThe best conditions for maximum cholesterol oxidase activity were found to be 105 min of incubation time, an initial pH of 7 and temperature of 37 °C. The optimum substrate concentration was found to be 0.4 mM. The higher thermal stability behavior of cholesterol oxidase was at 50 °C. Around 63.86% of the initial activity was retained by the enzyme after 20 min of incubation at 50 °C. The apparent molecular weight of the purified enzyme as sized by sodium dodecyl sulphate-polyacryalamide gel electrophoresis was approximately 46 KDa. On DEAE Sepharose CL-6B column cholesterol oxidase was purified to homogeneity with final specific activity of 16.08 U/mg protein and 3.14-fold enhancement. The amino acid analysis of the purified enzyme produced by Streptomyces aegyptia NEAE 102 illustrated that, cholesterol oxidase is composed of 361 residues with glutamic acid as the most represented amino acid with concentration of 11.49 μg/mL.ConclusionsTaking into account the extracellular production, wide pH tolerance, thermal stability and shelf life, cholesterol oxidase produced by Streptomyces aegyptia NEAE 102 suggested that the enzyme could be industrially useful.

Purification, characterization, and bioactivities of a polysaccharide from mycelial fermentation of Bjerkandera fumosa

In this work, a novel polysaccharide (named DBFM3) was isolated from mycelia of Bjerkandera fumosa by DEAE-32 and Sepharose CL–6B column chromatography. High-performance gel permeation chromatography showed that DBFM3 was homogeneous, with an average molecular weight of 1.8×105Da. Structural characteristics of the purified fraction were investigated by high-performance liquid chromatography (HPLC), FT-IR, and NMR. HPLC analysis indicated that DBFM3 was composed of mannose, galacturonic acid, and galactose at molar ratios of 1:18.16:0.702. Spectral analysis suggested that DBFM3 had (1→6), (1→3,6), (1→3) linkages and pyranose conformation. Antioxidant assay in vitro showed that DBFM3 exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical scavenging activity, and has protective effect against DNA damage and damage to SH-SY5Y cells induced by H2O2. Immunological tests indicated that DBFM3 significantly increased lymphocyte proliferation in vitro. Furthermore, DBFM3 increased the proliferation of lymphocytes in the presence of concanavalin A or lipopolysaccharide as mitogen.

PEGylated recombinant L-asparaginase from Erwinia carotovora: Production, properties, and potential applications

N-hydroxysuccinimide ester of monomethoxy polyethylene glycol hemisuccinate was synthesized. It acylated amino groups in a molecule of recombinant L-asparaginase from Erwinia carotovora. A method of L-asparaginase modification by the obtained activated polyethylene glycol derivative was developed. The best results were produced by modification of the enzyme with a 25-fold excess of reagent relative to the enzyme tetramer. The modified L-asparaginase was isolated from the reaction mixture by gel filtration on Sepharose CL-6B. The purified bioconjugate did not contain PEG unbound to the protein, demonstrated high catalytic activity, and exhibited antiproliferative action on cell cultures.

A simple approach for preparation of affinity matrices: Simultaneous purification and reversible immobilization of a streptavidin mutein to agarose matrix

SAVSBPM18 is an engineered streptavidin for affinity purification of both biotinylated biomolecules and recombinant proteins tagged with streptavidin binding peptide (SBP) tags. To develop a user-friendly approach for the preparation of the SAVSBPM18-based affinity matrices, a designer fusion protein containing SAVSBPM18 and a galactose binding domain was engineered. The galactose binding domain derived from the earthworm lectin EW29 was genetically modified to eliminate a proteolytic cleavage site located at the beginning of the domain. This domain was fused to the C-terminal end of SAVSBPM18. It allows the SAVSBPM18 fusions to bind reversibly to agarose and can serve as an affinity handle for purification of the fusion. Fluorescently labeled SAVSBPM18 fusions were found to be stably immobilized on Sepharose 6B-CL. The enhanced immobilization capability of the fusion to the agarose beads results from the avidity effect mediated by the tetrameric nature of SAVSBPM18. This approach allows the consolidation of purification and immobilization of SAVSBPM18 fusions to Sepharose 6B-CL in one step for affinity matrix preparation. The resulting affinity matrix has been successfully applied to purify both SBP tagged β-lactamase and biotinylated proteins. No significant reduction in binding capacity of the column was observed for at least six months.

Purification, structural features, antioxidant and moisture-preserving activities of an exopolysaccharide from Lachnum YM262

A water-soluble exopolysaccharide, designated as LEP-2a, was isolated from Lachnum YM262 and purified by DEAE-Cellulose 52 and Sepharose CL-6B chromatographic columns. LEP-2a was a homogeneous polysaccharide, with a molecular weight of 1.52×105 Da. It was composed of mannose and galactose in a molar ratio of 20.6:1.0. Its structural features were investigated and elucidated by methylation analysis, periodate oxidation and Smith degradation, FT-IR and NMR spectroscopy. Based on obtained data, the backbone of LEP-2a consisted of 1,2-linked-α-d-mannose, 1,3-linked-α-d-mannose, 1,2,6-linked-α-d-mannose and 1,3-linked-β-d-galactose and the side chains were attached to the backbone at O-6 position of 1,2,6-linked-α-d-mannose. In vitro antioxidant activity assay proved that LEP-2a possessed significant scavenging activities on superoxide, hydroxyl and DPPH radical. Furthermore, LEP-2a had strong in vitro moisture-absorption and -retention capacities as compared to chitosan and glycerol. These results suggested that LEP-2a might have a good potential to be applied as a multifunctional cosmetic additive in cosmetics.

Purification, characterization and functional analysis of the immune molecule lectin from the haemolymph of blue swimmer crab Portunus pelagicus and their antibiofilm properties

The present study reveals purification and characterization of immune molecule lectin from the haemolymph of blue swimmer crab Portunus pelagicus (Pp-Lec). The Pp-Lec was purified by affinity chromatography with mannose coupled sepharose CL-4B column and it exhibits single band with a molecular weight of 155 kDa in SDS-PAGE. The surface morphology of purified Pp-Lec displays the homogeneous nature of protein. A distinct peak with a retention time of 3.3 min was appeared in high performance liquid chromatography (HPLC) and X-ray diffraction (XRD) analysis expresses a single peak at 31.5° which shows the purity and crystalline nature of the protein respectively. Functional analysis of purified Pp-Lec exhibits encapsulation activity against sepharose beads and yeast agglutination activity against Saccharomyces cerevisiae. Moreover, the purified Pp-Lec has the ability to agglutinates with the human erythrocytes among tested and which was observed by light microscopy. In addition, purified Pp-Lec showed the broad spectrum of antibacterial activity against Gram-positive Bacillus pumulis, Bacillus thuringiensis, Enterococcus faecalis and Gram negative Citrobacter amalonaticus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Proteus vulgaris, Citrobacter murliniae, Citrobacter freundii, Morganella morganii. Antibiofilm potential of purified Pp-Lec against selective Gram-negative bacteria showed the disruption of biofilm architecture at the concentration of 50 μg ml−1.

Acid hydrolysis of xylan polysaccharides fractions isolated from argan (Argania spinosa) leaves

Xylan polysaccharides previously obtained from argan (Argania spinosa) leaves by sequential alkaline extractions and purified by chromatography on Sepharose CL-4B were fragmented by acid hydrolysis in 2 M HCl. The resulting fragments were separated by gel filtration on Biogel P2. The oligosaccharide fractions obtained were then characterized by gas–liquid chromatography (GLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results show the presence of acidic oligosaccharide motifs, some of which composed of xylose and 4-O-methylglucuronic acid and other ones comprising rhamnose and galacturonic acid.

Preparation and Bioactivity of Exopolysaccharide from an Endophytic Fungus Chaetomium sp. of the Medicinal Plant Gynostemma Pentaphylla.

Background:Many exopolysaccharides from the endophytes in medicinal plants possess various potential bioactivities.Materials and Methods:The endophytic fungus JY25 was isolated from the leave of the Chinese medicinal plant Gynostemma pentaphylla and identified as Chaetomium sp. by its phylogenetic and physiological analysis. One exopolysaccharide (EPS) fraction was isolated from the fermentation broth by ethanol precipitation and purified by gel filtration chromatography on Sepharose CL-6B. The molecular characteristics were examined by GC-MS, FT-IR, and multiangle laser light scattering (MALLS).Results:The monosaccharide composition analysis indicated that the purified EPS was mainly composed of glucose, mannose, arabinose, and galactose with the molecular ratio of 78.29: 8.99: 8.64: 4.08. FT-IR spectral analysis of the purified EPS revealed prominent characteristic groups, such as carbonyl bond, pyranose ring, and so on. The weight-average molar mass and the polydispersity ratio of the EPS were revealed to be 1.961×104 g/mol and 1.838, respectively. Furthermore, thermo gravimetric analysis (TGA) indicated that the degradation temperature of the purified EPS was 305° C. The purified EPS from the endophytic fungus Chaetomium sp. displayed antioxidant and antiproliferative activities.Conclusion:The results demonstrated that the EPS could be used as a healthful food and material source in pharmaceutical industries.SUMMARY An exopolysaccharides (EPS) with antioxidant and antiproliferative activities from an endophytic fungus Chaetomium sp. was reported. Abbreviations used: ANOVA: Analysis of variance; DPPH: 2,2-diphenyl-1-picrylhydrazyl; EPS: Exopolysaccharide; FT-IR: Fourier transform infrared spectroscopy; GC-MS: Gas chromatography–mass spectrometry; Mw: Mass weight; MALLS: Multiangle laser light scattering; SEC: Size Exclusion Chomatography; SPSS: Statistical Package of the Social Science; TGA: Thermo gravimetric analysis; TFA: Trifluoroacetic acid.

Partial Purification and Characterization of Cellulase Produced by <i>Bacillus sphaericus</i> CE-3

Cellulase is an enzyme produced by fungi, bacteria, protozoa and termite, that hydrolyze cellulose. They are known for their diverse applications in industry and medicine. The aim of this study is to purify and investigate cellulolytic properties of cellulase enzyme produced by Bacillus sphaericus CE-3 isolated from refuse dump in Nnamdi Azikiwe University, Awka, Nigeria. Enzyme was produced by submerged fermentation at 30°C for 30 h. The enzyme was purified to homogeneity by dialysis in 4M sucrose solution, ion-exchange chromatography on Q-Sepharose FF and by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The relative molecular mass of the enzyme was estimated using SDS-Polyacrylamide gel electrophoresis. Effects of temperature, pH and metals on enzyme activity and stability and the relative rate of hydrolysis of various substrates were also studied. The Purification fold for the enzyme was 7.8, with 66.4 μ/mg specific activity protein and overall yield of 35.8. The relative molecular mass range of the enzyme was estimated between 22.3 kDa - 26.3 kDa. The enzyme was optimally active at pH 9.0 and 40°C, stable at pH 9.0 and unusually retained over 90% activity between 50°C - 100°C after 30 min incubation. It was strongly activated by Mn2+ but inhibited by Ba2+, Co2+, Hg2+, Pb2+, Cu2+, Sr2+, Fe2+, Ca2+ and Zn2+. The cellulase displayed high catalytic activity with untreated sawdust, followed by carboxymethyl cellulose, while sodium hydroxide treated sawdust was the least hydrolyzed. Since the enzyme is thermo-stable, alkalophilic and could utilize natural wastes like sawdust as substrate, it is obvious that it would be of great use in textile, starch processing and pulp and paper industries.