DEAE-Sepharose Fast Flow Column Research Articles

Effective synthesis of theaflavin-3,3′-digallate with epigallocatechin-3-O-gallate and epicatechin gallate as substrates by using immobilized pear polyphenol oxidase

In the present study, pear polyphenol oxidase (PPO) was purified, immobilized and applied for the synthesis of theaflavin-3,3′-digallate (TF3). Firstly, PPO of pear (Pyrus bretschneideri Rehd cv. Huangguan) was purified 24.36-fold in specific activity with a recovery of 6.77% by sequential use of precipitation with 70% saturated ammonium sulfate and chromatography of DEAE-Sepharose Fast Flow column. The purified pear PPO, found to be a dimer of identical subunits of molecular mass 35kDa, was then successfully immobilized onto Fe3O4/chitosan nanoparticles via glutaraldehyde coupling reaction. Finally, TF3 was effectively synthesized by applying the immobilized PPO as biocatalyst with epicatechin gallate (ECG) and epigallocatechin-3-O-gallate (EGCG) as substrates, and the maximum yield of TF3 was 42.23% based on the amount of ECG added. In addition, the immobilized enzyme still remained 85% of its initial ability after successive usage of 8 cycles and could be stored for 30days with less loss of activity. All the results suggest that the developed procedure is quite useful and has great potential for synthesis of TF3 and other theaflavins.

Fractionation, preliminary structural characterization and bioactivities of polysaccharides from Sargassum pallidum

Sargassum pallidum polysaccharides were fractioned using a DEAE-Sepharose fast-flow column and four polysaccharide fractions (SP-P1, SP-P2, PV-P3 and SP-P4) were obtained. Structural analyses indicated that SP-P2 and SP-P4 had higher molecular weights than SP-P1 and SP-P3. SP-P2, SP-P3 and SP-P4 comprised of fucose, rhamnose, arabinose, galactose, glucose, xylose, and mannose in a similar molar ratio, while SP-P1 did not contain arabinose. SP-P2 and SP-P4 had a similar number of (1→6) or (1→) glycosidic linkages (1→2) or (1→4) glycosidic linkages and (1→3) glycosidic linkages, while SP-P1 and SP-P3 contained a small number of (1→3) glycosidic linkages. SP-P2 exhibited better bioactivities than SP-P1, SP-P3 and SP-P4, including antioxidant, anti-hemolysis inhibitory, α-amylase and α-glucosidase inhibitory and antiproliferative activities. These data suggest that S. pallidum has four polysaccharide fractions with different structural features and bioactivities and SP-P2 has potential to be explored as a functional food or complementary medicine.

Structural characteristics and antioxidant activity in vivo of a polysaccharide isolated from Phellinus linteus mycelia

In this study, a water-soluble polysaccharide (PLP1-I) was purified from Phellinuslinteus mycelia through hot water extraction, ethanol precipitation, and chromatography on DEAE Sepharose Fast Flow and Sephacryl S-400 HR columns. PLP1-I was characterized as a neutral heteropolysaccharide composed of glucose and galactose at a molar ratio of 8.9:1.0, with an average molecular weight (Mw) of ∼290,000kDa. The structure of PLP1-I consists of a (1→4)-α-D-glucopyranose backbone, whereas the (1→3)-α-D-galactopyranosyl residues formed branches at the O-6 position with 1-linked-α-d-glucopyranosyl terminal residues. Evaluation of the antioxidant activity in vivo showed that PLP1-I administration significantly increased the activity of antioxidant enzymes and reduced the levels of malondialdehyde in the serum and liver of aging mice induced with D-galactose. PLP1-I also effectively stimulated the immune system of aging mice. This study demonstrated that PLP1-I exhibited strong antioxidant activities in vivo and could be explored as a potential antioxidant for functional food and medicine.

Purification and characterization of a novel polysaccharide–peptide complex from Clinacanthus nutans Lindau leaves

A novel polysaccharide–peptide complex CNP-1-2 with molecular weight of 9.17×104Da was obtained from Clinacanthus nutans Lindau leaves by hot water extraction, ethanol precipitation, and purification with Superdex 200 and DEAE-Sepharose Fast Flow column chromatography. CNP-1-2 exhibited the highest growth inhibitory effect on human gastric cancer cells SGC-7901 with inhibition ratio of 92.34% and stimulated activation of macrophages with NO secretion level of 47.53μmol/L among the polysaccharide fractions. CNP-1-2 comprised approximately 87.25% carbohydrate and 9.37% protein. Monosaccharide analysis suggested that CNP-1-2 was composed of l-rhamnose, l-arabinose, d-mannose, d-glucose and d-galactose with a molar ratio of 1.30:1.00:2.56:4.95:5.09. Methylation analysis, FT-IR, and 1H NMR spectroscopy analysis revealed that CNP-1-2 might have a backbone consisting of 1,4-linked Glcp, 1,3-linked Glcp, 1,3-linked Manp, 1,4-linked Galp, 1,2,6-linked Galp and 1,2,6-linked Galp. Its side chain might be composed of 1-linked Araf, 1,6-linked Galp and 1-linked Rhap residues. AFM (atomic force micrograph) analysis revealed that CNP-1-2 had the molecular aggregation along with branched and entangled structure.

Purification and Characterization of a Membrane-Unbound Highly Thermostable Metalloprotease from Aeromonas Caviae

The main objective of the current study was to purify and characterize an alkaline thermostable metalloprotease from Aeromonas caviae. The enzyme was subjected to a two-step purification scheme using ammonium sulfate [(NH4)2SO4] fractionation and ion-exchange chromatography on a DEAE-sepharose fast flow column. The fraction which precipitated with 40–60 % (w/v) of [(NH4)2SO4] exhibited the highest enzyme activity. Anion-exchange chromatography resulted in approximately 51-fold purification, with a yield of 64.7 %. The enzyme was successfully purified to homogeneity, and this was confirmed on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). A single band with an approximate molecular mass of ∼42 kDa was observed. The maximum enzymatic activity was observed at pH 8 with azocasein as the substrate, and the enzyme was stable in the pH range of 6–10 after 5 h of incubation. The purified enzyme was active in the temperature range between 50 and 60 °C and showed 80 % loss in enzymatic activity upon heating at 70 °C. The K m and V max values for the purified protease, with azocasein as substrate under optimal reaction conditions (pH 8 and 50 °C) were 0.74 and 122.4 mg/ml, respectively. The protease was investigated for its application for the degradation of chicken feathers. The high enzymatic activity, pH, and thermal stability of AU04 protease make it an industrially important enzyme.

Extraction, isolation, characterization and antioxidant activity of polysaccharides from Astragalus membranaceus

In this study, optimization of combined enzyme extraction (CEE), isolation, characterizationand antioxidant activities of polysaccharides (APs) from Astragalus membranaceus (AM) were investigated. Response surface methodology (RSM) and orthogonal experiments were used to optimizethe CEE conditions, which are as follows: combined enzyme concentration of 1.5%, 1% and 0.5% of cellulase, pectase and papain, extraction time of 94.5min, extraction temperature of 49.9°C and pH of5.1. The APs were further fractionated using DEAE Sepharose Fast Flow and Sephadex G-200 column to afford three purified fractions of APs-1-1, APs-2-1 and APs-3-1. Their physicochemical properties were investigated by gas chromatography (GC), high-performance gel-permeation chromatography (HPGPC) and Fourier-transform infrared spectroscopy (FT-IR). The results indicated that their total sugar, uronic acid content, molecular weight and monosaccharide composition were significantly different. Antioxidant activities were evaluated by various antioxidant assays in vitro. The results showed that all the three polysaccharides possessed good antioxidant properties in a concentration-dependent manner, especially APs-3-1 with the highest uronic acid content and the lowest molecular weight had the strongest antioxidant capacity and free radicals scavenging activity. The present results suggested that APs-3-1 might be exploited as effective natural antioxidant for medical and food industries.

Purification and Characterization of Halophilic Organic Solvent Tolerant Protease from Marine Bacillus sp. APCMST-RS7 and Its Antioxidant Potentials

Halophilic organic solvent tolerant proteolytic bacterium was isolated from the marine sediment of Rajakkamankalam estuary and identified as Bacillus sp. APCMST-RS7. The alkaline protease synthesized by Bacillus sp. APCMST-RS7 was purified by using ammonium sulphate precipitation, dialysis and DEAE-Sepharose Fast Flow column. The purified protease showed 6.60-fold purity, 26.02 U/mg specific activity with 24.30 % yield. The molecular weight of the purified alkaline protease was 32 kDa. This purified protease registered maximum activity at pH 8 and it was stable between pH 8–9 after 1.30 h of incubation. The optimum temperature registered was 50 °C and it was stable between 30 and 40 °C even after 1.30 h of incubation. This enzyme also showed maximum activity at 1.5 M NaCl concentration. The Km and Vmax values registered were, 0.0002 g/l and 1428.57 U/ml, respectively. Further, magnesium chloride, barium chloride and copper sulphate influenced this enzyme activity remarkably and it was also found to be enhanced by many of the tested surfactants and solvents. Here, the serine protease inhibitor totally inhibited the enzyme activity; hence it is of serine protease family. The candidate bacterium effectively deproteinized the shrimp shell waste with maximum protease activity. The shrimp shell hydrolysate also exhibited maximum antioxidant activity.

Bioactivities and extraction optimization of crude polysaccharides from the fruits and leaves of Rubus chingii Hu

Polysaccharides of Rubus chingii Hu fruit and leaf were extracted to compare their antioxidant, anti-inflammatory, and anticancer activities against breast cancer cells MCF-7 and liver cancer cells Bel-7402. Results showed that all the tested bioactivities of polysaccharides from leaf (L-Ps) were better than those of polysaccharides from fruit (F-Ps). Response surface methodology was then used to optimize the extraction conditions of polysaccharides from leaf. Additionally, polysaccharides from fruit and leaf were characterized and their contents of total sugars, proteins and uronic acid were compared. It was found that polysaccharides from fruit and leaf were similar in IR and UV absorption, but significantly different in contents of total sugars, protein and uronic acid. Their elution profiles of DEAE-Sepharose fast flow column were different too. The main peak of polysaccharides from fruit was eluted with 0.3mol/l NaCl solution and the main peak of polysaccharides from leaf was eluted with deionized water. The differences between the two polysaccharides may be responsible for their differences in bioactivities. Further studies are required to explore their complete structural characteristics, structure-activity relationship and the mechanism of their activities.

Purification and partial characterization of a thermostable antimicrobial protein from Bacillus subtilis FB123.

Antimicrobial proteins/peptides have attracted much attention because of their potential use in the industrial setting. In the present study, a thermostable antimicrobial protein (BSAMP) was purified from the culture supernatant of Bacillus subtilis FB123 by ammonium sulfate precipitation, gel chromatography on Sephacryl S-200 High Resolution, and ion exchange chromatography on DEAE Sepharose Fast Flow column. The molecular weight of the purified BSAMP was 54 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis both in the absence and presence of β-mercaptoethanol. Its isoelectric point was determined to be 5.24 by isoelectric focusing electrophoresis. Periodic acid-Schiff staining revealed BSAMP to be a glycoprotein. Maximum activity was obtained at pH 6.0, with over 79% maximum activity retained at pH 3.0-5.0 and pH 7.0-9.0, respectively. BSAMP was shown to be highly thermostable, as its activity did not change obviously after treatment at 100 °C. However, it was partially sensitive to papain, trypsin, and alkali proteases. Finally, the bacterial protein exhibited broad-spectrum antimicrobial activity against several pathogenic organisms. These findings suggested that BSAMP should be further developed as a natural antibacterial agent for disease prevention in aquiculture and agriculture.

Isolation, purification and identification of polysaccharides from Gynura divaricata

The purpose of this study was to isolate and purify polysaccharide from Gynura divaricata and analyze its monosaccharide composition. A water-soluble crude polysaccharide was obtained by hot water extraction, ethanol precipitation and deproteinization after degreasing. The crude polysaccharide then purified with DEAE-Sepharose Fast Flow column chromatography and dialysis. The monosaccharide composition and structure were analyzed by HPLC, UV spectrophotometer and 1H-NMR. The results showed that the purity and molecular weight of GDPS-2 and GDPS-3 were 87.3%, 2.03 x 10(4) Da and 90.9%, 4.29 x 10(4) Da, respectively. The UV spectrophotometer and 1H-NMR data suggested that glycosidic bond of GDPS-2 and GDPS-3 were a type. Both GDPs-2 and GDPs-3 were homogeneous polysaccharides, and GDPs-2 was mainly composed of glucuronic acid and xylose at a molar ratio of 1.1:0.63. GDPs-3 was mainly composed of rhamnose, glucuronic acid, galactose, xylose and galacturonic acid at a molar ratio of 0.32:6.0:0.21:1.75:4.3.

Characterization, antioxidant and immunomodulatory activities of polysaccharides from Prunella vulgaris Linn.

Water-soluble polysaccharides from Prunella vulgaris Linn (P. vulgaris) were fractionated using DEAE-Sepharose fast-flow column to obtain several eluents of water (PV-P1), 0.1M NaCl (PV-P2) and 0.2M NaCl (PV-P3). Structural analyses showed that PV-P1 had a higher molecular weight and degree of branching as compared to PV-P2 and PV-P3. Tertiary structure analyses indicated that PV-P1, PV-P2 and PV-P3 did not have triple-helical conformation. PV-P2 and PV-P3 showed stronger antioxidant activities than PV-P1, as measured radical scavenging capacities. PV-P1 showed stronger immunomodulatory activities than PV-P2 and PV-P3 in term of stimulation of the production of pro-inflammatory cytokines, including nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in murine macrophage RAW 264.7 cells. PV-P1, PV-P2 and PV-P3 did not exhibit cytotoxicities against RAW 264.7 at the concentrations tested. These results suggest that P. vulgaris polysaccharides could be explored as potential antioxidant and immunomodulatory agents for the complementary medicine or functional foods.

Purification and enzymatic characterization of membrane-bound d-gluconate dehydrogenase from Arthrobacter globiformis

Arthrobacter globiformis C224 is an industrial 2-keto-gluconic acid (2KGA) producer and currently used for erythorbic acid production in China. In the present study, a membrane-bound gluconate dehydrogenase (GADH) with specific activity of 326.06U/mg and purification fold of 412 was purified from A. globiformis using a five-step procedure including ultrasonication, phase separation with Triton X-114, ammonium sulfate fractionation, DEAE-sepharose fast flow and hydroxyapatite column chromatography. The GADH was identified as to be flavin adenine dinucleotide (FAD)-dependent and consisted of three subunits with molecular mass of 66,000Da, 44,000Da and 23,000Da. The optimal pH and temperature of A. globiformis GADH were 5.0 and 40°C, respectively. It had the stable activity at pH of 5.0–7.0 or below 50°C, and the strict substrate specificity for d-gluconate with the Km of 3.15mmolL−1, 1.04mmolL−1 at pH 5.0 and pH 6.0, respectively. The GADH activity also was significantly influenced by metal ions, organic solvents, and organic acids. Our study will benefit for better understanding of 2KGA production process by A. globiformis C224.

Extraction and Purification of R-phycoerythrin from Marine Red Algae.

This chapter focuses on the recovery of an R-Phycoerythrin (R-PE)-enriched fraction from marine algae. Since R-PE is a proteinaceous pigment, we have developed a simple and rapid two-step method devoted to the extraction and purification of R-PE from marine red algae. Here we describe a phosphate buffer extraction followed by anion exchange chromatography carried on a DEAE Sepharose Fast Flow column. To ensure the quality and quantity of R-PE recovery, we also indicate different methods to monitor each fraction obtained, such as spectrophotometric indicators, gel filtration, and SDS-PAGE analysis.

Ultrahigh pressure extraction of polysaccharides from Cordyceps militaris and evaluation of antioxidant activity

Four polysaccharide fractions (CMP-1, CMP-2, CMP-3 and CMP-4) were extracted from cultured Cordyceps militaris (CM) using ultrahigh pressure extraction (UHPE), and purified using ultrafiltration, DEAE-Sepharose Fast Flow and Sephadex G-100 column chromatography. The effects on yields of crude polysaccharides (CCMP) were investigated by the response surface methodology (RSM) based on Box-Behnken Design (BBD). The optimal extraction condition for UHPE was: 5.18min, 424MPa, and 43.5°C. Under these conditions, the CCMP yield was 14.89±0.68%. The chemical characterizations of CMPs were determined by gas chromatography and high performance gel permeation chromatography (HPGPC). The antioxidant activities of CMPs were evaluated by ferric reducing antioxidant power (FRAP) and free radical scavenging assay. CMPs are composed of D-glucose, D-galactose and D-mannose, demonstrating noticeable antioxidant activities in a concentration-dependent manner. UHPE as an effective and environmental-friendly technique could be employed for extracting active ingredients from plants.

An alkaline and metallo-protein type endo xylanase from Streptomyces sp. CSWu-1

An alkaline xylanase (XynWu-1) from Streptomyces sp. CSWu-1 was isolated from the Korean soil sample, purified and biochemically characterized. The extracellular xylanase was purified 4.8 fold with a 16% yield using Sephadex G-50 followed by DEAE-Sepharose (fast flow) column chromatography. The molecular mass of the enzyme was approximately 37 kDa estimated by SDS-PAGE and xylan zymography. N-terminal amino acid sequence of XynWu-1 was AINVLVAALX. The enzyme was found to be stable in a broad range of pH (7.0 ∼ 13.0) and to 50°C and have an optimal pH and temperature of 11.0 and 60°C, respectively. XynWu-1 activity was found to be affected by Mn2+ ion with highest activity at 6 mM and produced xylose, xylobiose, and xylotetraose as major hydrolyzed end products. It was found to degrade agro waste materials like corncob and wheat bran by XynWu-1 (2,000 U/g) as shown by electron microscopy. As being stable in extreme alkaline pH, diverse peculiar biochemical characteristics, and ability to produce oligosaccharide shows that XynWu-1 has potential application in various bioindustries like probiotics, ethanol, etc.

Structural characterization of a water-soluble polysaccharide from the fruiting bodies of Agaricus bisporus.

An edible fungal polysaccharide termed as ABP was obtained by extraction with hot water, and followed successive chromatographic purification using DEAE-Sepharose Fast Flow column and Sephacryl S-300 High-Resolution column. A symmetrical peak was obtained on high-performance size-exclusion chromatography with an average molecular weight of 5.17 × 104 Da, which was named ABP, and its main components were d-glucose and d-mannose. Based on the study of methylation analysis, along with FT-IR, GC, GC-MS, 1D 1H and 13C NMR and 2D NMR (H-HCOSY, TOCSY, HMQC, and NOESY), its chemical structure was featured with a repeating unit (1→6) linking β-d-Glcp as the main backbone with (1→4)-linked α-d-Manp units. The structure of the mainly repeating units of ABP was established as: →6)-β-D-Glucp-(1→4)-α-D-Manp(1→6)-β-D-Glucp-(1→6)-β-D-Glucp-(1→

Antioxidant and anti-inflammatory activities of Radix Isatidis polysaccharide in murine alveolar macrophages

Radix Isatidis is an official herbal medicine for treatment of infection and inflammation in China. In this study, a novel heteropolysaccharide (RIWP) was isolated from R. Isatidis through DEAE Sepharose Fast Flow column and Sepharose CL-6B column. RIWP had a molecular weight of 57kDa and was mainly composed of glucose, galactose and arabinose with a relative molar ratio of 2.0:1.1:1.0. The cytoprotective, antioxidant and anti-inflammatory properties of RIWP in lipopolysaccharide (LPS) stimulated murine alveolar macrophages were first reported here. Pretreatment with RIWP was found to potently prolong cell survival and repress the generation of reactive oxygen species (ROS) and lipid peroxidation after LPS-stimulation in murine alveolar macrophages. Furthermore antioxidant status was significantly deteriorated in the LPS-treated alveolar macrophages, such as low superoxide dismutase (SOD) activity and G-SH content, which was effectively restored by RIWP supplementation. More importantly, RIWP significantly suppressed LPS-induced increase in nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production in murine alveolar macrophages. Additionally RIWP recovered mitochondrial membrane potential to normal conditions. All above response to LPS stimulation behaved in a concentration-dependent manner. This study provided evidences that RIWP appears to have the potential to prevent inflammatory disease in lung.

Structural characterisation of polysaccharides from Tricholoma matsutake and their antioxidant and antitumour activities

In this study, polysaccharides from Tricholoma matsutake (TM-P) were purified using a DEAE-Sepharose fast flow column and three polysaccharide fractions (TM-P1, TM-P2 and TM-P3) were obtained. The chemical composition and structural characteristics of TM-P2 were quite different from those of TM-P1 and TM-P3. TM-P2 consisted of glucose, galactose and mannose with a molar ratio of 5.9:1.1:1.0. The glycosidic linkages were mainly composed of 1,6- and 1-linked glucose. Furthermore, TM-P2 showed the strongest in vitro antioxidant and antitumour activities. The oxygen radical absorbance capacity (ORAC) of TM-P2 was 2100.44μmol Trolox/g. The antiproliferative activities of TM-P2 (4.0mg/ml) on the growth of HepG2 and A549 cells were 67.98% and 59.04%, respectively.

Ultrasound complex enzymes assisted extraction and biochemical activities of polysaccharides from Epimedium leaves

Ultrasound complex enzymes (UCE) were used to extract crude polysaccharides (CEP) from Epimedium leaves. The response surface methodology (RSM) based on Box–Behnken Design (BBD) was employed to further optimize the extraction conditions. The optimum conditions were: complex enzyme dose of 50, 250, 200, 100U/g (papain, pectase, cellulase and α-amylase), extraction temperature 46.8°C, ultrasound time 42.3min, pH 4.28, and ultrasound power 311W. Under these conditions, the experimental yield of CEP was 5.98%, which was well matched with the value predicted by the model. Three major polysaccharide fractions (EP-1, EP-2 and EP-3) were obtained by ultrafiltration, chromatography on DEAE-Sepharose Fast Flow and Sephadex G-100 column. Their chemical–physical characteristics were investigated using chemical methods, gas chromatography (GC) and high-performance size-exclusion chromatography (HPSEC). Biochemical characterization of the polysaccharide fractions was evaluated on the basis of free radical scavenging, ferric-reducing antioxidant power (FRAP), lymphocyte proliferation, macrophage phagocytosis and NK cell activity assay in vitro. The results showed that all fractions contained higher galacturonic acid (GalA) and galactose (Gal), possessed considerable antioxidant activity, and could promote proliferation of splenic lymphocytes, activate macrophages and enhance NK cells’ activity. UCE as a more effective and environment-friendly technique could be employed for extracting active ingredients from plant materials, extensively applied in pharmaceutical and food industries.

Antioxidant activities of five polysaccharides from Inonotus obliquus

Five polysaccharides (IOP1b, IOP2a, IOP2c, IOP3a and IOP4) were isolated and purified from Inonotus obliquus by DEAE-Sepharose fast flow and SepharoseCL-6B column chromatography. Their chemical and physical characteristics were determined and antioxidant activities were investigated on the basis of hydroxyl radical assay, superoxide radical assay and ferric-reducing antioxidant power assay. The results showed that five polysaccharides exhibited antioxidant activities, and the higher content of uronic acid and proteinous substances, the stronger antioxidant activities of polysaccharides. Besides, molecular weights of polysaccharides also influence their antioxidant activities. IOP3a and IOP4 showed higher antioxidant properties than IOP1b, IOP2a and IOP2c.