Sephadex G-100 Chromatography Research Articles

Thioredoxins induce oocyte maturation in holothuroids (Echinodermata)

Chromatographic fractions of a rough extract of echinoid spawn (REES) is demonstrated to efficiently induce oocyte maturation in aspidochirote holothuroids. The method is so efficient that it is currently used in holothuriculture to obtain fertilised eggs even outside the reproductive period of the aquacultivated species. In the present study, we isolated and identified from echinoid spawns the molecule responsible for the induction of the oocyte maturation in the Mediterranean holothuroid Holothuria tubulosa and the Indo-Pacific Holothuria scabra. The use of proteinase K and dialysing membranes suggests that the active molecule is a protein with a molecular weight superior to 12,000 kDa. The active molecule has been isolated on G-100 Sephadex chromatography column. Active chromatographic fractions include seven proteins identified with nanoLC-MS/MS technique. One of them, identified as a thioredoxin-2 and to which the name Trx-REES was given, gave levels of maturation similar to those obtained with the REES and with a commercial thioredoxin extracted from Escherichia coli. Occurrence of thioredoxin in REES was confirmed by immunoblot analysis, and the maturation-inducing properties of thioredoxin were positively checked using anti-thioredoxin antibodies. A peptide of 6 AAs corresponding to the active site of Trx-REES, composed of WCNPCK, was synthesised and its efficiency in holothuroid oocyte maturation was tested. At some concentrations, the isolated peptide was 1.2 times more active than the REES. This short peptide may be used as an effective tool for artificial induction of oocyte maturation in the sea cucumber aquaculture context.

Purification and characterization of peroxidase from zucchini ( Cucurbita pepo L.)

The purification and characterization of zucchini (Cucurbita pepo L.) peroxidase (POD) were investigated. POD from zucchini was purified using a combination of (NH4)2SO4 fractionation and Sephadex G-100 chromatography resulting in 4.7-fold enhancement of activity with a recovery of 10.3%. Molecular mass of zucchini POD was 76 kDa by SDS-PAGE. The optimal activity of the POD was achieved at pH 6.6 and 35°C with guaiacol as substrate. It was completely inactivated when incubated at 90°C for 3 min. L-Cys, ascorbic acid, citric acid, and NaHSO3 (10 mM) were effective POD inhibitors, whereas Cu2+, Zn2+, Fe3+, and Mg2+ could enhance the activity of POD. Practical applications Our paper studied the properties of peroxidase (POD) from zucchini (Cucurbita pepo L.), including crude enzyme extraction and purification, optimum temperature, optimum pH, thermal stability, and effect of inhibitors and metal ions, so as to select a reasonable method to restrain the activity of POD, then providing a theoretical basis for inhibiting enzymatic browning that occurred during the process of manufacturing and storage to improve the nutritional value and exterior quality.

Purification, characterization and tyrosinase inhibition activity of polysaccharides from chestnut (Castanea mollissima Bl.) kernel.

The present paper aimed to obtain the polysaccharides with potent tyrosinase inhibitory activity from chestnut kernel. The ultrasound extracted polysaccharide fractions 1-1 (UEP1-1) and UEP2-1 were obtained by successive ultrasound-assisted-extraction, DEAE-52 and Sephadex G-100 chromatography. The Fourier transform infrared spectrum revealed that both UEP1-1 and UEP2-1 had the characteristic absorption peaks of polysaccharides. The degree of esterification of UEP1-1 (52.4%) and UEP2-1 (49.0%) was higher than that of the crude UEP (33.0%), indicating that the gel properties of the polysaccharides changed after purification. The x-ray diffraction patterns of UEP1-1 and UEP2-1 suggested that they were semi-crystalline polymers with a degree of crystallinity of 23.9 and 35.8%, respectively. The weight-average molecular weight (Mw) of UEP1-1 and UEP2-1 was 75.4 and 59.9 kDA, respectively. The monosaccharide composition of UEP1-1 and UEP2-1 was glucose, galactose, arabinose, mannose, xylose, rhamnose and fructose with different proportions. UEP1-1 displayed a dose-dependent inhibitory effect on tyrosinase activity, and the inhibition mode was found to be competitive.

Extraction, purification and properties of water-soluble polysaccharides from mushroom Lepista nuda.

Lepista nuda has high nutritional and medicinal value, especially has high antioxidant, antitumor and antiviral activities. Based on results of single factor experiment, response surface methodology was used to optimize the extraction conditions of polysaccharides (LNP). Macroporous resin D301 was used to decolorization, and its technological conditions were determined by orthogonal experiment. In addition, two novel polysaccharides (LNP-1, LNP-2) were purified by DEAE Cellulose-52 and Sephadex G-150 chromatography. The molecular weight of LNP-1 is 11,703Da, mainly composed of mannose, glucose, galactose, xylose, arabinose and fucose, while the molecular weight of LNP-2 is 13,369Da, mainly composed of mannose, glucose, galactose, arabinose and fucose. The polysaccharide contents of crude LNP, LNP-1 and LNP-2 were 70.60%, 87.71% and 81.20% respectively, and the protein contents were 1.72%, 0.97% and 0.68% respectively. Their sulfuric contents were 3.39%, 5.02% and 8.64%, and uronic acid contents were 3.66%, 5.56% and 6.80%, respectively. Further studies showed that their ability to chelate iron ions, scavenge DPPH (1,1‑diphenyl‑2‑picrylhydrazyl) free radicals and scavenge superoxide anion radicals were 70.09%, 55.94% and 36.64% respectively. They showed strong ability to scavenge free radicals in a concentration-dependent manner. LNP is expected to be developed as a new efficacy factor in the food industry.

Isolation of a putative virulence agent, cytotoxic serine-elastase, from a newly isolated Pseudomonas aeruginosa ZuhP13

A 48 kDa ZuhP13 elastase from P. aeruginosa isolated from a urine sample was successfully purified to 8.8-fold and 39% recovery by DEAE-Sepharose CL-6B and Sephadex G-100 chromatography. Its ideal reaction values were pH 7.5 and 40°C. It showed stability at pH 6-9 for 1 h and up to 60°C for 30 min with midpoint temperature (Tm) at 61.3°C and isoelectric value (pI) at 5.6+/-0.2. Its Km and catalytic efficiency (Kcat/Km) for the substrate azocasein were 1.3 mg/mL and 4.629107 M-1s-1, respectively. On contrary to most P. aeruginosa proteases, Zn2+, EDTA, 2,2'-bipyridine and o-phenanthroline showed slight inhibition upon its activity, while, the elastase inhibitors (elastatinal and elastase inhibitor II) and the serine protease inhibitors (TLCK, PMSF, SBTI, and aprotinin) markedly decreased the enzymatic activity. Taken together, we suggest that ZuhP13 is a serine elastase-type. Interestingly, the tested enzyme showed both hemolytic and hemorrhagic activities in vivo. Furthermore, it induced nuclear lysis yielding hyperchromatism within leaky and malformed hepatocytes, suggesting ZuhP13 elastase as a high molecular weight potential pathological agent.

Structural features, antioxidant and acetylcholinesterase inhibitory activities of polysaccharides from stem of Physalis alkekengi L.

Physalis alkekengi L. (P. alkekengi) is an important medicinal plant in China. In this study, two polysaccharides (SPAP-1 and SPAP-2) were isolated from the stem of P. alkekengi using hot water extraction method. The extract was further purified by DEAE-cellulose and Sephadex G-200 chromatography. The chemical characteristics, surface features and thermal stability of SPAP-1 and SPAP-2 were evaluated. For the first time, high-performance liquid chromatography with refractive index detection and amino column (without sample derivatization) was performed to analyze the monosaccharide composition of polysaccharides obtained from the stem of P. alkekengi. As a result, SPAP-1 (Mw = 9.1 kDa) consisted of rhamnose, arabinose, galacturonic acid, mannose, and glucose in the ratio of 1.0:1.5:1.9:29.1:1.03, while SPAP-2 (Mw = 13.5 kDa) comprised of rhamnose, xylose, arabinose, mannose and glucose in the ratio of 31.8:14.8:1.0:6.5:22.6. Additionally, the antioxidant effects of SPAP-1 and SPAP-2 were evaluated by ferric reducing power, 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate acid) (ABTS) and 2.2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays. The results showed that the purified polysaccharides exerted significant scavenging activities. Subsequently, acetylcholinesterase inhibitory effects in vitro of the polysaccharides were investigated, which showed that, SPAP-1 and SPAP-2 showed moderate acetylcholinesterase inhibitory activities. Thus, the polysaccharide content in P. alkekengi stem as promising source of natural products possess good antioxidant activities and may exert beneficial effects on cognitive function.

Polysaccharides from pineapple pomace: new insight into ultrasonic-cellulase synergistic extraction and hypoglycemic activities

This study determined the optimal extraction conditions for ultrasonic-cellulase synergistic extraction of polysaccharides from pineapple pomace (PPP) using Plackett–Burman design and response surface methodology. The optimal hydrolysis temperature, ratio of material to water, pH value, hydrolysis time, ultrasonic power and the additive quantity of cellulase were 50 °C, 1:45 g/mL, 6.0, 100 min, 160 W and 2.0%, respectively, giving a extraction yield of 1.10 ± 0.03%. PPP was further isolated and purified by DEAE-52 cellulose and Sephadex G-100 chromatography columns, revealing four main elution peaks, named PPF0, PPF1, PPP2 and PPF3, were obtained. The molecular weight, monosaccharide compositions, structural features and appearance morphology of polysaccharide fractions (PPFs) were analyzed by high-performance liquid chromatography (HPLC), gel permeation chromatography (GPC), UV spectroscopy, fourier transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM). Furthermore, the hypoglycemic activities of PPFs with different concentrations were also investigated by insulin resistance HepG2 cells model in vitro. Results showed that PPF0, PPF1, PPF2 and PPF3 were composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose and fucose with molecular weight of 6.71 × 104, 1.11 × 104, 2.22 × 104 and 5.1 × 103 Da, respectively. All of them could alleviate the development of insulin resistance HepG2 cells with a dose-dependent relationship. The glucose consumption increased 46.4%, 50.5%, 82.1% and 53.6%; 86.8%, 81.6%, 86.8% and 84.2% at the concentration of 20 μg/mL, respectively, without or with insulin. These results suggested that PPFs can be explored as a potential hypoglycemic agent in biomedical and functional food.

Antioxidant and anti-diabetic effects of Auricularia auricular polysaccharides and their degradation by artificial gastrointestinal digestion - Bioactivity of Auricularia auricular polysaccharides and their hydrolysates.

Auricularia auricular polysaccharides (AAPs) derived from the dried fruit body of A. auricular are valuable compounds with many bioactivities. This research aimed to investigate the antioxidant and anti- diabetic activities of these polysaccharides and their artificial gastrointestinal fluid hydrolysates (AAPHs). Artificially simulated gastrointestinal fluid was used to obtain polysaccharide-de- rived fragments, and a rat model of type 2 diabetes mellitus (T2DM) using a high-fat diet and low-dose streptozotocin (STZ) was established to assess their antioxidant and anti-diabetic effects. It was found that AAPs and AAPHs were both heteropolysaccharides and were comprised of arab- inose, xylose, mannose, 2-deoxy-glucose, glucose and glucosamine, but at different mole ratios. AAPHs was purified by Sephadex G-100 chromatography to produce three fractions, namely, AAPHs1, AAPHs2, and AAPHs3. The molecular weights of these three fractions were 320, 169, and 62 kDa respectively. Both AAPs and AAPHs exhibited the evident ability to enhance the activities of antioxidant enzymes and the level of GSH, while increasing the content of liver glycogen and plasma C-peptide compared with the diabetic model group (p < 0.05). Furthermore, AAPHs could cause a marked improvement in glucose-stimulated GLP-1 secretion from 0 min to 30 min (p < 0.05). The possible mechanism was that AAPHs could partly restore the STZ-induced impairment of GLP-1 secretion, and inhibit the oxidative stress pathway, and thereby alleviate the progression of diabetes. This data demonstrated that the molecular mole ratio and molecular weight had a definite effect on antioxi- dant and anti-diabetic activities.

Extraction of polysaccharides from black mulberry fruit and their effect on enhancing antioxidant activity

The optimal extraction conditions with yield of 11.86 ± 0.21% for extraction of crude polysaccharide from black mulberry fruit were obtained as extraction temperature 87 °C, extraction time 3 h and ratio of water to raw material 39 mL/g. Two purified fractions of BMP-1-1 and BMP-2-1 with average molecular weights of 615 and 405 kDa, respectively, were obtained by chromatography of DEAE-52 cellulose and Sephadex G-100. BMP-1-1 consisted of mannose, rhamnose, glucuronic acid, galactose and arabinose, BMP-2-1 consisted of rhamnose, glucuronic acid, galacturonic acid, galactose and arabinose. Crude BMP exhibited the strongest reducing power and protective effect on H2O2-induced oxidative injury in PC12 cells and the most effective scavenging activities against DPPH, ABTS and hydroxyl radical.

Characterization and anti-tumor activity of a polysaccharide isolated from Dendrobium officinale grown in the Huoshan County

BackgroundPolysaccharides are carbohydrate chains composed of linked monosaccharide units. Accumulating studies report that polysaccharides isolated from Dendrobium officinale have a variety of functions. However, the composition and anti-tumor activity of D. officinale grown in the Huoshan area are largely unknown.MethodsA polysaccharide (DOPA-1) was isolated from D. officinale by hot water extraction and ethanol precipitation, followed by purification via DEAE-cellulose and Sephadex G-100 chromatography. DOPA-1 was analyzed by infrared and nuclear magnetic resonance and then characterized by periodate oxidation and Smith degradation. The anti-tumor activity of DOPA-1 was then tested in HepG-2 cells.ResultsOur results show that DOPA-1 is mainly comprised of mannose, glucose, and galactose at a molar ratio of 1:0.42:0.27 and has an average molecular weight of 2.29 × 105 Da. Additionally, DOPA-1 inhibited HepG-2 cell growth in a dose-dependent manner. DOPA-1-treated HepG-2 cells also had increased reactive oxygen species (ROS) levels and decreased mitochondrial membrane potential. Furthermore, apoptosis was observed in DOPA-1-treated HepG-2 cells along with Bcl-2 downregulation and Bax upregulation at the protein level.ConclusionsOur findings suggest that DOPA-1 induces apoptosis in tumor cells via altered mitochondrial function, ROS production, and altered apoptosis-related protein expression. This bioactive polysaccharide could, therefore, potentially be further developed as an anti-tumor adjuvant drug.

New xylanolytic enzyme from Geobacillus galactosidasius BS61 from a geothermal resource in Turkey

In this study, isolation, conventional and molecular characterizations of ten thermophilic bacteria from Rize/Ayder were carried out. Xylanase from Geobacillus galactosidasius BS61 (GenBank number: KX447660) was purified by acetone precipitation, Diethylaminoethyl-cellulose and Sephadex G-100 chromatographies. The xylanase of G. galactosidasius BS61 in clarifying fruit juice was also investigated. Enzyme was purified 29.80-fold with 75.18% yield; and molecular weight was determined as 78.15 kDa. The optimum temperature of xylanase was 60 °C. The enzyme activity was maintained fully after 24 h and over 50% after 168 h at pH 4.0–10.0, while optimum pH was 7.0. Km and Vmax for beech wood xylan were measured as 3.18 mg mL−1, 123 U mg protein−1. In addition, Ca2+, Na+, Al3+, Zn2+, Cd2+, Mg2+, Ni2+, Cu2+ had decreasing effect on enzyme activity, while enzyme activity had been protected against anions, especially HSO3− and HPO42− stimulated enzyme activity. Xylanase applications (with 15 U/mL enzyme activity) in orange and pomegranate juices were increased; and the sugar and turbidity amounts were reduced 17.36% ± 1.18 and 30.52 ± 1.23, respectively. These results indicated that the xylanase of G. galactosidasius BS61 has biotechnological potential in juice clarification due to its stability against metal ions, chemicals and high pH-values.

Hypoglycemic Effects of a Polysaccharide from Tetrastigma hemsleyanum Diels & Gilg in Alloxan-Induced Diabetic Mice.

Tetrastigma hemsleyanum Diels & Gilg, a well-known traditional Chinese medicine, possesses antitumor and anti-inflammatory activity, etc. However, the anti-diabetic effect has not been determined. In our present study, a water-soluble polysaccharide, named THP with molecular weight of 93307Da, was isolated from T. hemsleyanum by DEAE-52 ion-exchange and Sephadex G-100 chromatography. It contains rhamnose, arabinose, mannose, glucose, and galactose in the molar ratio of 0.07:0.14:0.38:0.21:0.31. Then anti-diabetic effects of THP were examined by treating alloxan-induced diabetic mice with different doses (100, 200, and 300mg/kg) of THP orally. The results showed that THP could decrease the blood glucose, TC, TG, LDL-C levels, increase the body weight, HDL-C, insulin levels, and enhance the activities of antioxidant enzyme system in alloxan-induced diabetic mice. Furthermore, the histopathological examination of pancreas, liver, and kidney indicated that THP could protect and reverse β-cells in diabetic mice with low damage to liver and kidney, which suggests that THP may stimulate pancreatic release of insulin and can be an effectively potential candidate for diabetes mellitus.

Purification, characterization and antioxidant activity of dextran produced by Leuconostoc pseudomesenteroides from homemade wine

An exopolysaccharide (EPS) was produced by Leuconostoc pseudomesenteroides DRP-5 isolated from homemade wine. The EPS was obtained with ethanol extraction, which was further purified by chromatography of Sephadex G-100 to get a purified fraction. The monosaccharide composition of the EPS was glucose, and its molecular weight (Mw) was 6.23 × 106 Da, as determined by gas chromatography (GC) and high-performance size-exclusion chromatography (HPSEC). Fourier transform infrared spectra (FT-IR) and nuclear magnetic resonance spectra (NMR) showed that the EPS was a linear glucan with α-(1→6)-linked glucosidic bonds. The water holding capacity (WHC), water solubility index (WSI) and emulsifying activity (EA) of DRP-5 EPS were 296.76 ± 18.93%, 98.62 ± 3.57% and 87.22 ± 2.18%, respectively. DRP-5 EPS have a higher degradation temperature of 278.36 °C, suggesting high thermal stability of the EPS. Also, DRP-5 EPS was found to have moderate 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical, superoxide anion radical, 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) radical, Fe2+ scavenging activities and reducing power. All these characteristics suggest that DRP-5 EPS might have potential applications in the pharmaceutical, cosmetic and food industries.

Purification and Characterization of Cellulase of Mold Isolated from Vermicomposting Process of Palm Oil Empty Fruit Bunches

In previous investigation of vermicomposting process of palm oil empty fruit bunches, five cellulase-producing microorganisms had been isolated. An isolated VTM1 identified as Aspergillus sp. as a cellulase producing mold, and proven produced extracellular cellulase in solid state fermentation of palm oil empty fruit bunches. The obtaining crude cellulase was purified to homogeneity by ammonium sulfate precipitation, gel filtration on Sephadex G-100 chromatography, and finally purified on ion exchange chromatography on DEAE Cellulofine A-500. The yield and purification fold were 12.89% and 107.50, respectively. The purified cellulase was easily released glucose when 5% CMC applied as substrate, as shown by TLC analysis. The purified cellulase had an optimal pH and temperature at 4.0 and 45°C, and was stable at pH 3-7 and 30-50°C, respectively.

Isolation, purification, characterization and antioxidant activity of polysaccharides from the stem barks of Acanthopanax leucorrhizus

A novel water-soluble polysaccharide (named ALP-1) was successfully isolated from the stem barks of Acanthopanax leucorrhizus by hot-water extraction, and further purified by Cellulose DEAE-52 and Sephadex G-100 chromatography. The structure of ALP-1 was characterized by HPLC, HPGPC, partial acid hydrolysis, periodate oxidation, Smith degradation, methylation, together with UV, IR and NMR spectral analysis. The antioxidant activities also were evaluated in vitro. Structural analysis revealed that ALP-1 was a homogeneous galactan with the average molecular weight of 169 kDa, composed of galactose, glucose, mannose and arabinose in a molar ratio of 6.1:2.1:1.1:1.0, owning a backbone structure of 1,6-linked α-d-Galp residues with some branches of α-d-Manp-(1 → 3)-α-l-Araf residues at O-3 and α-d-Galp residues at O-4 of 1,6-linked α-d-Galp. Antioxidant assay showed that ALP-1 exhibited strong DPPH and HO scavenging activities, as well as ferric-reducing antioxidant power. These results provide a scientific basis for the further use of polysaccharides from A. leucorrhizus.

Extraction, purification and antioxidant activity of novel polysaccharides from Dendrobium officinale by deep eutectic solvents

Two novel polysaccharides DOP1-DES and DOP2-DES with molecular weight of 297,911 Da and 30,400 Da were extracted by using DESs from Dendrobium officinale for the first time, and purified with macroporous resin AB-8, Sephadex G75 and Sephadex G200 chromatography. Besides, the effect of extraction temperature, molar ratio of DES, reaction time and compound enzymes concentration on the extraction yield of polysaccharides were investigated. Monosaccharide composition analysis showed DOP1-DES and DOP2-DES were composed of D-glucose and D-mannose with the ratio of 2.2:1 and 3.7:1, respectively. Structural features of DOP1-DES and DOP2-DES were investigated by a combination of FT-IR, methylation analysis and NMR, which indicated that both polysaccharides had a similarly backbone consisting of (1, 4)-linked β-D-Manp and (1, 4)-linked β-D-Glcp with different ratio. Simultaneously, the two fractions possessed considerable antioxidant activity.

Purification, characterization of Chondroitinase ABC from Sphingomonas paucimobilis and in vitro cardiocytoprotection of the enzymatically degraded CS-A

An extracellular chondroitinase ABC (ChSase ABC) produced by Sphingomonas paucimobilis was purified to homogeneity through ammonium sulfate precipitation, DEAE-Sepharose Fast Flow and Sephadex G-100 chromatography. The molecular weight was 82.3 kDa. It showed specific lyase activity toward chondroitin sulfate A (CS-A), CS-B, CS-C and hyaluronan (HA). Using CS-A as substrate, the specific activity was 98.04 U/mg, the maximal reaction rate (Vmax) and Michaelis-Menten constant (Km) were 0.49 μmol/min/ml and 0.79 mg/ml, respectively. Highest activity was obtained at pH 6.5 and 40 °C, and Hg2+ could strongly inhibit the enzyme activity. Mass spectrometry analysis indicated CS-A was degraded to unsaturated disaccharides by ChSase ABC. In vitro cytotoxic tests showed that CS-A oligosaccharide at the concentration of 50 and 100 μg/ml could promote the proliferation of normal H9c2 myocardial cells, decrease the damage induced by isoproterenol (ISO) and accelerate the recovery of cells injured by ISO. These findings suggested that ChSase ABC from Sphingomonas paucimobilis could be a promising tool for the structural analysis and bioactive oligosaccharide preparation of glucosaminoglycans.

Structural characterization and immunomodulating activities of a novel polysaccharide from Nervilia fordii

Nervilia fordii (Hance) Schltr. has been widely used as a medicinal and edible herb in Southwest China and Southeast Asia. In this study, NFP-1, a new water-soluble polysaccharidewith a purity of 97.8%, was purified from water extract of Nervilia fordii by DEAE-cellulose and Sephadex G-100 chromatography. NFP-1 has a relative molecular weight of 950 kDa determined by high performance gel-permeation chromatography (HPGPC). Its monosaccharide compositions were analyzed by high performance liquid chromatography (HPLC) after pre-column derivatizing its hydrolysate with 1-phenyl-3-methyl-5-pyrazolone (PMP). NFP-1 mainly consists of galactose, arabinose, rhamnose, and galacturonic acid. Based on FT-IR, methylation and GC–MS analysis, and NMR, the structure unit of NFP-1 was established as →4)-α-Rhap-(2→ 4)-α-GalpA-(1→2)-α-Rhap-(1→2)-α-Rhap-(4→1)-β-Galp-T containing two branch chains of →2,4)-α-Rhap-(1→5)-α-Araf-(1→3)-α-Araf-(1→, and →2,4)-α-Rhap-(1→4)-β-Galp-(1→. The immunomodulatory assays revealed the dual-functionalities of NFP-1. NFP-1 could significantly induce the secretion of nitric oxide (NO), and promote the secretions of TNF-α, IL-6, and IL-1β in RAW264.7 macrophages. NFP-1 could also significantly inhibit the production of NO, depress the secretions of TNF-α, IL-6 and IL-1β in RAW264.7 macrophages activated by lipopolysaccharide (LPS), and promote the production of IL-10 meanwhile. Our study suggested that Nervilia fordii could be an ideal medicinal or functional food due to its dual immunomodulatory activities.

Purification and biochemical characterization of two isolated laccase isoforms from Agaricus bisporus CU13 and their potency in dye decolorization

Agaricus bisporus CU13 laccase was purified using ammonium sulfate precipitation (40–80%), Sephadex G100, and DEAE Sephadex A50 anion exchange column chromatography, respectively. Two laccase isoenzymes (Lacc1 & Lacc2) with purification folds of 1.40 and 5.81 respectively, were obtained from DEAE Sephadex A50 column. Optimal temperature and pH were recorded at 55 °C and pH 5.0 for both laccase isoenzymes using ABTS as substrate. Lacc1 was more thermostable than Lacc2 with residual activity of 95, 80 and 6%, while Lacc2 only retained 72, 25 and 0.4% of its activity after incubation for 90 min. at 50, 60 and 70 °C, respectively. Lacc2 retained about 93 and 86% of the initial activity at pH 9.0 and 7.0, whereas Lacc1 was stable at pH 7.0 and 5.0 followed by pH 9.0 and retained about 87, 76, and 36% of its activity respectively, after 4 h of incubation. Lacc1 was activated by 40% in the presence of Cu2+ (10 mM). Km and Vmax values found to be 0.394 and 0.158 μM, and 0.1351 and 0.4755 μmol min−1 for Lacc1 and Lacc2, respectively. The efficiency of both isoenzymes to decolorize Acid blue dye, make the enzyme seems to be a prospective for further biotechnological applications.

Extraction Optimization, Purification, Antioxidant Activity, and Preliminary Structural Characterization of Crude Polysaccharide from an Arctic Chlorella sp.

The arctic strain of Chlorella sp. (Chlorella-Arc) exists in the coldest and driest arctic ecosystems, and it is a new resource of active polysaccharides. The extraction of crude polysaccharide from Chlorella-Arc was optimized using the response surface methodology. A crude polysaccharide yield of approximately 9.62 ± 0.11% dry weight was obtained under these optimized conditions. Three fractions (P-I, P-II, and P-III) were present after purification by 2-diethylaminoethanol Sepharose Fast Flow and Sephadex G-100 chromatography. The P-IIa fraction demonstrated significant antioxidant activities. Moreover, P-IIa was an α- and β-type heteropolysaccharide with a pyran group and contained variable amounts of rhamnose, arabinose, glucose, and galactose based on fourier-transform infrared spectroscopy, high-performance liquid chromatography, and 1H and 13C nuclear magnetic resonance imaging. Production of high amounts of polysaccharide may allow further exploration of the microalgae Chlorella-Arc as a natural antioxidant.