Sephadex G-100 Column Research Articles

Screening, characterization and anti-cancer application of purified intracellular MGL

L-methionine-γ-lyase (MGL) producing bacterial isolates were screened from soil samples that further characterized as ‘Klebsiella oxytoca BLM-1’ by biochemical and 16S rDNA sequencing. Intracellular MGL obtained from K. oxytoca BLM-1 by sonication was purified by Octyl-Sepharose and Sephadex G-200 column chromatography. MALDI-TOF-MS analysis of protein band (Mr ~ 63 kDa) confirmed the PLP-dependence and structural similarity with MGL enzyme. Purified MGL (1.1 μg) exhibited the maximum activity in potassium phosphate buffer (80 mM; with L-met 20 mM pH 7.0) at 37 °C. That further enhanced in the presence of NaCl (2 mM), Tween-80 (1.0 %; v/v) and EDTA (5 mM). Km and Vmax for purified MGL by using L-met as substrate was found to be 5.32 mM and 0.386 U/mL/min. The purified MGL showed PLP dependence and the half-life was 365.59 min. The MGL was effective against breast cancer (MCF7), gastric adenocarcinoma and human glioblastoma (U87MG) cancer cell lines with IC50 values of purified MGL 0.041 U/mL, 0.008 U/mL and 0.009 U/mL, respectively. The U87MG, greatly affected by MGL treatment, when cultured in DMEM medium (10 mL) with PLP, homocysteine and 10 % FCS as compared to control/untransformed mouse spleen cells.

Chemical Structure and Immune Activation of a Glucan From Rhizoma Acori Tatarinowii

Rhizoma Acori Tatarinowii is a traditional Chinese herb used to treat depression and coronary heart disease. Studies on its active components mainly focus on small molecular compounds such as asarone and other essential oil components, while the large molecular active components such as polysaccharides are ignored. In this study, we aimed to study the chemical structure and immune activation of polysaccharides from Rhizoma Acori Tatarinowii. In this study, a polysaccharide (RATAPW) was isolated and purified by DEAE-52 cellulose and Sephadex G-100 column chromatography from alkali extraction polysaccharide of Rhizoma Acori Tatarinowii. The average molecular weight of RATAPW was 2.51 × 104 Da, and the total carbohydrate contents of RATAPW were 98.23 ± 0.29%. The monosaccharide composition, methylation, and nuclear magnetic resonance (NMR) analysis results displayed that the polysaccharide was α-1,4-glucan with short α-1,6 branches. Immunofluorescence assay and inhibitor neutralization assay indicated that RATAPW could promote the TNF-α production of RAW264.7 macrophage through the nuclear factor kappa B (NF-κB) molecular signaling pathway. Treatment with 200 μg/ml of RATAPW enhanced a 38.77% rise in the proliferation rate of spleen lymphocytes. RATAPW also enhances ConA-induced T cells and lipopolysaccharide (LPS)-induced B cell proliferation in a dose-dependent effect. Our study lays a foundation for the discovery of natural polysaccharide immune modulators or functional food from Rhizoma Acori Tatarinowii.

Antioxidant Activities of Aqueous Extracts and Protein Hydrolysates from Marine Worm Hechong (Tylorrhynchus heterochaeta).

Hechong (Tylorrhynchus heterochaeta) is an edible marine worm widely distributed in the estuary area. The objective of this study is to determine the antioxidant activities of extracts and protein hydrolysates from Hechong. Results showed that the aqueous extracts of steamed Hechong had the highest antioxidant values using the methods of DPPH, ABTS, and FRAP testing (76.29 μmol TE/g, 181.04 μmol TE/g, and 10.40 mmol Fe2+/100 g, respectively). Furthermore, protein hydrolysates of Hechong were observed significant antioxidant activities when compared to crude Hechong. The purification was carried out by DEAE-52 cellulose and Sephadex G-100 column chromatography. The microspatial structure of glycoprotein showed fibrous shapes and cracks with uniform distribution. The study has concluded that the extract and protein hydrolysates of Hechong have significant antioxidant activities, which is merited to be further investigated in the food and pharmaceutical fields.

Comparison of structural and antioxidant activity of polysaccharide extracted from truffles.

As the main component of truffles, polysaccharides have a variety of biological activities such as anti-oxidation, anti-tumor, and hypoglycemic activity, and these activities are closely related to its structure. In this study, Tuber Aestivum crude polysaccharide (TACP) and Tuber Melanosporum crude polysaccharide (TMCP) were obtained from Tuber Aestivum and Tuber Melanosporum by using microwave-assisted hot water, and then the Sephadex G-200 column was utilized to further separate and purify Tuber Aestivum polysaccharide (TAP) and Tuber Melanosporum polysaccharide (TMP) from TACP and TMCP. The structural characterization results showed that the molecular weight of TAP was 2.18 × 104 kDa, while TMP was 8.79 × 103 kDa. Although the two polysaccharide components were mainly composed of mannose (Man) and glucose (Glc), the molar ratio of Man and Glc in TAP was 14.76: 12.31, with a molar ratio of 5.43:10.94 in TMP. Furthermore, the antioxidant activity of two polysaccharide components was evaluated. TAP and TMP could protect porcine jejunal epithelial (IPEC-J2) cells from oxidative damage by H2 O2 , but TAP exhibited stronger antioxidant effects. It was mainly reflected that TAP could increase the secretion level of intracellular antioxidant enzymes (superoxide dismutase and catalase) in IPEC-J2 cells, and had a significant effect on the total antioxidant capacity of cells. The reactive oxygen species and malondialdehyde had better scavenging ability at the concentration of 20µg/ml.Thedifference between TAP and TMP may be due to the dissimilar structure. Its structure-activity relationship needs further study. PRACTICAL APPLICATION: The structure of TAP and TMP were different, and TAP had higher molecular weight. Besides, TAP and TMP can protect IPEC-J2 cells from oxidative stress, providing a theoretical basis for developing potential antioxidant drugs of practical significance.

Enzyme mediated transformation of CO2 into calcium carbonate using purified microbial carbonic anhydrase

In this study, a bacterial carbonic anhydrase (CA) was purified from Corynebacterium flavescens for the CO2 conversion into CaCO3. The synthesized CaCO3 can be utilized in the papermaking industry as filler material, construction material and in steel industry. Herein, the CA was purified by using a Sephadex G-100 column chromatography having 29.00 kDa molecular mass in SDS-PAGE analysis. The purified CA showed an optimal temperature of 35 °C and pH 7.5. In addition, a kinetic study of CA using p-NPA as substrate showed Vmax (166.66 μmoL/mL/min), Km (5.12 mM), and Kcat (80.56 sec−1) using Lineweaver Burk plot. The major inhibitors of CA activity were Na2+, K+, Mn2+, and Al3+, whereas Zn2+ and Fe2+ slightly enhanced it. The purified CA showed a good efficacy to convert the CO2 into CaCO3 with a total conversion rate of 65.05 mg CaCO3/mg of protein. In silico analysis suggested that the purified CA has conserved Zn2+ coordinating residues such as His 111, His 113, and His 130 in the active site center. Further analysis of the CO2 binding site showed conserved residues such as Val 132, Val 142, Leu 196, Thr 197, and Val 205. However, a substitution has been observed where Trp 208 of its closest structural homolog T. ammonificans CA is replaced with Arg 207 of C. flavescens. The presence of a hydrophilic mutation in the CO2 binding hydrophobic region is a further subject of investigation.

Optimization of alkali extraction, structure, and antioxidant activity of protein-bound polysaccharide from seeds of Plantago ovata Forssk

In this study, the effects of different solvents on the viscosity of the seeds of Plantago ovata Forssk (POF) were compared to select the extraction solvent; then, a three-level and four-variable Box–Behnken design (BBD) was applied to determine the optimum extraction conditions for the maximum yields of polysaccharides from POF. Two novel protein-bound heteropolysaccharides (POFG-1a and POFG-2a) with molecular weights of 118 kDa and 113 kDa were isolated and purified by ethanol fractionation precipitation, DEAE-650 M anion exchange column chromatography, and Sephadex G-100 column chromatography. POFG-1a and POFG-2a contain 49.6% and 17.2% of polysaccharide, 26.9% and 56.4% of protein, 9.1% and 5.0% of uronic acid, respectively; they are rich in monosaccharides, Ara, Gal, and Rha, and amino acids, Ser, Pro, Ile, and Phe. Based on FT-IR, β-elimination, methylation analysis, NMR spectroscopy, and some literature data, it was found that POFG-1a is a homogeneous acidic protein-bound heteropolysaccharide with O-glycosidic linkage. The main chain of POFG-1a is connected by 1→4 Araf and 1→5 Araf with a high degree of branching, connected to the branch at the O-2 and O-3 positions of 1→5 Araf. The POFGs exhibited strong scavenging activity on DPPH, ABTS, and NO free radicals, which is rarely observed in other plant polysaccharides. These results indicate that POFGs might have potential applications in the medical and food industries as novel natural antioxidants.

Purification of wild type and recombinant xylanases and evaluation of their arabinoxylan hydrolysis ability

Arabinoxylan hydrolyzing enzymes are very rich and diversified groups, among these, endo-1,4-β-xylanase is the most important group which catalyzes the cleavage of xylan main skeleton to release arabinoxylan oligosaccharides such as D-xylose, xylobiose, L-arabinose, xylotetraose, xylopentose. This group of enzyme was efficiently produced from natural microorganism including bacteria, yeast and fungi and recombinant strains. In this study, we have purified wild-type and recombinant xylanase to compare the difference in hydrolysis products of these two enzymes. Using sephadex G-100 and DEAE-sephadex column chromatography, we have purified xylanase from wild-type Aspergillus niger DSM1957 with molecular weight of 25 kDa, specific activity of 3240 IU/mL, the purity increases 1.68 folds compare to crude extract, and a recovery rate of 36%. The recombinant enzyme from Pichia pastoris GS115/pPXlnA was purified by ProbondTM affinity chromatography column, with a molecular weight of 36 kDa; the purity increased 3.2 folds than the crude extract, and with the recovery efficiency of 21.1%. The activity of commercial wild-type xylanase was better than that of our recombinant and wild-type enzymes. Our wild-type xylanase could hydrolyze arabinoxylan into L-arabinose in 50 mM CH3COONa pH 5.0. Our results showed that xylanases purified from different sources were stable and highly specific to xylan. Xylanase has potential application in the production of high quality biotechnological products.

Structural characterization of a novel polysaccharide from sweet corncob that inhibits glycosylase in STZ-induced diabetic rats : Structural characterization of a novel polysaccharide.

In the current study, we extracted a polysaccharide from sweet corncob and evaluated its hypoglycemic function. After collection in water, alcohol precipitation, and purification by DEAE-52 and Sephadex G-100 columns, we obtained a polysaccharide (SCP50) that was composed primarily of mannose and glucose (9.73:190.27), with a molecular weight of 9280.33Da. We demonstrated that SCP50 exhibited significant inhibition of α-glucosidase activity, with an IC50 of 4.866mg/mL, Km of 1.297 × 10-3, and Vmax of 0.076mol/L·min-1 in vitro. We also observed that SCP50 markedly attenuated disaccharidase (maltase, sucrase, and lactase) activity in a rat model of T2DM. We conclude that SCP50 exerts a hypoglycemic effect via inhibition of intestinal glycosylase. These results thus provide new insight into the hypoglycemic action underlying sweet corncob polysaccharide's effects.

Structural characteristics and immune-enhancing activity of fractionated polysaccharides from Athyrium Multidentatum (Doll.) Ching.

Polysaccharides coded as CP were extracted from Athyrium Multidentatum (Doll.) Ching and then fractionated into five fractions (FP-1, FP-2, FP-3, FP-4 and FP-5). A purified polysaccharide designated as FP-3-4 was prepared from FP-3 by Sephadex G-100 column chromatography. Chemical analysis disclosed that CP and these fractions were heteropolysaccharides and mainly composed of glucose, galactose, arabinose, mannose, rhamnose, xylose, fucose, ribose and uronic acid with different molar ratios. They presented different images of SEM. FP-3-4 was highly branched polymers with sixteen types of linkages. The in vitro immunomodulatory results stated that CP and these fractions could promote macrophage proliferation, enhance macrophage phagocytosis and increase the production of NO, TNF-α, IFN-γ, IL-1β, IL-6, IL-10 and IL-2, indicating remarkable immune enhancement activities. RNA sequencing analysis revealed that CP and FP-3 induced macrophage activation mainly through MAPK and alternative NF-κΒ signaling pathways via CD14/TLR4 and Dectin-2 receptors, which were verified by RT-qPCR and western blot.

PURIFICATION AND CHARACTERIZATION OF L-GLUTAMINASE ENZYME FROM SYMBIONT ENDOPHYTIC BACTERIA OF MARINE PLANT Sargassum sp. AS ANTIMICROBIAL ADJUVANTS

Endophytic bacterial symbiont of marine plant seaweed is a potential source of various bioactive compounds as a raw material for drugs. This research aimed to study the antimicrobial potential of L-glutaminase enzyme isolated from seaweed bacterial symbiont collected from Cikoang Island, South Sulawesi Indonesia. The production of L-glutaminase of bacterial symbiont marine plant Sargassum sp was optimum in nutrient broth using a shaker at 37 °C for 3 d. L-glutaminase was obtained from extraction and fractionation with ammonium sulphate at increased saturation levels of 0-20%, 20-40%, 40-60% and 60-80%, continued by dialysis and column chromatography. Antimicrobial activity assessment was conducted by an agar diffusion method using paper discs. Enzyme fractions were produced by purification using DEAE-Cellulose and Sephadex G-100 column chromatography with a purity of 12.23 and 16.95 times, respectively. The enzyme characterization showed that the optimum condition was at pH 8.0 and 37oC, where at this condition, enzyme activity showed a slight increase after the addition of K+ and Na+ ions and however, significant decrease in enzyme activity after the addition of Cu2+ and Fe2+ ions. The results of the antimicrobial activity evaluation revealed that the L-glutaminase of sephadex G-100 fraction had the highest activity against Escherchia coli and Staphylococcus aureus with the inhibition zone of 12.1 ± 0.29 and 17.4 ± 0.21mm, respectively. These results demonstrated that the environmentally friendly bacterial symbiont of the marine plant Sargassum binderi could be a new alternative as a source to produce the antimicrobial L-glutaminase enzyme. This work probably provides an essential finding for further research on the antimicrobial action of protein fraction from bacterial symbiont of marine plant Sargassum sp. or the other species.

Structural characterization and immunomodulatory activity of exopolysaccharide from Aureobasidium pullulans CGMCC 23063

Exopolysaccharide (EPS-1) was isolated from Aureobasidium pullulans CGMCC 23063 and purified with DEAE 650M and Sephadex G-100 column chromatography. The structural characteristics and immunomodulatory activity of EPS-1 were investigated. The results showed EPS-1 (294.9 KDa) was β-glucan consisting of the backbone (1→3)-linked β-Glcp and β-(1,6)-branches by Ion chromatography, gas chromatography–mass spectrometry, and 1D/2D NMR spectroscopy. The SEM and AFM showed that EPS-1 composed spheres joined by triple-helix conformation into chains and circles. In vitro cell experiments, EPS-1 displayed immunological activity on the RAW264.7 cells. After being treated with EPS-1 (400 μg/mL), the concentration of NO, TNF-α, and IL-6 could reach 17.05 μM, 448.46 pg/mL, and 15.18 pg/mL, respectively. In addition, the cytokines mRNA levels also demonstrated the above results at the molecular level. The western-blot results demonstrated that the expression of phosphorylated p65 and p-erk associated with cytokine release, was increased with the dose of EPS-1.

Structure characterization, antioxidant and hypoglycemic activity of an arabinogalactoglucan from Scutellaria baicalensis Georgi

An arabinogalactoglucan SBP-1 was purified from Scutellaria baicalensis by DEAE-52 and Sephadex G-100 column chromatography. The structure of SBP-1 was characterized using HPLC, IR, GC–MS, 1-D and 2-D NMR. The antioxidant and hypoglycemic activity of SBP-1 was investigated by vitro evaluation. The results showed that SBP-1 was composed of arabinose, glucose and galactose in a molar ratio of 1.0:5.9:1.1 and its Mw were 91,156. The backbone of SBP-1 was mainly composed of repeating →1)-α-D-Glcp-(4 → 1)-α-D-Glcp-(3 → 1)-α-D-Galp-(4→. The braches were composed of →2)-α-L-Araf-(1→, →3)-β-D-Glcp-(1→ and α-D-Glcp-(1→, which mainly substituted at O-6 of Glc, while terminal residue was α-L-Araf-(1→ and α-D-Glcp-(1→. Vitro bioactivity showed that SBP-1 had dose-dependent antioxidant and hypoglycemic activity. The scavenging rate on ABTS, DPPH, hydroxyl and superoxide radicals was all beyond 60% as SBP-1 concentration reached 4 mg/mL, and the inhibition rate on α-glucosidase and α-amylase was both more than 80%, which was closely to that of acarbose.

Acid-Catalyzed Water Extraction of Two Polysaccharides from Artemisia argyi and Their Physicochemical Properties and Antioxidant Activities.

In this study, two purified polysaccharide fractions, Artp1 and Artp2, were obtained using acid-catalyzed water extraction, and then purified by DEAE-52 cellulose and Sephadex G-200 column chromatography from the crude polysaccharides of Artemisia argyi. Their physicochemical properties were investigated by gel permeation chromatography (GPC), high-performance anion exchange chromatography (HPAEC), Fourier transform infrared (FT-IR), scanning electron microscope (SEM), thermal analysis, and methylation analysis. The average molecular weight (Mw) of Artp1 and Artp2 were estimated to be 42.17 kDa and 175.22 kDa, respectively. Monosaccharide composition analysis revealed that the Rha, Gal, and GalA occupied main proportion in Artp1 with the molar ratio of 25.1:24.7:40.4, while the Rha, Gal, Xly, and GalA occupied the main proportion in Artp2 with the molar ratio of 16.7:13.5:12.8:38.7. Due to the high yield and the relatively high carbohydrate content, the Artp1 was determined by the methylation analysis and NMR. The results of Artp1 indicated that 1,4-GalpA and 1,2,4-Rhap formed the backbone with some 1,2-Rhap, 1,3-Galp, and 1,6-Galp in the backbone or the side chains. Artp1 and Artp2 exhibited effective antioxidant activities by DPPH radical scavenging assay and hydroxyl radical scavenging assay in a dose-dependent manner. These investigations of the polysaccharides from A. argyi. provide a scientific basis for the uses of Artp1 and Artp2 as ingredients in functional foods and medicines.

Structural characterization and immunoregulatory activity of polysaccharides from Dendrobium officinale leaves.

In this study, two kinds of polysaccharides from leaves of Dendrobium officinale, namely DLP-1 and DLP-2, were obtained by hot water extraction, ethanol sedimentation, and chromatographic separation using DEAE-52 cellulose and Sephadex G-100 columns. They were composed of different monosaccharides and the content of monosaccharides varied significantly while DLP-1 (Mw 1.38×106 Da) was mainly composed of mannose (71.69%) and glucose (22.89%), and DLP-2 (Mw 1.93×106 Da) was constituted by rhamnose (35.05%), arabinose (24.12%), and galactose (25.65%). A triple-helical conformation was exhibited by both of them. The scanning electron microscope image of DLP-1 showed an irregular and large lamellar shape, as well as a smooth surface and a porous interior, illustrating they had an amorphous structure. In contrast, DLP-2 revealed a rough, loose, and uneven surface consisting of large sponge-like particles. Nuclear magnetic resonance analysis showed that (1→4)-β-D-Manp, (1→4)-β-D-Glcp, and (1→4)-2-O-acetyl-β-D-Manp were the main linkage types of DLP-1, whereasDLP-2 was constituted by a large amount of (1→4)-β-D-Manp, (1→4)-β-D-Glcp, and other residues. Besides, DLP-1 and DLP-2 stimulated the proliferation and phagocytic capacities of RAW 264.7 cells and improved the production of nitric oxide, interleukin-6, TNF-α, and IL-1β. These results proved that both DLP-1 and DLP-2 possessed excellent immunoregulatory bioactivities and could be functional food or adjuvant drug. PRACTICAL APPLICATIONS: The leaf of Dendrobium officinale is a by-product with huge biomass. The lack of systematic research on its chemical composition and pharmacologic effect, leading to a great waste of resources. In order to maximize the value of D. officinale, this study aimed to investigate the structural characteristics and immunologic effects of two polysaccharide fractions (DLP-1 and DLP-2) from D. officinale leaves, showing that DLP-1 and DLP-2 in D. officinale leaves could be used as anti-inflammatory agents to avoid wasting.

Protein Diversity and Immune Specificity of Hemocyanin From Shrimp Litopenaeus vannamei.

Hemocyanin is an important non-specific innate immune defense molecule with phenoloxidase, antiviral, antibacterial, hemolytic, and antitumor activities. To better understand the mechanism of functional diversity, proteomics approach was applied to characterize hemocyanin (HMC) expression profiles from Litopenaeus vannamei. At first, hemocyanin was purified by Sephadex G-100 and DEAE-cellulose (DE-52) columns from shrimp serum, and 34 protein spots were identified as HMC on the 2-DE gels. Furthermore, we found that 9 HMC spots about 75 or 77 kDa were regulated by Streptococcus agalactiae and Vibrio parahaemolyticus infection at 6, 12, and 24 h. In addition, 6 different pathogen-binding HMC fractions, viz., HMC-Mix, HMC-Vp, HMC-Va, HMC-Vf, HMC-Ec, and HMC-Sa, showed different agglutinative and antibacterial activities. Moreover, lectin-blotting analysis showed significant differences in glycosylation level among HMC isomers and bacteria-binding HMC fractions. Particularly, the agglutinative activities of the HMC fractions were almost completely abolished when HMC was deglycosylated by O-glycosidase, which suggest that O-linked sugar chains of HMC played important roles in the innate immune recognition. Our findings demonstrated for the first time that L. vannamei HMC had molecular diversity in protein level, which is closely associated with its ability to recognize diverse pathogens, whereas glycan modification probably contributed to HMC’s diversity and multiple immune activities.

Purification, structural characterization and antioxidant activity of a new arabinogalactan from Dorema ammoniacum gum

Gum ammoniacum is a polymer obtained from Dorema ammoniacum and its medicinal use was already known to the ancient times. In this study, a new D. ammoniacum carbohydrate (DAC-1) with a molecular weight of 27.1 kDa was extracted by hot water and then purified on DEAE-52-cellulose and Sephadex G-100 columns. The structural features of DAC-1 were investigated by partial acid hydrolysis, fourier-transform infrared spectroscopy (FT-IR), methylation, gas chromatography–mass spectrometry (GC–MS), gas chromatography-flame ionization detection (GC-FID), and 1D and 2D nuclear magnetic resonance spectroscopy (1D & 2D NMR). The results indicated that DAC-1 was an arabinogalactan including galactose, arabinose, rhamnose, glucuronic acid and 4-O-methyl-β-d-glucopyranosyl uronic acid (meGlcpA) with a relative percentage of 44.63%, 23.30%, 13.46%. 12.47%, and 6.14%. The structure units of DAC-1 were elucidated as 3,1)-β-D-Galp-(6 → 1)-β-D-Galp-(3,6 → containing four branch chains of →1,6)-β-D-Galp-(3 → 1)-α-L-Araf-(5 → 1)-β-D-GlcpA-(4 → 1)-α-L-Rhap-T (two times), →1,6)-β-D-Galp-(3→1)-β-D-Galp-(3 → 1)-β-D-Galp-(3 → 1)-β-D-Galp-(3 → 1)-α-L-Araf-T and →1,6)-β-D-Galp-(3 → 1)-α-L-Araf-(5 → 1)-β-D-meGlcpA-T. X-ray diffraction (XRD) pattern indicated a semi-crystalline structure. Thermal behavior of the polysaccharide was evaluated by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) and revealed temperatures higher than 200 °C as dominant region of weight loss. DAC-1 showed acceptable antioxidant activity when analyzed by DPPH, ABTS, FRAP, and OH radical removal methods.

A Multifaceted Fungus Pig’s Ears: Gomphus clavatus Gray

Gomphus clavatus, commonly known as pig’s ears, is a species of fungi in the genus Gomphus, family Gomphaceae native to Eurasia and North America. Pig’s ear derives its name from the funnel-shaped and folded fruiting body, which resembles a pig’s ear in shape and texture. Gomphus clavatus mushrooms are edible; these are used as soup, sauce and baking dishes. Gomphus clavatus Gray is not only used as a nutritious food but also might be an important source of biologically active compounds with potential additional medical value. In recent research, the fruit bodies of Gomphus clavatus Gray were collected from the wild. A novel heteropolysaccharide, namely ‘GCG-1’, from the fruit bodies of Gomphus clavatus Gray was isolated through Sephadex G-200 and DEAEcellulose columns. Fungal polysaccharide is a type of active organic compound that is composed of long chains of monosaccharide units bound together by glycosidic linkages. Recently, an increasing number of fungal polysaccharides have been identified to show a variety of biological activities, including antitumor and antioxidant properties. Antioxidation test in GCG-1 in vitro showed that it has strong free radical scavenging activity. It suggests that Gomphus clavatus can be considered as a medicinal food with antioxidant activities.

Characterization of Antioxidant Peptides from Thai Traditional Semi-Dried Fermented Catfish

Herein, the antioxidant peptides from a Thai traditional semi-dried fermented farmed hybrid catfish (Clarias macrocephalus × Clarias gariepinus) catfish, Pla Duk Ra, were characterized. After extraction and deproteinization, Pla Duk Ra crude peptide extract (CPE) was fractioned using 2 connected Hitrap Sephadex-G25 columns, yielding two significant fractions, F1 with higher browning intensity (A420) and F2. CPE, F1, and F2 had different amino acid profiles, contents, and sequences evaluated by LC-MS/MS, which could be responsible for their antioxidant properties. F2 contained the highest numbers of hydrophobic amino acid (HBA) (47.45%) and aromatic amino acid (27.31%), followed by F1, and CPE. The peptides with 8–24 amino acid residues were detected in CPE and its fractions. In CPE, F1, and F2, there were 69, 68, and 85 peptides with varied HBA content, respectively. ARHSYGMLYCSCPPND (50% HBA), ALRKMGRK (37.5% HBA), and ANWMIPLM (87.5% HBA) were the most prevalent peptides found in CPE, F1, and F2. Overall, F2 was the most effective at inhibiting free radicals (DPPH● and ABTS●+) and reactive oxygen species (hydroxyl radical, singlet oxygen, and hydrogen peroxide), followed by F1 and CPE. The metal chelation of F1 was, however, superior to that of F2 and CPE. For the stability test, the effects of pH, heating temperature, and in vitro digestion on the DPPH● scavenging activity of F2 were investigated. The activity was boosted by lowering the pH and raising the heating temperature. In the gastrointestinal tract model system, however, roughly 50% of DPPH● scavenging activity reduced after digesting.

Purification and characterization of a novel bacteriocin produced by Acinetobacter movanagherensis AS isolated from goat rectum

AbstractThe present investigation focuses on the purification and characterization of a novel bacteriocin produced by Acinetobacter movanagherensis AS isolated from goat rectum, which demonstrated high antimicrobial activity against both gram‐positive and gram‐negative bacteria. It was identified as A. movanagherensis AS using biochemical and 16S rDNA sequencing. Tryptic soy broth supplemented with 1% yeast extract with pH 6.5 and incubation temperature of 37°C for 32 h were the optimal conditions for bacteriocin production. Bacteriocin was purified in three steps: 70% ammonium sulfate precipitation, ion‐exchange chromatography with diethylaminoethyl‐cellulose resin, and size exclusion chromatography with a Sephadex G75 column. Purified bacteriocin had a recovery of 7.93% with 23.43 purification fold, and a molecular weight of 12.11 kDa was determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis. Its biochemical characterization showed good stability at a broad range of temperature (40–80°C) as well as pH (3–9). Inhibition of antimicrobial activity was observed after treatment with trypsin, pepsin, and protease, which confirmed the proteinaceous nature of the purified bacteriocin. Bioinformatic analysis suggests that the purified novel bacteriocin from A. movanagherensis AS may belong to the colicin family. Considering the antimicrobial potential of purified bacteriocin coated on low‐density polyethylene film, we assume that it could be potentially exploited for food preservation in food processing industries.

Purification and kinetic properties of a novel β-amylase from Penicillum citrinum AS-9

β-amylase-rich preparation produced by the soil fungal strain Penicillium citrinum AS-9 was completely purified within the succeeding steps; ultrafiltration, acetone fractionation and gel filtration on Sephadex G-150 column. Acetone fractionation recovered the highest β-amylase activity and the 85% acetone fraction exhibited 3.5-fold activity that of the crude enzyme. The column affected 8.6-fold purification for β-amylase, which produced maltose as the major product of starch hydrolysis. The pure enzyme showed a Km value of 17.5 mM and Vmax of 17.5 Umg-1 protein, applying Woolf plot and exhibited its maximum velocity at pH 7.1 and 50°C. In absence of substrate, thermal treatments of the enzyme solution at pH 5.2 and 5.5 had the most adverse effects on the enzyme activity. CaCl2 (1mM) activated the enzyme, while each of Ca2+, Fe3+, cysteine, cystine, I2 and p-chloromercuribenzoate (PCMB) had different inhibitory effects. Maltose as the enzyme product at final concentration of 80 mM strongly inhibited the enzyme.