DEAE-Sepharose Fast Flow Research Articles

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.

Development of a Highly Specific IgM Enzyme-Linked Immunosorbent Assay for Bartonella henselae Using Refined N-Lauroyl-Sarcosine-Insoluble Proteins for Serodiagnosis of Cat Scratch Disease.

The conventional anti-Bartonella henselaeIgM enzyme-linked immunosorbent assay (IgM-ELISA) methods for diagnosing cat scratch disease (CSD) remain poor in both sensitivity and specificity. We sought to develop an IgM-ELISA with improved accuracy in the serodiagnosis of CSD by exploring the antigens that are most suitable for an ELISA. We prepared 5 different protein antigens: antigen I (sonicatedB. henselaewhole-cell antigen), antigen II (N-lauroyl-sarcosine-insoluble antigen), antigen III (processed sarcosine-soluble antigen), and antigen IV and antigen V (sarcosine-insoluble and sarcosine-soluble antigens refined by DEAE-Sepharose Fast Flow ion-exchange chromatography). The IgM antibodies in the sera of 47 patients with clinically suspected CSD (24 definite, 23 suspected) and of 85 healthy individuals were examined by ELISAs using the 5 antigens, and the results were compared with those of an IgM indirect fluorescent antibody assay (IgM-IFA). In a reference panel, which consisted of 5 positive and 5 negative sera, antigen I and antigen III failed to distinguish between the two statuses, whereas the other three antigens succeeded in distinguishing between them. When the cutoff value was set at the 98th percentile of the ELISA index for healthy individuals, the sensitivity of IgM-IFA for the 24 cases of definite CSD was 54%, whereas the sensitivities of the IgM-ELISAs with antigen II, IV, and V were 75%, 83%, and 75%, respectively. The sensitivities of these three IgM-ELISAs for all 47 of the clinically suspected cases were 49%, 64%, and 51%, respectively. In contrast, the sensitivity of IgM-IFA was 28%. These results indicate that the refined sarcosine-insoluble proteins (antigen IV), which possessed the highest specificity among the 5 antigens, are the most appropriate for developing an IgM-ELISA for the highly specific serodiagnosis of CSD.

Bioactive exopolysaccharides from a S. thermophilus strain: Screening, purification and characterization

A lactic acid bacteria strain AR333 with high-production of exopolysaccharides (EPS) was screened out from 350 bacteria strains isolated from naturally fermented dairy products. It was identified as Streptococcus thermophilus by morphological observation, physiological and biochemical characteristics, and 16S rDNA sequence determination. The EPS from AR333 were purified through DEAE-Sepharose Fast Flow and Sepharose CL-6B, and the purified fraction was designated as EPS333. In vitro test showed that EPS333 could stimulate macrophage RAW 264.7 to release NO significantly (p<0.05). The further work tried to elucidate the structural features of EPS333 via FT-IR spectrum, high-performance anion exchange chromatography (HPAEC) and high-performance size exclusion chromatography (HPSEC). The results showed that EPS333 was a pure neutral polysaccharide with monosaccharide composition of galactose and glucose in a molar ratio of 6:5. A certain amount of acetyl groups might exist in EPS333 according to the FT-IR spectrum. The molecular weight (Mw) was estimated to be 332kDa. Current study suggested that the lactic bacteria strain AR333 could be a potential source of immunoregulatory polysaccharide.

Biochemical and Thermodynamic Characterization of a Novel, Low Molecular Weight Xylanase from Bacillus Methylotrophicus CSB40 Isolated from Traditional Korean Food.

A low molecular weight xylanase from Bacillus strain CSB40, isolated from traditional Korean food and produced in beechwood xylan, was biochemically and thermodynamically characterized. It was purified 8.12-fold with a 15.88% yield using DEAE sepharose fast flow, and it was determined to have a mass of ∼27kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and xylan zymography. The purified xylanase was optimally active at 50°C and pH 6 and stable over a wide range of pH (4.5-12.5). The N-terminal amino acid sequence of xylanase was GIQQGDDGKL. The activation energy for beechwood xylan hydrolysis was 29.39kJmol(-1) with k cat value of 927.582 × 10(2)s(-1). K m and V max were 0.080mg/ml and 794.63mmolmin(-1)mg(-1). The analysis of other thermodynamic parameters like ∆H, ∆G, ∆S, Q10, ∆GE-S, and ∆GE-T also supported the spontaneous formation of products, greater hydrolytic efficiency, and feasibility of enzymatic reaction, which also ratifies the novelty of this xylanase. The enzyme was strongly activated by Zn(2+) and inhibited by Cu(2+). The principal hydrolyzed end-products of this xylanase are xylobiose, xylotriose, and xylotetrose, which can be used in the pharmaceutical industry and as prebiotic in food.

Structural Characterization and Immunological Activities of a Novel Water-Soluble Polysaccharide from the Fruiting Bodies of Culinary-Medicinal Winter Mushroom, Flammulina velutipes (Agaricomycetes).

A water-soluble polysaccharide, designated FVPA2, was isolated from the fruiting bodies of Flammulina velutipes using DEAE Sepharose Fast Flow and gel-permeation chromatography. Its structure was elucidated by monosaccharide composition and methylation analysis, ultraviolet, Fourier transform infrared spectrometry, and nuclear magnetic resonance spectroscopy. Results showed that FVPA2 was a homogeneous heteropolysaccharide containing galactose, fucose, and mannose in a molar ratio of 5:1:1. High-performance liquid chromatography indicated its molecular weight as 3.4 × 104 Da. FVPA2 also has a repeating unit. In vitro immunomodulatory studies showed that Raw264.7 cells were stimulated to secret nitric oxide upon administration of 200-500 µg/mL FVPA2. FVPA2 also stimulated the proliferation of mouse spleen lymphocytes and B lymphocytes.

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.

Investigation on lignocellulosic saccharification and characterization of haloalkaline solvent tolerant endo-1,4 β-d-xylanase from Halomonas meridiana APCMST-KS4

The bacterial strain Halomonas meridiana isolated from marine sediment sample was used for the production of xylanase enzyme and it was also purified and characterized. Xylanase was purified to its homogenecity by using ammonium sulphate precipitation and DEAE Sepharose Fast Flow anion exchange chromatography with 17.23% total enzyme recovery and 15.83 fold purification. The molecular weight of the purified xylanase was 41 kDa and it was observed as an alkaline thermophilic. The optimum enzyme activity was found at pH 8 and also at 50 °C temperature. Further this xylanase was stable at wide pH range between 6 and 9 and temperature between 40 and 50 °C. This enzyme was active at 1.5 M NaCl. Metal ions such as manganese sulphate, zinc sulphate, zinc chloride, copper sulphate and magnesium chloride showed the highest influence on enhancing xylanase activity. The xylanase activity was also influenced by surfactants such as Tween 20, Tween 40 and Tween 60 and it was resistant to SDS. The enzyme metallo-xylanase was highly inhibited by EDTA. The other additives like beechwood xylan, birchwood xylan and all the tested organic solvents were positively influenced the enzyme activity when compared to the control. The enzyme registered maximum saccharification rate on pretreated rice bran, wheat straw and Ulva respectively.

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.

Vanadium-binding protein from vanadium-enriched sea cucumber Apostichopus japonicus inhibits adipocyte differentiation through activating WNT/β-catenin pathway

A vanadium-binding protein (VBP) was isolated from the intestine of edible sea cucumber using a procedure which involved precipitating the extract by the addition of (NH4)2SO4, ion exchange chromatography on DEAE-Sepharose Fast Flow, and gel filtration chromatography on Sephacryl S-300 HR. The inhibitory effect of VBP on adipocyte differentiation was then examined. VBP up-regulated the mRNA expression of WNT/β-catenin related factors, namely FZ1, LRP5 and LRP6, while it had no effect on Wnt10b and GSK3β. qRT-PCR and western blotting results showed that VBP enhanced the level of total β-catenin, nuclear β-catenin and exhibited marked inhibitory effect on adipocyte differentiation. The lipid accumulation of cells treated with VBP was decreased by 24.07%. VBP decreased the expression of PPARγ and C/EBPα, further indicating the inhibitory effect on adipocyte differentiation. These findings suggest that VBP exhibits marked inhibitory effect on adipocyte differentiation and this effect is mediated by activating the WNT/β-catenin pathway.

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.

One-step purification of R-phycoerythrin from the red edible seaweed Grateloupia turuturu.

A one-step chromatographic method for the purification of R-phycoerythrin (R-PE) of Grateloupia turuturu Yamada is described. Native R-PE was obtained with a purity index of 2.89 and a recovery yield of 27% using DEAE-Sepharose Fast Flow chromatography with a three-step increase in ionic strength. The analysis by SDS electrophoresis showed a broad band between 18 and 21kDa in size corresponding to subunits α and β and a low intensity band of 29kDa corresponding to the γ subunit. Two forms of R-PE were identified by gel filtration chromatography: a native form with a molecular weight of 260±5kDa and a dissociated form with a molecular weight of 60±2kDa. The native form presented the characteristic absorption spectrum of R-PE with three absorbance maxima at 498, 540 and 565nm, whereas the dissociated form presented only the 498 and 540nm peaks. Moreover, the two forms displayed two different fluorescence maxima.

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.

Isolation and purification of monosialotetrahexosylgangliosides from pig brain by extraction and liquid chromatography.

Monosialotetrahexosylganglioside (GM1), one of glycosphingolipids containing sialic acid, plays particularly important role in fighting against paralysis, dementia and other diseases caused by brain and nerve damage. In this work, a simple and highly efficient method with high yield was developed for isolation and purification of GM1 from pig brain. The method consisted of an extraction by chloroform-methanol-water and a two-step chromatographic separation by DEAE-Sepharose Fast Flow anion-exchange medium and Sephacryl S-100 HR size-exclusion medium. The purified GM1 was proved to be homogeneous and had a purity of >98.0% by high-performance anion-exchange and size-exclusion chromatography. The molecular weight was 30.0 kDa by high-performance size-exclusion chromatography and 1546.9 Da by electrospray ionization mass spectrometry. The chromogenic reaction by resorcinol-hydrochloric acid solution indicated that the purified GM1 showed a specific chromogenic reaction of sialic acid. Through this isolation and purification program, ~1.0 mg of pure GM1 could be captured from 500 g wet pig brain tissue and the yield of GM1 was around 0.022%, which was higher than the yields by other methods. The method may provide an alternative for isolation and purification of GM1 in other biological tissues.

Characteristics of an R-phycoerythrin with two γ subunits prepared from red macroalga Polysiphonia urceolata.

An R-phycoerythrin (R-PE) was isolated by gel filtrations on Sepharose CL-4B and Sephadex G-150 from the phycobiliprotein extract of the marine red macroalga Polysiphonia urceolata Grev and further purified by ion exchange chromatography on DEAE-Sepharose Fast Flow. The purified R-PE showed three absorption peaks at 498 nm, 538 nm, 566 nm and one fluorescent emission maximum at 577 nm. Although the R-PE showed a single band on the examination by native PAGE, it exhibited two very close bands at pH about 4.7 in native isoelectric focusing (IEF). Polypeptide analysis of the R-PE demonstrated that it contained four chromophore-carrying subunits, α18.2, β20.6, γ31.6 (γ'), γ34.6 (γ), and no colorless polypeptide; its subunit composition was 6α18.2:6β20.6:1 γ31.6:2γ34.6. The α and β subunits were distributed within a acidic pH range from 5.0 to 6.0 in denaturing IEF and the γ subunits were in a basic pH range from 7.6 to 8.1. These results reveal that the prepared R-PE may exist in two hexamers of γ (αβ)3 γ (αβ)3γ' and γ (αβ)3 γ'(αβ)3 γ and that the R-PE participate in the rod domain assembly of P. urceolata phycobilisomes by stacking each of its trimer (αβ)3 face-to-face with the aid of one γ subunit (γ or γ').

Extraction, characterization and antioxidant activities of Se-enriched tea polysaccharides

Se-polysaccharides from Se-enriched tea leaves were purified by DEAE–sepharose fast flow gel column (2.5×60cm) and three polysaccharide fractions (Se-TPS1, Se-TPS2, and Se-TPS3) were isolated and purified with yields of 6.5, 37.14, and 8.57%, respectively. The average sizes of Se-TPS1 and Se-TPS2 were determined by HPGPC system, with molecular weights of 1.1×105 and 2.4×105Da, respectively. Se-TPS3 was a polysaccharide polymer with two peaks with molecular weights of 9.2×105 and 2.5×105Da. Monosaccharide components analysis by ion chromatography revealed Se-polysaccharides were acidic polysaccharoses and different from each other in monosaccharide kinds and molar ratio. Elements of Se, C, H, N, S, and 14 kinds of mineral elements were analyzed by AFS, EA, and ICP-AES, respectively. Spectral analysis (IR and UV) indicated Se-polysaccharides were typical glycoproteins. Morphological analyses of the samples were determined by SEM and AFM. In addition, the DPPH and superoxide radicals scavenging activities were also discussed to assess antioxidant activities of the samples, and Se-polysaccharides showed higher antioxidant activities compared to the ordinary polysaccharides.

Purification and characterization of a novel cold-adapted phytase from Rhodotorula mucilaginosa strain JMUY14 isolated from Antarctic.

A yeast producing a cold-adapted phytase was isolated from Antarctic deep-sea sediment and identified as a Rhodotorula mucilaginosa strain JMUY14 of basidiomycetous yeasts. It was cultured in fermentation optimized by a response surface methodology based on the Box-Behnken design. The maximum activity of phytase reached 205.447 U ml(-1), which was close to the predicted value of 201.948 U ml(-1) and approximately 3.4 times higher than its initial activity. The extracellular phytase was purified by 15.2-fold to homogeneity with a specific activity of 31,635 U mg(-1) by (NH4 )2 SO4 precipitation, and a combination of DEAE Sepharose Fast Flow, SP Sepharose Fast Flow, and Sephadex G-100. The molecular weight of the purified enzyme was estimated to be 63 kDa and its pI was 4.33. Its optimal temperature and pH were 50 °C and 5.0, respectively. Its activity was 85% at 37 °C, and showed good stability at pH 3.0 ∼ 7.0. When compared with mesophilic counterparts, the phytase not only exhibited a higher activity during 20 ∼ 30 °C but also had a low Km (247 µM) and high kcat (1394 s(-1)). The phytase activity was slightly stimulated in the presence of Mg(2+), Fe(2+), Fe(3+), K(+), Na(+), Ca(2+), EDTA, and EGTA and moderately inhibited by Cu(2+), Zn(2+), Mn(2+), Ag(+), PMSF, SDS, and phenylgloxal hydrate. It was resistant to both pepsin and trypsin. Since the phytase produced by the R. mucilaginosa JMUY14 showed a high specific activity, good pH stability, strong protease resistance, and high activity at low temperature, it has great potential for feed applications, especially in aquaculture.

Chemical and biological characterization of polysaccharides from the bark of Avicennia marina

A water-soluble polysaccharide named BGR-S2 from the bark of Avicennia marina was isolated by ultrasonic-assisted extraction with hot water followed by successive chromatographic purification using DEAE-Sepharose Fast-flow and Sephacryl S-300 columns. A symmetrical peak with an average molecular weight of 119 kDa was obtained by high-performance size exclusion chromatography. Chemical analysis revealed that BGR-S2 is mainly composed of galactose, arabinose, rhamnose and glucose with a molar ratio of 6.2:2.3:1.0:0.8. Based on gas chromatographic, infrared spectral, methylation and NMR analysis, BGR-S2 was structurally characterized as 1,4-d-GalpA interspersed with 1,2-l-Rhap and 1,2,4-l-Rhap residues. The branches are composed of 1,5-l-Araf, 1,3,5-l-Araf, 1,3-l-Araf, 1,6-d-Galp, 1,4,6-d-Galp and 1,4-d-Glcp, and attached to the backbone at the O-4 position of Rhap residues. BGR-S2 showed significant anti-complementary activity by using pectic polysaccharide HAM-3-IIb-II from the stem of A. marina and heparin sodium as positive control. This study provides a foundation for the exploration and utilization of the bark of A. marina in possible therapeutic development.