DEAE-cellulose Ion-exchange Chromatography Research Articles

Purification and Characterization of Recombinant Levansucrase From Bacillus lichniformans MJ8

The ability to produce levansucrase was estimated in fifty Bacillus spp. isolates obtained from various sources. One isolate was found to be the higher producer of this enzyme through primary and secondary screening, which was performed by estimating levansucrase activity. It was found to be 6.05 U/ml. The results show, after identification by the Vitek 2 compact system with 92% probability and 16S ribosomal RNA gene sequencing, that this isolate is Bacillus licheniformis, designated in this study as MJ8, and registered in the Gene Bank under the accession number OM672244.1. Then, the gene of (SacB) was studied in detail. It contained 1449 nucleotides (accession number ON811641.1), expressed for 482 amino acids and 29 amino-acid as signal peptides. A recombinant plasmid, pET28a(+), with the levansucrase (SacB) gene, was over expressed in high efficiency E. coli BL21 (DE3). Then the enzyme was purified in three steps; firstly, it was precipitated with ammonium sulfate 40-80%. Secondly, DEAE cellulose ion exchange chromatography was applied then the gel filtration chromatography. The activity of levansucrase at the final purification steps reached 48.65 U/ml with a purification fold of 18.0 and a recovery of 48.9%. The levansucrase characterization was also studied. It was found that the molecular weight by SDS-PAGE and gel filtration methods was 43 kDa and 47 kDa, respectively, and the optimum temperature activity and stability were 45 ◦C, 30-35 ◦C, respectively while the pH activity and stability were 6.0, and 7.0, The levan was produced from cloned (SacB) levansucrase, separated, purified, and identified by NMR, AFM, and SEM analyses showed that its structure mostly consists of fructose residues.

Purification and characterization of pectinase from gut-associated Klebsiella oxytoca af-G4 of dwarf honey bee, Apis florea

ObjectivesPectinases are enzymes that hydrolyze pectin or pectic compounds. Microorganisms in the gut of bees are involved in the breakdown of these macromolecules. In this study, we aimed to optimize the physicochemical parameters for enhancing enzyme pectinase production by bacteria Klebsiella oxytoca af-G4 isolated from Apis florea gut; in addition, purification and characterization of purified enzyme were carried out. MethodsOptimization of growth conditions for pectinase production using different synthetic and agro-industrial wastes by Klebsiella oxytoca af-G4 were performed. Purification and characterization of the pectinase were carried out. Application of the pectinase in juice extraction was evaluated. ResultsPectinase activity was maximum at 30 °C at pH 5.0, in media supplemented with lactose (1 % w/v) and ammonium sulfate (1 % w/v) as carbon and nitrogen sources, respectively, with a growth period of 72 h. The pectinase production with agro-industrial wastes showed higher production with pomegranate peel (1 % w/v) as substrate at a growth condition of 40 °C, pH 6.0, and a growth period of 92 h. The enzyme was purified to homogeneity (4.8-fold) using DEAE cellulose ion-exchange chromatography. The purified enzyme showed the molecular weight of ∼ 60 kDa on SDS-PAGE with an activity of 23.26 U/ml and specific activity of 14.36 U/mg. The pectinolytic activity was confirmed on the zymogram. The purified enzyme was more active at an optimum pH of 6.0, and stable at a pH range between 5 and 8. The enzyme was stable at temperatures ranging from 30 °C to 80 °C, with 60 °C being the optimum. The enzyme showed Km and Vmax values of 6.04 mg/ml and 4.92 U/ml, respectively. The enzyme showed maximum activity with the addition of metal ion Mn2+ at pH 6 and 60 °C. The enzyme enhanced the extraction of apple juice and clarification significantly. ConclusionThe enzyme characteristics and kinetics as well as the higher production with agro-waste support its pectin-based industrial application. The enzyme properties prove that it is a good candidate for the hydrolysis of pectin and juice clarification for industrial applications.

Study the characteristics of partially purified arginase from the serum of patients with diabetes mellitus type two (DMT2) in the Nineveh Governorate

Arginase plays an essential role in diabetes mellitus type 2 (DMT2). The study aimed to isolate and partially purify the serum arginase from patients with diabetes mellitus type 2, estimate its molecular weight, and determine the optimal conditions for the enzyme's action. The study included 35 patients of both sexes of 16 males and 19 females. Three purification steps were used to purify arginase are the precipitation of the protein with ammonium sulfate at a saturation of 65%, DEAE-cellulose ion-exchange chromatography, and Sephadex G-100 gel scattering technique. The results showed that the specific activity of unpurified arginine is equal to 0.6 and the specific activity increased 19 times when purified while the specific activity was equal to 11.4 (U/mg). The number of purification times was 19-fold, and recovery of 59.2 %. And the molecular weight of purified arginase equals 96050 ± 1414.2 Daltons. The highest activity of purified arginase in 250 µL serum as a source of enzyme,100 mM of buffer solution sodium barbitone, pH = 9.5, and time incubation for 45 minutes at 37 C, and 200 mM of arginine as a substrate for arginase. The study concluded that the specific activity of arginase from serum of diabetic patients increased 19 times after purification. The properties of arginine and the optimal conditions for its action are differ according to the source of the enzyme purified that from it.

Biochemical Characterization of Thermostable Carboxymethyl Cellulase and β-Glucosidase from Aspergillus fumigatus JCM 10253

Second-generation biofuel production has emerged as a prominent sustainable and alternative energy. The biochemical properties of cellulolytic enzymes are imperative for cellulosic biomass conversion into fermentable sugars. In the present study, thermostable CMCase and β-glucosidase were purified and characterized from Aspergillus fumigatus JCM 10253. The enzymes were purified through 80% ammonium sulfate precipitation, followed by dialysis and DEAE-cellulose ion-exchange chromatography. The molecular masses of the purified CMCase and β-glucosidase were estimated to be 125kDa and 90kDa, respectively. The CMCase and β-glucosidase demonstrated optimum activities at pH6.0 and 5.0, respectively. Their respective maximum temperatures were 50 and 60°C. The cellulase activities were stimulated by 10mM concentration of Ca2+, Ni2+, Fe2+, Mg2+, Cu2+, Mn2+, Zn2+, and Pb2+ ions. The CMCase activity was enhanced by surfactant Triton X-100 but marginally influenced by most inhibitors. The β-glucosidase retained its activity in the presence of organic solvents (30%) isoamyl alcohol, heptane, toluene, and ethyl acetate, while CMCase was retained with acetone during a prolonged incubation of 168h. TheKm and Vmaxvaluesof the two cellulases were studied. The properties of high thermostability and good tolerance against organic solvents could signify its potential use in biofuel production and other value-added products.

Purification and characterization of chitinase produced by thermophilic fungi Thermomyces lanuginosus

Background In the past few years, the production of shrimp shell waste from the seafood processing industries has confronted a significant surge. Furthermore, insignificant dumping of waste has dangerous effects on both nature and human well-being. This marine waste contains a huge quantity of chitin which has several applications in different fields. The chitinase enzyme can achieve degradation of chitin, and the chitin itself can be used as the substrate as well for production of chitinase. In the current study, the chitinase enzyme was produced by Thermomyces lanuginosus. The extracellular chitinase was purified from crude extract using ammonium sulfate precipitation followed by DEAE-cellulose ion-exchange chromatography and Sephadex G-100 gel filtration chromatography. The stability and activity of chitinase with different pH, temperature, different times for a reaction, in the presence of different metal ions, and different concentration of enzyme and substrate were analyzed. Result The chitinase activity was found to be highest at pH 6.5, 50 °C, and 60 min after the reaction began. and the chitinase showed the highest activity and stability in the presence of β-mercaptoethanol (ME). The SDS-PAGE of denatured purified chitinase showed a protein band of 18 kDa. Conclusion The characterization study concludes that Cu2+, Hg2+, and EDTA have an inhibitory effect on chitinase activity, whereas β-ME acts as an activator for chitinase activity. The utilization of chitin to produce chitinase and the degradation of chitin using that chitinase enzyme would be an opportunity for bioremediation of shrimp shell waste.

Method for Isolation of Myxozoan Proliferative Stages from Fish at High Yield and Purity: An Essential Prerequisite for In Vitro, In Vivo and Genomics-Based Research Developments.

Myxozoans are a diverse group of microscopic cnidarian parasites and some representatives are associated with important diseases in fish, in both marine and freshwater aquaculture systems. Research on myxozoans has been largely hampered by the inability to isolate myxozoan parasites from their host tissues. In this study, we developed and optimized a method to isolate the myxozoan proliferative stages of different size and cellularity from fish blood, using DEAE-cellulose ion exchange chromatography. We optimized several parameters and obtained 99–100% parasite purity, as well as high survival and infectivity. Using polyclonal pan-carp blood cell-specific antibodies, we further developed a rapid cytometric assay for quantification of the proliferative stages, not only in highly concentrated DEAE-C isolates but also in dilute conditions in full blood. Early developmental stages of myxozoans are key to parasite proliferation, establishment, and pathology in their hosts. The isolation of these stages not only opens new possibilities for in vivo and in vitro studies, but also for obtaining purified DNA and protein extracts for downstream analyses. Hence, we provide a long-desired tool that will advance the functional research into the mechanisms of host exploitation and immune stimulation/evasion in this group, which could contribute greatly to the development of therapeutic strategies against myxozoans.

Xylogalacturonan-enriched pectin from the fruit pulp of Adansonia digitata: Structural characterization and antidepressant-like effect

The low methyl-esterified and acetylated xylogalacturonan (DM 20 %, DA 2 %, Mw ∼ 58 kDa) was isolated by water extraction for 4 h × 2 at 50 °C (yield 23 %) from the pulp of baobab fruit (Adansonia digitata L.). Subsequent tightening of the conditions for water extraction by mean increasing the temperature to 70 °C and time to 12 h led to the co-extraction of small amounts of starch components and RG I with xylogalacturonan. Structural analysis (DEAE-cellulose ion-exchange chromatography, HPSEC, monosaccharide analysis, NMR spectroscopy) revealed that about 12 mol. % of 1,4-linked α-GalpA residues were substituted by single β-Xylp residues at the O-3 position. The xylogalacturonan was found to possess an antidepressant-like effect in mice. The study offers using the baobab fruit as a rich source of soluble dietary fiber – water-soluble pectin with beneficial physiological effect.

Purification and Characterization of a Noble Thermostable Alpha-amylase from Anoxybacillus tengchongensis RA1-2-1 Isolated from Geothermal Spring of Nepal

A thermophilic amylolytic strain, Anoxybacillus tengchongensis RA1-2-1 was isolated from geothermal spring of Rasuwagadi district of Nepal. The BLAST alignment of the 16s rRNA sequence revealed 99.3% similarity with the type strain Anoxybacillus tengchongensis T-11. The morphological, physiological and biochemical properties were similar to the type strain. The enzyme from the strain was purified to 40-fold purification by DEAE-cellulose ion exchange chromatography. The Km value of the enzyme was 0.68±0.05 mg/ml. The optimum pH and temperature were 7.0 and 70 °C. SDS-PAGE analysis showed a single band at 69 kDa. The half-life of the enzyme at 70°C and 80°C were 85.01min and 51.96 min respectively. TLC analysis of the hydrolysis product showed that the enzyme is maltogenic amylase. The calcium independent enzyme was completely inhibited by Hg2+ but showed inhibitory effect in the range of 100 %-30 % in the presence of other salts at 1-10 mM concentrations.

Purification and characterization of a noble thermostable algal starch liquefying alpha-amylase from Aeribacillus pallidus BTPS-2 isolated from geothermal spring of Nepal

A thermophilic strain, Aeribacillus pallidus BTPS-2 was isolated from Bhurung geothermal spring of Nepal. The 16 s rRNA sequence showed 99.8 % similarity with the type strain Aeribacillus pallidus DSM 3670. The morphological, physiological and biochemical properties were similar to the type strain. Alpha-amylase from A. pallidus BTPS-2 was purified to 19-fold purification by DEAE-Cellulose ion exchange chromatography. The Km value of amylase on starch was 0.51 ± 0.05 mg/mL. The optimum pH and temperature were 7.0 and 70 °C. SDS-PAGE analysis showed a single band at 100 kDa. The half-life of the enzyme at 80 °C was 2.81 h. The enzyme showed an inhibitory effect in the presence of Fe2+, Pb2+, Sn2+ and Hg2+ at 10 mM concentrations. TLC analysis showed that the enzyme is a liquifying alpha-amylase. The enzyme reduced the viscosity of algal biomass suspension up to 74.2 ± 0.17 % which was more efficient than Bacillus amyloliquefaciens alpha-amylase (80.5 ± 0.2 %).

Purification and characterization of thermostable α-amylase produced from Bacillus licheniformis So-B3 and its potential in hydrolyzing raw starch

AimsThis work was achieved to obtain the optimum culture conditions of the thermostable alpha-amylase produced by thermophilic Bacillus licheniformis SO-B3. Furthermore, the α-amylase was purified and then characterized, and also its kinetic parameters were determined. Materials and methodsA new thermotolerant bacteria called Bacillus licheniformis SO-B3 employed in this work was isolated from a sample of thermal spring mud in Şırnak (Meyremderesi). Several parameters such as the impact of temperature, time, and pH on enzyme production were examined. Thin-Layer Chromatography (TLC) was employed to analyze the end-products of soluble starch hydrolysis, and the utilization of purified α-amylase in the clarification of unripe apple juices was studied. Key findingsThe highest enzyme production conditions were determined as 35 °C, 36th hour, and pH 7.0. Thermostable α-amylase was purified by 70% ammonium sulfate precipitation, DEAE-cellulose ion-exchange chromatography, and dialysis, with a 51-purification fold and 30% yield recovery. The Km and Vmax values for this enzyme were 0.004 mM and 3.07 μmol min−1 at 70 °C, respectively. The α-amylase's molecular weight was found as 74 kDa. In addition, α-amylase showed a good degradation rate for raw starch. SignificanceIt was hypothesized that Bacillus licheniformis SO-B3 could be used as an α-amylase source. These findings displayed that purified enzyme could be utilized in fruit juice industries for clarification of apple juice and raw starch hydrolyzing.

Chemical composition diversity of fucoidans isolated from three tropical brown seaweeds (Phaeophyceae) species

Abstract. Lutfia FN, Isnansetyo A, Susudarti RA, Nursid M. 2020. Chemical composition diversity of fucoidans isolated from three tropical brown seaweeds (Phaeophyceae) species. Biodiversitas 21: 3170-3177. Fucoidan is a polysaccharide with high sulfate content, found in the cell walls matrix of brown seaweed. Its bioactivities vary depending on the algae species and the chemical structure. The aims of this research were to extract, purify, and characterize fucoidan from three Indonesian brown seaweed, Sargassum sp., Turbinaria sp., and Padina sp. The extraction was carried out using acid method followed by precipitation with ethanol and CaCl2, while its purification using DEAE-cellulose ion-exchange chromatography. Characterization was performed by FTIR and 1H-NMR spectroscopic analyses. Chemical components of fucoidan determined were total carbohydrates, sulfate residue, uronic acid residue, and monosaccharide components. The results revealed that the Turbinaria sp. produced highest yield of fucoidan (4.8% dry matter), followed by Sargassum sp. (2.7% dry matter) and Padina sp. (2.6% dry matter). The carbohydrate contents of Sargassum sp., Turbinaria sp. and Padina sp. fucoidan were 64.55±0.12%, 67.42 ± 0.03% and 62.90 ± 0.04% with sulfate residues of 0.013% ± 4.71; 19.47±0.002% and 0.016%±8.81; and uronic acid residues of 25.19±0.03%; 12.69±0.03% and 12.91±0.01%, respectively. Sargassum sp., Turbinaria sp., and Padina sp. fucoidans consist of fucose and some other minor monosaccharides.

Fibrinolytic protease from Bacillus cereus S46: Purification, characterization, and evaluation of its in vitro thrombolytic potential.

Intravascular thrombosis is a prime cause of cardiac complications worldwide. Microbial fibrinolytic proteases are of clinical significance in thrombosis treatment. The present study discusses the purification and characterization of a protease from Bacillus cereus S46, ascertaining its in vitro thrombolytic activity against a blood clot. By the three-step purification involving precipitation, dialysis, and diethylaminoethyl-cellulose ion-exchange chromatography, a 12.37-fold purification of the enzyme to homogeneity was achieved. The apparent molecular mass of the protease was 30 kDa, as found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme was observed at pH 8.0 and 40°C. The enzyme retained an 82.19% residual activity at pH 8.0 and 40°C for 1 h. The Km and Vmax values of the protease with casein were 0.0027 mM and 9.712 µmol/min, respectively. In an in vitro assay, the purified protease resulted in 97.02% lysis of the blood clot. The fibrinolytic potential of the enzyme, together with its characteristics of being active and stable under near-physiological conditions, may suggest its application as a therapeutic agent.

A novel fibrinolytic enzymes from the Korean traditional fermented food-Jotgal: Purification and characterization.

The fibrinolytic activity in Korean traditional fermented food, Jotgal (pickled fish) was identified. Though the fibrinolytic activity could vary in different kinds of Jotgal, this activity seems to be produced by microorganisms during the natural fermentation stage. From Gonjaengijot (pickled opossum shrimp), two novel fibrinolytic enzymes named by JP-I and JP-II, have been purified by ethanol precipitation, Bio-GEL P-100 gel filtration, and DEAE-cellulose ion-exchange chromatography. Compared to the crude enzyme extract, the specific activity of the JP-I and JP-II increased 258, 85-fold with the recovery of 22.1, 8.5%, respectively. The molecular weights of both enzymes were estimated as 36kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal condition for fibrinolytic activity of JP-I was at 50°C and pH 8.1, while that of JP-II was at 45°C and 9.9. Both enzymes were stable at a broad range of pH (5.0 to 10.5) and have metalloprotease nature. From these results, it concludes that these enzymes could be a novel potent thrombolytic agent. PRACTICAL APPLICATIONS: The fibrinolytic enzyme is one of the clinical agents for cardiovascular diseases which is the leading cause of morbidity and mortality worldwide with 17 million deaths every year. A variety of fibrinolytic enzymes are found and characterized from various sources such as plants, animals, and microorganisms, and new sources for fibrinolytic enzymes continue to be explored. Jotgal, widely used in Korean people's diet, is a traditional Korean seafood prepared from many different types of fishes, fish eggs, fish intestines, and shellfishes. Through an amount of research, some of fibrinolytic enzymes were found and purified from Jotgal, however, no studies have been done on fibrinolytic enzyme from opossum shrimp. In this study, the purification, enzymatic characteristics, and fibrinolytic activity of the proteases, originated from Korean traditional fermented food, Jotgal were reported. These enzymes could be novel potent thrombolytic agent.

Purification and characterization of low molecular weight alkali stable xylanase from Neosartorya spinosa UZ-2-11

Abstract Alkaliphilic xylanase from Neosartorya spinosa UZ-2-11 was purified using a three-step of purification scheme of ammonium sulphate precipitation followed by Sephadex G-100 gel filtration and DEAE-cellulose ion-exchange chromatography, and compared its properties with N. tatenoi KKU-CLB-3-2-4-1 of our previous report. The purified xylanase from N. spinosa UZ-2-11 exhibited maximum activity at pH 9.0 and 45 °C which was similar to endo-xylanase from N. tatenoi KKU-CLB-3-2-4-1. However, this enzyme was stable in a range of pH 6.0–11.0. It was also more stable at a high temperature of 50 °C where the activity was still up to 50% after heating for 120 min. The xylanase was purified 7.89-fold with 3.0% of yield to obtain a specific activity of 11.88 U/mg. The molecular weight of xylanase from this fungus was 27.68 kDa. The Km and Vmax values of the purified xylanase were 0.24 mg/mL and 15.85 μmol/min/mg, respectively. The xylanase activity was moderately inhibited by Hg2+ at a concentration of 10 mM, which was different to the case of N. tatenoi KKU-CLB-3-2-4-1 where Hg2+ was a strong inhibitor. In addition, the hydrolysed birchwood xylan was obtained mailnly xylobiose, xylotriose, xylotetraose and xylopentaose as end products, suggesting that it was an endo-xylanase.

Protease from Courgette (Luffa Acutangula L (Roxb)): Isolation, Purification, and Some Characteristics

The Courgette or oyong (Luffa acutangula L. (Roxb)) is member of Cucurbitaceae mainly used as vegetable. Beside used as vegetables, courgette aslo used as keratolytic agent. This fact is supposed that this vegetables contain protease. This research is succeed to purified courgette’s protease by four step. That was precipitate by 70% ammonium sulphate saturation, purification using DEAE cellulose ion exchange chromatography and gel filtration chromatography using sephadex G-100 and G-75. Purified courgette’s protease had 81,922 U/mg for specific activity and 34 kDa molecular weight. This enzyme had the characteristic such as activated optimally at 37oC, pH 7 and 10 minute duration time. This enzyme activity can decrease by PMSF and H2O2, its remarkable that courgette protease is serine protease and had the thiol group in its structure. The ability to digest food proteins materials like boiled meat and boiled white egg by courgette protease proves that the courgette protease enzyme is could be used in enzyme replacement therapy in mild digestion problem.

Properties of arabinogalactan-proteins in European pear (Pyrus communis L.) fruits

Arabinogalactans (AGs) and arabinogalactan-proteins (AGPs) were partially purified from an extract of fruits of the European pear (Pyrus communis L.) by DEAE-cellulose ion-exchange and Sepharose 6B gel-filtration chromatography. Among 7 AG(P)-containing fractions, a neutral AGP (SE-1) was confirmed to be highly purified (Mr 67,000) and rich in L-Ara and Gal; this fraction included a small amount (2.6%, w/w) of protein and showed the highest reactivity forming precipitate with β-Glc Yariv reagent among the 7 fractions, the intensity of which was comparable to that of gum arabic, a standard AGP. Another accompanying minor low-Mr neutral AGP (SE-2; Mr approx. 7200) still contained other polysaccharide (starch fragments) and did not show Yariv reactivity. The carbohydrate moieties of SE-1 consisted of consecutive (1 → 3)-linked β-galactosyl backbone chains substituted with side chains of (1 → 6)-linked β-galactosyl residues at O-6, to which mainly single α-l-arabinofuranosyl residues were attached through O-3. This structural feature was also observed for SE-2. Successive digestion of SE-1 with α-l-arabinofuranosidase and exo-β-(1 → 3)-galactanase with the aid of endo-β-(1 → 3)-galactanase released most (more than 98%, w/w) of the carbohydrate moieties as low-Mr fragments. These consisted of free L-Ara and Gal, and a series of β-(1 → 6)-galactooligosaccharides with degree of polymerization (dp) up to at least 17, indicative of attachment of (1 → 6)-linked β-galactosyl side chains of varying length along the (1 → 3)-linked β-galactosyl backbone chains.

Exopolysaccharides from the fungal endophytic Fusarium sp. A14 isolated from Fritillaria unibracteata Hsiao et KC Hsia and their antioxidant and antiproliferation effects

Exopolysaccharides (EPSs) are high-molecular-weight carbohydrates with a wide range of biophysiological activities, such as antioxidant activity, immunostimulatory activity, antitumor activity, hepatoprotective activity, and antifatigue effects. In the present work, two water-soluble EPSs, namely, A14EPS-1 and A14EPS-2, were isolated and purified from the fungal endophytic strain A14 using ethanol precipitation, DEAE-cellulose ion exchange chromatography and Sepharose G-150 gel filtration chromatography. A14EPS-1 (∼2.4× 104Da, the major fraction) was mainly composed of mannose, rhamnose, glucose, galactose, xylose and arabinose with a molar ratio of 0.31:0.55:10.00:0.34:0.03:0.06. The major monosaccharide of A14EPS-1 was pyranose, which was connected by α-glycosidic linkages. And the side chains of A14EPS-1 may be composed of rhamnose, arabinose, glucose and galactose; moreover, the backbone of A14EPS-1 may be composed of rhamnose, xylose, arabinose and glucose. A14EPS-2 (∼0.5× 104Da) was mainly composed of mannose, rhamnose, glucose, galactose, xylose and arabinose in a ratio of 0.16:0.88:10.00:0.39:0.06:0.06. Pyranose was observed in both the α- and β-configurations in A14EPS-2, and the α configuration was dominant. In addition, the results of the bioactivity assays indicated that both A14EPS-1 and A14EPS-2 had moderate antioxidant activity invitro, and A14EPS-2 showed a moderate antiproliferation effect on human hepatocellular carcinoma HepG2 cells.

The Compositional Analysis and Antihypertensive Activity of Polysaccharides from White Mulberry Fruit

This study summarizes investigations on the structure and antihypertensive activity of polysaccharides extracted from white mulberry fruits. Polysaccharides were extracted in hot water and the purified polysaccharides were obtained following diethylaminoethyl cellulose ion exchange and Sephacryl S-100 gel filtration chromatography. The molecular weight of the polysaccharides was determined by multi-angle laser light scattering and the monosaccharide compositional analysis by gas Chromatography–Mass Spectroscopy. The antihypertensive activity of white mulberry fruit polysaccharides was assessed by inhibition of the angiotensin-converting enzyme. The results obtained indicate that these preparations consist of pure polysaccharides with a low dispersion and homogeneous molecular weight (Mw = 7.892 × 104 Da), and the main sugar composition of White mulberry fruit polysaccharides included galactose, arabinose, rhamnose, glucose, mannose and xylose with molar ratios of 5.329:3.614:1.476:1.321:1.059:1. Both galactose and arabinose represented the main components, constituting the backbone of the polysaccharide chain. Furthermore, white mulberry fruit polysaccharides exhibited a dose-dependent antihypertensive activity and angiotensin-converting enzyme inhibition. Overall, the results from this research may provide a reference for the further development of white mulberry fruit polysaccharides as a product to be used in hypertension treatment.

Investigation of antioxidant and anticancer potential of fucoidan from Sargassum polycystum

The present study was aimed to evaluate the antioxidant and anticancer potential of fucoidan isolated from Sargassum polycystum. The isolated fucoidan was successfully purified by DEAE cellulose-ion exchange chromatography and dialysis. Totally four active fractions (F1–F4) were collected and explored its chemical constitution by calorimetric assays. Among them, fraction 2 (F2) showed the higher yield percentage, fucose and sulphate content. Further, monosaccharide composition, structural and functional properties of the F2 was analyzed by HPLC, FTIR and NMR. F2 shows highest DPPH radical scavenging activity (55.94 ± 0.69%), reducing power (0.33 absorbance rate), hydrogen peroxide scavenging activity (71.76 ± 2.14%) and nitric oxide radical scavenging activity (51.81 ± 1.04%) at 1000 μg/ml. The cell viability of MCF-7 and HCT-15 cell lines was proportionate to the concentration of F2 with an estimated IC50 was 20 and 50 μg/ml respectively. The fluorescence and confocal laser scanning microscopic analysis demonstrated the apoptotic morphological changes and cell mediated death in F2 treated cancer cells. Higher amount of LDH release was found in the F2 treated cancer cells than the control group. Thus, the present finding proved that the isolated F2 encompasses significant antioxidant and anticancer property.

Purification and Characterization of Agarase from Marine Bacteria Acinetobacter sp. PS12B and Its Use for Preparing Bioactive Hydrolysate from Agarophyte Red Seaweed Gracilaria verrucosa.

Acinetobacter strain PS12B was isolated from marine sediment and was found to be a good candidate to degrade agar and produce agarase enzyme. The extracellular agarase enzyme from strain PS12B was purified by ammonium sulfate precipitation followed by DEAE-cellulose ion-exchange chromatography. The specific activity of the crude enzyme which was 1.52U increased to 45.76U, after two-stage purification, with an enzyme yield of 9.76%. Purified enzyme had a molecular mass of 24kDa. The optimum pH and temperature for activity of purified agarase were found to be 8.0 and 40°C, respectively. The Km and Vmax values for agarase were 4.69mg/ml and 0.5μmol/min, respectively. Treatment with EDTA reduced the agarase activity by 58% at 5mM concentration. The enzyme activity was stimulated by the presence of Fe2+, Mn2+, and Ca2+ ions while reducing reagents (β-mercaptoethanol and dithiothreitol, DTT) enhanced its activity by 30-40%. The purified agarase exhibited tolerance to both detergents and organic solvents. Major hydrolysis products of agar were DP4 and also a mixture of longer oligosaccharides DP6 and DP7. The enzyme hydrolysed seaweed (Gracilaria verrucosa) exhibited strong antioxidant activity in vitro. Successful hydrolysis of seaweed indicates the potential use of the enzyme to produce seaweed hydrolysate having health benefits as well as the industrial application like the production of biofuels.