Sephacryl S-200 Gel Filtration Research Articles

Identification of potent antioxidant bioactive peptides from the soluble proteins of chicken egg yolk

Background: Eggs are an excellent nutrient-dense food containing proteins, fats, carbohydrates, minerals, and vitamins. While many proteins are present in egg yolk, there arefew studies on their health benefits. Objective: The aim of this study was to explore the antioxidant peptides derived from the soluble protein fractions of egg yolk. Lipids and lipoproteins were removed with activated carbon and centrifugation after water dilution at acidic pH. Materials and Methods: Compared with 3.6 grams of protein in egg white, egg yolk contains 2.7 grams of protein in a single large egg. The egg yolk soluble protein (EYsP) was digested with pepsin for 2h at pH 3.0 (P-EYsP), followed by digestion with trypsin (P-EYsP-T), α-chymotrypsin (P-EYsP-α), or both enzymes (P-EYsP-T/α). The most active digest was fractionated using sephacryl S-100 gel filtration, followed by reversed phase-HPLC of the most active fraction. The antioxidant activities were evaluated using a superoxide anion-generating system of xanthine oxidase, DPPH-scavenging assay, and yeast cells as an oxidative-stress tolerance cellular model. Results: The intact proteins (EYsP) showed antioxidant activity, but pepsin hydrolysate (P-EYsP) exhibited greater superoxide-scavenging activities than EYsP, while P-EYsP-T, P-EYsP-α and P-EYsP-T/α lacked activities. The P-EYsP and its subsequent proteases (P-EYsP-T, P-EYsP-α and P-EYsP-T/α) exhibited significant DPPH reduction, but P-EYsP-T, P-EYsP-α and P-EYsP-T/α exhibited the strongest DPPH reduction activities. MALDI-TOF-MS analysis revealed five major antioxidant peptides, two derived from yolk glycoprotein 40 (859 Da, 883Da), two from lipovitelline (901Da, 945 Da), and one from livetin (1089 Da). The peptides exhibited potent superoxide anion as well as DPPH scavenging activities and markedly enhanced the tolerance of yeast cells against peroxide-induced oxidative stress.Conclusion: The results show that these bioactive peptides hold a fascinating opportunity for their potential as nutraceuticals in prevention and combating oxidative stress-associated diseases.Keywords: Egg yolk proteins; bioactive peptides; antioxidant; superoxide-scavenging; DPPH-reduction; yeast tolerance

Characterization, antimicrobial and antitumor activity of superoxide dismutase extracted from Egyptian honeybee venom (Apis mellifera lamarckii)

BackgroundSuperoxide dismutase is an important antioxidative stress enzyme which is found in honeybee venom and has a wide pharmaceutical and medical applications. ResultsWe reported the purification and characterization of venom SOD from Egyptian honeybee Apis mellifera lamarckii and termed BVSOD. It was purified to homogeneity from the Egyptian honeybee venom. The purification procedures included crude extraction, DEAE-cellulose anion exchange column chromatography, and Sephacryl S-300 gel filtration column chromatography. The purified BVSOD is found to be homogeneous as investigated by native PAGE. It exhibited homodimeric structure with a molecular weight of native form of 32 kDa and subunits of 16.0 kDa. It displayed the maximum activity at pH 7.4. CuCl2, ZnCl2, and MgCl2 and elevated the activity of BVSOD, while CoCl2, FeCl2, and NiCl2 inhibited BVSOD activity. Potassium cyanide and hydrogen peroxide were most potent inhibitors for BVSOD activity suggesting that it is a Cu/Zn-SOD type. ConclusionsThe purified BVSOD is found to have antimicrobial and antitumor activities which can be used for various medical and clinical applications.

Identification and Characterization of a Bacteriocin from the Newly Isolated Bacillus subtilis HD15 with Inhibitory Effects against Bacillus cereus.

Natural antimicrobial substances are needed as alternatives to synthetic antimicrobials to protect against foodborne pathogens. In this study, a bacteriocin-producing bacterium, Bacillus subtilis HD15, was isolated from doenjang, a traditional Korean fermented soybean paste. We sequenced the complete genome of B. subtilis HD15. This genome size was 4,173,431 bp with a G + C content of of 43.58%, 4,305 genes, and 4,222 protein-coding genes with predicted functions, including a subtilosin A gene cluster. The bacteriocin was purified by ammonium sulfate precipitation, Diethylaminoethanol-Sepharose chromatography, and Sephacryl gel filtration, with 12.4-fold purification and 26.2% yield, respectively. The purified protein had a molecular weight of 3.6 kDa. The N-terminal amino acid sequence showed the highest similarity to Bacillus subtilis 168 subtilosin A (78%) but only 68% similarity to B. tequilensis subtilosin proteins, indicating that the antimicrobial substance isolated from B. subtilis HD15 is a novel bacteriocin related to subtilosin A. The purified protein from B. subtilis HD15 exhibited high antimicrobial activity against Listeria monocytogenes and Bacillus cereus. It showed stable activity in the range 0-70°C and pH 2-10 and was completely inhibited by protease, proteinase K, and pronase E treatment, suggesting that it is a proteinaceous substance. These findings support the potential industrial applications of the novel bacteriocin purified from B. subtilis HD15.

Two Novel Polysaccharides From Clitocybe squamulosa: Their Isolation, Structures, and Bioactivities.

The crude polysaccharides from the fruiting bodies of Clitocybe squamulosa (CSFP) were isolated by hot-water extraction. Two novel polysaccharides, CSFP1-β and CSFP2-α, were further purified by DEAE-52 anion exchange and Sephacryl S-400 gel filtration chromatography, and the purities reached 98.44 and 97.83%, respectively. The structural characteristics and bioactivities of CSFP, CSFP1-β, and CSFP2-α were identified by the combination of chemical and instrumental analysis. Results showed that CSFP was formed by the aggregation of honeycomb spherical materials; CSFP1-β and CSFP2-α were interwoven by reticular and fibrous structures, respectively. Purified components of both CSFP1-β and CSFP2-α showed typical infrared absorption peaks of polysaccharides, and contents of nucleic acid and protein decreased significantly. Simultaneously, CSFP with a molecular weight (Mw) of 1.948 × 104 Da were composed mainly of glucose, mannose, galactose, and rhamnose. CSFP1-β was composed mainly of glucose, galactose, and mannose, while CSFP2-α was composed of glucose, and both their Mw distributions were uneven. Compared with CSFP, the antioxidant activities of CSFP1-β and CSFP2-α were significantly improved (p < 0.05), and they both showed good abilities to bind free cholesterol and bile acid salts in vitro. The binding abilities of the two compounds were found to be 68.62 and 64.43%, and 46.66 and 45.05 mg/g, respectively. CSFP, CSFP1-β, and CSFP2-α had good bacteriostatic effects with a linear increasing relationship to increasing concentration. In addition, CSFP promoted the growth of RAW264.7 cells and has potential immunomodulatory, anti-inflammatory, and anti-tumor activities.

In silico and biochemical analysis on a newly isolated Trichoderma asperellum l-asparaginase

Microbial l-asparaginase enzyme has been considered as a good source of potential anticancer drugs. The enzyme purified from different microbes has been shown different biochemical and kinetics properties. Thus, our study aims to investigate l-asparaginase from Trichoderma asperellum (MN960163), including in silico characterization of the l-asparaginase encoding genes as well as the biochemical characters of the purified enzyme. In silico, analysis of the T. asp genome database revealed the presence of six protein encoding genes with N-terminal asparaginase domain that has asparaginase production potential as indicated from their physico-chemical properties, verification of three of them was confirmed. T. asperellum l- asparaginase with a specific activity of 200 U/mg and 61.7 fold purification was achieved by ammonium sulphate precipitation (60%) followed by DEAE-sepharose ion exchange and Sephacryl S-200 gel filtration chromatography. The purified enzyme has a molecular mass of 65 kDa and exhibited its maximum activity at pH 8 and 50 °C. The purified enzyme showed high thermal stability up to 50 °C and a high affinity to l- asparagine with a Km of 0.35 mM. The activity was significantly enhanced by Cu, K and Ca ions while Fe and EDTA was completely inhibited l-asparginase activity. In conclusion, the current data highlight new possible genes that could be employed as a promising source in asparaginase production for medical purposes. Additionally, the feature of the purified asparaginase enzyme could help to elucidate the unique properties of T. asp asparaginase.

Characterization of the C-type lectin from the marine sponge (Stylissa flexibilis)

A lectin from the marine sponge Stylissa flexibilis, designated as SFL, was purified by cold ethanol precipitation followed by ion exchange chromatography on DEAE Sepharose column and Sephacryl S-200 gel filtration. SFL is a dimeric glycoprotein of 32 kDa subunits linked by a disulfide bridge with a molecular mass of 64 kDa by SDS-PAGE and 65 kDa by Sephacryl S-200 gel filtration chromatography. The lectin preferentially agglutinated enzyme treated human A erythrocytes, whereas it did not agglutinate any type of rabbit, human B and O erythrocytes, irrespective of treatment with enzymes. The hemagglutination activity of lectin was strongly inhibited by monosaccharide, D-galactose and glycoproteins, asialo-porcine stomach mucin and asialo-fetuin, indicating that lectin is specific for O-glycans. Activity of SFL was stable over a range of pH from 5 to 8, up to 60 °C for 30 min and its activity was Ca2+ dependent, indicating that SFL was belonged to the C-type lectin family and requires metal for biological activity. SFL caused agglutination of Vibrio alginolyticus and V. parahaemolyticus in a dose dependent manner and inhibited the growth rate of these bacterial strains, suggesting that the lectin caused the agglutination through binding to the target receptor(s) on the surface of Vibrios. Thus, SFL can be considered as a good source of lectin(s) being useful as carbohydrate probe and antibacterial reagent.

Purification and Biochemical Characterization of a Lipase Produced by Aspergillus Niger: Interfacial Binding, Activity of Lipase at the Lipid-Water Interface Using Monomolecular Film Techniques

Aspergillus Niger lipase (ANL) was purified from a culture medium. Pure ANL was obtained after ammonium sulfate fractionation, Sephacryl S-200 gel filtration and cation exchange chromatography (Mono-Q). The pure lipase, which is not a glycoprotein, was presented as a monomer having a molecular mass of about 99 kDa. The lipase activity was maximal at pH 8 and at 39°C. ANL hydrolyses the long chains of triacylglycerols more efficiently than the short ones. A specific activity of 3779 U/mg was measured on olive oil as substrate at 39°C and at pH 8 in the presence of 4 mM NaTDC. ANL was inactivated when the enzyme was incubated at 75°C or at pH less than 4. Natural detergent (NaTDC), pH dependence of the catalytic activity of ANL was at pH 8, which confirm that ANL present one pH profile.

Isolation of Nile crocodile (Crocodylus niloticus) serum immunoglobulin M and Y (IgM and IgY)

Crocodile immunity has not been fully characterised with more studies on crocodile innate immunity than cell-mediated or humoral immunity. Crocodile immunoglobulin genes have been described but immunoglobulin proteins have not been isolated or studied biochemically. Two large proteins proposed to be crocodile IgM and IgY were isolated and purified from Crocodylus niloticus sera using two different protocols. A 50% (w/v) ammonium sulfate and a 15% (w/v) polyethylene glycol precipitation step was followed by Cibacron blue F3GA affinity- and Sephacryl-S300 gel filtration chromatography. An alternate purification protocol, with only two steps, involved thiophilic affinity- and Sephacryl-S300 gel filtration chromatography. The purified crocodile IgM resolved on reducing SDS-PAGE with an apparent mass of 180 kDa. Purified crocodile IgY resolved at 180 kDa alongside chicken IgY on a non-reducing SDS-PAGE gel, and is deduced to consist of two 66 kDa heavy and two 23 kDa light chains under reducing conditions. The thiophilic/gel filtration two-step protocol gave three-fold higher yields of isolated protein than the four-step precipitation/chromatography protocol. Antibodies against the isolated crocodile IgM and IgY were raised in chickens and affinity purified. The chicken antibodies differentiated between crocodile IgM and IgY and have the potential for use in the diagnosis of crocodile infections. The purified crocodile antibodies can be biochemically characterised and compared to mammalian and avian antibodies to give a better understanding of crocodile humoral immunity.

Biodegradation of polyhydroxybutyrate by Pseudomonas sp. DSDY0501 and purification and characterization of polyhydroxybutyrate depolymerase.

Strain DSDY0501 with polyhydroxybutyrate (PHB)-degrading ability was isolated from activated sludge. The morphological, physiological, and biochemical properties of the strain and phylogenetic analysis indicated that the strain belongs to Pseudomonas sp. The strain used PHB as the sole carbon source and degraded PHB films completely in 21h in liquid culture. An extracellular PHB depolymerase was purified from the supernatant of the culture by ultrafiltration and Sephacryl S-200 gel filtration. The specific activity of the purified enzyme increased 24.2-fold, and the recovery yield was 16.61%. Extracellular PHB depolymerase, a monomeric enzyme with a molecular weight of approximately 57.9kDa, showed optimum activity at 60°C and pH 9.0, and was stable in the temperature range of 10-60°C and a pH range of 6.0-10.0. The secondary structure of the enzyme contained approximately 60% α-helix and 40% β-pleated sheet according to the circular dichroism spectrum. Mass spectrum analysis showed that the main degradation product of the enzyme was PHB monomer, indicating the exo-type action of this PHB depolymerase.

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.

Molecular Mechanism of Apoptosis Signal‐Regulating Kinase I Oligomerization and Auto‐activation

Apoptosis Signal-Regulating Kinase I (ASK1) is a member of the Mitogen Activated Protein Kinase (MAPK) pathway. Responding to various stimulations, ASK1 activates four MAPK kinase (MKK) substrates including MKK4/7 and MKK3/7, which phosphorylate the c – Jun N terminal Kinase (JNK) and p38, respectively. The dysfunction and misregulation of ASK1 lead to various diseases such as cancers, cardiovascular diseases, neurodegenerative disorders, inflammatory diseases and diabetes. The activation of ASK1 is highly regulated by many regulatory factors. It is widely believed that the oligomerization of ASK1 is essential for its activation. However, the molecular mechanism of ASK1 activation largely remains unclear. We recently found out that the purified ASK1 is capable of auto-activating itself and it exists as a huge oligomer instead of a homodimer as previous studies suggested. To explore the mechanism of ASK1 oligomerization and auto-activation, we have generated a series of ASK1 truncates and hoping to identify the responsible domains/motifs for its oligomerization and auto-activation. We have purified two ASK1 truncates - ASK1-NK (ASK1 protein expressing N terminal and Kinase domains) and ASK1-K (ASK1 protein expressing Kinase domain only). After performing gel filtration, it was found out that ASK1-NK forms monomer and ASK1-K forms dimer in native state. We are currently developing protocols of kinase assay for different ASK1 truncates to analyze its auto-activation mechanism. In conclusion, this study will help us to unveil the detailed mechanism of ASK1 oligomerization which will surely enrich our knowledge in understanding the activation process of ASK1. Support or Funding Information This project is supported by Faculty Research Grant to Xuanzhi Zhan and Student Research Grant to Adua Rahman awarded by Tennessee Technological University. ASK1 mediated pathway Purification of ASK1 truncates. (A) ASK1-K domain was purified using Heparin and Q-sepharose column chromatography. The purified ASK1-K domain (~2μg) is shown in SDS-PAGE; (B) ASK1-NK domain was purified using Q – Sepharose and Phenyl – Sepharose column chromatography. The purified ASK1-NK domain (~2μg) is shown in SDS-PAGE. Sephacryl gel filtration chromatography of ASK1 truncates. (A) ASK1-NK is observed as monomer (116KDa). (B) Predominant form of ASK1-K is dimer (77 KDa). (C) The chromatogram of gel filtration standard and log of molecular weight vs. elution volume plot is generated to determine the molecular weights of the ASK1 truncates. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Purification and characterization of a novel high molecular weight alkaline protease produced by an endophytic Bacillus halotolerans strain CT2

A protease-producing strain CT2 isolated from Tunisian potatoes, exhibiting a potent protease activity (prot CT2), was identified as Bacillus halotolerans according to 16S ribosomal DNA sequence analysis. Maximum prot CT2 production was obtained in medium supplemented with bean seed proteins. Proteolytic activity was purified by ammonium sulphate precipitation, Sephacryl S-200 gel filtration and SP-sepharose cation-exchange chromatography. Optimal enzyme activity was reached at pH 9 and temperature of 50 °C. Proteolytic activity was enhanced by Ca2+ and Mn2+ ions, completely inhibited by PMSF suggesting a serine protease nature and exhibited high stability in the presence of commercial detergents. Prot CT2 showed broad substrate specificity towards both synthetic and natural substrates, with a high capacity to hydrolyze legume seed proteins. Using electrophoretic analysis, its molecular weight was around 250 kDa with two major subunit showing important homologies with serine proteases belonging to the subtilisin-like serine proteases. Based on the Lineweaver–Burk plots Km and Vmax values were 10 mg/ml and 50,000 U/mg respectively. This newly described prot CT2 displays relevant properties which highlight its potential use in various industrial and biotechnological applications.

Purification and thermal denaturation kinetics of serum albumin in bactrian camel milk

Serum albumin is a non-specific transporter protein. It is the 2nd most abundant whey protein present in camel milk, with significantly higher content than in milk from other animals. In this study, we isolated and purified camel serum albumin (CSA) from milk by DEAE-Sepharose FF and Sephacryl S-100 gel filtration chromatography and checked its purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ultra-performance liquid chromatography (UPLC). The main band of the purified product on SDS-PAGE corresponded with the location of a bovine serum albumin standard. In UPLC, the purified product presented a single sharp peak, which indicated it was a homogenous preparation. Amino acid sequence analysis of the purified protein confirmed it was CSA. Subsequently, we analysed the thermal denaturation of CSA from 70 to 95°C for 10, 20, 30, 40 and 50 min. CSA had upper stability under 75°C, the residual rate range over 70.25% to 87.12%. The degree of denaturation increased with temperature and time, but small range of fluctuations between denaturation rate. The residual rate of CSA, heated 10 min at 95°C, was about 58.34%. After CSA was heated 50 min at 95°C, it was not entirely denatured. These results suggested that CSA could maintain high stability in dairy products treated by low-temperature long-time or high-temperature short-time heating treatment and the reaction order of heat denaturation was 1.9. This study provides reference and application data for further research on CSA.

Purification and Characterization of Active Aggregates of an Organic Solvent Tolerant Lipase from Marinobacter sp. EMB5

Background: Marinobacter is a halophilic bacterial genus and a bio-geochemically important group in sea water, is generally associated with the bioremediation of hydrocarbon-rich effluents released into the salt-rich environment by various anthropogenic activities. It is a well-known lipase producer-the NCBI Protein database shows nearly 520 Marinobacter lipases. In this context, its lipase and esterase are considered important. However, their enzymatic properties in purified form have been scantily studied. Since hydrocarbons are acted upon on solvent water interface, the stability and catalytic behavior of Marinobacter lipase in non-aqueous media needs to be more specifically investigated. Aim: Marinobacter sp. EMB5 was isolated from Kozhikode, Kerala, India. It was found to be potent lipase producer. The aim of the present work was to obtain a purified preparation and undertake its detailed characterization with respect to above aspects. Work: The crude Marinobacter sp. lipase was purified by Sephacryl S200 gel filtration chromatography. The lipase eluted as a single activity peak in void volume and had a very high molecular mass. Aggregation has not been reported in case of Marinobacter lipase, so far, although it is known in other lipases. The aggregated lipase was mixed with 2-propanol (70%, v/v) and disaggregated lipase was obtained as a single activity peak in Sephacryl S200 gel filtration chromatography corresponding to 82 kDa molecular mass. This monomeric species was less active than aggregated purified preparation. Hence, further characterization and studies were carried out on purified active aggregate lipase. It was stable in a wide range of organic solvents at high concentration up to 24 h. The organic solvent stability was further investigated by Far UV CD spectroscopy and fluorescence spectroscopy. Conclusion: Due to the fact that Marinobacter sp. EMB5 lipase remained stable in organic solvents for prolonged incubation periods, it has potential to be used in industrial organic synthesis.

Purification of Endoxylanase from Bacillus pumilus B20 for Production of Prebiotic Xylooligosaccharide Syrup; An In vitro Study.

Background: The need for more cost-effective compounds is imperative because the demand for prebiotic compounds is ever on the rise. Objective: The focus of this study is the purification of the endoxylanase from Bacillus pumilus B20 and its application in a cost-effective production of the prebiotic xylooligosaccharide (XOS) syrup having a high concentration of oligosaccharides. Materials and Methods: The extracellular endoxylanase was purified using ammonium sulphate fractionation, DEAE anion exchange, and Sephacryl gel filtration chromatography. The enzymatically produced XOS was used in the preparation of XOS syrup adopting the method of ultrafiltration with 10 and 3 kDa molecular weight cut-off (MWCO) membranes. Culture-dependent technique for the bacterial enumeration using selective probiotic microorganisms in an in vitro analysis was employed to confirm the prebiotic nature of XOS syrup. Results: The molecular mass of the purified xylanase (XylB) was found to be approximately 85 kDa with the optimum pH and temperature of 6.5 and 60 °C, respectively. XylB hydrolyzed the xylan and produced short-chain xylooligosaccharides (XOS). At the end of the two-step ultrafiltration process, the hydrolysate was refi ned to form XOS syrup (44.4%) consisting of XOS with a degree of polymerization (DP) between 2 and 5, and >5. Among all the tested probiotic strains, Lactobacillus brevis exhibited maximum growth in the presence of 0.5% XOS syrup with a specific growth rate of 1.2 h-1. Conclusion: Through this study, we have identified a method to produce XOS syrup that can be used as an effective prebiotic supplement for the growth of several probiotic strains. Human gut probiotics was used as a model system for in vitro analysis of prebiotic oligosaccharide XOS, but for further confirmation of the prebiotic activity, in vivo feeding studies using animal models are needed to be carried out.

Peroxidase characterization isolated from germinated peanut embryos (GPE) and application of the freeze-dried GPE powder as enzyme source for biomimetic production of δ-viniferin

Novel enzyme source is essential in biomimetic synthesis. Use of peanut peroxidase in mediation of oxidative polymerization of phenolic compounds is meager. In this study, the 5 day-germinated peanut embryos were homogenized, centrifuged to collect supernatant and (NH4)2SO4 fractionated, the 40–75% saturated fraction exhibited most peroxidase activities. After UF-concentration between MWCO 300 and 10 kDa, Sephacryl S300 gel filtration and DEAE-Sephadex A25 ion-exchange, a 50.6-fold-purified peroxidase with 32 kDa in SDS-PAGE analysis was obtained. The freeze-dried and pulverized into powder (GPEP) bearing potent peroxidase activities was used in biomimetic resveratrol dimerization. In a semi-continuous system mediated by 0.5 g GPEP suspended in phosphate buffer (pH 7.4) with continuous feedings of resveratrol dissolved in acetone and aqueous H2O2 solution for 150 min, all added resveratrol reacted. After subjecting the reactant to SPE C18 cartridge clean-up, semi-HPLC purification and NMR 1- and 2D spectroscopic elucidation, a dimeric product was identified as δ-viniferin with ca. 60% of recovery. The δ-viniferin exhibited potent trolox equivalent antioxidant capacity as did BHT. It is of merit to demonstrate GPEP as a potential peroxidase source enabling scalable biomimetic production of δ-viniferin.

Purification, characterization and biological effect of lectin from the marine sponge Stylissa flexibilis (Lévi, 1961).

SFL, a lectin from the marine sponge Stylissa flexibilis was purified by cold ethanol precipitation followed by ion exchange chromatography on DEAE Sepharose column and Sephacryl S-200 gel filtration. SFL is a dimeric glycoprotein of 32kDa subunits linked by a disulfide bridge with a molecular mass of 64kDa by SDS-PAGE and 65kDa by Sephacryl S-200 gel filtration. SFL preferentially agglutinated enzyme treated human A erythrocytes. The activity of lectin was strongly inhibited by monosaccharide d-galactose and glycoproteins asialo-porcine stomach mucin and asialo-fetuin. The lectin was Ca2+ dependent, stable over a range of pH from 5 to 8, and up to 60°C for 30min. The N-terminal amino acid sequence of SFL was also determined and a blast search on amino acid sequences revealed that the protein showed similarity only with lectins from the marine sponge Spheciospongia vesparia. SFL caused agglutination of Vibrio alginolyticus and V. parahaemolyticus in a dose dependent manner and inhibited the growth rates of the virulent bacterial strains. Growth inhibition of V. alginolyticus and V. parahaemolyticus with SFL was not observed in the presence of d-galactose or asialo-porcine stomach mucin, suggesting that the lectin caused the agglutination through binding to the target receptor(s) on the surface of Vibrios. Thus, the marine sponge S. flexibilis could promise to be a good source of a lectin(s) that may be useful as a carbohydrate probe and an antibacterial reagent.

Purification and characterization of two novel peroxidases from the dye-decolorizing fungus Bjerkandera adusta strain CX-9

Two extracellular peroxidases from Bjerkandera adusta strain CX-9, namely a lignin peroxidase (called LiP BA45) and manganese peroxidase (called MnP BA30), were purified simultaneously by applying successively, ammonium sulfate precipitation-dialysis, Mono-S Sepharose anion-exchange and Sephacryl S-200 gel filtration and biochemically characterized. The sequence of their NH2-terminal amino acid residues showed high homology with those of fungi peroxidases. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI–TOF/MS) analysis revealed that the purified enzymes MnP BA30 and LiP BA45 were a monomers with a molecular masses 30125.16 and 45221.10Da, respectively. While MnP BA30 was optimally active at pH 3 and 70°C, LiP BA45 showed optimum activity at pH 4 and 50°C. The two enzymes were inhibited by sodium azide and potassium cyanide, suggesting the presence of heme-components in their tertiary structures. The Km and Vmax for LiP BA45 toward 2,4-Dichlorolphenol (2,4-DCP) were 0.099mM and 9.12U/mg, respectively and for MnP BA30 toward 2,6-Dimethylphenol (2,6-DMP), they were 0.151mM and 18.60U/mg, respectively. Interestingly, MnP BA30 and LiP BA45 demonstrated higher catalytic efficiency than that of other tested peroxidases (MnP, LiP, HaP4, and LiP-SN) and marked organic solvent-stability and dye-decolorization efficiency. Data suggest that these peroxidases may be considered as potential candidates for future applications in distaining synthetic-dyes.

Interaction of tubulin and protein kinase CK2 in Trypanosoma equiperdum.

A polypeptide band with an apparent molecular weight of 55,000 was phosphorylated in vitro in whole-cell lysates of Trypanosoma equiperdum. This band corresponds to tubulin as demonstrated by immunoprecipitation of the phosphorylated polypeptide from T. equiperdum extracts when anti-α and anti-β tubulin monoclonal antibodies were employed. A parasite protein kinase CK2 was in charge of modifying tubulin given that common mammalian CK2 inhibitors such as emodin and GTP, hindered the phosphorylation of tubulin and exogenously added casein. Interestingly, a divalent cation-dependent translocation of the T. equiperdum tubulin and the CK2 responsible for its phosphorylation was noticed, suggesting a direct interaction between these two proteins. Additionally, this fraction of tubulin and its kinase coeluted using separations based on parameters as different as charge (DEAE-Sepharose anion-exchange chromatography) and size (Sephacryl S-300 gel filtration chromatography). Analyses by non-denaturing polyacrylamide gel electrophoresis and immunoblot of the purified and radioactively labeled fraction containing both tubulin and the CK2 enzyme, established the phosphorylation of a single band that was recognized by anti-CK2 α-subunit and anti-tubulin antibodies. All these findings revealed a physical association between a pool of tubulin and a CK2 in T. equiperdum.

Lactobacillus brevis Lipase: Purification, Immobilization onto Magnetic Florosil NPs, Characterization and Application as a Detergent Additive

Abstract In this study, a thermo-tolerant and alkaline lipase enzyme was purified from Lactobacillus brevis and immobilized onto modified γ-Fe3O4 florisil nanoparticles (γ-Fe3O4 MF NFs) and the usability of free lipase (FL) and immobilized lipases (IML) as detergent additives was investigated. Lipase enzyme was purified by fractional precipitation using 20% ammonium sulfate, DEAE-Sephadex ion-exchange chromatographic column, and Sephacryl S200 gel filtration chromatographic techniques. Then, the enzyme was purified, which resulted in 135.2-fold purification. Its molecular mass was determined to be 57 kDa by SDS-PAGE. The covalent immobilization of purified lipase was done using γ-Fe3O4 MF NPs. γ-Fe3O4 MF NPs and IML were characterized by using SEM, TEM, FT-IR, and XRD. IML showed a good thermo-stability and its activities were calculated as 80% at 60°C. The free and IML enzymes were most stable at alkaline pHs in the range of 7.0–10.0. Also, IML is more stable towards metal ions compared to free lipase enzyme. Washing performances of some detergent formulations were investigated in the presence and absence of Lipase. Olive oil was removed by the detergent alone and by the detergent and IML at ratios of 45% and 72%, respectively. The study on removal of oil stain from cotton cloths indicated that the removal of oil was superior in the presence of IML and IML with detergent, when compared to the detergent alone.