JOURNAL OF FOOD BIOCHEMISTRY, Issue 5 2010
AJAY PAL
ABSTRACT Bacteriocin from Weissella paramesenteroides DFR-8 isolated from cucumber (Cucumis sativus) was purified by using only two steps, viz., pH-mediated cell adsorption,desorption method and gel permeation chromatography. A single peak observed in the purity check by analytical Reverse Phase-High Performance Liquid Chromatography (Waters 600 analytical HPLC system, Milford, MA) and a single band (molecular weight,3.74 kDa) shown on SDS-PAGE analysis strongly indicated the homogeneity of the bacteriocin preparation. Treatment with proteolytic enzymes abolished the antimicrobial activity indicating the proteinaceous nature of bacteriocin. The purified bacteriocin exhibited a broad inhibitory spectrum against foodborne pathogens and spoilage microorganisms, including gram-negative bacteria such as Salmonella typhimurium, Vibrio parahaemolyticus, Aeromonas hydrophila and Listeria monocytogenes. Response surface methodology was employed to study the interactive effect of temperature and pH on bacteriocin activity, and a regression equation was developed. The bacteriocin retained full activity after storage at,20C for 90 days, while partial and complete activity loss was observed when stored at 4 and 37C, respectively. PRACTICAL APPLICATION In recent years, bacteriocins of lactic acid bacteria have gained much attention as food biopreservatives because of their origin from generally regarded as safe organisms. In spite of various bacteriocins studied worldwide, studies on bacteriocins of Weissella paramesenteroides remain rare. The present work involves the purification of bacteriocin up to absolute homogeneity from W. paramesenteroides, an isolate first time reported from cucumber (Cucumis sativus). The purified bacteriocin (molecular weight ,3.74 kDa) was found to inhibit a large number of foodborne pathogens, including Listeria monocytogenes, which is resistant to commercially available bacteriocin, i.e., nisin. The application of central composite rotatable design enabled us to design a regression equation from which the residual activity of bacteriocin can be predicted at any given conditions of temperature and pH within the experimental domain. The broad inhibitory spectrum and thermostability of bacteriocin suggest its potential application in food preservation. [source]

PURIFICATION AND CHARACTERIZATION OF ,-CARRAGEENASE FROM MARINE BACTERIUM MUTANT STRAIN PSEUDOALTEROMONAS SP.

JOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2010
AJ5-13 AND ITS DEGRADED PRODUCTS
ABSTRACT A ,-carrageenan-degrading bacterial strain AJ5 isolated from the intestine of Apostichopus japonicus was identified as Pseudoalteromonas sp. based on the phenotypic characters and 16S rRNA gene sequencing. The mutant Pseudoalteromonas sp. AJ5-13 with ,-carrageenase activity of 61 U/mg protein was obtained from Pseudoalteromonas sp. AJ5 using mutagenesis technique. An extracellular ,-carrageenase was purified from Pseudoalteromonas sp. AJ5-13 cultural supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex G-200) and cation-exchange chromatography (CM-cellulose 52). The purified enzyme yielded a single band on SDS-PAGE with the molecular mass of 35 kDa. Data of the N-terminal amino acid sequence indicated that this protein might be a novel ,-carrageenase. The pI and Km of the enzyme were 8.5 and 9.8 ± 0.2 mg/mL, respectively. The enzyme exhibited maximal activity at pH 8.0 and 55C. It hydrolyzed the ,-1, 4-glycosidic linkages of ,-carrageenan yielding ,-neocarrabiose, -tetraose, -hexaose, -octaose and -decaose sulfates as the main end-products. PRACTICAL APPLICATIONS ,-Carrageenases degrade ,-carrageenan by hydrolyzing the ,-1,4 linkages to a series of oligosaccharides. Thus, it is expected that like other ,-carrageenases, the ,-carrageenase isolated from Pseudoalteromonas sp. AJ5-13 would also be useful in seaweed biotechnology, pharmacy and immunology. ,-Carrageenases can be applied to study the composition and structure of carrageenans from different red alga, and to study the bacterial ,-carrageenan metabolism. They also provide the opportunity to investigate the structure-function relationship of the hydrolases that degrade self-associating sulfated polysaccharides. Examples of the practical applications of ,-carrageenases include their use in degrading the cell walls of seaweeds to obtain protoplasts, and in hydrolyzing ,-carrageenan to produce oligosaccharides. ,-Carrageenan-oligosaccharides have various potential biological properties, such as antiviral, antitumor, antioxidant activities, cytoprotection, immunomodulation, etc. [source]

HIGH YIELD PURIFICATION OF DEXTRANSUCRASE FROM LEUCONOSTOC MESENTEROIDES NRRL B-512F BY PHASE PARTITIONING

JOURNAL OF FOOD BIOCHEMISTRY, Issue 1 2006
MANISHA NIGAM
ABSTRACT The extracellular dextransucrase (sucrose: 1,6-,-D-glucan 6-,-D-glucosyltransferase EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F was purified by phase partitioning using poly(ethylene) glycol (PEG) and dextran generated by dextransucrase. Three steps of repeated phase partitioning by PEG 6000 and PEG 400 showed that the purification of dextransucrase by PEG 6000 was much greater than that obtained by PEG 400. Both the specific activity of 42.1 U/mg and the overall yield of 84% of dextransucrase activity obtained after the third step of phase partitioning by PEG 6000 were significantly higher than that of 23.8 U/mg and 46% overall yield, respectively, by PEG 400 or of that previously reported. The successive three steps, two-phase partitioning with a final concentration of 5% PEG 6000, reproducibly yielded a homogeneous preparation of dextransucrase. [source]

PURIFICATION AND PROPERTIES OF CYCLODEXTRIN GLUCANOTRANSFERASE FROM AN ALKALOPHILIC BACILLUS sp.

JOURNAL OF FOOD BIOCHEMISTRY, Issue 6 2004

ABSTRACT Cyclodextrin glucanotransferases (EC 2.4.1.19) (CGTase) are industrially important enzymes for production of cyclodextrin (CD) from starch. ,-CD yield of CGTase from alkalophilic Bacillus species is usually much lower than ,-CD, while from alkalophilic Bacillus sp. 7-12. ,-CD yield is close to ,-CD. A CGTase from alkalophilic Bacillus sp. 7-12 was purified and characterized. When purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography and Sepharose CL-6B column chromatography, the enzyme obtained consisted of a single band that did not dissociate into subunits by SDS polyacrylamide gel electrophoresis. Molecular weight of the purified enzyme was determined to be 69,000 Da by SDS-PAGE. The enzyme showed a Kmof 1.24 mg/mL and Vmax0.101 µM/min when potato starch was used as substrate. The enzyme was stable below 70C with an optimum activity at 60C, and stable at pH range 6,10 with an optimum pH at 8.5. The enzyme activity was strongly inhibited by Ag+, Cu2+, Mg2+, Al3+, Co2+, Zn2+, Fe2+and slightly inhibited by Sn2+, Mn2+. The ions Ca2+and K+, EDTA and DTT had no influence on the enzyme activity. [source]

PARTIAL PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE TRYPSIN FROM PYLORIC CAECA OF TAMBAQUI (COLOSSOMA MACROPOMUM)

JOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2001
RANILSON S. BEZERRA
ABSTRACT A 38.5 kDa alkaline protease from pyloric caeca of tambaqui (Colossoma macropomumj, a tropical freshwater fish, was partially purified in three steps: thermal treatment (45Cfor 30 min), salting-out (ammonium sulfate at 40,80% of saturation) and gel filtration (Sephadex G-75), The purification and yield were 51.2-fold and 40%, respectively. The effects of pH, temperature, inhibitors, and substrates on proteolytic activities of partially purified enzyme were investigated. The optimum pH was 9.5, while the optimum temperature was 60C. This alkaline proteolytic activity remained unaltered after 30 min incubation at 55C. Active site inhibition provided additional evidence that this activity is attributed to a trypsin-like enzyme. [source]

PARTIAL PURIFICATION AND CHARACTERIZATION OF NEUTRAL TREHALASE FROM COMMERCIAL BAKER'S YEAST, SACCHAROMYCES CEREVISIAE

JOURNAL OF FOOD BIOCHEMISTRY, Issue 6 2000
SANIYE YARAR
ABSTRACT The neutral trehalase of a commercial baker's yeast (S. cerevisiae) strain has been partially purified using ammonium sidfate fractionation and DEAE-cellulose column chromatography techniques. Trehalase was precipitated between 35,50% ammonium sulfate saturation and approximately 5,8 fold purification was achieved. The yeast cAMP-dependent protein kinase was also precipitated in the same fraction and these two proteins were separated by DEAE-cellulose column chromatography. Trehalase became totally inactive after ion exchange chromatography, "cryptic trehalase" (tre-c), but was later activated with the addition of partially purified protem kinase together with cAMP and ATP. A 215 fold purification was obtained after DEAE-ceUulose column chromatography. One mM EDTA caused complete inhibition of the enzyme in crude extract, however the inhibition levels in ammonium sulfate and DEAE-cellulose fractions were 73.5% and 50%, respectively. Optimal pH range and temperature of the enzyme were determined as pH 6,6.8 and 30C, respectively. The kinetic parameters, Km and Vmax, were estimated as 11.78 mM trehalose and 12.47 ,mole glucose/min-mg protein, respectively. [source]

CLARIFICATION AND PURIFICATION OF AQUEOUS STEVIA EXTRACT USING MEMBRANE SEPARATION PROCESS

JOURNAL OF FOOD PROCESS ENGINEERING, Issue 3 2009
M.H.M. REIS
ABSTRACT Stevia rebaudiana Bertoni is a native plant from South America and its active constituents have been considered the "sweeteners of the future."Stevia is a natural diet-sweetening source, safe to health and without calories. However, the obtained raw extract is foul smelling, bitter tasting, dark brown colored, and presents suspension matter due to organic and inorganic compounds. Therefore, further purification/clarification is essential in order to get a product of commercial quality. In this work ceramic membranes were applied in the stevia extract clarification process. The process was carried out under different membrane pore sizes and at different pressure values. The best clarification result was obtained with the membrane of 0.1 µm at 4 bar. On the other hand, the best condition for the flux was obtained with the membrane of 0.2 µm at 6 bar. The process with all the tested membranes and conditions achieved recovery of sweeteners higher than 90%. Finally, a filtration mathematical model was applied to describe the flux behavior, showing that the main fouling phenomenon during the process occurred because of the complete blocking of pores. PRACTICAL APPLICATION Stevia is the world's only all-natural sweetener with zero calories, zero carbohydrates and a zero glycemic index. However, the obtained stevia extract has a dark brown appearance, mainly because of the presence of impurities. In this work the membrane separation process was studied for stevia extract clarification and purification in order to get a product with higher commercial acceptability. The obtained results showed that total clarification and recuperation of sweeteners was almost achieved. Nonetheless, membrane fouling is an inevitable problem during membrane filtration. The mathematical analysis of the fouling occurrences showed that the complete blocking of pores is the main cause for the membrane permeability decrease. [source]

PURIFICATION OF AMYLASE FROM TILAPIA BY MAGNETIC PARTICLE

JOURNAL OF FOOD PROCESSING AND PRESERVATION, Issue 1 2010
MING CHANG WU
ABSTRACT Recent development in magnetic carrier technology involves the use of nonmagnetic substrates attached to superparamagnetic particles forming functionally modified magnetic support to isolate a particular enzyme in a controllable magnetic field. In this study, the superparamagnetic particles were modified by epichlorohydrin and other agents to cross-link with starch to form the purification support. This affinity support was able to bind the amylase and was used in the purification of amylase from Taiwan tilapia. After ammonium sulfate precipitation of amylase from Taiwan tilapia, the modified superparamagnetic particles were able to purify the crude amylase by 20.78-fold with recovery of activity of 75.6%. The molecular weight of the amylase was estimated to be 66.1 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both crude and purified amylase reached an optimum at a pH of 8.0 and temperature of 50C, and the enzyme was stable between 20 and 50C. PRACTICAL APPLICATIONS Because of the rapid development of high technology such as carrier supports for enzyme purification, the development, research and application of magnetic carriers are timely needed. The present study demonstrated that the affinity superparamagnetic particles could be used as a carrier support to absorb and purify the amylase and that technology of affinity purification can be widely used in protein purification. Compared with the traditional chromatography used in the purification of proteins, this novel affinity superparamagnetic particle technology is rapid, has low operation cost, requires simple facilities, and involves easy separation and recovery of the enzymes. [source]

PURIFICATION AND CHARACTERIZATION OF A LECTIN, BRYOHEALIN, INVOLVED IN THE PROTOPLAST FORMATION OF A MARINE GREEN ALGA BRYOPSIS PLUMOSA (CHLOROPHYTA) ,

JOURNAL OF PHYCOLOGY, Issue 1 2006
Gwang Hoon Kim
When the coenocytic green alga Bryopsis plumosa (Huds.) Ag. was cut open and the cell contents were expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. Aggregation of cell organelles in seawater was mediated by a lectin,carbohydrate complementary system. Two sugars, N -acetyl- d -glucosamine and N -acetyl- d -galactosamine inhibited aggregation of cell organelles. The presence of these sugars on the surface of chloroplasts was verified with their complementary fluorescein isothiacyanate-labeled lectins. An agglutination assay using human erythrocytes showed the presence of lectins specific for N -acetyl- d -galactosamine and N -acetyl- d -glucosamine in the crude extract. One-step column purification using N -acetyl- d -glucosamine-agarose affinity chromatography yielded a homogeneous protein. The protein agglutinated the cell organelles of B. plumosa, and its agglutinating activity was inhibited by the above sugars. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results showed that this protein might be composed of two identical subunits cross-linked by two disulfide bridges. Enzyme and chemical deglycosylation experiments showed that this protein is deficient in glycosylation. The molecular weight was determined as 53.8 kDa by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The N-terminal 15 amino acid sequence of the lectin was Ser,Asp,Leu,Pro,Thr,X,Asp,Phe,Phe,His,Ile,Pro,Glu,Arg,Tyr, and showed no sequence homology to those of other reported proteins. These results suggest that this lectin belongs to a new class of lectins. We named this novel lectin from B. plumosa"bryohealin." [source]

Purification and crystal growth of TlBr for application as a radiation detector

CRYSTAL RESEARCH AND TECHNOLOGY, Issue 10 2004
I. B. Oliveira
Abstract Thallium bromide is a semiconductor compound with high atomic number and density. It has a CsCl-type simple cubic crystal structure and it is non-hygroscopics. The TlBr crystals are relatively soft with a knoop hardness number of 12. In this work, the TlBr commercial powder was purified by zone refining and the purest material section was used for crystal growth by Bridgman method. Efforts have been concentrated on the purification of the TlBr. The purification efficiency has been evaluated (NAA and ICP-MS) by impurities reduction results after zone refining passes. The crystalline quality was evaluated by X-ray diffraction. The characterized TlBr crystal as a detector has shown good response to gamma radiation. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source]

Purification and cDNA Cloning of Lysozyme II from Cabbage Butterfly, Artogeia rapae Larvae

ENTOMOLOGICAL RESEARCH, Issue 4 2005
BANG In Seok
ABSTRACT Last instar larvae of cabbage butterfly Artogeia rapae respond to injection of bacteria with a set of inducible antibacterial peptides/proteins. The inducible peptides/proteins are related to the known hinnavins (I and II) and lysozymes (I and II). The lysozyme II has been isolated by heat treatment, cation exchange, and reversed-phase chromatography from immunized hemolymph of last instar larvae. The lysozyme II gene of A. rapae was isolated and its nucleotide sequence was determined by the RACE-PCR from immunized fat body with E. coli. It has an open reading frame of 414 bp nucleotide corresponding to 138 amino acids including an 18 amino acid signal sequence. The molecular weight and the isoelectric point of Artogeia lysozyme II without a signal peptide were 13,649.38 Da and 9.11, respectively. It is great similarity with Manduca lysozyme among other lepidopteran. [source]

Purification and Characterization of Acid Phosphatase from the Egg of the Lady Beetle, Harmonia axyridis (Coccinellidae: Coleoptera)

ENTOMOLOGICAL RESEARCH, Issue 1 2004
Jun Hyuk LEE
ABSTRACT Acid phosphatase (AP) in the egg of the lady beetle, Harmonia axyridis, was purified and characterized. Ammonium sulfate precipitation, CM column and isoelectrofocusing (IEF) were applied to purify an estimated molecular weight of 66 kDa AP. The purity was checked by SDS PAGE, native PAGE and Western blot. AP was detected in the hemolymph of the female and the egg, but not in the male on the blotting. Km of AP for a substrate, p -nitrophenyl phosphate (p -NPP), was 1.64 x 10 -4 M. AP had the optimum enzymatic activity at pH 3.5. In inhibition tests performed with various chemicals, ammonium molybdate suppressed 99% of the enzyme activity of AP even at the concentration of 5 x 10 -4 mM. AP was stable up to 50°C. [source]

Extraction, purification and characterization of wax from flax (Linum usitatissimum) straw

EUROPEAN JOURNAL OF LIPID SCIENCE AND TECHNOLOGY, Issue 7 2009
Yasantha Athukorala
Abstract The chemical composition and selected physical parameters of wax extracted from flax straw with supercritical CO2 (SC-CO2) and hexane have been determined. From the GC/MS results, clear variations in composition and component distributions were observed between SC-CO2 - and hexane-extracted samples. The major components of the SC-CO2 and hexane extracts from three flax cultivars were: fatty acids (36,49%), fatty alcohols (20,26%), aldehydes (10,14%), wax esters (5,12%), sterols (7,9%) and alkanes (4,5%). Purification of SC-CO2 -extracted wax with silica gel chromatography yielded 0.4,0.5% (dry matter) and was composed primarily of wax esters (C44, C46 and C48) and alkanes (C27, C29 and C31). UV-Vis scans of the purified wax samples exhibited two main peaks indicating the presence of conjugated dienes and carotenoids or related compounds. Fourier transform infrared results showed prominent peaks at 2918 (-C-H), 2849 (-C-H), 1745 (-C=O), 1462 (-C-H), 1169 (-C-O) and 719,cm,1 (-(CH2)n -), with NorLin wax showing a slightly deviating pattern compared to the other samples. Thermal analysis by differential scanning calorimetry revealed a mean melting point of 55,56,°C and oxidation temperatures of 146,153,°C for purified wax from flax straw processed using different procedures. [source]

Multilayer Amorphous-Si-B-C-N/,-Al2O3/,-Al2O3 Membranes for Hydrogen Purification,,

ADVANCED ENGINEERING MATERIALS, Issue 6 2010
Ravi Mohan Prasad
Abstract The hydrogen and carbon monoxide separation is an important step in the hydrogen production process. If H2 can be selectively removed from the product side during hydrogen production in membrane reactors, then it would be possible to achieve complete CO conversion in a single-step under high temperature conditions. In the present work, the multilayer amorphous-Si-B-C-N/,-Al2O3/,-Al2O3 membranes with gradient porosity have been realized and assessed with respect to the thermal stability, geometry of pore space and H2/CO permeance. The ,-Al2O3 support has a bimodal pore-size distribution of about 0.64 and 0.045 µm being macroporous and the intermediate ,-Al2O3 layer,deposited from boehmite colloidal dispersion,has an average pore-size of 8,nm being mesoporous. The results obtained by the N2 -adsorption method indicate a decrease in the volume of micropores,0.35 vs. 0.75,cm3,g,1,and a smaller pore size ,6.8 vs. 7.4 Å,in membranes with the intermediate mesoporous ,-Al2O3 layer if compared to those without. The three times Si-B-C-N coated multilayer membranes show higher H2/CO permselectivities of about 10.5 and the H2 permeance of about 1.05,×,10,8 mol m,2 s,1 Pa,1. If compared to the state of the art of microporous membranes, the multilayer Si-B-C-N/,-Al2O3/,-Al2O3 membranes are appeared to be interesting candidates for hydrogen separation because of their tunable nature and high-temperature and high-pressure stability. [source]

Purification of three aminotransferases from Hydrogenobacter thermophilus TK-6 , novel types of alanine or glycine aminotransferase

FEBS JOURNAL, Issue 8 2010
Enzymes, catalysis
Aminotransferases catalyse synthetic and degradative reactions of amino acids, and serve as a key linkage between central carbon and nitrogen metabolism in most organisms. In this study, three aminotransferases (AT1, AT2 and AT3) were purified and characterized from Hydrogenobacter thermophilus, a hydrogen-oxidizing chemolithoautotrophic bacterium, which has been reported to possess unique features in its carbon and nitrogen anabolism. AT1, AT2 and AT3 exhibited glutamate:oxaloacetate aminotransferase, glutamate:pyruvate aminotransferase and alanine:glyoxylate aminotransferase activities, respectively. In addition, both AT1 and AT2 catalysed a glutamate:glyoxylate aminotransferase reaction. Interestingly, phylogenetic analysis showed that AT2 belongs to aminotransferase family IV, whereas known glutamate:pyruvate aminotransferases and glutamate:glyoxylate aminotransferases are members of family I,. In contrast, AT3 was classified into family I, distant from eukaryotic alanine:glyoxylate aminotransferases which belong to family IV. Although Thermococcus litoralis alanine:glyoxylate aminotransferase is the sole known example of family I alanine:glyoxylate aminotransferases, it is indicated that this alanine:glyoxylate aminotransferase and AT3 are derived from distinct lineages within family I, because neither high sequence similarity nor putative substrate-binding residues are shared by these two enzymes. To our knowledge, this study is the first report of the primary structure of bacterial glutamate:glyoxylate aminotransferase and alanine:glyoxylate aminotransferase, and demonstrates the presence of novel types of aminotransferase phylogenetically distinct from known eukaryotic and archaeal isozymes. [source]

Purification and characterization of cathepsin B-like cysteine protease from cotyledons of daikon radish, Raphanus sativus

FEBS JOURNAL, Issue 21 2008
Akihiko Tsuji
Plant cathepsin B-like cysteine protease (CBCP) plays a role in disease resistance and in protein remobilization during germination. The ability of animal cathepsin B to function as a dipeptidyl carboxypeptidase has been attributed to the presence of a dihistidine (His110-His111) motif in the occluding loop, which represents a unique structure of cathepsin B. However, a dihistidine motif is not present in the predicted sequence of the occluding loop of plant CBCP, as determined from cDNA sequence analysis, and the loop is shorter. In an effort to investigate the enzymatic properties of plant CBCP, which possesses the unusual occluding loop, we have purified CBCP from the cotyledons of daikon radish (Raphanus sativus) by chromatography through Sephacryl S-200, DEAE,cellulose, hydroxyapatite and organomercurial,Sepharose. The molecular mass of the enzyme was estimated to be 28 kDa by SDS/PAGE under reducing conditions. The best synthetic substrate for CBCP was t -butyloxycarbonyl Leu-Arg-Arg-4-methylcoumaryl 7-amide, as is the case with human cathepsin B. However, the endopeptidase activity of CBCP towards glucagon and adrenocorticotropic hormone showed broad cleavage specificity. Human cathepsin B preferentially cleaves model peptides via its dipeptidyl carboxypeptidase activity, whereas daikon CBCP displays both endopeptidase and exopeptidase activities. In addition, CBCP was found to display carboxymonopeptidase activity against the substrate o -aminobenzoyl-Phe-Arg-Phe(4-NO2). Daikon CBCP is less sensitive (1/7000) to CA-074 than human cathepsin B. Expression analysis of CBCP at the protein and RNA levels indicated that daikon CBCP activity in cotyledons is regulated by post-transcriptional events during germination. [source]

A Graphene Oxide,Streptavidin Complex for Biorecognition , Towards Affinity Purification

ADVANCED FUNCTIONAL MATERIALS, Issue 17 2010
Zunfeng Liu
Abstract In our postgenomic era, understanding of protein-protein interactions by characterizing the structure of the corresponding protein complex is becoming increasingly important. An important problem is that many protein complexes are only stable for a few minutes. Dissociation will occur when using the typical, time-consuming purification methods such as tandem affinity purification and multiple chromatographic separations. Therefore, there is an urgent need for a quick and efficient protein-complex purification method for 3D structure characterization. The graphene oxide (GO)·streptavidin complex is prepared via a GO·biotin·streptavidin strategy and used for affinity purification. The complex shows a strong biotin recognition capability and an excellent loading capacity. Capturing biotinylated DNA, fluorophores and Au nanoparticles on the GO·streptavidin complexes demonstrates the usefulness of the GO·streptavidin complex as a docking matrix for affinity purification. GO shows a high transparency towards electron beams, making it specifically well suited for direct imaging by electron microscopy. The captured protein complex can be separated via a filtration process or even via on-grid purification and used directly for single-particle analysis via cryo-electron microscopy. Therefore, the purification, sample preparation, and characterization are rolled into one single step. [source]

Purification, characterization, cDNA cloning and nucleotide sequencing of a cellulase from the yellow-spotted longicorn beetle, Psacothea hilaris

FEBS JOURNAL, Issue 16 2003
Masahiro Sugimura
A cellulase (endo-,-1,4-glucanase, EC 3.2.1.4) was purified from the gut of larvae of the yellow-spotted longicorn beetle Psacothea hilaris by acetone precipitation and elution from gels after native PAGE and SDS/PAGE with activity staining. The purified protein formed a single band, and the molecular mass was estimated to be 47 kDa. The purified cellulase degraded carboxymethylcellulose (CMC), insoluble cello-oligosaccharide (average degree of polymerization 34) and soluble cello-oligosaccharides longer than cellotriose, but not crystalline cellulose or cellobiose. The specific activity of the cellulase against CMC was 150 µmol·min,1·(mg protein),1. TLC analysis showed that the cellulase produces cellotriose and cellobiose from insoluble cello-oligosaccharides. However, a glucose assay linked with glucose oxidase detected a small amount of glucose, with a productivity of 0.072 µmol·min,1·(mg protein),1. The optimal pH of P. hilaris cellulase was 5.5, close to the pH in the midgut of P. hilaris larvae. The N-terminal amino-acid sequence of the purified P. hilaris cellulase was determined and a degenerate primer designed, which enabled a 975-bp cDNA clone containing a typical polyadenylation signal to be obtained by PCR and sequencing. The deduced amino-acid sequence of P. hilaris cellulase showed high homology to members of glycosyl hydrolase family 5 subfamily 2, and, in addition, a signature sequence for family 5 was found. Thus, this is the first report of a family 5 cellulase from arthropods. [source]

Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoides

FEBS JOURNAL, Issue 22 2002
Nicolas Sauvageot
The three genes pduCDE encoding the diol dehydratase of Lactobacillus collinoides, have been cloned for overexpression in the pQE30 vector. Although the three subunits of the protein were highly induced, no activity was detected in cell extracts. The enzyme was therefore purified to near homogeneity by ammonium sulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity, three main bands were present after SDS/PAGE with molecular masses of 63, 28 and 22 kDa, respectively. They were identified by mass spectrometry to correspond to the large, medium and small subunits of the dehydratase encoded by the pduC, pduD and pduE genes, respectively. The molecular mass of the native complex was estimated to 207 kDa in accordance with the calculated molecular masses deduced from the pduC, D, E genes (61, 24.7 and 19,1 kDa, respectively) and a ,2,2,2 composition. The Km for the three main substrates were 1.6 mm for 1,2-propanediol, 5.5 mm for 1,2-ethanediol and 8.3 mm for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Km for the cofactor was 8 µm. Inactivation of the enzyme was observed by both glycerol and cyanocobalamin. The optimal reaction conditions of the enzyme were pH 8.75 and 37 °C. Activity was inhibited by sodium and calcium ions and to a lesser extent by magnesium. A fourth band at 59 kDa copurified with the diol dehydratase and was identified as the propionaldehyde dehydrogenase enzyme, another protein involved in the 1,2-propanediol metabolism pathway. [source]

Purification, characterization, and cDNA cloning of a novel soluble saxitoxin and tetrodotoxin binding protein from plasma of the puffer fish, Fugu pardalis

FEBS JOURNAL, Issue 22 2001
Mari Yotsu-Yamashita
Some species of puffer fish have been reported to possess both of tetrodotoxin and saxitoxin, which share one binding site on sodium channels. We purified a novel soluble glycoprotein that binds to these toxins from plasma of the puffer fish, Fugu pardalis, and named puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP). PSTBP possessed a binding capacity of 10.6 ± 0.97 nmol·mg,1 protein and a Kd of 14.6 ± 0.33 nm for [3H]saxitoxin in equilibrium binding assays. [3H]Saxitoxin (10 nm) binding to PSTBPs was half-inhibited by the presence of tetrodotoxin and saxitoxin at 12 µm and 8.5 nm, respectively. From the results of gel filtration chromatography (200 kDa) and SDS/PAGE (104 kDa), PSTBP was suggested to consist of noncovalently linked dimers of a single subunit. PSTBP was completely deglycosylated by glycopeptidase F, producing a single band at 42 kDa. Two highly homologous cDNAs to each other coding PSTBP (PSTBP1 and PSTBP2, the predicted amino-acid identity 93%), were obtained from a cDNA library of F. pardalis liver. These proteins consisted to two tandemly repeated homologous domains. The predicted amino-acid sequences of PSTBP1 and 2 were not homologous to that of saxiphilin, a reported saxitoxin binding protein, or sodium channels, but their N-terminus sequences were homologous to that of the reported tetrodotoxin binding protein from plasma of Fugu niphobles, which has not been fully characterized. The partially homologous cDNA sequences to PSTBP1 and 2 were also found in expressed sequence tag clones of nontoxic flounders liver. Presumably, PSTBP is involved in accumulation and/or excretion of toxins in puffer fish. [source]

Purification and characterization of the heterologously expressed trehalose/maltose ABC transporter complex of the hyperthermophilic archaeon Thermococcus litoralis

FEBS JOURNAL, Issue 14 2001
Gerhard Greller
We report the purification of the maltose/trehalose transporter complex MalFGK of the hyperthermophilic archaeon Thermococcus litoralis. The complex was expressed in Escherichia coli, solubilized in dodecyl maltoside and purified with the aid of a histidine tag on one of the membrane proteins. One hundred grams of cells yielded 3 mg of pure complex. The final product showed ATPase activity at 70 °C and was soluble at low detergent concentration. ATPase activity was not due to dissociation of the MalK subunit from the integral membrane proteins MalF and MalG but could not be further stimulated by trehalose/maltose binding protein (TMBP), be it the native protein as isolated from T. litoralis or the soluble engineered protein. The purified native TMBP was identified as a glycoprotein. [source]

Purification and cDNA cloning of nitric oxide reductase cytochrome P450nor (CYP55A4) from Trichosporon cutaneum

FEBS JOURNAL, Issue 11 2001
Li Zhang
Cytochrome P450nor is involved in fungal denitrification as nitric oxide (NO) reductase. Although the heme protein has been known to occur in restricted species of fungi that belong to ascomycotina, we have previously suggested that it would also occur in the yeast Trichosporon cutaneum, which is phylogenetically far from those P450nor-producing ascomycetous fungi. Here we isolated and characterized the heme protein from the basidiomycetous yeast T. cutaneum. P450nor of the yeast (TcP450nor) exhibited properties in terms of catalysis, absorption spectrum and molecular mass that are almost identical to those of its counterparts in ascomycetous fungi. We also isolated and sequenced its cDNA. The predicted primary structure of TcP450nor showed high sequence identities (around 65%) to those of other P450nors, indicating that they belong to the same family. TcP450nor protein cofractionated with cytochrome c oxidase by subcellular fractionation and its predicted primary structure contained an extension on its amino terminus that is characteristic of a mitochondrial-targeting signal, indicating that it is a mitochondrial protein like some of the isoforms of other fungi. On the other hand, TcP450nor was unique in that inducers such as nitrate, nitrite, or NO were not required for its production in the cells. The occurrence of P450nor across the subdivisions of eumycota suggests that P450nor and denitrification are distributed more universally among fungi than was previously thought. [source]

Purification and structure of the major product obtained by reaction of NADPH and NMNH with the myeloperoxidase/hydrogen peroxide/chloride system

FEBS JOURNAL, Issue 10 2001
Françoise Auchère
The first spectrophotometric study of the reaction of the myeloperoxidase/H2O2/Cl, system with NADPH and NMNH showed that the reaction products were not the corresponding oxidized nucleotides and that modifications would take place on the nicotinamide part of the molecule [Auchère, F. & Capeillère-Blandin, C. (1999) Biochem. J. 343, 603,613]. In this report, in order to obtain more precise information on the structural modifications and mechanism of the reaction, we focus on the purification and isolation of products derived from NADPH and NMNH by RP-HPLC. Electrospray ionization mass spectra indicated that the relative height of the peaks reflected that of the natural isotopic abundance of 35Cl and 37Cl, providing evidence that the products derived from NADPH and NMNH were monochlorinated. Moreover, calculated masses revealed the 1 : 1 addition of HOCl to the molecule. Various 1D and 2D NMR experiments provided data for the assignments of the chemical shifts of protons and carbons and the coupling constants of the protons of the chlorinated nucleotides. Further NOESY experiments allowed the characterization of the spatial structure of the chlorinated product and showed that trans HOCl addition occurred at the C5=C6 carbon double bond of the nicotinamide ring, leading to a chlorohydrin. [source]

Purification, characterization and amino-acid sequence analysis of a thermostable, low molecular mass endo-,-1,4-glucanase from blue mussel, Mytilus edulis

FEBS JOURNAL, Issue 16 2000
Bingze Xu
A cellulase (endo-,-1,4- d -glucanase, EC 3.2.1.4) from blue mussel (Mytilus edulis) was purified to homogeneity using a combination of acid precipitation, heat precipitation, immobilized metal ion affinity chromatography, size-exclusion chromatography and ion-exchange chromatography. Purity was analyzed by SDS/PAGE, IEF and RP-HPLC. The cellulase (endoglucanase) was characterized with regard to enzymatic properties, isoelectric point, molecular mass and amino-acid sequence. It is a single polypeptide chain of 181 amino acids cross-linked with six disulfide bridges. Its molecular mass, as measured by MALDI-MS, is 19 702 Da; a value of 19 710.57 Da was calculated from amino-acid composition. The isoelectric point of the enzyme was estimated by isoelectric focusing in a polyacrylamide gel to a value of 7.6. According to amino-acid composition, the theoretical pI is 7.011. The effect of temperature on the endoglucanase activity, with carboxymethyl cellulose and amorphous cellulose as substrates, respectively, was studied at pH 5.5 and displayed an unusually broad optimum activity temperature range between 30 and 50 °C. Another unusual feature is that the enzyme retains 55,60% of its maximum activity at 0 °C. The enzyme readily degrades amorphous cellulose and carboxymethyl cellulose but displays no hydrolytic activity towards crystalline cellulose (Avicel) and shows no cross-specificity for xylan; there is no binding to Avicel. The enzyme can withstand 10 min at 100 °C without irreversible loss of enzymatic activity. Amino-acid sequence-based classification has revealed that the enzyme belongs to the glycoside hydrolase family 45, subfamily 2 (B. Henrissat, Centre de Recherches sur les Macromolecules Végétales, CNRS, Joseph Fourier Université, Grenoble, France, personal communication). [source]

Purification and characterization of the single-strand-specific and guanylic-acid-preferential deoxyribonuclease activity of the extracellular nuclease from Basidiobolus haptosporus

FEBS JOURNAL, Issue 16 2000
Neelam A. Desai
An extracellular nuclease from Basidiobolus haptosporus (designated as nuclease Bh1) was purified to homogeneity by ammonium sulfate precipitation, heat treatment, negative adsorption on DEAE-cellulose, and chromatography on phenyl-Sepharose followed by FPLC on phenyl-Superose. The overall yield was 26%. The Mr of the purified enzyme, determined by gel filtration, was 41 000 whereas by SDS/PAGE (after deglycosylation) it was 30 000. It is a glycoprotein with a pI of 6.8. The optimum pH and temperature for DNA hydrolysis were 8.5 and 60 °C, respectively. Nuclease Bh1 is a metalloprotein but has no obligate requirement for metal ions to be active, nor is its activity stimulated in the presence of metal ions. The enzyme was inhibited by Zn2+, Ag2+, Hg2+, Fe3+ and Al3+, inorganic phosphate, pyrophosphate, dithiothreitol, 2-mercaptoethanol, NaCl and KCl. It was stable to high concentrations of organic solvents and urea but susceptible to low concentrations of SDS and guanidine hydrochloride. Nuclease Bh1 is a multifunctional enzyme and its substrate specificity is in the order of ssDNA , 3,AMP , RNA > dsDNA. Studies on its mode of action showed that it cleaved supercoiled pUC 18 DNA and phage M13 DNA, endonucleolytically, generating single base nicks. The enzyme hydrolyzed DNA with preferential liberation of 5,dGMP, suggesting it to be a guanylic acid preferential endoexonuclease. 5,dGMP, the end product of hydrolysis, was a competitive inhibitor of the enzyme. The absence of 5,dCMP as a hydrolytic product, coupled with the resistance of (dC)10 and deoxyribodinucleoside monophosphates having cytosine either at the 3, or the 5, end, indicates that C-linkages are resistant to cleavage by nuclease Bh1. [source]

Purification and properties of a new Brevibacterium sterolicum cholesterol oxidase produced by E. coli MM294/pnH10

FEMS MICROBIOLOGY LETTERS, Issue 2 2002
Kinya Fujishiro
Abstract A gene encoding a cholesterol oxidase from Brevibacterium sterolicum nov. sp. ATCC21387 was isolated by an expression cloning method and highly expressed by a recombinant strain Escherichia coli MM294/pnH10. The purified cholesterol oxidase was a typical flavoprotein with a molecular mass of 46.5 kDa, absorption peaks at 280, 360, and 450 nm. Optimum pH and temperature were found at pH 6.5 and 55°C, respectively. The enzyme acted on 3,-hydroxysteroids such as cholesterol, pregnenolone, and ,-sitosterol at high rates, but on dehydro- epi -androsterone to a lesser degree. The molecular and catalytic properties were different from those of cholesterol oxidase I, which was initially discovered in B. sterolicum nov. sp. ATCC21387. The new enzyme, designated cholesterol oxidase II, was distinguished by its high affinity toward cholesterol (Km= 30,M). [source]

Purification and characterization of a glutathione S -transferase from the fungus Cunninghamella elegans

FEMS MICROBIOLOGY LETTERS, Issue 2 2001
Chang-Jun Cha
Abstract Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S -transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120,000×g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate,polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of Mr 27,000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian ,-, ,-, and ,-class GSTs, although it showed a small degree of cross-reactivity with a ,-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs. [source]

Men's Recollections of a Women's Rite: Medieval English Men's Recollections Regarding the Rite of the Purification of Women after Childbirth

GENDER & HISTORY, Issue 2 2002
Becky R. Lee
This study examines the recollections of medieval English men, found in proof,of,age inquests, regarding their participation in the rite of the purification of women after childbirth. Because the rite of purification was reserved to women, scant attention has been paid to how this rite and the customs surrounding it played in the lives of medieval men. These men's recollections situate postpartum purification within the festivities celebrating the birth of a man's heir. For them, it is a public event celebrating paternity and lineage, and a forum for the negotiation of social relationships. [source]

Laser Ablation (193 nm), Purification and Determination of Very Low Concentrations of Solar Wind Nitrogen Implanted in Targets from the GENESIS Spacecraft

GEOSTANDARDS & GEOANALYTICAL RESEARCH, Issue 2 2009
Laurent Zimmermann
azote; ablation laser; purification; spectrométrie de masse; mission GENESIS The GENESIS space mission recovered ions emitted by the Sun during a 27 month period. In order to extract, purify and determine the very low quantities of solar nitrogen implanted in the GENESIS targets, a new installation was developed and constructed at the CRPG (Nancy, France). It permitted the simultaneous determination of nitrogen and noble gases extracted from the target by laser ablation. The extraction procedure used a 193 nm excimer laser that allowed for surface contamination in the outer 5 nm to be removed, followed by a step that removed 50 nm of the target material, extracting the solar nitrogen and noble gases implanted in the target. Following purification using Ti and Zr getters for noble gases and a Cu-CuO oxidation cycle for N2, the extracted gases were analysed by static mode (pumps closed) mass spectrometry using electron multiplier and Faraday cup detectors. The nitrogen blanks from the purification section and the static line (30 minutes) were only 0.46 picomole and 0.47 picomole, respectively. La mission GENESIS a récupéré des ions émis par le soleil pendant une période de 27 mois. Afin d'extraire, purifier et analyser de très faibles quantités d'azote solaire implantés dans des cibles GENESIS, une nouvelle installation a été développée et construite au CRPG. Elle a permis l'analyse simultanée de l'azote et des gaz nobles extraits de la couche d'or par ablation. La procédure d'extraction a utilisé un laser Excimer 193 nm qui a permis une étape d'extraction à 5 nm pour éliminer la pollution à la surface, suivie d'une étape qui a extrait jusqu'à une profondeur de 50 nm l'azote et les gaz rares solaires implantés dans la cible. Après une purification à l'aide de getters Ti et Zr pour les gaz rares et un cycle d'oxydation Cu-CuO pour N2, les gaz extraits ont été analysés en mode statique (pompage fermé) par spectrométrie de masse à l'aide d'un multiplicateur d'électrons et d'une cage de Faraday. Les blancs d'azote provenant de la partie purification et de la ligne en statique (30 minutes) étaient de seulement 0.46 et 0.47 picomole, respectivement. [source]

Large-Scale Synthesis, Annealing, Purification, and Magnetic Properties of Crystalline Helical Carbon Nanotubes with Symmetrical Structures,

ADVANCED FUNCTIONAL MATERIALS, Issue 9 2007
J. Tang
Abstract Crystalline helical carbon nanotubes (HCNTs) are synthesized as the main products in the pyrolysis of acetylene at 450,°C over Fe nanoparticles generated by means of a combined sol,gel/reduction method. Transmission electron microscopy (TEM) images reveal that there are two HCNTs attached to each Fe3C nanoparticle, and that the two HCNTs are mirror images of each other. Annealing in Ar at 750,°C and purification by immersion in hot (90,°C) HCl solution do not significantly change the structure of the HCNTs, despite the partial removal of Fe nanoparticles by the latter treatment. The magnetic properties of the as-prepared, annealed, and purified HCNTs have been systematically examined. The annealed sample shows relatively high magnetization due to the ferromagnetic ,-Fe nanoparticles encapsulated in the HCNT nodes. In the case of HCl treatment, relatively pure HCNTs are obtained by the removal of ferromagnetic nanoparticles from the double-HCNT nodes. The effects of the amount of catalyst used in the synthesis process on the morphology and yield of the carbon products have also been investigated. [source]