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Sample records for purification method enzyme

  1. Agarase: Review of Major Sources, Categories, Purification Method, Enzyme Characteristics and Applications

    Directory of Open Access Journals (Sweden)

    Sang Moo Kim

    2010-01-01

    Full Text Available Agarases are the enzymes which catalyze the hydrolysis of agar. They are classified into α-agarase (E.C. 3.2.1.158 and β-agarase (E.C. 3.2.1.81 according to the cleavage pattern. Several agarases have been isolated from different genera of bacteria found in seawater and marine sediments, as well as engineered microorganisms. Agarases have wide applications in food industry, cosmetics, and medical fields because they produce oligosaccharides with remarkable activities. They are also used as a tool enzyme for biological, physiological, and cytological studies. The paper reviews the category, source, purification method, major characteristics, and application fields of these native and gene cloned agarases in the past, present, and future.

  2. Purification of highly chlorinated dioxins degrading enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Ishii, K.; Furuichi, T.; Koike, K.; Kuboshima, M. [Hokkaido Univ. (Japan). Division of Environment Resource Engineering, Graduate School of Engineering

    2004-09-15

    Soil contamination caused by dioxins in and around sites of incinerators for municipal solid waste (MSW) is a concern in Japan. For example, scattering wastewater from a wet gas scrubber at an MSW incinerator facility in Nose, Osaka caused soil and surface water contamination. The concentration of dioxins in the soil was about 8,000 pg-TEQ/g. Other contamination sites include soils on which fly ash has been placed directly or improperly stored and landfill sites that have received bottom and fly ash over a long period. Some countermeasures are required immediately at these dioxins-contaminated sites. We have previously developed bioreactor systems for dioxin-contaminated water and soil. We have shown that a fungus, Pseudallescheria boydii (P. boydii), isolated from activated sludge treating wastewater that contained dioxins, has the ability to degrade highly chlorinated dioxins. A reaction product of octachlorinated dibenzo-p-dioxin (OCDD) was identified as heptachlorinated dibenzo-p-dioxin. Therefore, one of the pathways for degradation of OCDD by this fungus was predicted to be as follows: OCDD is transformed by dechlorination and then one of the remaining aromatic rings is oxidized. To apply P. boydii to on-site technologies (e.g., bioreactor systems), as well as in situ technologies, enzyme treatment using a dioxin-degrading enzyme from P. boydii needs to be developed because P. boydii is a weak pathogenic fungus, known to cause opportunistic infection. As a result, we have studied enzyme purification of nonchlorinated dioxin, namely, dibenzo-pdioxin (DD). However, we did not try to identify enzymes capable of degrading highly chlorinated dioxins. This study has elucidated a method of enzyme assay for measuring OCDD-degrading activity, and has attempted to purify OCDD-degrading enzymes from P. boydii using enzyme assay. In addition, as first step toward purifying 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), 2,3,7,8-TCDD degradation tests were carried out

  3. Affinity purification of polysaccharide degrading enzymes with crosslinked substrates

    NARCIS (Netherlands)

    Rozie, H.J.

    1992-01-01

    The aim of this work was to find economically favourable, affinity based, purification methods for several polysaccharide splitting bulk enzymes. The framework in which this study is done is described in Chapter 1.

    Chapter 2 describes the adsorption of endo-polygalacturonase (endoPG

  4. Affinity purification of polysaccharide degrading enzymes with crosslinked substrates

    NARCIS (Netherlands)

    Rozie, H.J.

    1992-01-01

    The aim of this work was to find economically favourable, affinity based, purification methods for several polysaccharide splitting bulk enzymes. The framework in which this study is done is described in Chapter 1.

    Chapter 2 describes the adsorption of endo-polygalacturonase

  5. A new affinity method for purification of bovine testicular hyaluronidase enzyme and an investigation of the effects of some compounds on this enzyme.

    Science.gov (United States)

    Kaya, Mustafa Oguzhan; Arslan, Oktay; Guler, Ozen Ozensoy

    2015-01-01

    In this study, a new affinity gel for the purification of bovine testicular hyaluronidase (BTH) was synthesized. L-Tyrosine was added as the extension arm to the Sepharose-4B activated with cyanogen bromide. m-Anisidine is a specific inhibitor of BTH enzyme. m-Anisidine was clamped to the newly formed Sepharose-4B-L-tyrosine as a ligand. As a result, an affinity gel having the chemical structure of Sepharose-4B-L-tyrosine-m-anisidine was obtained. BTH purified by ammonium sulfate precipitation and affinity chromatography was obtained with a 16.95% yield and 881.78 degree of purity. The kinetic constants K(M) and V(Max) for BTH were determined by using hyaluronic acid as a substrate. K(M) and V(Max) values obtained from the Lineweaver-Burk graph were found to be 2.23 mM and 19.85 U/mL, respectively. In vitro effects of some chemicals were determined on purified BTH enzyme. Some chemically active ingredients were 1,1-dimethyl piperidinium chloride, β-naphthoxyacetic acid and gibberellic acid. Gibberellic acid showed the best inhibition effect on BTH.

  6. Purification, characterization of phytase enzyme from Lactobacillus ...

    African Journals Online (AJOL)

    SAM

    2014-06-04

    Jun 4, 2014 ... Based on these results, the phytase enzyme of L. plantarum is considered ... first found in rice bran (Suzuki et al., 1907) and in the blood of .... Bradford method was used to determine the amount of protein in the enzyme ...

  7. Extraction and Purification of Collagenase Enzymes: A Critical Review

    Directory of Open Access Journals (Sweden)

    Said M. Daboor

    2010-01-01

    Full Text Available Problem statement: Enzymes have vital roles in several industrial processes (foods, cosmetics, nutraceuticals and pharmaceuticals due to their highly selective nature and high activity at very low concentrations. Recent efforts to identify new sources of useful enzymes have been concentrated on the marine environment because of the potential to make use of processing wastes. About 35-50% of the mass of the fish caught is a waste that is disposed off at sea or in landfills. The extraction of enzymes from fish processing waste can reduce environment problems and improve the economics of the fish industry. Collagenases are a group of enzymes that can be extracted from fish waste. Approach: Comprehensive reviews of the literature on the extraction, purification, characterization and use of collagenases was carried out. Results: Collagenases have different molecular weights based on their types and sources. They have the ability to break down the peptide bonds in collagen at physiological pH. They are classified into two types: serine and metallocollagenase. Collagenolytic activities have been shown at a wide range of temperatures (20-40°C and pH (6-8. Many activators can be used to achive collagenase activity including 4-Aminophenylmercuric Acetate (APMA, trypsin, potassium or sodium thiocyanate, iodoacetamide and potassium iodide. Dithiothreitol (DTT, mercaptoethanol, ethylendiaminetetracetic acid, o-phenanthroline and cysteine inactivate the enzyme. Collagenases enzymes can be extracted with a variety of techniques using different buffering systems (tris-HCl, sodium bicarbonate, calcium chloride and cacodylate. All techniques involve the use of ammonium sulphate fractionation and centrifugation to precipitate the enzyme. Collagenases are normally purified using chromatographic techniques such as gel-filtration, ion-exchange and affinity column chromatography. Collagenase can be assayed with a number of methods, including: colorimetric absorbance

  8. Economic Methods of Ginger Protease'sextraction and Purification

    Science.gov (United States)

    Qiao, Yuanyuan; Tong, Junfeng; Wei, Siqing; Du, Xinyong; Tang, Xiaozhen

    This article reports the ginger protease extraction and purification methods from fresh ginger rhizome. As to ginger protease extraction, we adapt the steps of organic solvent dissolving, ammonium sulfate depositing and freeze-drying, and this method can attain crude enzyme powder 0.6% weight of fresh ginger rhizome. The purification part in this study includes two steps: cellulose ion exchange (DEAE-52) and SP-Sephadex 50 chromatography, which can purify crude ginger protease through ion and molecular weight differences respectively.

  9. PURIFICATION OF CATALASE ENZYME FROM PLEUROTUS OSTREATUS

    Directory of Open Access Journals (Sweden)

    Susmitha.S

    2014-03-01

    Full Text Available The oyster mushroom Pleurotus ostreatus is the most commonly cultivated mushroom, and are effective for antitumor, antibacterial, anti viral and hematological agents and in immune modulating treatments. Several compounds from oyster mushrooms, potentially beneficial for human health have been isolated and studied. The aim of this research is to purify an enzyme catalase from Pleurotus ostreatus through Sephadox G-75 column, its molecular weight was determined by polyacrylamide gel electrophoresis and the catalase enzyme stability were observed at various temperature and different pH condition. Under denaturing conditions, polyacrylamide gel electrophoresis revealed dissociation of a major component of molecular weight 62,000 kDa, which constituted 90% of the total protein of the stained gel, suggesting that the native enzyme is tetrameric. The optimum temperature and pH for the purified enzyme catalase from Pleurotus ostreatus enzymatic reaction were 30°C and pH 7.5.

  10. Expression, purification, and characterization of a carbohydrate-active enzyme: A research-inspired methods optimization experiment for the biochemistry laboratory.

    Science.gov (United States)

    Willbur, Jaime F; Vail, Justin D; Mitchell, Lindsey N; Jakeman, David L; Timmons, Shannon C

    2016-01-01

    The development and implementation of research-inspired, discovery-based experiences into science laboratory curricula is a proven strategy for increasing student engagement and ownership of experiments. In the novel laboratory module described herein, students learn to express, purify, and characterize a carbohydrate-active enzyme using modern techniques and instrumentation commonly found in a research laboratory. Unlike in a traditional cookbook-style experiment, students generate their own hypotheses regarding expression conditions and quantify the amount of protein isolated using their selected variables. Over the course of three 3-hour laboratory periods, students learn to use sterile technique to express a protein using recombinant DNA in E. coli, purify the resulting enzyme via affinity chromatography and dialysis, analyze the success of their purification scheme via SDS-PAGE, assess the activity of the enzyme via an HPLC-based assay, and quantify the amount of protein isolated via a Bradford assay. Following the completion of this experiment, students were asked to evaluate their experience via an optional survey. All students strongly agreed that this laboratory module was more interesting to them than traditional experiments because of its lack of a pre-determined outcome and desired additional opportunities to participate in the experimental design process. This experiment serves as an example of how research-inspired, discovery-based experiences can benefit both the students and instructor; students learned important skills necessary for real-world biochemistry research and a more concrete understanding of the research process, while generating new knowledge to enhance the scholarly endeavors of the instructor.

  11. Affinity chromatography purification of cytochrome c binding enzymes.

    OpenAIRE

    Azzi, A; Bill, K; Broger, C

    1982-01-01

    An efficient affinity chromatography procedure for the isolation of mitochondrial cytochrome c oxidase and reductase is described. Saccharomyces cerevisiae cytochrome c was used as a ligand, bound to a thiol-Sepharose 4B gel through cysteine-107. In this way, the site of interaction of cytochrome c with cytochrome oxidase and reductase remained unmodified and available for binding to a number of partner enzymes. The procedure is adequate for the purification of all those proteins having in co...

  12. Expression and purification of a human, soluble Arylsulfatase A for Metachromatic Leukodystrophy enzyme replacement therapy.

    Science.gov (United States)

    Martino, Sabata; Consiglio, Antonella; Cavalieri, Cristina; Tiribuzi, Roberto; Costanzi, Egidia; Severini, Giovanni Maria; Emiliani, Carla; Bordignon, Claudio; Orlacchio, Aldo

    2005-05-25

    The production of active Arylsulfatase A is a key step in the development of enzyme replacement therapy for Metachromatic Leukodystrophy. To obtain large amounts of purified Arylsulfatase A for therapeutic use, we combined a retroviral expression system with a versatile and rapid purification protocol that can easily and reliably be adapted to high-throughput applications. The purification method consists of an initial ion-exchange DEAE-cellulose chromatography step followed by immuno-affinity purification using a polyclonal antibody against a 29-mer peptide of the Arylsulfatase A sequence. Immuno-adsorbed protein was eluted with a combination of acidic pH and an optimal concentration of the 29-mer peptide. This protocol reproducibly yielded approximately 100 microg of >99% pure human Arylsulfatase A, corresponding to 152 mU of enzyme activity, per liter of culture medium with properties similar to those of human non-recombinant protein.

  13. Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing Enzyme Tag

    Science.gov (United States)

    Shen, Aimee; Lupardus, Patrick J.; Morell, Montse; Ponder, Elizabeth L.; Sadaghiani, A. Masoud; Garcia, K. Christopher; Bogyo, Matthew

    2009-01-01

    We introduce a new method for purifying recombinant proteins expressed in bacteria using a highly specific, inducible, self-cleaving protease tag. This tag is comprised of the Vibrio cholerae MARTX toxin cysteine protease domain (CPD), an autoprocessing enzyme that cleaves exclusively after a leucine residue within the target protein-CPD junction. Importantly, V. cholerae CPD is specifically activated by inositol hexakisphosphate (InsP6), a eukaryotic-specific small molecule that is absent from the bacterial cytosol. As a result, when His6-tagged CPD is fused to the C-terminus of target proteins and expressed in Escherichia coli, the full-length fusion protein can be purified from bacterial lysates using metal ion affinity chromatography. Subsequent addition of InsP6 to the immobilized fusion protein induces CPD-mediated cleavage at the target protein-CPD junction, releasing untagged target protein into the supernatant. This method condenses affinity chromatography and fusion tag cleavage into a single step, obviating the need for exogenous protease addition to remove the fusion tag(s) and increasing the efficiency of tag separation. Furthermore, in addition to being timesaving, versatile, and inexpensive, our results indicate that the CPD purification system can enhance the expression, integrity, and solubility of intractable proteins from diverse organisms. PMID:19956581

  14. Simplified, enhanced protein purification using an inducible, autoprocessing enzyme tag.

    Directory of Open Access Journals (Sweden)

    Aimee Shen

    Full Text Available We introduce a new method for purifying recombinant proteins expressed in bacteria using a highly specific, inducible, self-cleaving protease tag. This tag is comprised of the Vibrio cholerae MARTX toxin cysteine protease domain (CPD, an autoprocessing enzyme that cleaves exclusively after a leucine residue within the target protein-CPD junction. Importantly, V. cholerae CPD is specifically activated by inositol hexakisphosphate (InsP(6, a eukaryotic-specific small molecule that is absent from the bacterial cytosol. As a result, when His(6-tagged CPD is fused to the C-terminus of target proteins and expressed in Escherichia coli, the full-length fusion protein can be purified from bacterial lysates using metal ion affinity chromatography. Subsequent addition of InsP(6 to the immobilized fusion protein induces CPD-mediated cleavage at the target protein-CPD junction, releasing untagged target protein into the supernatant. This method condenses affinity chromatography and fusion tag cleavage into a single step, obviating the need for exogenous protease addition to remove the fusion tag(s and increasing the efficiency of tag separation. Furthermore, in addition to being timesaving, versatile, and inexpensive, our results indicate that the CPD purification system can enhance the expression, integrity, and solubility of intractable proteins from diverse organisms.

  15. A New Method for the Purification of Restriction Enzyme Not Ⅰ%限制性内切酶NotI提纯的新工艺

    Institute of Scientific and Technical Information of China (English)

    张巧; 叶贤龙; 任桂萍; 张楠; 李璐; 李德山

    2011-01-01

    BR322-Eag ⅠM. Then transformed this plasmid into E. coli 2566. Secondly,extracted the genome DNA from Nocardia otitidis-caviarum as template and obtained the restriction enzyme NotⅠR gene by PCR. After ligating the NotⅠR gene to pACYC184-PT7 ,the pACYC184-PT7-NotⅠR plasmid was transformed into the ER2566 which was protected through the methylation by pBR322-EagⅠM recombinant plasmid. The engineered strain ER2566 [ pBR322-EagⅠM,pACYC184-PT7-NotⅠR]could be induced to express restriction enzymes NotⅠ by IPTG and the induction conditions were optimized to make its expression mostly in soluble form. The enzyme was purified by (A)KTA purifier 100 protein purification system.Through DEAE Sephrose FF,phenyl HP and Superdex 75 10/300 GL molecular sieve chromatography,the Not Ⅰ enzyme was purified 35-fold, the yield was 9. 8 × 106Units / g wet cell which was up to17. 8% of the crude enzyme and the specific activity of the purified NotⅠwas 1. 37 × 106U/mg. Digestion results showed that the enzyme was purified to homogeneity and was free of detectable contamination by other DNase ( exo and endo) . After optimization of the expression and purification conditions, the yield and efficiency of NotⅠ enzyme were greatly improved in comparison with that previously reported.

  16. Recombinant Plasmodium falciparum glutamate rich protein; purification and use in enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Dziegiel, M; Borre, Mette; Jepsen, S

    1991-01-01

    A method for purification of a recombinant Plasmodium falciparum protein produced in E. coli and its use in an enzyme-linked immunosorbent assay (ELISA) is described. The cloned gene fragment encodes GLURP,489-1271 the carboxy-terminal 783 amino acid residue portion of a 1271 amino acid residue P...... of the immunogenicity of a possible future P. falciparum vaccine utilizing epitopes from GLURP....

  17. Utilizing Isolation, Purification, and Characterization of Enzymes as Project-Oriented Labs for Undergraduate Biochemistry

    Science.gov (United States)

    Deal, S. Todd; Hurst, Michael O.

    1997-02-01

    Senior-level biochemistry labs are mostly verification-type laboratories with little chance for exploration. We have developed a project-based biochemistry laboratory which gives them a chance to carry out a major biochemistry project. In the first quarter it is based on the purification of the enzyme lysozyme. The students are given some basic information, and then work out the details of their own procedures, make up their own solutions, and work at their own pace. Students use centrifugation, ion-exchange chromatography, spectral enzyme assays, and SDS-gel electrophoresis to purify and characterize the protein. In the second quarter students are given acid phosphatase and the basic assay for the enzyme, and then develop and carry out a method for determining the kinetic parameters of the enzyme. These experiments continue the development of laboratory independence of the students which steadily progresses in most curriculum

  18. Purification and Properties of Clostridium perfringens Spore Lytic Enzymes.

    Science.gov (United States)

    1983-01-01

    sacs was effective. Further purification was obtained using carboxymethylcellulose and Sephadex G-100 chromatography. At this point the purified produce...Concentrated culture supernatant fluid (CSF) containing the initiation protein (IP) was prepared from 7 h cultures of C perfringens NCTC 8798 grown in DS...four different methods (a) 0.05 M DTT, (b) 0.05 M DTT plus 0.5% (w/v) SDS, both prepared in 0.05 M glycine-NaOH buffer, with the pH adjusted to 10.0

  19. Purification of Carbon Nanotubes: Alternative Methods

    Science.gov (United States)

    Files, Bradley; Scott, Carl; Gorelik, Olga; Nikolaev, Pasha; Hulse, Lou; Arepalli, Sivaram

    2000-01-01

    Traditional carbon nanotube purification process involves nitric acid refluxing and cross flow filtration using surfactant TritonX. This is believed to result in damage to nanotubes and surfactant residue on nanotube surface. Alternative purification procedures involving solvent extraction, thermal zone refining and nitric acid refiuxing are used in the current study. The effect of duration and type of solvent to dissolve impurities including fullerenes and P ACs (polyaromatic compounds) are monitored by nuclear magnetic reasonance, high performance liquid chromatography, and thermogravimetric analysis. Thermal zone refining yielded sample areas rich in nanotubes as seen by scanning electric microscopy. Refluxing in boiling nitric acid seem to improve the nanotube content. Different procedural steps are needed to purify samples produced by laser process compared to arc process. These alternative methods of nanotube purification will be presented along with results from supporting analytical techniques.

  20. Expression, Purification, and Characterization of a Carbohydrate-Active Enzyme: A Research-Inspired Methods Optimization Experiment for the Biochemistry Laboratory

    Science.gov (United States)

    Willbur, Jaime F.; Vail, Justin D.; Mitchell, Lindsey N.; Jakeman, David L.; Timmons, Shannon C.

    2016-01-01

    The development and implementation of research-inspired, discovery-based experiences into science laboratory curricula is a proven strategy for increasing student engagement and ownership of experiments. In the novel laboratory module described herein, students learn to express, purify, and characterize a carbohydrate-active enzyme using modern…

  1. Expression, Purification, and Characterization of a Carbohydrate-Active Enzyme: A Research-Inspired Methods Optimization Experiment for the Biochemistry Laboratory

    Science.gov (United States)

    Willbur, Jaime F.; Vail, Justin D.; Mitchell, Lindsey N.; Jakeman, David L.; Timmons, Shannon C.

    2016-01-01

    The development and implementation of research-inspired, discovery-based experiences into science laboratory curricula is a proven strategy for increasing student engagement and ownership of experiments. In the novel laboratory module described herein, students learn to express, purify, and characterize a carbohydrate-active enzyme using modern…

  2. Simple and Economic Purification Method of Berberine

    OpenAIRE

    SHU-HUA, LU; Xu, Li; GUI-BAO, LU; TOYOKAZU, KISHI; Setsuko, SEKITA; Motoyoshi, SATAKE; Tianjin Institute for Drug Control; Japan International Corporation Agency; National Institute of Health Sciences

    1998-01-01

    The authers examined for the purification and quantitative analysis of berberine, thin layer chromatography, centrifugal round plate thin layer chromatography, recrystallization, dry silica gel column chromatography, wet silica gel column chromatography, solvent partition and preparative thin layer chromatography. We established a simple and economic method which is a combination of solvent partition and preparative thin layer chromatography.

  3. Application of two types of CIM tube column for purification of microbial enzymes.

    Science.gov (United States)

    Isobe, Kimiyasu; Kawakami, Yoshimitsu

    2005-02-11

    Chromatography conditions for two types of convection interaction media (CIM) tube monolithic column, DEAE-8 and C4-8, were investigated using three enzymes from different microorganisms. The enzymes were adsorbed on a CIM DEAE-8 tube column under the same conditions as conventional DEAE columns. The CIM C4-8 tube column required a high concentration of ammonium sulfate compared to the conventional C4 column for adsorbing the enzymes. The separation of enzymes on the CIM tube column chromatography was not affected at flow rates between 0.15 and 1.25 volumes of the column per min. Both columns were successfully applied to the purification of enzymes from crude enzyme solution. Thus, both CIM tube monolithic columns proved useful in greatly reducing the purification time, and could be used at any stage of enzyme purification.

  4. Enzymic synthesis of gastrodin through microbial transformation and purification of gastrodin biosynthesis enzyme.

    Science.gov (United States)

    Zhu, Hongli; Dai, Penggao; Zhang, Wei; Chen, Erfang; Han, Wenxian; Chen, Chao; Cui, Yali

    2010-01-01

    Gastrodin, a major bioactive component of a famous Chinese herb Gastrodia elata B1., has diverse pharmaceutical functions. It is usually obtained by extraction from a plant or through chemical synthesis. However, traditional extraction from Gastrodia elata B1. is time and money consuming, while chemical synthesis is a complicated procedure and always leads to very serious environmental pollution. Thus it is urgent to explore a new gastrodin source which is more economical and environmental. The present study reports a novel approach to the production of gastrodin through biosynthesis and microbial transformation. Rhizopus chinensis SAITO AS3.1165 was screened from about 50 fungal and bacterial strains and found capable of biotransforming p-hydroxybenzaldehyde into gastrodin for use in gastrodin production. A series of purification steps including (NH(4))(2)SO(4) precipitation, ion exchange chromatography and gel filtration column chromatography was successfully used for purification of the gastrodin biosynthesis enzyme (GBE). The purity of GBE was above 95% and its molecular weight was about 63.2 kDa. We further characterized GBE's function condition, and found that the optimal temperature was 50 °C and the optimum pH 6.0. The enzyme was stable at a temperature lower than 50 °C and a pH between 6.0 and 9.0. The result indicated that gastrodin could be successfully synthesized by microbial transformation, providing a new approach for gastrodin production.

  5. Simplified purification method for Clostridium difficiletoxin A

    Institute of Scientific and Technical Information of China (English)

    Si-Wu Fu; Jing Xue; Ya-Li Zhang; Dian-Yuan Zhou

    2004-01-01

    AIM: To establish the purification method for Clostridiumdifficile ( C. difficile) toxin A.METHODS: C. difficile VPI 10463 filtrate was cultured anaerobically by the dialysis bag methods. And then the toxin A was purified by precipitation with 500 g/L (NH4)2SO4and acid precipitation at pH 5.5, followed by ion-exchange chromatography on DEAE-Toyopearl.RESULTS: Purified toxin A exhibited only one band on nativepolyacrylamide gel electrophoresis (native-PAGE) andOuchterlony double immunodiffusion. The molecular weight of toxin A was estimated to be 550 000. The purified toxin A had a protein concentration of 0.881 mg/mL. The minimum lethal dose was 1×106 MLD/mL (i.p.mice). The cytotoxictiter was 107 CU/mg. The haemagglutinate activity was ata concentration of 1.72 μg/mL. The ratio of fluid volume (mL)accumulated to the length (cm) of the loop was 2.46. CONCLUSION: The modified method for purification of toxin A of C. difficile was simple and convenient. It may be even more suitable for purification of toxin A on large scales.

  6. Enzymic synthesis of indole-3-acetyl-1-O-beta-d-glucose. I. Partial purification and characterization of the enzyme from Zea mays

    Science.gov (United States)

    Leznicki, A. J.; Bandurski, R. S.

    1988-01-01

    The first enzyme-catalyzed reaction leading from indole-3-acetic acid (IAA) to the myo-inositol esters of IAA is the synthesis of indole-3-acetyl-1-O-beta-D-glucose from uridine-5'-diphosphoglucose (UDPG) and IAA. The reaction is catalyzed by the enzyme, UDPG-indol-3-ylacetyl glucosyl transferase (IAA-glucose-synthase). This work reports methods for the assay of the enzyme and for the extraction and partial purification of the enzyme from kernels of Zea mays sweet corn. The enzyme has an apparent molecular weight of 46,500 an isoelectric point of 5.5, and its pH optimum lies between 7.3 and 7.6. The enzyme is stable to storage at zero degrees but loses activity during column chromatographic procedures which can be restored only fractionally by addition of column eluates. The data suggest either multiple unknown cofactors or conformational changes leading to activity loss.

  7. Enzymic synthesis of indole-3-acetyl-1-O-beta-d-glucose. I. Partial purification and characterization of the enzyme from Zea mays

    Science.gov (United States)

    Leznicki, A. J.; Bandurski, R. S.

    1988-01-01

    The first enzyme-catalyzed reaction leading from indole-3-acetic acid (IAA) to the myo-inositol esters of IAA is the synthesis of indole-3-acetyl-1-O-beta-D-glucose from uridine-5'-diphosphoglucose (UDPG) and IAA. The reaction is catalyzed by the enzyme, UDPG-indol-3-ylacetyl glucosyl transferase (IAA-glucose-synthase). This work reports methods for the assay of the enzyme and for the extraction and partial purification of the enzyme from kernels of Zea mays sweet corn. The enzyme has an apparent molecular weight of 46,500 an isoelectric point of 5.5, and its pH optimum lies between 7.3 and 7.6. The enzyme is stable to storage at zero degrees but loses activity during column chromatographic procedures which can be restored only fractionally by addition of column eluates. The data suggest either multiple unknown cofactors or conformational changes leading to activity loss.

  8. 贵州豆豉纤溶酶发酵液中活性酶的盐析分离纯化%Separation and Purification of Active Enzyme from Fibrinolytic Enzyme Fermentation Broth of Guizhou Lobster Sauce with Salting Fractionation Method

    Institute of Scientific and Technical Information of China (English)

    张敏; 宋智魁; 王鹏娇; 王清忱; 彭安堂; 舒阳; 高秀丽

    2013-01-01

    Objective:To culture fibrinolytic enzyme strains (DCK-B)extracted from Guizhou characteristic lobster sauce with liquid ferment method,and to develop a method for primary purification of active fibrinolytic enzyme from the fermentation products.Methods:Ammonium sulphate (AS)salting out method was used for primary purification,and the effects of one step salting out method with 0% ~ 80% AS and two step salting out method with AS of 0% ~ 30% and 30% ~ 60% were compared.Results:The precipitate was impure protein when AS concentration was lower than 30%.A lot of target protein precipitated when AS concentration was higher than 30%.When the concentration was up to 50%,almost all the active protein precipitated.It indicated that a few active protein was salted out when AS concentration was between 0 ~ 30% and most of active protein salted out when AS concentration was between 30% ~ 60%.Conclusions:The two step AS salting out method for separation and purification of active enzyme from fibrinolytic enzyme fermentation broth of Guizhou lobster sauce is established.%目的:对贵州豆豉发酵液中纤溶酶分离纯化.方法:贵州特色药食两用的豆豉中提取得到的纤溶酶菌株(DCK-B),用液体发酵法将其发酵,采用硫酸铵盐析法进行粗提纯,并对0~80%浓度的一级硫酸铵梯度盐析与0~30%、30%~ 60%两段硫酸铵梯度盐析的结果进行比较.结果:硫酸铵梯度盐析在30%以前出现的沉淀为杂蛋白,且较复杂;30%梯度以后的目标蛋白大量沉淀出来,到50%时活性蛋白几乎沉淀完全;经两段盐析,当0~30%盐析时有少量活性蛋白盐析出来,绝大部分蛋白在30%~ 60%这个盐析梯度沉降下来.结论:确定了贵州豆豉纤溶酶发酵液中活性酶的两段硫酸铵盐析分离纯化方法.

  9. A simple method of catalase purification for the undergraduate experimental course.

    Science.gov (United States)

    Chen, Qian; Cheng, Meng; Wang, Yinnan; Yao, Ming; Chen, Yongchun; Gao, Yuan; Ding, Wenyuan

    2015-02-01

    Catalase is a characteristic enzyme of peroxisomes, of which it is the most abundant protein. This enzyme serves as a typical example of a peroxisomal enzyme and is important in the teaching of biochemistry and molecular biology. Although there is substantial information regarding catalase purification, purifying catalase for the junior‑grade undergraduate experimental course face challenges in obtaining materials and increasingly expensive purification equipment. This study presents a simple method for the purification of mouse liver catalase using ethanol‑chloroform treatment, sodium sulfate fractionation, dialysis and Sephadex G‑200 gel filtration chromatography. Catalase was purified 31.8‑fold with an 18.3% yield. The advantages of this method were its low operating environment requirements, simple procedure and reduced cost. Furthermore, the method was designed to improve students' comprehensive ability and manipulative ability and to introduce a sense of innovation in the fields of biochemistry and molecular biology during their junior year.

  10. Purification and characterization of a hexanol-degrading enzyme extracted from apple

    Science.gov (United States)

    An enzyme having activity towards n-hexanol was purified from apple and its biochemical characteristics were analyzed. The purification steps consisted of sedimentation with ammonium sulfate, DEAE Sepharose Fast Flow ion exchange chromatography and Sephadex G-100 column. The obtained enzyme had a yi...

  11. A Versatile and Inexpensive Enzyme Purification Experiment for Undergraduate Biochemistry Labs.

    Science.gov (United States)

    Farrell, Shawn O.; Choo, Darryl

    1989-01-01

    Develops an experiment that could be done in two- to three-hour blocks and does not rely on cold room procedures for most of the purification. Describes the materials, methods, and results of the purification of bovine heart lactate dehydrogenase using ammonium sulfate fractionation, dialysis, and separation using affinity chromatography and…

  12. Purification and Characterization of Enzymes from Yeast: An Extended Undergraduate Laboratory Sequence for Large Classes

    Science.gov (United States)

    Johanson, Kelly E.; Watt, Terry J.; McIntyre, Neil R.; Thompson, Marleesa

    2013-01-01

    Providing a project-based experience in an undergraduate biochemistry laboratory class can be complex with large class sizes and limited resources. We have designed a 6-week curriculum during which students purify and characterize the enzymes invertase and phosphatase from bakers yeast. Purification is performed in two stages via ethanol…

  13. Purification and Characterization of Enzymes from Yeast: An Extended Undergraduate Laboratory Sequence for Large Classes

    Science.gov (United States)

    Johanson, Kelly E.; Watt, Terry J.; McIntyre, Neil R.; Thompson, Marleesa

    2013-01-01

    Providing a project-based experience in an undergraduate biochemistry laboratory class can be complex with large class sizes and limited resources. We have designed a 6-week curriculum during which students purify and characterize the enzymes invertase and phosphatase from bakers yeast. Purification is performed in two stages via ethanol…

  14. Enzymic synthesis of 1-O-(indol-3-ylacetyl)-beta-D-glucose. Purification of the enzyme from Zea mays, and preparation of antibodies to the enzyme.

    Science.gov (United States)

    Kowalczyk, S; Bandurski, R S

    1991-10-15

    The enzyme indol-3-ylacetylglucose synthase (UDP-glucose:indol-3-ylacetate beta-D-glucosyltransferase) catalyses the reaction: [formula: see text] This is the first step in the series of reactions leading to the indol-3-ylacetic acid conjugates found in maize. Previous attempts to purify this enzyme from the liquid endosperm of kernels of Zea mays (sweet corn) were not entirely successful owing to the lability of partially purified preparations during column chromatography. Thus this enzyme has not previously been purified to homogeneity. During the present study it was found that retention of enzyme activity required the combined presence of glycerol and dithiothreitol. Adding these requirements permitted purification of the enzyme to homogeneity with retention of catalytic activity. These purified preparations were used for preparation of rabbit polyclonal antibodies to the enzyme. Antibodies to the Zea mays endosperm enzyme cross-react with the enzyme from Zea mays vegetative tissues and with an enzyme from the liquid endosperm of oak acorns (Quercus sp). In this paper we report a simplified purification procedure adaptable to the preparation of milligram amounts of the enzyme.

  15. Purification and properties of elastolytic enzyme from Flavobacterium immotum.

    Science.gov (United States)

    Ozaki, H; Shiio, I

    1975-01-01

    Elastolytic enzyme was purified and crystallized from culture fluid of Flavobacterium immotum No. 9-35. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight was determined by Sephadex G-100 gel filtration to be 13,000. The isoelectric point was between pH 8.3 and 8.9. The optimum pH of the enzyme was 7.2 for elastolytic activity. The purified enzyme showed not only elastolytic activity, but also non-specific proteolytic activity against various other proteins. Milk-clotting activity was also observed. The enzyme did not act on keratin, collagen, or fourteen amino acid esters, including N-benzoyl-L-alanine methyl ester, N-benzoyl-L-arginine ethyl ester, and N-acetyl-L-tyrosine ethyl ester, which were typical substrates of pancreatic elastase [EC 3.4.21.11], trypsin [EC 3.4.21.4], and chymotrypsin [EC 3.4.21.1], respectively. However, the enzyme selectively hydrolyzed elastin when both elastin and albumin were present in the reaction mixture. The enzyme was inhibited by o-phenanthroline and various heavy metals such as cadmium, lead, zinc, and mercury. Various inhibitors, such as diisopropyl phosphofluoridate, tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, trypsin inhibitor, iodoacetamide, etc., had no effect on the elastolytic activity.

  16. Single-step affinity purification of enzyme biotherapeutics: a platform methodology for accelerated process development.

    Science.gov (United States)

    Brower, Kevin P; Ryakala, Venkat K; Bird, Ryan; Godawat, Rahul; Riske, Frank J; Konstantinov, Konstantin; Warikoo, Veena; Gamble, Jean

    2014-01-01

    Downstream sample purification for quality attribute analysis is a significant bottleneck in process development for non-antibody biologics. Multi-step chromatography process train purifications are typically required prior to many critical analytical tests. This prerequisite leads to limited throughput, long lead times to obtain purified product, and significant resource requirements. In this work, immunoaffinity purification technology has been leveraged to achieve single-step affinity purification of two different enzyme biotherapeutics (Fabrazyme® [agalsidase beta] and Enzyme 2) with polyclonal and monoclonal antibodies, respectively, as ligands. Target molecules were rapidly isolated from cell culture harvest in sufficient purity to enable analysis of critical quality attributes (CQAs). Most importantly, this is the first study that demonstrates the application of predictive analytics techniques to predict critical quality attributes of a commercial biologic. The data obtained using the affinity columns were used to generate appropriate models to predict quality attributes that would be obtained after traditional multi-step purification trains. These models empower process development decision-making with drug substance-equivalent product quality information without generation of actual drug substance. Optimization was performed to ensure maximum target recovery and minimal target protein degradation. The methodologies developed for Fabrazyme were successfully reapplied for Enzyme 2, indicating platform opportunities. The impact of the technology is significant, including reductions in time and personnel requirements, rapid product purification, and substantially increased throughput. Applications are discussed, including upstream and downstream process development support to achieve the principles of Quality by Design (QbD) as well as integration with bioprocesses as a process analytical technology (PAT).

  17. Nitrile-synthesizing enzyme: Screening, purification and characterization.

    Science.gov (United States)

    Kumano, Takuto; Suzuki, Takahisa; Shimizu, Sakayu; Kobayashi, Michihiko

    2016-09-12

    Cyanide is known as a toxic compound for almost all living organisms. We have searched for cyanide-resistant bacteria from the soil and stock culture collection of our laboratory, and have found the existence of a lot of microorganisms grown on culture media containing 10 mM potassium cyanide. Almost all of these cyanide-resistant bacteria were found to show β-cyano-L-alanine (β-CNAla) synthetic activity. β-CNAla synthase is known to catalyze nitrile synthesis: the formation of β-CNAla from potassium cyanide and O-acetyl-L-serine or L-cysteine. We found that some microorganisms were able to detoxify cyanide using O-methyl-DL-serine, O-phospho-L-serine and β-chloro-DL-alanine. In addition, we purified β-CNAla synthase from Pseudomonas ovalis No. 111 in nine steps, and characterized the purified enzyme. This enzyme has a molecular mass of 60,000 and appears to consist of two identical subunits. The purified enzyme exhibits a maximum activity at pH 8.5-9.0 at an optimal temperature of 40-50°C. The enzyme is specific for O-acetyl-L-serine and β-chloro-DL-alanine. The Km value for O-acetyl-L-serine is 10.0 mM and Vmax value is 3.57 μmol/min/mg.

  18. Purification and Characterization of Melanogenic Enzyme Tyrosinase from Button Mushroom

    Directory of Open Access Journals (Sweden)

    Kamal Uddin Zaidi

    2014-01-01

    Full Text Available Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A key enzyme, tyrosinase, catalyzes the first and only rate-limiting steps in melanogenesis. Since the discovery of its melanogenic properties, tyrosinase has been in prime focus and microbial sources of the enzyme are sought. Agaricus bisporus widely known as the common edible mushroom, it’s taking place in high amounts of proteins, enzyme, carbohydrates, fibers, and low fat contents are frequently cited in the literature in relation to their nutritional value. In the present study tyrosinase from Agaricus bisporus was purified by ammonium sulphate precipitation, dialysis followed by gel filtration chromatography on Sephadex G-100, and ion exchange chromatography on DEAE-Cellulose; the enzyme was purified, 16.36-fold to give 26.6% yield on total activity in the crude extract and final specific activity of 52.19 U/mg. The SDS-PAGE electrophoresis showed a migrating protein band molecular weight of 95 kDa. The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C. The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM. This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications.

  19. Production and Purification of Pharmaceutically Important Fibrinolytic Enzyme from Bacillus Species

    Directory of Open Access Journals (Sweden)

    Sharav A. Desai

    2015-11-01

    Full Text Available The medicinal and pharmaceutical importance of currently available thrombolytic agents like urokinase, t-PA, streptokinase, staphylokinase and others, demonstrated repeatedly since 1970s, however sometimes they cause undesirable side effects like bleeding and allergic responds. The present findings reports isolation, screening and identification of soil bacterium for production of fibrinolytic enzyme. Samples for the study were collected from different locations were first screened for proteolytic activity using skimmed milk agar plate and lastly fibrin plate method was used to evaluate fibrinolytic activity. The strain capable of producing fibrinolytic protein was identified as Bacillus Spp. Using both Bergery’s manual of systemic bacteriology and biochemical characterization simultaneously. Selected strain was than subjected to the process of fermentation using basal media for 5 days, 37°C and at 180rpm. Protein content and fibrinolytic activity were measured by Biuret method using bovine serum albumin as standard and fibrinolytic assay respectively. Three stage purification was done, that includes salting out with ammonium sulphate, followed by gel filtration chromatography and finally separated by RP-HPLC, proteins were eluted in peaks with a retention time of 2.092, 3.188, 5.178, 7.295, and 11.32 minutes. The fraction with retention time 7.295 minutes shows a maximum activity. The enzyme found to be having an optimum pH between 7.0 and 7.5. Enzyme is also stable at the optimum pH and found to lose its activity on higher side of acidity or alkalinity. It is more active at 40°C and is stable at 37°C to 43°C with slight modification in activity.

  20. A novel liquid/liquid extraction process composed of surfactant and acetonitrile for purification of polygalacturonase enzyme from Durio zibethinus.

    Science.gov (United States)

    Amid, Mehrnoush; Manap, Yazid; Azmira, Farhana; Hussin, Muhaini; Sarker, Zaidul Islam

    2015-07-01

    Polygalacturonase is one of the important enzymes used in various industries such as food, detergent, pharmaceutical, textile, pulp and paper. A novel liquid/liquid extraction process composed of surfactant and acetonitrile was employed for the first time to purify polygalacturonase from Durio zibethinus. The influences of different parameters such as type and concentration of surfactants, concentrations of acetonitrile and composition of surfactant/acetonitrile on partitioning behavior and recovery of polygalacturonase was investigated. Moreover, the effect of pH of system and crude load on purification fold and yield of purified polygalacturonase were studied. The results of the experiment indicated the polygalacturonase was partitioned into surfactant top rich phase with impurities being partitioned into acetonitrile bottom rich phase in the novel method of liquid/liquid process composed of 23% (w/w) Triton X-100 and 19% (w/w) acetonitrile, at 55.6% of TLL (tie line length) crude load of 25% (w/w) at pH 6.0. Recovery and recycling of components also was measured in each successive step of liquid/liquid extraction process. The enzyme was successfully recovered by the method with a high purification factor of 14.3 and yield of 97.3% while phase components were also recovered and recycled above 95%. This study demonstrated that the novel method of liquid/liquid extraction process can be used as an efficient and economical extraction method rather than the traditional methods of extraction for the purification and recovery of the valuable enzyme.

  1. Purification and properties of the inducible enzyme cyanase.

    Science.gov (United States)

    Anderson, P M

    1980-06-24

    Cyanase (cyanate hydrolase EC 3.5.5.3) has been purified 270-fold to a high state of purity from Escherichia coli B. The native enzyme has a molecular weight of approximately 150 000 as estimated by sucrose density gradient centrifugation and gel-filtration chromatography on Bio-Gel P-300. The enzyme is an oligomer composed of apparently identical subunits which have a molecular weight of approximately 15 000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analyses showed that the enzyme contains no tryptophan and a single histidine residue, based on a subunit molecular weight of 14 661. Catalytic hydrolysis of cyanate was found to be dependent on the patience of bicarbonate and to be affected by ionic strength. The concentration of bicarbonate required to give half-maximal activity in the presence of 2 mM potassium cyanate was 0.1 mM. The apparent Km for cyanate in the presence of 3 mM bicarbonate is 0.6 mM. The initial product of the reaction is carbamate (or a related, unstable compound and/or carbamate precursor) which subsequently decomposes to ammonia and bicarbonate.

  2. Evaluation, partial characterization and purification of acetylcholine esterase enzyme and antiangiogenic activity from marine sponges

    Institute of Scientific and Technical Information of China (English)

    Maushmi Shailesh Kumar; Sukanya Gopalkrishnan

    2014-01-01

    Objective: To test three marine sponges Halichondria glabrata Keller, 1891; Spirastrellapachyspira (S. pachyspira) Levi, 1958 and Cliona lobata Hancock, 1849 for the presence of the acetylcholinesterase (AChE) in both young and developed samples from western coastal area of India. S. pachyspira methanolic extract was selected for anti/pro angiogenic activity. Methods:They were evaluated for AChE activity using Ellman’s assay based on production of yellow colored 5-thio-2-nitrobenzoate. Purification of the enzyme was planned using ammonium sulphate precipitation and characterization by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Chorioallantoic membrane (ChAM) assay model was used for angiogenic/antiangiogenic testing. Results:All the three sponges showed good specific enzyme activity and S. pachyspira contained maximum specific enzyme activity. Sixty percent of ammonium sulphate precipitation of crude protein sample gave single band at 66 kDa corresponding to the true AChE. ChAM assay was performed at 62.5, 125.0 and 250.0 µg/mL. Dosage beyond 250 µg/mL extract showed toxic response with anti angiogenic activity at all the concentrations. Conclusions:AChE activity was detected in all samples. Extract showed good anti-angiogenic response at 62.5 µg/mL. Extract was highly toxic affecting microvasculature of ChAM as well as normal growth and development of the embryo at 500 µg/mL. With further characterization of bioactive compounds from the extract of S. pachyspira, the compounds can be developed for anti tumor activity.

  3. Evaluation, partial characterization and purification of acetylcholine esterase enzyme and antiangiogenic activity from marine sponges

    Directory of Open Access Journals (Sweden)

    Maushmi Shailesh Kumar

    2014-11-01

    Full Text Available Objective: To test three marine sponges Halichondria glabrata Keller, 1891; Spirastrella pachyspira (S. pachyspira Levi, 1958 and Cliona lobata Hancock, 1849 for the presence of the acetylcholinesterase (AChE in both young and developed samples from western coastal area of India. S. pachyspira methanolic extract was selected for anti/pro angiogenic activity. Methods: They were evaluated for AChE activity using Ellman’s assay based on production of yellow colored 5-thio-2-nitrobenzoate. Purification of the enzyme was planned using ammonium sulphate precipitation and characterization by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Chorioallantoic membrane (ChAM assay model was used for angiogenic/ antiangiogenic testing. Results: All the three sponges showed good specific enzyme activity and S. pachyspira contained maximum specific enzyme activity. Sixty percent of ammonium sulphate precipitation of crude protein sample gave single band at 66 kDa corresponding to the true AChE. ChAM assay was performed at 62.5, 125.0 and 250.0 µg/mL. Dosage beyond 250 µg/mL extract showed toxic response with anti angiogenic activity at all the concentrations. Conclusions: AChE activity was detected in all samples. Extract showed good anti-angiogenic response at 62.5 µg/mL. Extract was highly toxic affecting microvasculature of ChAM as well as normal growth and development of the embryo at 500 µg/mL. With further characterization of bioactive compounds from the extract of S. pachyspira, the compounds can be developed for anti tumor activity.

  4. The efficiency study of different purification methods for liquid scintillator

    CERN Document Server

    Hu, Wei; Yu, Boxiang; Zhang, Xuan; Zhou, Li; Cai, Xiao; Sun, Lijun

    2016-01-01

    JUNO is an experiment aimed at detecting neutrino mass hierarchy. The innermost part of the JUNO detector is formed by 20,000 tons of liquid scintillator which should have very low level of radioactive materials, such as 238U, 232Th, and 40K. Since the radioactive level of raw LAB(the solvent of LS)cannot reach so stringent requirements of JUNO, the purification for LAB plays an extremely important role in LS production. This article studies the efficiency of several different purification methods for LS, like distillation, water extraction and Al2O3 purification.

  5. A practical method of expression, purification and identification of beta-site app-cleaving enzyme%一种实用的β-分泌酶表达、纯化和活性鉴定方法

    Institute of Scientific and Technical Information of China (English)

    朱瑞; 伍巧燕; 张兴梅; 高小芳; 卫佩如; 张馨宇; 王方

    2015-01-01

    Objective To introduce a practical method that can be used to efficiently express,purify and identify Alzheimer's disease (AD) related beta-site app-cleaving enzyme 1 (BACE1) in common eukaryotic cells.Methods BACE1 cDNA was fished out from human brain cDNA library and ligated into the pEGFP-c3 expression vector,and then,the recombinant plasmid was transfected into the HEK293 cells.The BACE1 protein was purified with TALON Mental Affinity Resins column.The target protein was identified by Western blotting and fluorescence resonance energy transfer (FRET).BACE1 Activity Assay Kit was employed to test the activity of purified BACE1 in vitro.The recombinant BACE1/pEGFP-c3 plasmid and amyloid precusor protein (APP)/pDsRed-Monomer-N1 plasmid were co-transfected to the HEK293 cells and the cleavage activity of BACE1 in the cells was identified by Western blotting.Results The sequencing data of the obtained BACE1 gene were identical with those in GenBank.Activity test showed that the fluorescent values of blank controls,expressed BACE1 and standard BACE1 were 55.013±3.597,1836.629±154.195 (n=3) and 2639.548±207.1901 (n=3),respectively;as compared with the control group,significant differences were noted in both of the two groups (F=78.681,P=0.000);however,there is no significant difference between expressed BACE1 and standard BACE1 groups (P>0.05).Westem blotting showed the co-transfected BACE1 could cleave APP in HEK293 cells and the CTF-APP band was detectable.Conclusion A practical protocol is established for high expression,purification and identification of BACE1 in HEK293 cells,which is helpful to obtain BACE1,an important molecular target in AD research and treatment.%目的 探讨一种在常用真核细胞系中表达、纯化和鉴定阿尔茨海默病(AD)相关蛋白-β-分泌酶(BACE1)的方法. 方法 在从人脑基因库中获取BA CE1序列并成功扩增的基础上,将其插入带有增强绿色荧光蛋白(EGFP)的表达载体pEGFP-c3中.将BACE1/p

  6. Recommended methods for purification of solvents and tests for impurities

    CERN Document Server

    Coetzee, J F

    1982-01-01

    Recommended Methods for Purification of Solvents and Tests for Impurities is a compilation of recommended procedures for purification of solvents and tests for solvent impurities. Ten solvents are covered: acetonitrile, sulfolane, propylene carbonate, dimethyl sulfoxide, dimethylformamide, hexamethylphosphoramide, pyridine, ethylenediamine, N-methylacetamide, and N-methylpropionamide. This book is comprised of 12 chapters and opens with an introduction to general aspects of impurity effects. The rationale for the selection of solvent is explained, and the relative reactivities of solutes in di

  7. Biodiesel Production Using Wet and Dry Purification Methods

    OpenAIRE

    DEMIR, Veli Gokhan

    2017-01-01

    Inbiodiesel production via transesterification, after removing glycerol fromcrude biodiesel, purification process must be performed before using biodieselas a fuel that meets the EN 14214 standard. In the literature, variousprocesses are presented for purification of biodiesel however; dry and wetwashing methods are mostly recommended because of their higher efficiencies andeasier applicabilities. In this study, methyl esters (biodiesel) derived fromwaste frying oil (WFO) and sunflower oil we...

  8. A rapid and economic in-house DNA purification method using glass syringe filters.

    Directory of Open Access Journals (Sweden)

    Yun-Cheol Kim

    Full Text Available BACKGROUND: Purity, yield, speed and cost are important considerations in plasmid purification, but it is difficult to achieve all of these at the same time. Currently, there are many protocols and kits for DNA purification, however none maximize all four considerations. METHODOLOGY/PRINCIPAL FINDINGS: We now describe a fast, efficient and economic in-house protocol for plasmid preparation using glass syringe filters. Plasmid yield and quality as determined by enzyme digestion and transfection efficiency were equivalent to the expensive commercial kits. Importantly, the time required for purification was much less than that required using a commercial kit. CONCLUSIONS/SIGNIFICANCE: This method provides DNA yield and quality similar to that obtained with commercial kits, but is more rapid and less costly.

  9. Partial purification and characterization of protease enzyme from Bacillus subtilis and Bacillus cereus.

    Science.gov (United States)

    Orhan, Elif; Omay, Didem; Güvenilir, Yüksel

    2005-01-01

    The aim of this experimental study was to isolate and partially purify protease enzyme from Bacillus cereus and Bacillus subtilis. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species in suitable nutrient plates. The partial purification was realized by applying, respectively, ammonium sulfate precipitation, dialysis, and DEAE-cellulose ion-exchange chromatography to the supernatant that was produced later. Optimum pH, optimum temperature, pH stability, and temperature stability were determined, as well as the effects of pH, temperature, substrate concentration, reaction time, and inhibitors and activators on enzyme activity. In addition, the molecular mass of the obtained enzyme was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of partially purified enzyme from B. subtilis was determined to be 84 U/mg. The final enzyme preparation was eight-fold more pure than the crude homogenate. The molecular mass of the partially purified enzyme was found to be 45 kDa by using SDS-PAGE. The protease enzyme that was partially purified from B. cereus was purified 1.2-fold after ammonium sulfate precipitation. The molecular mass of the partially purified enzyme was determined to be 37 kDa by using SDS-PAGE.

  10. Separation and purification of enzymes by continuous pH-parametric pumping

    Energy Technology Data Exchange (ETDEWEB)

    Huang, S.Y.; Lin, C.K.; Juang, L.Y.

    1985-10-01

    Trypsin and chymotrypsin were separated from porcine pancreas extract by continuous pH-parametric pumping. CHOM (chicken ovomucoid) was convalently bound to laboratory-prepared crab chitin with glutaraldehyde to form an affinity adsorbent of trypsin. The pH levels of top and bottom feeds were 8.0 and 2.5, respectively. Similar inhibitor, DKOM (duck ovomucoid), and pH levels 8.0 and 2.0 for top and bottom feeds, respectively, were used for separation and purification of chymotrypsin. e-Amino caproyl-D-tryptophan methyl ester was coupled to chitosan to form an affinity adsorbent for stem bromelain. The pH levels were 8.7 and 3.0. Separation continued fairly well with high yield, e.g., 95% recovery of trypsin after continuous pumping of 10 cycles. Optimum operational conditions for concentration and purification of these enzymes were investigated. The results showed that the continuous pH-parametric pumping coupled with affinity chromatography is effective for concentration and purification of enzymes. 19 references.

  11. A simple method for purification of herpesvirus DNA

    DEFF Research Database (Denmark)

    Christensen, Laurids Siig; Normann, Preben

    1992-01-01

    A rapid and reliable method for purification of herpesvirus DNA from cell cultures is described. The method is based on the isolation of virus particles and/or nucleocapsids by differential centrifugation and exploits the solubilizing and denaturing capabilities of cesium trifluoroacetate during ...... isopycnic centrifugation, so that phenol/chloroform extractions can be omitted. The method was used for the purification of DNA from several members of the Alfaherpesvirinae subfamily.......A rapid and reliable method for purification of herpesvirus DNA from cell cultures is described. The method is based on the isolation of virus particles and/or nucleocapsids by differential centrifugation and exploits the solubilizing and denaturing capabilities of cesium trifluoroacetate during...

  12. Purification of Pseudomonas sp. Lipase by Continuous Elution Electrophoresis Based on Pb2+ Precipitation Method

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hua-li; WANG Zhi; LIU Bin; WANG Xue-li; CAO Shu-gui; LI Zheng-qiang

    2005-01-01

    A Pb2+ precipitation method was designed to get rid of the impure proteins in a lipase. The results show that it was a simple way in the primary treatment of the crude samples and about 20% impure proteins were removed in the precipitation step. Further, continuous elution electrophoresis was also applied as a preparative technique for attaining the highly pure lipase. During the continuous elution electrophoresis, the enzyme was eluted as a single peak and 5.7-fold purification was achieved in a yield of 54.3%. The two steps finally yielded an electrophoretically homogeneous enzyme.

  13. Enhanced method for microbial community DNA extraction and purification from agricultural yellow loess soil.

    Science.gov (United States)

    Kathiravan, Mathur Nadarajan; Gim, Geun Ho; Ryu, Jaewon; Kim, Pyung Il; Lee, Chul Won; Kim, Si Wouk

    2015-11-01

    In this study, novel DNA extraction and purification methods were developed to obtain high-quantity and reliable quality DNA from the microbial community of agricultural yellow loess soil samples. The efficiencies of five different soil DNAextraction protocols were evaluated on the basis of DNA yield, quality and DNA shearing. Our suggested extraction method, which used CTAB, EDTA and cell membrane lytic enzymes in the extraction followed by DNA precipitation using isopropanol, yielded a maximum DNA content of 42.28 ± 5.59 µg/g soil. In addition, among the five different purification protocols, the acid-treated polyvinyl polypyrrolidone (PVPP) spin column purification method yielded high-quality DNA and recovered 91% of DNA from the crude DNA. Spectrophotometry revealed that the ultraviolet A 260/A 230 and A 260/A 280 absorbance ratios of the purified DNA were 1.82 ± 0.03 and 1.94 ± 0.05, respectively. PCR-based 16S rRNA amplification showed clear bands at ~1.5 kb with acid-treated PVPP-purified DNA templates. In conclusion, our suggested extraction and purification protocols can be used to recover high concentration, high purity, and high-molecular-weight DNA from clay and silica-rich agricultural soil samples.

  14. Efficient and inexpensive method for purification of heparin binding proteins.

    Science.gov (United States)

    Batra, Sumit; Sahi, Nilesh; Mikulcik, Kristen; Shockley, Heather; Turner, Camille; Laux, Zachary; Badwaik, Vivek D; Conte, Eric; Rajalingam, Dakshinamurthy

    2011-08-15

    Heparin binding (HB) proteins mediate a wide range of important cellular processes, which makes this class of proteins biopharmaceutically important. Engineering HB proteins may bring many advantages, but it necessitates cost effective and efficient purification methodologies compared to currently available methods. One of the most important classes of HB proteins are fibroblast growth factors (FGFs) and their receptors (FGFRs). In this study, we report an efficient off-column purification of FGF-1 from soluble fractions and purification of the D2 domain of FGFR from insoluble inclusion bodies, using a weak Amberlite cation (IRC) exchanger. FGF-1 and the D2 domain have been expressed in Escherichia coli and purified to homogeneity using IRC resin. This approach is an alternative to conventional affinity column chromatography, which exhibits several disadvantages, including time-consuming experimental procedures for purification and regeneration and results in the expensive production of recombinant proteins. Results of the heparin binding chromatography and steady state fluorescence experiments show that the FGF-1 and the D2 are in a native conformation. The findings of this study will not only aid an in-depth investigation of this class of proteins but will also provide avenues for inexpensive and efficient purification of other important biological macromolecules.

  15. A convenient method for lecithin purification from fresh eggs

    Directory of Open Access Journals (Sweden)

    Flavio A. Maximiano

    2008-01-01

    Full Text Available The increasing demand for fatty acid-free lecithin required modifications in existing purification methods. In this technical note we describe a purification procedure with the following steps: a homogenization and extraction of yolks obtained from fresh eggs with acetone, b solubilization with ethanol and solvent elimination and c repeated solubilization/precipitation with petroleum ether/acetone. This crude extract was chromatographed on neutral alumina, which was exhaustively washed with chloroform before elution with chloroform:methanol, allowing the sequential separation of fatty acids and lecithin. Chromatographic behavior and mass spectra of the product are presented. This fast procedure yields fatty acid-free lecithin at a competitive cost.

  16. Partial purification of chlorophyll degrading enzymes from cavendish banana (Musa Cavendishi).

    Science.gov (United States)

    Janave, Machhindra T; Sharma, Arun

    2004-08-01

    Cavendish banana (Musa Cavendishi, subgroup AAA) remains green upon ripening at tropical temperature (25-30 degrees C), due to incomplete degradation of chlorophyll (Chl). Earlier, evidence for the existence of two distinct degradative pathways--chlorophyllase and chlorophyll oxidase pathways in these bananas was provided. Here, an attempt has been made to understand further the mechanism of inhibition of Chl degradation at different stages of ripening and detecting various enzyme activities by partial purification. Soluble and Triton-solubilized protein fractions obtained from peel acetone powder from green-unripe, green-ripe and yellow-ripe bananas efficiently degraded Chl a. About 2-fold increase in Chl hydrolyzing/oxidizing and magnesium-dechelatase activities was observed in ripe, as compared to green-unripe bananas. The electrophoretic pattern of the soluble and detergent-solubilized proteins from the three stages of ripening revealed that the latter fraction contained only three slow moving proteins, which were found to be glycoproteins, as revealed in PAS staining. The soluble enzyme fraction contained all other bands along with the above three bands, as observed in the Native-PAGE of DEAE-Sepharose purified fractions. Only soluble fraction from 'green-ripe' bananas, catalyzed formation of an unknown intermediate (retention time 8.6 min), which was formed by the action of Triton-solubilized enzyme fractions, obtained from 'green-unripe' and 'yellow-ripe' bananas. The enzyme responsible for the formation of this intermediate might be involved in the stay-green character and could be a component of Chl oxidase pathway. Partial purification of soluble protein fraction by DEAE-Sepharose showed the presence of chlorophyllase, magnesium-dechelatase, pheophorbide a oxygenase, red fluorescent catabolite reductase and Chl oxidase. Native PAGE of pooled fractions showed separation of proteins in different bands. Pooled fractions IV and VI showed the presence of a

  17. Partial purification of new milk-clotting enzyme produced by Nocardiopsis sp.

    Science.gov (United States)

    Cavalcanti, M T H; Teixeira, M F S; Lima Filho, J L; Porto, A L F

    2004-05-01

    Numerous attempts have been made to replace calf rennet with other milk clotting proteases because of limited supply and increasingly high prices. The aim of this work was to investigate the characteristic of the milk-clotting enzyme from Nocardiopsis sp. The partial purification extract was obtained by fractional precipitation with ammonium sulphate. Of the fractions obtained by precipitation, 40-60% possessed the milk-clotting activity (156.25 U/mg). The chromatography of 40-100% ammonium sulphate fraction in DEAE-cellulose yielded four fractions (F4, F5, F6, F7) with milk-clotting activity. The F5 yielded the best milk-clotting activity (20 U/ml). Both crude and partially purified extract were active at the range pH 4.5-11.0, however, optimum activity was displayed at pH 11.0 and pH 7.5, respectively. The milk-clotting activity was highest at 55 degrees C for both crude and partially purified extract. The crude and partial purification extract were inactivated at 65 and 75 degrees C after 30 min.

  18. DYNAMIC MODELLING AND ADVANCED PREDICTIVE CONTROL OF A CONTINUOUS PROCESS OF ENZYME PURIFICATION

    Directory of Open Access Journals (Sweden)

    Dechechi E.C.

    1997-01-01

    Full Text Available A dynamic mathematical model, simulation and computer control of a Continuous Affinity Recycle Extraction (CARE process, a protein purification technique based on protein adsorption on solid-phase adsorbents is described in this work. This process, consisting of three reactors, is a multivariable process with considerable time delay in the on-line analyses of the controlled variable. An advanced predictive control configuration, specifically the Dynamic Matrix Control (DMC, was applied. The DMC algorithm was applied in process schemes where the aim was to maintain constant the enzyme concentration in the outlet of the third reactor. The performance of the DMC controller was analyzed in the feed-flow disturbances and the results are presented.

  19. Purification, kinetic behavior, and regulation of NAD(P)+ malic enzyme of tumor mitochondria.

    Science.gov (United States)

    Moreadith, R W; Lehninger, A L

    1984-05-25

    The purification and kinetic characterization of an NAD(P)+-malic enzyme from 22aH mouse hepatoma mitochondria are described. The enzyme was purified 328-fold with a final yield of 51% and specific activity of 38.1 units/mg of protein by employing DEAE-cellulose chromatography and an ATP affinity column. Sephadex G-200 chromatography yielded a native Mr = 240,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major subunit with Mr = 61,000, suggesting a tetrameric structure, and also showed that the preparation contained less than 10% polypeptide impurities. Use of the ATP affinity column required the presence of MnCl2 and fumarate (an allosteric activator) in the elution buffers. In the absence of fumarate, the Michaelis constants for malate, NAD+, and NADP+ were 3.6 mM, 55 microM, and 72 microM, respectively; in the presence of fumarate (2 mM), the constants were 0.34 mM, 9 microM, and 13 microM, respectively. ATP was shown to be an allosteric inhibitor, competitive with malate. However, the inhibition by ATP displayed hyperbolic competitive kinetics with a KI (ATP) of 80 microM (minus fumarate) and 0.5 mM (plus 2 mM fumarate). The allosteric properties of the enzyme are integrated into a rationale for its specific role in the pathways of malate and glutamate oxidation in tumor mitochondria.

  20. Chromatographic and electrophoretic methods for nanodisc purification and analysis

    DEFF Research Database (Denmark)

    Justesen, Bo Højen; Günther-Pomorski, Thomas

    2014-01-01

    Soluble nanoscale lipid bilayers, termed nanodiscs, are widely used in science for studying the membrane-anchored and integral membrane protein complexes under defined experimental conditions. Although their formation occurs by a self-assembly process, nanodisc purification and the verification...... of proper reconstitution are still major challenges during the sample preparation. This review gives an overview of the methods used for purifying and analyzing nanodiscs and nanodisc-reconstituted membrane proteins, with an emphasis on the chromatographic and electrophoretic approaches....

  1. A novel aqueous micellar two-phase system composed of surfactant and sorbitol for purification of pectinase enzyme from Psidium guajava and recycling phase components.

    Science.gov (United States)

    Amid, Mehrnoush; Murshid, Fara Syazana; Manap, Mohd Yazid; Hussin, Muhaini

    2015-01-01

    A novel aqueous two-phase system composed of a surfactant and sorbitol was employed for the first time to purify pectinase from Psidium guajava. The influences of different parameters, including the type and concentration of the surfactant and the concentration and composition of the surfactant/sorbitol ratio, on the partitioning behavior and recovery of pectinase were investigated. Moreover, the effects of system pH and the crude load on purification fold and the yield of purified pectinase were studied. The experimental results indicated that the pectinase was partitioned into surfactant-rich top phase, and the impurities were partitioned into the sorbitol-rich bottom phase with the novel method involving an ATPS composed of 26% (w/w) Triton X-100 and 23% (w/w) sorbitol at 54.2% of the TLL crude load of 20% (w/w) at pH 6.0. The enzyme was successfully recovered by this method with a high purification factor of 15.2 and a yield of 98.3%, whereas the phase components were also recovered and recycled at rates above 96%. This study demonstrated that this novel ATPS method can be used as an efficient and economical alternative to the traditional ATPS for the purification and recovery of the valuable enzyme.

  2. Partial purification of saccharifying and cell wall-hydrolyzing enzymes from malt in waste from beer fermentation broth.

    Science.gov (United States)

    Khattak, Waleed Ahmad; Kang, Minkyung; Ul-Islam, Mazhar; Park, Joong Kon

    2013-06-01

    A number of hydrolyzing enzymes that are secreted from malt during brewing, including cell wall-hydrolyzing, saccharide-hydrolyzing, protein-degrading, lipid-hydrolyzing, and polyphenol and thiol-hydrolyzing enzymes, are expected to exist in an active form in waste from beer fermentation broth (WBFB). In this study, the existence of these enzymes was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, after which enzyme extract was partially purified through a series of purification steps. The hydrolyzing enzyme activity was then measured under various conditions at each purification step using carboxymethyl cellulose as a substrate. The best hydrolyzing activities of partially purified enzymes were found at pH 4.5 and 50 °C in a citrate buffer system. The enzymes showed highest thermal stability at 30 °C when exposed for prolonged time. As the temperature increased gradually from 25 to 70 °C, yeast cells in the chemically defined medium with enzyme extract lost their cell wall and viability earlier than those without enzyme extract. Cell wall degradation and the release of cell matrix into the culture media at elevated temperature (45-70 °C) in the presence of enzyme extract were monitored through microscopic pictures. Saccharification enzymes from malt were relatively more active in the original WBFB than supernatant and diluted sediments. The presence of hydrolyzing enzymes from malt in WBFB is expected to play a role in bioethanol production using simultaneous saccharification and fermentation without the need for additional enzymes, nutrients, or microbial cells via a cell-free enzyme system.

  3. Purification of pectinase from mango (Mangifera indica L. cv. Chokanan) waste using an aqueous organic phase system: a potential low cost source of the enzyme.

    Science.gov (United States)

    Amid, Mehrnoush; Abdul Manap, Mohd Yazid; Mustafa, Shuhaimi

    2013-07-15

    As a novel method of purification, an aqueous organic phase system (AOPS) was employed to purify pectinase from mango waste. The effect of different parameters, such as the alcohol concentration (ethanol, 1-propanol, and 2-propanol), the salt type and concentration (ammonium sulfate, potassium phosphate and sodium citrate), the feed stock crude load, the aqueous phase pH and NaCl concentration, were investigated in the recovery of pectinase from mango peel. The partition coefficient (K), selectivity (S), purification factor (PF) and yield (Y, %) were investigated in this study as important parameters for the evaluation of enzyme recovery. The desirable partition efficiency for pectinase purification was achieved in an AOPS of 19% (w/w) ethanol and 22% (w/w) potassium phosphate in the presence of 5% (w/w) NaCl at pH 7.0. Based on the system, the purification factor of pectinase was enhanced 11.7, with a high yield of 97.1%. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Simple method for purification of enterotoxigenic Escherichia coli fimbriae.

    Science.gov (United States)

    Curtis, Brittany; Grassel, Christen; Laufer, Rachel S; Sears, Khandra T; Pasetti, Marcela F; Barry, Eileen M; Simon, Raphael

    2016-03-01

    Enterotoxigenic Escherichia coli (ETEC) are endemic pathogens in the developing world. They frequently cause illness in travelers, and are among the most prevalent causes of diarrheal disease in children. Pathogenic ETEC strains employ fimbriae as adhesion factors to bind the luminal surface of the intestinal epithelium and establish infection. Accordingly, there is marked interest in immunoprophylactic strategies targeting fimbriae to protect against ETEC infections. Multiple strategies have been reported for purification of ETEC fimbriae, however none is ideal. Purification has typically involved the use of highly virulent wild-type strains. We report here a simple and improved method to purify ETEC fimbriae, which was applied to obtain two different Class 5 fimbriae types of clinical relevance (CFA/I and CS4) expressed recombinantly in E. coli production strains. Following removal from cells by shearing, fimbriae proteins were purified by orthogonal purification steps employing ultracentrifugation, precipitation, and ion-exchange membrane chromatography. Purified fimbriae demonstrated the anticipated size and morphology by electron microscopy analysis, contained negligible levels of residual host cell proteins, nucleic acid, and endotoxin, and were recognized by convalescent human anti-sera. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Excretion of laccase by sycamore (Acer pseudoplatanus L.) cells. Purification and properties of the enzyme.

    Science.gov (United States)

    Bligny, R; Douce, R

    1983-02-01

    A laccase-type polyphenol oxidase is excreted by sycamore cells (Acer pseudoplatanus L.) cells. The enzyme has been purified by classical purification techniques. It is a blue copper protein of Mr 97 000, containing 45% carbohydrate and 0.24% copper. This protein consists of one single unit and the copper content corresponds to four copper atoms per protein molecule. The specific activity of the purified extracellular sycamore-cell laccase measured at pH 6.6 (optimum pH) and in the presence of 20mM-4-methhylcatechol (optimum substrate conditions) corresponded to an oxygen uptake of 32 000 nmol of O2/min per mg of protein. Under these conditions, the catalytic-centre activity of the enzyme reached 100 s-1. The excretion of laccase by sycamore cells is significant, being about 2% of the total protein synthesized by the cells during the exponential phase of growth, and is independent of cell growth. The physiological significance and the problems raised by the passage of this protein across the cytoplasmic membrane are discussed.

  6. Fermentation, fractionation and purification of streptokinase by chemical reduction method

    Directory of Open Access Journals (Sweden)

    M Niakan

    2011-05-01

    Full Text Available Background and Objectives: Streptokinase is used clinically as an intravenous thrombolytic agent for the treatment of acute myocardial infarction and is commonly prepared from cultures of Streptococcus equisimilis strain H46A. The objective of the present study was the production of streptokinase from strain H46A and purification by chemical reduction method."nMaterials and Methods: The rate of streptokinase production evaluated under the effect of changes on some fermentation factors. Moreover, due to the specific structure of streptokinase, a chemical reduction method employed for the purification of streptokinase from the fermentation broth. The H46A strain of group C streptococcus, was grown in a fermentor. The proper pH adjusted with NaOH under glucose feeding in an optimum temperature. The supernatant of the fermentation product was sterilized by filtration and concentrated by ultrafiltration. The pH of the concentrate was adjusted, cooled, and precipitated by methanol. Protein solution was reduced with dithiothreitol (DTT. Impurities settled down by aldrithiol-2 and the biological activity of supernatant containing streptokinase was determined."nResults: In the fed -batch culture, the rate of streptokinase production increased over two times as compared with the batch culture and the impurities were effectively separated from streptokinase by reduction method."nConclusion: Improvements in SK production are due to a decrease in lag phase period and increase in the growth rate of logarithmic phase. The methods of purification often result in unacceptable losses of streptokinase, but the chemical reduction method give high yield of streptokinase and is easy to perform it.

  7. Fibrinolytic enzyme from newly isolated marine bacterium Bacillus subtilis ICTF-1: media optimization, purification and characterization.

    Science.gov (United States)

    Mahajan, Prafulla M; Nayak, Shubhada; Lele, Smita S

    2012-03-01

    Fibrinolytic enzymes are important in treatment of cardiovascular diseases. The present work reports isolation, screening and identification of marine cultures for production of fibrinolytic enzymes. A potent fibrinolytic enzyme-producing bacterium was isolated from marine niches and identified as Bacillus subtilis ICTF-1 on the basis of the 16S rRNA gene sequencing and biochemical properties. Further, media optimization using L(18)-orthogonal array method resulted in enhanced production of fibrinolytic enzyme (8814 U/mL) which was 2.6 fold higher than in unoptimized medium (3420 U/mL). In vitro assays revealed that the enzyme could catalyze blood clot lysis effectively, indicating that this enzyme could be a useful thrombolytic agent. A fibrinolytic enzyme was purified from the culture supernatant to homogeneity by three step procedures with a 34.42-fold increase in specific activity and 7.5% recovery. This purified fibrinolytic enzyme had molecular mass of 28 kDa, optimal temperature and pH at 50 °C and 9, respectively. It was stable at pH 5.0-11.0 and temperature of 25-37 °C. The enzyme activity was activated by Ca(2+) and obviously inhibited by Zn(2+), Fe(3)(+), Hg(2+) and PMSF. The purified fibrinolytic enzyme showed high stability towards various surfactants and was relatively stable towards oxidizing agent. Considering these properties purified fibrinolytic enzyme also finds potential application in laundry detergents in addition to thrombolytic agent. The gene encoding fibrinolytic enzyme was isolated and its DNA sequence was determined. Compared the full DNA sequence with those in NCBI, it was considered to be a subtilisin like serine-protease.

  8. Partial purification and characterization of an enzyme involved in the formation of beta-aspartyl dipeptides in rat kidney.

    Science.gov (United States)

    Tanaka, T; Hirai, M; Nakajima, T

    1978-11-01

    The formation of beta-aspartyl-glycine from asparagine and glycine was demonstrated in the supernatant of rat kidney. The enzyme involved in this process was partially purified. Based on the properties of the enzyme reaction and the coincidence of purification rates of this activity and asparaginase, it can be speculated that the enzyme is a kind of asparaginase. Examination of the preference for beta-aspartyl donors and acceptors showed that asparagine and glycine were the preferred donor and acceptor, respectively. beta-Aspartyl dipeptides also transferred their aspartyl residues to amino acids. Amino acids other than glycine also accepted the aspartyl moiety from the donors.

  9. Purification and Characterisation of a Fibrinolytic Enzyme from Rhizopus micro sporus var. tuberosus

    Directory of Open Access Journals (Sweden)

    Shuli Zhang

    2015-01-01

    Full Text Available Extracellular fibrinolytic enzyme from Rhizopus microsporus var. tuberosus was purified and characterised. The microorganism was isolated in a distillery from daqu, a fermentative agent used in the production of Chinese liquor and vinegar at diff erent temperatures. The fibrinolytic enzyme was partially purifi ed by ammonium sulphate precipitation, dialysis, DEAE Sepharose® Fast Flow ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The molecular mass of the fi brinolytic enzyme was estimated to be 24.5 kDa by SDS-PAGE. The purified enzyme showed optimal activity at pH=7.0 and 37 °C by fibrin plate method. It showed stronger resistance to the inhibition by trypsin and was stable at 37 °C retaining 96.1 % residual activity aft er 4 h of incubation. The fibrinolytic activity of the enzyme was enhanced by Na+, Ca2+, Mg2+ and Mn2+. Conversely, Zn2+ and Cu2+ partly inhibited enzymatic activity. Using fibrin plate method, we found that the enzyme not only degrades fibrin directly, but also activates plasminogen into plasmin to degrade fibrin. The results indicate that the pure enzyme has a potential in dissolving blood clot, and the possibility for application in the treatment of thrombosis.

  10. Modern Methods for Isolation, Purification, and Cultivation of Soil Cyanobacteria.

    Science.gov (United States)

    Temraleeva, A D; Dronova, S A; Moskalenko, S V; Didovich, S V

    2016-07-01

    Up-to-date methods for isolation of cyanobacteria from soil samples, removal of accompanying microflora, obtaining axenic strains, and -conditions and media for subsequnt cultivation are reviewed. Char acterization of soil as a specific habitat for cyanobacteria is provided. Comparative analysis of pH and ele- mental composition of the liquid phase of most soil types with the media for cultivating cyanobacteria is car- ried out. The functional role of the major components required for the cultivation of cyanobacteria is de- scribed. The problems associated with isolation, purification, and cultivation of soil cyanobacteria, as well as the relevant solutions, are discussed.

  11. Developing Unconstrained Methods for Enzyme Evolution

    Science.gov (United States)

    2014-09-19

    methods fail to produce catalytically efficient enzymes. This study has broad application in many technologies from chemical synthesis to human health and...enzymes. This study has broad application in many technologies from chemical synthesis to human health and the environment. Our work centers around the...minimal media with N-15 labeled ammonia . After several months of screening, we finally identified conditions that allowed us to obtain labeled protein in

  12. PURIFICATION OF ANGIOTENSIN CONVERTING ENZYME INHIBITORY PEPTIDE DERIVED FROM KACANG GOAT MEAT PROTEIN HYDROLYSATE

    Directory of Open Access Journals (Sweden)

    J. Jamhari

    2014-10-01

    Full Text Available The objective of this study was to identify the Angiotensin Converting Enzyme (ACE inhibitorypeptide derived from Kacang goat meat protein hydrolysate. Kacang goat meat loin section washydrolyzed with pepsin, trypsin and chymotrypsin. Protein hydrolysate of Kacang goat meat was thentested the protein concentration and ACE inhibitory activity. ACE inhibitory peptide of the proteinhydrolysate was purified through several steps of purification by column SEP-PAK Plus C18 Cartridgeand RP-HPLC using a Cosmosil column 5PE-SM, 4.6 x 250 mm. The sequence of amino acid of ACEinhibitory peptide was identified by amino acid sequencer. The results showed that amino acidssequence of ACE inhibitory peptide derived from protein hydrolysate of Kacang goat meat was leu-thrglu-ala-pro-leu-asn-pro-lys-ala-arg- asn-glu-lys. It had a molecular weight (MW of 1581 and occurredat the position of 20th to 33rd residues of b-actin of goat meat protein (Capra hircus. The ACE inhibitoryactivity (IC50 of the peptide was 190 mg/mL or 120 mM.

  13. Simple optimization method for partitioning purification of hydrogen networks

    Directory of Open Access Journals (Sweden)

    W.M. Shehata

    2015-03-01

    Full Text Available The Egyptian petroleum fuel market is increasing rapidly nowadays. These fuels must be in the standard specifications of the Egyptian General Petroleum Corporation (EGPC, which required lower sulfur gasoline and diesel fuels. So the fuels must be deep hydrotreated which resulted in increasing hydrogen (H2 consumption for deeper hydrotreating. Along with increased H2 consumption for deeper hydrotreating, additional H2 is needed for processing heavier and higher sulfur crude slates especially in hydrocracking process, in addition to hydrotreating unit, isomerization units and lubricant plants. Purification technology is used to increase the amount of recycled hydrogen. If the amount of recycled hydrogen is increased, the amount of hydrogen that is sent to the furnaces with the off gas will decrease. In this work, El Halwagi et al. (2003 and El Halwagi (2012 optimization methods which are used for recycle/reuse integration systems have been extended to be used in the partitioning purification of hydrogen networks to minimize the hydrogen consumption and the hydrogen discharge. An actual case study and two case studies from the literature are solved to illustrate the proposed method.

  14. Binding of tRNA nucleotidyltransferase to Affi-Gel Blue: rapid purification of the enzyme and binding studies.

    Science.gov (United States)

    Deutscher, M P; Masiakowski, P

    1978-06-01

    Rabbit liver tRNA nucleotidyldransferase bound to columns of Affi-Gel Blue and could be specifically eluted with tRNA. This observation led to development of a rapid purification procedure for the enzyme. The adsorbent was also used to assess interaction of tRNA nucleotidyltransferase with various polynucleotides and substrates. The enzyme could be efficiently desorbed from Affi-Gel Blue by low concentrations of tRNA-C-C, less well by tRNA-C-C-A, and not at all by poly(A), poly(C), ATP or CTP.

  15. [Extraction, Purification and Identification of a Dexamethasone-degrading Enzymes Generated by Pseudomonas Alcaligenes].

    Science.gov (United States)

    Zhu, Lili; Yang, Zhibang; Yang, Qian; Shi, Zhongquan; Deng, Xichuan

    2015-10-01

    In this research a strain of isolated Pseudomonas alcaligenes which causes degradation of dexamethasone was acclimated further and its proteins of every position in the bacterium were separated by the osmotic shock method. The separated intracellular proteins which had the highest enzyme activity were extracted by the salting out with ammonium sulfate and were purified with the cation exchange chromatography and gel chromatography. The purified proteins which was active to cause degradation of dexamethasone had been detected were cut with enzyme and were analyzed with mass spectrometry. The results showed that the degradation rate to dexamethasone by acclimated Pseudomonas alcaligenes were increased from 23.63% to 52.84%. The degrading enzymes were located mainly in the intracellular of the bacteria and its molecular weight was about 41 kD. The specific activity of the purified degrading enzymes were achieved to 1.02 U x mg(-1). Its 5-peptide amino acid sequences were consistent with some sequences of the isovaleryl-CoA dehydrogenase. The protein enzyme may be a new kind degrading enzyme of steroidal compounds. Our experimental results provided new strategies for cleanup of dexamethasone in water environment with microbial bioremediation technique.

  16. [Extraction and purification method of rice DNA from rice powder containing Konjak flour].

    Science.gov (United States)

    Minematsu, Kazuhiko; Nakamura, Kosuke; Akiyama, Hiroshi; Harikai, Naoki; Nakajima, Osamu; Kitta, Kazumi; Teshima, Reiko; Iizuka, Tayoshi

    2010-01-01

    Rice powder containing Konjak flour made with tuberous roots of Amorphophallus konjac is imported as a rice-processed product from China to Japan. An improved DNA purification method for the polymerase chain reaction (PCR) analysis of rice in such products is necessary, since Konjak flour constituents absorb the DNA purification buffer to form a gel, and cause problems in the subsequent purification steps. Here, we present a simple preparative system for isolation of the rice and a purification method of the rice DNA from the product. The purified DNA was confirmed to be a good template for both PCR and real-time PCR.

  17. Large-scale purification of synaptophysin and quantification with a newly established enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Schlaf, G; Göddecke, M; Wolff, J R; Felgenhauer, K; Mäder, M

    1996-09-01

    Synaptophysin (SYP I), an integral membrane protein, was purified on a large scale (0.55 - 2.7 mg) from isolated small synaptic vesicles (SSV) of porcine cortex. In order to achieve this, a conventional purification procedure which consists of size exlusion chromatography, hydrophobic interaction chromatography and chromatofocusing has been developed. This procedure was compared with purification of SYP I by immunoaffinity chromatography. The elution patterns of both procedures were monitored using sodium dodecylsulfate gel electrophoresis (SDS-PAGE) with subsequent Coomassie blue staining of proteins and simultaneous immunoblotting with SYP I-specific antibody. Contaminating proteins with relative molecular masses (M(r)) very similar to SYP I could be removed during the process of purification, demonstrating that the 38 kDa protein found after Triton X-100 lysis of enriched SSV does not exclusively represent SYP I. A specific antiserum was raised in rabbits using a highly purified preparation of SYP I. This antiserum was used in combination with a monoclonal antibody to establish a specific and sensitive enzyme-linked immunosorbent assay (ELISA) which allowed rapid and reliable quantification of this hydrophobic membrane protein in all purification steps, starting with Triton X-100-lysed brain homogenates. Using this ELISA, the concentration of SYP I in highly purified SSV was determined to be 5.8% of solubilized protein.

  18. PURIFICATION OF YEAST CYTOCHROME-C-OXIDASE WITH A SUBUNIT COMPOSITION RESEMBLING THE MAMMALIAN ENZYME

    OpenAIRE

    Taanman, J.W.; Capaldi, R A

    1992-01-01

    Yeast cytochrome c oxidase has been isolated by ion exchange chromatography using lauryl maltoside (n-dodecyl beta-D-maltoside) as the solubilizing detergent. The enzyme prepared in this way has a heme aa3 concentration of 8-9 nmol/mg of protein and a turnover number in the range of 180-210 s-1 at pH 6.2 in 0.01% lauryl maltoside at 20-degrees-C. Yeast cytochrome c oxidase prepared by any of several previously published methods which use Triton X-100 contains nine subunits. The enzyme isolate...

  19. A Novel Aqueous Two Phase System Composed of a Thermo-Separating Polymer and an Organic Solvent for Purification of Thermo-Acidic Amylase Enzyme from Red Pitaya (Hylocereus polyrhizus Peel

    Directory of Open Access Journals (Sweden)

    Mehrnoush Amid

    2014-05-01

    Full Text Available The purification of thermo-acidic amylase enzyme from red pitaya (Hylocereus polyrhizus peel for the first time was investigated using a novel aqueous two-phase system (ATPS consisting of a thermo-separating copolymer and an organic solvent. The effectiveness of different parameters such as molecular weight of the thermo-separating ethylene oxide-propylene oxide (EOPO copolymer and type and concentration of organic solvent on the partitioning behavior of amylase was investigated. In addition, the effects of phase components, volume ratio (VR, pH and crude load of purification factor and yield of amylase were evaluated to achieve the optimum partition conditions of the enzyme. In the novel ATPS method, the enzyme was satisfactorily partitioned into the polymer-rich top phase in the system composed of 30% (w/w EOPO 2500 and 15% (w/w 2-propanol, at a volume ratio of 1.94 and with a crude load scale of 25% (w/w at pH 5.0. Recovery and recycling of components was also measured in each successive step of the ATPS process. The enzyme was successfully recovered by the method with a high purification factor of 14.3 and yield of 96.6% and copolymer was also recovered and recycled at a rate above 97%, making the method was more economical than the traditional ATPS method.

  20. A novel aqueous two phase system composed of a thermo-separating polymer and an organic solvent for purification of thermo-acidic amylase enzyme from red pitaya (Hylocereus polyrhizus) peel.

    Science.gov (United States)

    Amid, Mehrnoush; Manap, Yazid; Zohdi, Nor Khanani

    2014-05-22

    The purification of thermo-acidic amylase enzyme from red pitaya (Hylocereus polyrhizus) peel for the first time was investigated using a novel aqueous two-phase system (ATPS) consisting of a thermo-separating copolymer and an organic solvent. The effectiveness of different parameters such as molecular weight of the thermo-separating ethylene oxide-propylene oxide (EOPO) copolymer and type and concentration of organic solvent on the partitioning behavior of amylase was investigated. In addition, the effects of phase components, volume ratio (VR), pH and crude load of purification factor and yield of amylase were evaluated to achieve the optimum partition conditions of the enzyme. In the novel ATPS method, the enzyme was satisfactorily partitioned into the polymer-rich top phase in the system composed of 30% (w/w) EOPO 2500 and 15% (w/w) 2-propanol, at a volume ratio of 1.94 and with a crude load scale of 25% (w/w) at pH 5.0. Recovery and recycling of components was also measured in each successive step of the ATPS process. The enzyme was successfully recovered by the method with a high purification factor of 14.3 and yield of 96.6% and copolymer was also recovered and recycled at a rate above 97%, making the method was more economical than the traditional ATPS method.

  1. A novel purification method for histidine-tagged proteins containing a thrombin cleavage site

    NARCIS (Netherlands)

    Hefti, M.H.; Vugt-Toorn, van der C.J.; Dixon, R.; Vervoort, J.J.M.

    2001-01-01

    A general procedure for the purification of histidine-tagged proteins has been developed using immobilized metal-ion affinity chromatography. This two-step purification method can be used for proteins containing a hexahistidine tag and a thrombin cleavage site, yielding high amounts of purified prot

  2. Method for Rapid Purification of Class IIa Bacteriocins and Comparison of Their Activities

    OpenAIRE

    Guyonnet, D.; Fremaux, C; Cenatiempo, Y; Berjeaud, J. M.

    2000-01-01

    A three-step method was developed for the purification of mesentericin Y105 (60% yield) from the culture supernatant of Leuconostoc mesenteroides Y105. The same procedure was successfully applied to the purification of five other anti-Listeria bacteriocins identified by mass spectrometry. Specific activities of the purified bacteriocins were compared.

  3. Purification and Study of Proteolytic Activity of Ficin Enzyme of Fig (Ficus Carica

    Directory of Open Access Journals (Sweden)

    A. Mostafaie

    2013-07-01

    Full Text Available Introduction & Objective: Ficin is a member of plant cystein proteases that is abundant in fig. This enzyme has many pharmacological and industrial uses. In the present study, the enzyme was purified by a simple procedure and its proteolytic activity was assayed on several plant and animal proteins. Materials & Methods: Ficin was extracted from unripe fig, precipitated by ammonium sulfate and purified using ion-exchange chromatography on a Carboxymethyl Sepharose column. Proteolytic activities of the purified enzyme were determined in 4 buffering conditions on casein, alpha lactalbumin, beta lactoglobulin and gelatin proteins. Results: Purified enzymes include two bands with molecular mass of 24 and 26 KDa. Re-sults of proteolytic activity showed that ficin can digest casein. It has moderate hydrolytic activity on beta lactoglobulin and gelatin but ficin can not hydrolyze alpha lactalbumin. Conclusion: It seems ficin has selective effects on some proteins so it can be a good candi-date for digestion of casein and making related drugs. (Sci J Hamadan Univ Med Sci 2013; 20 (2:126-132

  4. Substrates and method for determining enzymes

    Science.gov (United States)

    Smith, Robert E.; Bissell, Eugene R.

    1981-01-01

    A method is disclosed for determining the presence of an enzyme in a biological fluid, which includes the steps of contacting the fluid with a synthetic chromogenic substrate, which is an amino acid derivative of 7-amino-4-trifluoromethylcoumarin; incubating the substrate-containing fluid to effect enzymatic hydrolysis; and fluorometrically determining the presence of the free 7-amino-4-trifluoromethylcoumarin chromophore in the hydrolyzate.

  5. Substrates and method for determining enzymes

    Science.gov (United States)

    Smith, R.E.; Bissell, E.R.

    1981-10-13

    A method is disclosed for determining the presence of an enzyme in a biological fluid, which includes the steps of contacting the fluid with a synthetic chromogenic substrate, which is an amino acid derivative of 7-amino-4-trifluoromethylcoumarin; incubating the substrate-containing fluid to effect enzymatic hydrolysis; and fluorometrically determining the presence of the free 7-amino-4-trifluoromethylcoumarin chromophore in the hydrolyzate. No Drawings

  6. Enzyme-functionalized gold-coated magnetite nanoparticles as novel hybrid nanomaterials: synthesis, purification and control of enzyme function by low-frequency magnetic field.

    Science.gov (United States)

    Majouga, Alexander; Sokolsky-Papkov, Marina; Kuznetsov, Artem; Lebedev, Dmitry; Efremova, Maria; Beloglazkina, Elena; Rudakovskaya, Polina; Veselov, Maxim; Zyk, Nikolay; Golovin, Yuri; Klyachko, Natalia; Kabanov, Alexander

    2015-01-01

    The possibility of remotely inducing a defined effect on NPs by means of electromagnetic radiation appears attractive. From a practical point of view, this effect opens horizons for remote control of drug release systems, as well as modulation of biochemical functions in cells. Gold-coated magnetite nanoparticles are perfect candidates for such application. Herein, we have successfully synthesized core-shell NPs having magnetite cores and gold shells modified with various sulphur containing ligands and developed a new, simple and robust procedure for the purification of the resulting nanoparticles. The carboxylic groups displayed at the surface of the NPs were utilized for NP conjugation with a model enzyme (ChT). In the present study, we report the effect of the low-frequency AC magnetic field on the catalytic activity of the immobilized ChT. We show that the enzyme activity decreases upon exposure of the NPs to the field.

  7. Large-scale isolation, fractionation, and purification of soluble starch-synthesizing enzymes: starch synthase and branching enzyme from potato tubers.

    Science.gov (United States)

    Mukerjea, Rupendra; Falconer, Daniel J; Yoon, Seung-Heon; Robyt, John F

    2010-07-19

    Soluble starch-synthesizing enzymes, starch synthase (SSS) and starch-branching enzyme (SBE), were isolated, fractionated, and purified from white potato tubers (Solanum tuberosum) on a large scale. Five steps were used: potato tuber extract from 2 kg of peeled potatoes, two acetone precipitations, and two fractionations on a large ultrafiltration polysulfone hollow fiber 100 kDa cartridge. Three kinds of fractions were obtained: (1) mixtures of SSS and SBE; (2) SSS, free of SBE; and (3) SBE, free of SSS. Contaminating enzymes (amylase, phosphorylase, and disproportionating enzyme) and carbohydrates were absent from the 2nd acetone precipitate and from the column fractions, as judged by the Molisch test and starch triiodide test. Activity yields of 122% (300,000-400,000 units) of SSS fractions and 187% (40,000-50,000 units) of SBE fractions were routinely obtained from the cartridge. Addition of 0.04% (w/v) polyvinyl alcohol 50K and 1 mM dithiothreitol to the glycine buffer (pH 8.4) gave long-term stability and higher yields of SSS and SBE, due to activation of inactive enzymes. Several SSS and SBE fractions from the two fractionations had very high specific activities, indicating high degrees of purification. Polyacrylamide gel electrophoresis of selected SSS and SBE fractions gave two to five SSS and/or SBE activity bands, corresponding to the one to five protein bands present in the 2nd acetone precipitate.

  8. Affinity purification and characterization of a biodegradable plastic-degrading enzyme from a yeast isolated from the larval midgut of a stag beetle, Aegus laevicollis.

    Science.gov (United States)

    Suzuki, Ken; Sakamoto, Hironori; Shinozaki, Yukiko; Tabata, Jun; Watanabe, Takashi; Mochizuki, Atsushi; Koitabashi, Motoo; Fujii, Takeshi; Tsushima, Seiya; Kitamoto, Hiroko K

    2013-09-01

    Two yeast strains, which have the ability to degrade biodegradable plastic films, were isolated from the larval midgut of a stag beetle, Aegus laevicollis. Both of them are most closely related to Cryptococcus magnus and could degrade biodegradable plastic (BP) films made of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA) effectively. A BP-degrading enzyme was purified from the culture broth of one of the isolated strains employing a newly developed affinity purification method based on the binding action of the enzyme to the substrate (emulsified PBSA) and its subsequent degradative action toward the substrate. Partial amino acid sequences of this enzyme suggested that it belongs to the cutinase family, and thus, the enzyme was named CmCut1. It has a molecular mass of 21 kDa and a degradative activity for emulsified PBSA which was significantly enhanced by the simultaneous presence of Ca(2+) or Mg(2+) at a concentration of about 2.5 mM. Its optimal pH was 7.5, and the optimal temperature was 40 °C. It showed a broad substrate specificity for p-nitrophenyl (pNP)-fatty acid esters ranging from pNP-acetate (C2) to pNP-stearate (C18) and films of PBSA, PBS, poly(ε-caprolactone), and poly(lactic acid).

  9. Purification and characterization of the enzymes involved in nicotinamide adenine dinucleotide degradation by Penicillium brevicompactum NRC 829.

    Science.gov (United States)

    Ali, Thanaa Hamed; El-Ghonemy, Dina Helmy

    2016-06-01

    The present study was conducted to investigate a new pathway for the degradation of nicotinamide adenine dinucleotide (NAD) by Penicillium brevicompactum NRC 829 extracts. Enzymes involved in the hydrolysis of NAD, i.e. alkaline phosphatase, aminohydrolase and glycohydrolase were determined. Alkaline phosphatase was found to catalyse the sequential hydrolysis of two phosphate moieties of NAD molecule to nicotinamide riboside plus adenosine. Adenosine was then deaminated by aminohydrolase to inosine and ammonia. While glycohydrolase catalyzed the hydrolysis of the nicotinamide-ribosidic bond of NAD+ to produce nicotinamide and ADP-ribose in equimolar amounts, enzyme purification through a 3-step purification procedure revealed the existence of two peaks of alkaline phosphatases, and one peak contained deaminase and glycohydrolase activities. NAD deaminase was purified to homogeneity as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with an apparent molecular mass of 91 kDa. Characterization and determination of some of NAD aminohydrolase kinetic properties were conducted due to its biological role in the regulation of cellular NAD level. The results also revealed that NAD did not exert its feedback control on nicotinamide amidase produced by P. brevicompactum.

  10. Three-phase partitioning as a rapid and easy method for the purification and recovery of catalase from sweet potato tubers (Solanum tuberosum).

    Science.gov (United States)

    Duman, Yonca Avcı; Kaya, Erdem

    2013-07-01

    Three-phase partitioning (TPP) was used to purify and recover catalase from potato crude extract. The method consists of ammonium sulfate saturation, t-butanol addition, and adjustment of pH, respectively. The best catalase recovery (262 %) and 14.1-fold purification were seen in the interfacial phase in the presence of 40 % (w/v) ammonium sulfate saturation with 1.0:1.0 crude extract/t-butanol ratio (v/v) at pH 7 in a single step. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the enzyme showed comparatively purification and protein molecular weight was nearly found to be 56 kDa. This study shows that TPP is a simple, economical, and quick method for the recovering of catalase and can be used for the purification process.

  11. [Assessment of schemes for sewage purification from petroleum products, by using various flotation methods].

    Science.gov (United States)

    Zabuga, G A; Filippova, T M; Sivkov, A A

    2010-01-01

    Petroleum products are the most common pollutants in petroleum refinery wastewater and are freed from the latter by flotation that is one of the most frequently applied physicochemical methods. The existing petroleum refinery OAO "Angara Petroleum Company" scheme for sewage purification from petroleum products, by using pressure flotation and proposed as a competitive purification scheme by applying electrical and impeller flotations underwent a comparative ecologoeconomic analysis. The use of electrical flotation instead of pressure flotation and that of an impeller flotation-electrical flotation system instead of a mechanical purification-pressure flotation one can considerably lower the concentration of petroleum products at the wastewater outlet into the Angara river.

  12. Expression and purification of the modification-dependent restriction enzyme BisI and its homologous enzymes.

    Science.gov (United States)

    Xu, Shuang-Yong; Klein, Pernelle; Degtyarev, Sergey Kh; Roberts, Richard J

    2016-06-29

    The methylation-dependent restriction endonuclease (REase) BisI (G(m5)C ↓ NGC) is found in Bacillus subtilis T30. We expressed and purified the BisI endonuclease and 34 BisI homologs identified in bacterial genomes. 23 of these BisI homologs are active based on digestion of (m5)C-modified substrates. Two major specificities were found among these BisI family enzymes: Group I enzymes cut GCNGC containing two to four (m5)C in the two strands, or hemi-methylated sites containing two (m5)C in one strand; Group II enzymes only cut GCNGC sites containing three to four (m5)C, while one enzyme requires all four cytosines to be modified for cleavage. Another homolog, Esp638I cleaves GCS ↓ SGC (relaxed specificity RCN ↓ NGY, containing at least four (m5)C). Two BisI homologs show degenerate specificity cleaving unmodified DNA. Many homologs are small proteins ranging from 150 to 190 amino acid (aa) residues, but some homologs associated with mobile genetic elements are larger and contain an extra C-terminal domain. More than 156 BisI homologs are found in >60 bacterial genera, indicating that these enzymes are widespread in bacteria. They may play an important biological function in restricting pre-modified phage DNA.

  13. A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells.

    Science.gov (United States)

    Zongyi, Yin; Funian, Zou; Hao, Li; Ying, Cheng; Jialin, Zhang; Baifeng, Li

    2017-01-01

    Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods). Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P 1000 islets). In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized.

  14. Separation and Purification of Thrombin-like Enzymes by Affinity Adsorbents

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    An affinity adsorbent, benzamidineSepharose 4B, was used to separate and purify thrombinlike enzymes. The paminobenzamidine as a specific ligand was coupled to the matrix-Sepharose 4B. The recombinant thrombinlike enzyme-defibrase was used as a model in order to evaluate the efficiency of this biospecific affinity adsorbent. The homogeneity of the enzyme preparation was comfirmed as one band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis.

  15. Purification and Characterization of a Nonylphenol (NP)-degrading Enzyme from Bacillus cereus.Frankland

    Institute of Scientific and Technical Information of China (English)

    YANG Ge; ZHANG Ying; BAI Yanfen

    2011-01-01

    An extracellular NP-degrading enzyme secreted by Bacillus cereus.Frankland was purified to homogeneity by a combination of ammonium sulfate precipitation,Phenyl-Sepharose hydrophobic-interaction chromatography and DEAE anion-exchange chromatography.On SDS(sodium dodecyl sulfate)-polyacrylamide gel electrophoresis analysis,the purified enzyme showed a relative molecular mass of 58.3 kDa.The depolymerzation of subunits was accompanied with the loss of NP-degrading enzyme activity,and removing denaturing factors by dialysis could restore the dimer structure and enzymatic activity.The enzyme had an isoelectric point of 5.5 and an optimal temperature of 60℃,and was the most active at pH 6.0.The enzymatic activity was stable at pH 4-8 and inhibited by Cu2+.TenN-terminal amino acids were determined to be ASVNSIKIGY,demonstrating that the purified enzyme was a novel one.The hydrolysis pattern of the purified enzyme indicated that the NP-degrading enzyme was an endo NP-degrading enzyme.The extraordinary thermo-stability provided the enzyme with a good prospect to be used as a new tool for clean-production process for textile industry.

  16. Purification and characterization of a novel fibrinolytic enzyme from culture supernatant of Pleurotus ostreatus.

    Science.gov (United States)

    Liu, Xiao-Lan; Zheng, Xi-Qun; Qian, Peng-Zhi; Kopparapu, Narasimha-Kumar; Deng, Yong-Ping; Nonaka, Masanori; Harada, Naoki

    2014-02-28

    A fibrinolytic enzyme was produced by an edible mushroom of Pleurotus ostreatus using submerged culture fermentation. The enzyme was purified from the culture supernatant by applying a combination of freeze-thaw treatment, ammonium sulfate precipitation, hydrophobic interaction, and gel filtration chromatographies. The enzyme was purified by a 147-fold, with a yield of 7.54%. The molecular masses of the enzyme an determined by gel filtration and SDSPAGE were 13.6 and 18.2 kDa, respectively. The isoelectric point of the enzyme was 8.52. It hydrolyzed fibrinogen by cleaving the α and β chains of fibrinogen followed by the γ chains, and also activated plasminogen into plasmin. The enzyme was optimally active at 45°C and pH 7.4. The enzyme activity was completely inhibited by EDTA, whereas protease inhibitors of TPCK, SBTI, PMSF, aprotinin and pepstatin showed no inhibition on its activity. The partial amino acid sequences of the enzyme as determined by Q-TOF2 were ATFVGCSATR, GGTLIHESSHFTR, and YTTWFGTFVTSR. These sequences showed a high degree of homology with those of metallo-endopeptidases from P. ostreatus and Armillaria mellea. The purified enzyme can also be applied as a natural agent for oral fibrinolytic therapy or prevention of thrombosis.

  17. Partial purification and characterization of arginine decarboxylase from avocado fruit, a thermostable enzyme.

    Science.gov (United States)

    Winer, L; Vinkler, C; Apelbaum, A

    1984-09-01

    A partially purified preparation of arginine decarboxylase (EC 4.1.1.19), a key enzyme in polyamine metabolism in plants, was isolated from avocado (Persea americana Mill. cv Fuerte) fruit. The preparation obtained from the crude extract after ammonium sulfate precipitation, dialysis, and heat treatment, had maximal activity between pH 8.0 and 9.0 at 60 degrees C, in the presence of 1.2 millimolar MnCl(2), 2 millimolar dithiothreitol, and 0.06 millimolar pyridoxal phosphate. The K(m), of arginine for the decarboxylation reaction was determined for enzymes prepared from the seed coat of both 4-week-old avocado fruitlet and fully developed fruit, and was found to have a value of 1.85 and 2.84 millimolar, respectively. The value of V(app) (max) of these enzymes was 1613 and 68 nanomoles of CO(2) produced per milligram of protein per hour for the fruitlet and the fully developed fruit, respectively. Spermine, an end product of polyamine metabolism, caused less than 5% inhibition of the enzyme from fully developed fruit and 65% inhibition of the enzyme from the seed coat of 4-week-old fruitlets at 1 millimolar under similar conditions. The effect of different inhibitors on the enzyme and the change in the nature of the enzyme during fruit development are discussed.

  18. PARTIAL PURIFICATION OF MILK-CLOTTING ENZYME FROM THE SEEDS OF MORINGA OLEIFERA

    Directory of Open Access Journals (Sweden)

    Amna E. Tajalsir

    2014-08-01

    Full Text Available The aim of the present study was to search for milk clotting substitute from different parts (flowers, seeds, stem, leaves, ripe and unripe fruits of Moringa oleifera. The samples were blended and extracted using different types of extracting solutions. The most reliable, quick and efficient enzyme extracting solution was found to be 5% NaCl in 100 mM sodium acetate buffer, pH 5.0, which was used throughout the study. The milk clotting activity was only observed in the seeds extract while the other parts were either deficient or has very low milk clotting activity. Thus, the moringa seeds were used as source of milk clotting enzyme. The extracted proteins were fractionated with ammonium sulfate at concentration of 20, 30, 40, 50 and 60 %. Highest milk clotting activity was observed in the 20 % fraction. This fraction was assumed to contain the clotting enzymes and characterized for its heating stability (30 – 90°C and optimum temperature (30 – 90°C. The results demonstrated that moringa seeds milk clotting enzyme is stable up to 50°C with an optimum milk clotting activity of 70°C. The high ratio of milk-clotting to proteolytic activity of the partially purified enzyme indicates the potential of this enzyme as suitable rennet substitute in dairy industry. However, further study is needed to completely purify and characterize this promising milk clotting enzyme from moringa seeds.

  19. Purification and characterisation of a novel amylase enzyme from red pitaya (Hylocereus polyrhizus) peel.

    Science.gov (United States)

    Amid, Mehrnoush; Abd Manap, Mohd Yazid

    2014-12-15

    An amylase enzyme from pitaya peel was purified 234.2-folds with 72.1% recovery using ammonium sulphate precipitation, gel filtration and ion exchange chromatography. Gel filtration chromatography and SDS-PAGE revealed that the enzyme is monomeric with a molecular weight of 42.1kDa. The apparent Km and Vmax of the amylase were 2.7 mg/ml and 34.30 u/min/mg of protein, respectively. The enzyme was highly active and stable over a wide pH range from pH 3 to pH 11.0, with optimum activity being observed at pH 5.0. The enzyme was highly selective for soluble starch, amylopectin, glycogen and pulullan. The purified amylase did not require calcium and displayed extreme stability with regard to surfactants and oxidising agents. EDTA, a powerful chelating agent, did not have any significant effect on the stability of the enzyme. Such characteristics have not been previously reported for this type of enzyme from fruit peel. This enzyme, which possesses unique properties, could be widely used in different types of industries, especially in food and biotechnological applications.

  20. Glutamine synthetase. IX. Purification and characterization of the enzyme from sheep spleen.

    Science.gov (United States)

    Wu, C

    1977-04-01

    Glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) has been purified about 550-fold from sheep spleen. The subunit weight of the enzyme is estimated to be 48 000. Sedimentation coefficient determination by density gradient centrifugation gives a value of 15.0 S. The approximate molecular weight calculated from the S value is 378500. In addition, electron micrographs of the enzyme show an "H" shape. Hence, the protein appears to have eight subunits. In sheep spleen, the enzyme resides chiefly in the soluble fraction of the cell. The amino acid composition of the enzyme from spleen shows similarity to that from other sources. The enzyme activity is nearly five times as high in Mg2+ as in Mn2+. ATP inhibits the enzyme; the inhibition is competitive with respect to Mg2+ATP. A number of compounds, such as D-alanine, AMP, creatine phosphate, arsenite in combination with 2,3-dimercaptopropanol, and 2-amino-4-phosphonobutyrate, also inhibit the enzyme. The inhibition by the last compound is competitive with respect to glutamate. D-Glutamate and alpha-methyl-DL-glutamate can serve as substrates in the synthesis reaction, but N-methyl-DL-glutamate cannot. On the other hand, neither D-glutamine nor N-acetyl-L-glutamine can replace L-glutamine as a substrate in the gamma-glutamyl transfer reaction of the enzyme. Inhibition of Mn2+ and ATP and its reversal by Mg2+ have been discussed as a means of regulating the enzyme activity in mammalian tissues.

  1. Non-complexed four cascade enzyme mixture: simple purification and synergetic co-stabilization.

    Directory of Open Access Journals (Sweden)

    Suwan Myung

    Full Text Available Cell-free biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it was essential to produce (purified enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. We studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate isomerase (TIM from Thermus thermophiles, fructose bisphosphate aldolase (ALD from Thermotoga maritima, fructose bisphosphatase (FBP from T. maritima, and phosphoglucose isomerase (PGI from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by gradient ammonia sulfate precipitation. In contrast, PGI was not stable enough for heat treatment. In addition, the stability of a low concentration PGI was enhanced by more than 25 times in the presence of 20 mg/L bovine serum albumin or the other three enzymes. At a practical enzyme loading of 1000 U/L for each enzyme, the half-life time of free PGI was prolong to 433 h in the presence of the other three enzymes, resulting in a great increase in the total turn-over number of PGI to 6.2×10(9 mole of product per mole of enzyme. This study clearly suggested that the presence of other proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme PGI due to in vitro macromolecular crowding effect. Also, this result could be used to explain why not all enzymes isolated from thermophilic microorganisms are stable in vitro because of a lack of the macromolecular crowding environment.

  2. CTAB-silica Method for DNA Extraction and Purification from Castanea mollissima and Ginkgo biloba

    Institute of Scientific and Technical Information of China (English)

    Shen Yongbao; Shi Jisen

    2003-01-01

    A new method CTAB-silica for DNA extraction and purification from the leaves and buds of Castanea mollissima and Ginkgo biloba was tested. The method is based on the silica-based purification protocol developed by Boom et al. (1990). By modifying the protocol, plant genome DNA could be extracted easily from dormant buds, mature leaves, and other parts of plant. Our results showed that the purified DNA was of high purity and could be analyzed by PCR. Furthermore, this CTAB-silica method took much less time for a successful DNA purification process compared to the traditional methods (CTAB and SDS). By our method, the suitable DNA can be extracted and purified from over 10 plant samples by one person in an hour.

  3. Modified methods for culturing myoblasts of rats: Combination of multi-enzymatic digestion and double purification

    Institute of Scientific and Technical Information of China (English)

    Li Zhang; Wei Wang; Ming Fan; Xiaoping Chen; Shuhong Liu; Liang Sun

    2007-01-01

    and enzyme digestion so as to observe morphological characteristics and growth, draw growth curve, analyze surface structure under scanning electron microscope, and evaluate with Desmin immunohistochemical staining.MAIN OUTCOME MEASURES: Morphological characteristics and growth of myoblasts cultured in vitro.RESULTS: ① Growth of myoblasts of skeletal muscle: Primary cells had well growth, mature and differentiation. The positive rate of Desmin was 94% and purification of cells was ideal. Growth curve of cells demonstrated that myoblasts which were characterized by high purification started proliferation plentiful through transient growth lag phase (about at one or two days after inoculation). If myoblasts were not dealt with any interventions, they might become sarcotubule gradually at 3 - 5 days after proliferative phase. During this period, myoblasts maintained a monocaryon-bipolarity state under inverted phase contrast microscope. Furthermore, the growth of cells was the strongest and reproductive activity was the most powerful. This suggested that myotube started to form; in addition, muscle fiber of contractility might form under a well culturing condition. ② Immunocytochemical stain with desmin antibody: Interzonal fiber of desmin from myoblasts showed strongly positive reaction. Positive staining existed in cytoplasm had a high nucleus-cytoplasm ratio. However, myoblasts showed negative or mildly positive reaction.CONCLUSION: It is ideal for modified multi-enzymatic digestion and double purification method to dissociate and purify myoblasts of skeletal muscle; meanwhile, these two methods are both the effective ways to provide convenient conditions to obtain seed cells for neural regeneration researches.

  4. RESEARCH METHODS OF WATER PURIFICATION FROM POLLUTION WITH PETROLEUM AND PETROLEUM PRODUCTS

    Directory of Open Access Journals (Sweden)

    Privalova N. M.

    2015-11-01

    Full Text Available This article provides an overview of the currently existing methods of purification of waters from pollution with petroleum and petroleum products. The most popular cleaning ways and new emerging technologies are considered. For each method of combating with petroleum pollution the circumstances and the factors are given, under which the application of this method is the most efficient and cost-effective. The article briefly describes the technology of each method, and its main strengths and weaknesses, particularly the use and quality of water purification

  5. Magnetite-alginate beads for purification of some starch degrading enzymes.

    Science.gov (United States)

    Teotia, Sunita; Gupta, M N

    2002-03-01

    Starch degrading enzymes, viz., beta-amylase, glucoamylase, and pullulanase, were purified using magnetite-alginate beads. In each case, the enzyme activity was eluted by using 1.0 M maltose. beta-Amylase (sweet potato), glucoamylase (Aspergillus niger), and pullulanase (Bacillus acidopullulyticus) from their crude preparations were purified 37-, 31-, and 49-fold with 86, 87, and 95% activity recovery, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed single band in each case.

  6. Purification and characterization of a collagenolytic enzyme from a pathogen of the great barrier reef sponge, Rhopaloeides odorabile.

    Directory of Open Access Journals (Sweden)

    Joydeep Mukherjee

    Full Text Available BACKGROUND: In recent years there has been a global increase in reports of disease affecting marine sponges. While disease outbreaks have the potential to seriously impact on the survival of sponge populations, the ecology of the marine environment and the health of associated invertebrates, our understanding of sponge disease is extremely limited. METHODOLOGY/PRINCIPAL FINDINGS: A collagenolytic enzyme suspected to enhance pathogenicity of bacterial strain NW4327 against the sponge Rhopaloeides odorabile was purified using combinations of size exclusion and anion exchange chromatography. After achieving a 77-fold increase in specific activity, continued purification decreased the yield to 21-fold with 7.2% recovery (specific activity 2575 collagen degrading units mg(-1protein possibly due to removal of co-factors. SDS-PAGE of the partially pure enzyme showed two proteins weighing approximately 116 and 45 kDa with the heavier band being similar to reported molecular weights of collagenases from Clostridium and marine Vibrios. The enzyme degraded tissue fibres of several sponge genera suggesting that NW4327 could be deleterious to other sponge species. Activity towards casein and bird feather keratin indicates that the partially purified collagenase is either a non-selective protease able to digest collagen or is contaminated with non-specific proteases. Enzyme activity was highest at pH 5 (the internal pH of R. odorabile and 30 degrees C (the average ambient seawater temperature. Activity under partially anaerobic conditions also supports the role of this enzyme in the degradation of the spongin tissue. Cultivation of NW4327 in the presence of collagen increased production of collagenase by 30%. Enhanced enzyme activity when NW4327 was cultivated in media formulated in sterile natural seawater indicates the presence of other factors that influence enzyme synthesis. CONCLUSIONS/SIGNIFICANCE: Several aspects of the sponge disease etiology were

  7. Purification and characterization of an acidothermophilic cellulase enzyme produced by Bacillus subtilis strain LFS3.

    Science.gov (United States)

    Rawat, Rekha; Tewari, Lakshmi

    2012-07-01

    In the present investigation, a microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated and identified as Bacillus subtilis strain LFS3 by 16S rDNA sequence analysis. The carboxymethylcellulase (CMCase) enzyme produced by the B. subtilis strain LFS3 was purified by (NH₄)₂SO₄ precipitation, ion exchange and gel filtration chromatography, with an overall recovery of 15 %. Native-PAGE analysis of purified CMCase revealed the molecular weight of enzyme to be about 185 kDa. The activity profile of CMCase enzyme showed the optimum activity at temperature 60 °C and pH 4.0, respectively. The enzyme activity was induced by Na⁺, Mg²⁺, NH₄⁺, and EDTA, whereas strongly inhibited by Hg²⁺ and Fe³⁺. The purified enzyme hydrolyzed CMC, filter paper, and xylan, but not p-nitrophenyl β-D-glucopyranoside and cellulose. Kinetic analysis of purified enzyme showed the K(m) value of 2.2 mg/ml. Thus, acidophilic as well as thermophilic nature makes this cellulase a suitable candidate for current mainstream biomass conversion into fuel and other industrial processes.

  8. Expression, purification and immobilization of recombinant AiiA enzyme onto magnetic nanoparticles.

    Science.gov (United States)

    Beladiya, Chirag; Tripathy, Rajan K; Bajaj, Priyanka; Aggarwal, Geetika; Pande, Abhay H

    2015-09-01

    AiiA is a "28-kDa lactonase" from Gram-positive Bacillus sp. 240B1. The enzyme can hydrolyze and inactivate a variety of acyl homoserine lactones (AHLs), quorum sensor molecules involve in bacterial quorum sensing (QS). AiiA is a strong candidate for the development of bio-decontaminating agent that can disrupt QS in industrial and environmental samples. However, commercial application of AiiA suffer from several limitations including high cost of production of enzyme and lack of efficient recovery mean(s) of enzyme from the application environment for its reuse. In this study we have cloned, expressed and purified recombinant AiiA (r-AiiA) enzyme. The purified enzyme was covalently immobilized onto magnetic nanoparticles (MNPs) and the quorum quenching ability of r-AiiA-MNP nanobiocatalyst was evaluated in aqueous buffer. Our results show that r-AiiA-MNPs (a) can hydrolyze 3O-C10AHL and inhibit QS in aqueous buffer, (b) can be recovered from the reaction mixture using external magnetic field, and (c) can be reused multiple times to hydrolyze 3O-C10AHL in aqueous buffer. Results of this study can be used to develop a formulation of AiiA enzyme for industrial applications.

  9. Purification and Some Properties of a Tetrathionate Decomposing Enzyme from Thiobacillus thiooxidans.

    Science.gov (United States)

    Tano, T; Kitaguchi, H; Harada, M; Nagasawa, T; Sugio, T

    1996-01-01

    A tetrathionate-decomposing enzyme that catalyzes the decomposition of tetrathionate into thiosulfate and sulfate was purified to homogeneity from tetrathionate-grown Thiobacillus thiooxidans. The enzyme had an apparent molecular weight of 104,000, and was composed of two identical subunits (MW = 58,000) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had an isoelectric point at 9.6 and was most active at pH 3.0-3.5 and 40°C. Enzyme activity was increased approximately 100-fold in the presence of 400 mm sulfate ion. The Michaelis constant of this enzyme for tetrathionate in the presence of 20, 50, and 200 mm of sulfate ion was 2.4 mm. Mercuric and ferric ions completely inhibited the enzyme activity at 1 mm. Though cupric ion up to 0.01 mm markedly stimulated the activity in the presence of 20 mm sulfate ion, a higher concentration (1 mm) rather strongly inhibited the activity. Ethylenediaminetetraacetic acid (EDTA) strongly inhibited the activity, but this inhibiton was completely restored by cupric ion.

  10. Purification and immunochemical detections of ?-naphthoflavone- and phenobarbital-induced avian cytochrome P450 enzymes

    Science.gov (United States)

    Brown, R.L.; Levi, P.E.; Hodgson, E.; Melancon, M.J.

    1996-01-01

    Livers from mallards (Anas platyrhynchos) were treated with either -naphthoflavone (50 mg/kg) or phenobarbital (70 mg/kg). Purification of induced hepatic cytochrome P450 was accomplished using both DEAE and hydroxyapatite columns, as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis separation. Polyclonal antibodies to these proteins were then produced in young male New Zealand White rabbits. ?-naphthoflavone (?NF)- and phenobarbital(PB)-treated red-winged blackbird, screech owl, European starling and lesser scaup liver microsomes were analyzed in western blots for species cross-reactivity. Although all four of these avian species exhibited cross-reactivity with antibodies to ?NF-induced mallard P450, all but the lesser scaup revealed a protein of higher molecular weight than that of the ?NF-induced mallard. In addition, only the lesser scaup exhibited cross-reactivity with the anti-PB-induced mallard P450 antibodies.

  11. High oleic sunflower biodiesel: quality control and different purification methods

    Directory of Open Access Journals (Sweden)

    Pighinelli, Anna L.M.T.

    2011-06-01

    Full Text Available The objective of the present work is to evaluate the production of biodiesel using ethanol and sunflower oil. The extraction of the sunflower oil was evaluated first. An experimental design was used to estimate the influence of the independent variables grain temperature (25º to 110ºC and expeller rotation (85 to 119rpm on the crude oil. The best result obtained was 68.38%, achieved with a rotation from 100 to 115rpm, grain temperature ranging from 25º to 30ºC and moisture content of around 7%. The next study consisted of transesterification, evaluating the influence of the ethanol, oil molar ratio and the catalyst concentration (sodium methylate on the ester-rich phase yield. The highest yield was 98.39% obtained with a molar ratio of 9:1 and 3% catalyst. An experiment was then carried out on a small reactor and the biodiesel produced was purified by three different methods: acidified water, silica and distillation. The quality aspects of the purified biodiesel samples were evaluated according to the Brazilian specifications for biodiesel, and distillation was shown to be the best method of purification.

    El objetivo del presente trabajo es evaluar la producción de biodiesel usando etanol y aceite de girasol. La extracción del aceite de girasol fue evaluada primero. Un diseño experimental fue usado para estimar la influencia de las variables independientes: temperatura del grano (25º a 110ºC y rotación del expeller (85 a 119 rpm en la obtención del aceite crudo. El mejor resultado obtenido fue un 68,38%, conseguido con una rotación de 100 a 115 rpm, una temperatura del grano de 25º a 30ºC y un contenido de humedad de alrededor del 7%. El siguiente estudio mediante transesterificación, evaluó la influencia de la relación molar etanol: aceite y concentración de catalizador (metilato sódico en el rendimiento de la fase rica en esteres. El rendimiento más alto fue 98,39% obtenido con una relación molar de 9.1 y 3% de

  12. Trace Conserving Purification for Linear Scaling [O(N)] Methods: A First Enhancement to CP2K

    Science.gov (United States)

    2014-09-01

    purification scheme times in CP2K. Timings are normalized to TRS4 for each band gap. 5 Fig. 2 Graphical representation of the 1024 water box...Trace Conserving Purification for Linear Scaling [O(N)] Methods: A First Enhancement to CP2K by Jonathan Mullin ARL-CR-0746 September...Proving Ground, MD 21005-5069 ARL-CR-0746 September 2014 Trace Conserving Purification for Linear Scaling [O(N)] Methods: A First

  13. Cloning, sequencing and functional expression of cytosolic malate dehydrogenase from Taenia solium: Purification and characterization of the recombinant enzyme.

    Science.gov (United States)

    Nava, Gabriela; Laclette, Juan P; Bobes, Raúl; Carrero, Julio C; Reyes-Vivas, Horacio; Enriquez-Flores, Sergio; Mendoza-Hernández, Guillermo; Plancarte, Agustín

    2011-07-01

    We report herein the complete coding sequence of a Taenia solium cytosolic malate dehydrogenase (TscMDH). The cDNA fragment, identified from the T. solium genome project database, encodes a protein of 332 amino acid residues with an estimated molecular weight of 36517Da. For recombinant expression, the full length coding sequence was cloned into pET23a. After successful expression and enzyme purification, isoelectrofocusing gel electrophoresis allowed to confirm the calculated pI value at 8.1, as deduced from the amino acid sequence. The recombinant protein (r-TscMDH) showed MDH activity of 409U/mg in the reduction of oxaloacetate, with neither lactate dehydrogenase activity nor NADPH selectivity. Optimum pH for enzyme activity was 7.6 for oxaloacetate reduction and 9.6 for malate oxidation. K(cat) values for oxaloacetate, malate, NAD, and NADH were 665, 47, 385, and 962s(-1), respectively. Additionally, a partial characterization of TsMDH gene structure after analysis of a 1.56Kb genomic contig assembly is also reported.

  14. Purification and functional characterization of two fibrinogenolytic enzymes from Bothrops alternatus venom

    Directory of Open Access Journals (Sweden)

    J. O. Costa

    2007-01-01

    Full Text Available Two fibrinogenolytic enzymes, Bothrops alternatus metalloprotease isoform (BaltMP-I and II, were purified from Bothrops alternatus venom using Diethylaminoethyl (DEAE Sephacel, Sephadex G-75 and Heparin-Agarose column chromatography. Purified BaltMP-I and II ran as single protein bands on analytical polyacrylamide gel electrophoresis and showed molecular weights of 29000 and 36000, respectively, under reducing conditions in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE. BaltMP-II, but not BaltMP-I, displayed blood-clotting activity in bovine plasma, which was about 10-fold higher than that of the crude venom. Both enzymes were proteolytically active against bovine fibrinogen as substrate. When fibrinogen and each enzyme were incubated at 37°C, at a ratio of 1:100 (w/w, BaltMP-II cleaved preferentially the Aalpha -chain and more slowly the Bbeta -chain. The action of BaltMP-I was similar, but lower. None of the proteases degraded the gamma-chain of fibrinogen. The fibrinogenolytic activity of the enzymes was inhibited by 1,10-phenanthroline, suggesting they are metalloproteases. Since both enzymes were found to cause defibrinogenation when intraperitoneally (i.p. administered to mice, they can be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.

  15. A Simple and Efficient Method for Purification of Egg White Major Proteins Using Ion Exchange Chromatography

    Directory of Open Access Journals (Sweden)

    Sh. Veisi

    2008-04-01

    Full Text Available Introduction & Objective: Egg white contains four high-quantity proteins which have numerous applications. In this research, a simple and efficient method for the purification of those proteins was designed and performed based on ion exchange chromatography.Materials & Methods: In this experimental study egg white was initially separated from insoluble substances by acidic pH. The resulting extract was isolated after two steps of ion exchange chromatography using CM-Sepharose and DEAE-Sepharose columns, respectively. Purification degree and yield of each fraction were analyzed by electrophoresis densitometry.Results: The results showed that purification degrees of ovalbumin, ovotransferrin, ovomucoid and lysozyme were 97, 98, 85 and 99 percent and their yields were 98, 98 95 and 99 percent, respectively.Conclusion: High yields, reproducibility and feasibility on low or high scales are considered as the strengths of this method.

  16. Malate Utilization by a Group D Streptococcus: Physiological Properties and Purification of an Inducible Malic Enzyme

    Science.gov (United States)

    London, Jack; Meyer, Eleanor Y.

    1969-01-01

    Growth of Streptococcus faecalis in the presence of l-malate resulted in the induction of a “malic enzyme” [l-malate:nicotinamide adenine dinucleotide (NAD) oxidoreductase (decarboxylating), E.C. 1.1.1.39]. Synthesis of the malic enzyme did not appear to be subject to catabolite repression by intermediate products of glucose or fructose dissimilation. However, malate utilization was inhibited during growth in the presence of glucose or fructose. The purified enzyme was specific for malate as substrate and NAD as cofactor. Mn+2 or Mg+2 was required for optimal activity and NH4Cl stimulated the reaction rate. Several lines of indirect evidence suggested that the streptococcal malic enzyme was involved primarily with energy production and not biosynthesis. Images PMID:4306540

  17. A Simple Method for Synthesis, Purification and concentration Stabilized Goldnanoparticles

    Directory of Open Access Journals (Sweden)

    Dr. Fatin F.M. AL-Kazazz

    2013-11-01

    Full Text Available The greatest barriers to biological nanoparticles in use are issues of particle stability in shape and size control. Simple process is desirable for several reasons likewide range of sample volumes, gentle conditions and inexpensive equipment. Gold nanoparticles(GNPs which are produced by reducing gold chloride with TSC have very limited time storage. In order to get highly stable nanoparticles, purification has been performed for industrial applications. Removal of the side product of the reaction between HAuCl4 with Tri-Sodium Citrate (TSCis considered to make GNPs more stable. The result demonstrates that GNPs are more stable after the purification process. Low degree of polydispersity can be achieved during gold nanoparticles synthesis, however, the particles need to be purified post-synthesis. The present research relates to single process for the removal of small-molecule impurities and the isolation of small nanoparticles from larger nanostructures through dialysis and microfiltration then concentration these small nanoparticles through polymer adsorbent materials by a factor of 100 or more to storage in the room temperature prolongs the stability of the purified gold nanoparticles suspension up to one year.The size and shape of the GNps is measured by atomic force microscopy (AFM, and the optical properties by UV-Vis spectrometer.

  18. Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae.

    Science.gov (United States)

    Maeda, Hiroshi; Yamagata, Youhei; Abe, Keietsu; Hasegawa, Fumihiko; Machida, Masayuki; Ishioka, Ryoji; Gomi, Katsuya; Nakajima, Tasuku

    2005-06-01

    We used biodegradable plastics as fermentation substrates for the filamentous fungus Aspergillus oryzae. This fungus could grow under culture conditions that contained emulsified poly-(butylene succinate) (PBS) and emulsified poly-(butylene succinate-co-adipate) (PBSA) as the sole carbon source, and could digest PBS and PBSA, as indicated by clearing of the culture supernatant. We purified the PBS-degrading enzyme from the culture supernatant, and its molecular mass was determined as 21.6 kDa. The enzyme was identified as cutinase based on internal amino acid sequences. Specific activities against PBS, PBSA and poly-(lactic acid) (PLA) were determined as 0.42 U/mg, 11 U/mg and 0.067 U/mg, respectively. To obtain a better understanding of how the enzyme recognizes and hydrolyzes PBS/PBSA, we investigated the environment of the catalytic pocket, which is divided into carboxylic acid and alcohol recognition sites. The affinities for different substrates depended on the carbon chain length of the carboxylic acid in the substrate. Competitive inhibition modes were exhibited by carboxylic acids and alcohols that consisted of C4-C6 and C3-C8 chain lengths, respectively. Determination of the affinities for different chemicals indicated that the most preferred substrate for the enzyme would consist of butyric acid and n-hexanol.

  19. Purification and some properties of a keratinolytic enzyme from an alkaliphilic Nocardiopsis sp. TOA-1.

    Science.gov (United States)

    Mitsuiki, Shinji; Sakai, Masashi; Moriyama, Yasushi; Goto, Masatoshi; Furukawa, Kensuke

    2002-01-01

    A novel alkaliphilic Nocardiopsis sp., strain TOA-1, was isolated from a tile-joint of a bathroom. Strain TOA-1 produced a variety of alkaline hydrolytic enzymes. An alkaline protease, designated NAPase, was purified and characterized. NAPase had a very high keratinolytic activity and high stability under acidic conditions.

  20. A new method for high yield purification of type beta transforming growth factor from human platelets

    NARCIS (Netherlands)

    Eijnden-van Raaij, A.J.M. van den; Koornneef, I.; Zoelen, E.J.J. van

    1988-01-01

    A new method was developed for the purification of type beta transforming growth factor from human platelets. This method is a three-step procedure including gel filtration, weak cation exchange HPLC and reverse phase HPLC. All steps are carried out at low pH using exclusively volatile acidic buffer

  1. A Quick, Cost-Free Method of Purification of DNA Fragments from Agarose Gel

    Directory of Open Access Journals (Sweden)

    Yuan Sun, Kannappan Sriramajayam, Dianzhong Luo, D. Joshua Liao

    2012-01-01

    Full Text Available In this short communication we report a quick, cost-free method of purification of DNA fragments from agarose gel. Unlike those procedures that involve commercial kits, this method uses glass wool or absorbent cotton to filter agarose gel during a quick spinning-down of DNA, thus significantly simplifying the routine practice of many molecular biologists and decreasing the cost.

  2. Fast and reliable production, purification and characterization of heat-stable, bifunctional enzyme chimeras.

    Science.gov (United States)

    Neddersen, Mara; Elleuche, Skander

    2015-12-01

    Degradation of complex plant biomass demands a fine-regulated portfolio of glycoside hydrolases. The LE (LguI/Eco81I)-cloning approach was used to produce two enzyme chimeras CB and BC composed of an endoglucanase Cel5A (C) from the extreme thermophilic bacterium Fervidobacterium gondwanense and an archaeal β-glucosidase Bgl1 (B) derived from a hydrothermal spring metagenome. Recombinant chimeras and parental enzymes were produced in Escherichia coli and purified using a two-step affinity chromatography approach. Enzymatic properties revealed that both chimeras closely resemble the parental enzymes and physical mixtures, but Cel5A displayed lower temperature tolerance at 100°C when fused to Bgl1 independent of the conformational order. Moreover, the determination of enzymatic performances resulted in the detection of additive effects in case of BC fusion chimera. Kinetic measurements in combination with HPLC-mediated product analyses and site-directed mutation constructs indicated that Cel5A was strongly impaired when fused at the N-terminus, while activity was reduced to a slighter extend as C-terminal fusion partner. In contrast to these results, catalytic activity of Bgl1 at the N-terminus was improved 1.2-fold, effectively counteracting the slightly reduced activity of Cel5A by converting cellobiose into glucose. In addition, cellobiose exhibited inhibitory effects on Cel5A, resulting in a higher yield of cellobiose and glucose by application of an enzyme mixture (53.1%) compared to cellobiose produced from endoglucanase alone (10.9%). However, the overall release of cellobiose and glucose was even increased by catalytic action of BC (59.2%). These results indicate possible advantages of easily produced bifunctional fusion enzymes for the improved conversion of complex polysaccharide plant materials.

  3. Application of protein purification methods for the enrichment of a cytotoxin from Campylobacter jejuni

    Directory of Open Access Journals (Sweden)

    Gatsos Xenia

    2012-12-01

    Full Text Available Abstract Background Campylobater jejuni, a major foodborne diarrhoeal pathogen is reported to produce a number of cytotoxins of which only a cytolethal distending toxin (CDT has been characterised so far. One or more additional cytotoxins other than CDT, including a Chinese hamster ovary (CHO cell active, Vero cell inactive cytotoxin, may mediate inflammatory diarrhoea. Our objective was to develop a method to enrich and thus partially characterise this cytotoxin, as a pathway to the eventual identification and characterisation of the toxin. Results A number of biochemical methods including cation- and anion-exchange chromatography were evaluated to enrich the cytotoxin from a cell lysate of a known cytotoxin-producing C. jejuni, C31. The cytotoxin in crude lysate was initially prepared by size-exclusion desalting and then subjected to high pressure liquid chromatography (HPLC ion-exchange fractionation. One pooled fraction (pool B was cytotoxic for CHO cells equivalent to crude toxin (tissue culture infectivity dose 50 [TCID50] of 1–2 μg/ml. The proteins of pool B were identified by mass spectrometry (MS after separation by SDS-PAGE and trypsin digestion. Also, pool B was directly digested with trypsin and then subjected to liquid chromatography tandem mass spectrometry (LCMS analysis for identification of lesser abundant proteins in the fraction. A total of 41 proteins were found in the fraction, which included enzymes involved in metabolic and transport functions. Eighteen non-cytoplasmic proteins including 2 major antigenic peptide proteins (PEB2 and PEB3 and 3 proteins of unknown function were also identified in the screen. Cytotoxicity in pool B was trypsin-sensitive indicating its protein nature. The cytotoxic activity was heat-stable to 50°C, and partially inactivated at 60-70°C. The pool B fraction also induced fluid accumulation in the adult rabbit ileal loop assay with cytotoxicity for mucosa confirming the presence of the

  4. A grey mullet enzyme displaying both lipase and phospholipase activities: purification and characterization.

    Science.gov (United States)

    Smichi, Nabil; Gargouri, Youssef; Miled, Nabil; Fendri, Ahmed

    2013-07-01

    A lipase from the golden grey mullet viscera was purified to homogeneity by ammonium sulphate precipitation, gel filtration, anionic and cation exchange chromatographies. The pure enzyme tentatively named grey mullet digestive lipase (GmDL) is a monomer having a molecular mass of about 35 kDa, as determined by SDS-PAGE analysis. No similarity was found between the NH2-terminal amino acid residues of GmDL and those of other known digestive lipases. GmDL is a serine enzyme, like all known lipases from different origins. Interestingly, GmDL has not only lipase activity but also a phospholipase activity which requires the presence of Ca(2+) and bile salts. Specific activities of 64 U/mg, 55 U/mg and 63 U/mg were measured using tributyrin, olive oil emulsion or phosphatidylcholine as substrate, respectively at pH 8 and at 50°C. GmDL is therefore a thermo-active enzyme as compared to other fish lipases studied so far. It is worth to notice that grey mullet lipase was active in the presence of salt concentrations as high as 0.8M.

  5. Extraction and purification methods in downstream processing of plant-based recombinant proteins.

    Science.gov (United States)

    Łojewska, Ewelina; Kowalczyk, Tomasz; Olejniczak, Szymon; Sakowicz, Tomasz

    2016-04-01

    During the last two decades, the production of recombinant proteins in plant systems has been receiving increased attention. Currently, proteins are considered as the most important biopharmaceuticals. However, high costs and problems with scaling up the purification and isolation processes make the production of plant-based recombinant proteins a challenging task. This paper presents a summary of the information regarding the downstream processing in plant systems and provides a comprehensible overview of its key steps, such as extraction and purification. To highlight the recent progress, mainly new developments in the downstream technology have been chosen. Furthermore, besides most popular techniques, alternative methods have been described.

  6. Production of Brazilian human norovirus VLPs and comparison of purification methods

    Directory of Open Access Journals (Sweden)

    Thais Alves da Costa Lamounier

    2015-01-01

    Full Text Available AbstractNoroviruses (NVs are responsible for most cases of human nonbacterial gastroenteritis worldwide. Some parameters for the purification of NV virus-like particles (VLPs such as ease of production and yield were studied for future development of vaccines and diagnostic tools. In this study, VLPs were produced by the expression of the VP1 and VP2 gene cassette of the Brazilian NV isolate, and two purification methods were compared: cesium chloride (CsCl gradient centrifugation and ion-exchange chromatography (IEC. IEC produced more and purer VLPs of NV compared to CsCl gradient centrifugation.

  7. 酶及蛋白质分离纯化技术研究进展%Research Advance in the Separation and Purification Technology of Enzyme and Protein

    Institute of Scientific and Technical Information of China (English)

    白利涛; 张丽萍

    2012-01-01

    蛋白质的分离纯化技术是实现酶和蛋白质研究及产品工业化的关键技术之一.该研究就适用于酶和蛋白质分离纯化的各项技术的原理、分类和特点进行了简要的阐述,并对各分离技术的应用进展和应用中应注意问题进行了重点综述.%Separation and purification technology of enzyme and protein is one of the most key processes in the research and industrialized production of protein and enzyme, The paper briefly introduced the principles, classification and characteristics of various separation and purification technologies, and reviewed their application advances and problems.

  8. Evaluation of automated and manual DNA purification methods for detecting Ricinus communis DNA during ricin investigations.

    Science.gov (United States)

    Hutchins, Anne S; Astwood, Michael J; Saah, J Royden; Michel, Pierre A; Newton, Bruce R; Dauphin, Leslie A

    2014-03-01

    In April of 2013, letters addressed to the President of United States and other government officials were intercepted and found to be contaminated with ricin, heightening awareness about the need to evaluate laboratory methods for detecting ricin. This study evaluated commercial DNA purification methods for isolating Ricinus communis DNA as measured by real-time polymerase chain reaction (PCR). Four commercially available DNA purification methods (two automated, MagNA Pure compact and MagNA Pure LC, and two manual, MasterPure complete DNA and RNA purification kit and QIAamp DNA blood mini kit) were evaluated. We compared their ability to purify detectable levels of R. communis DNA from four different sample types, including crude preparations of ricin that could be used for biological crimes or acts of bioterrorism. Castor beans, spiked swabs, and spiked powders were included to simulate sample types typically tested during criminal and public health investigations. Real-time PCR analysis indicated that the QIAamp kit resulted in the greatest sensitivity for ricin preparations; the MasterPure kit performed best with spiked powders. The four methods detected equivalent levels by real-time PCR when castor beans and spiked swabs were used. All four methods yielded DNA free of PCR inhibitors as determined by the use of a PCR inhibition control assay. This study demonstrated that DNA purification methods differ in their ability to purify R. communis DNA; therefore, the purification method used for a given sample type can influence the sensitivity of real-time PCR assays for R. communis. Published by Elsevier Ireland Ltd.

  9. The metallo-. beta. -lactamases of Bacillus cereus 5/B/6: Expression in Echierichia coli, purification, and characterization of the purified recombinant enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Shaw, R.W.; Clark, S.D.; Hilliard, N.P.; Harman, J.G. (Texas Tech Univ., Lubbock (United States))

    1991-03-11

    The gene for the B. cereus 5/B/6 metallo-{beta}-lactamase was subcloned into the E. coli expression vector pRE-2. The resultant recombinants displayed a low level of enzyme activity. Generation of a site-directed mutant of the {beta}-lactamase gene containing both a Nde I site and an initiator codon allowed us to separate the {beta}-lactamase structural gene from its leader sequence. When only the structural gene was cloned into pRE-2, the B. cereus {beta}-lactamase activity was increased 9.8-fold. Purification of the recombinant enzyme from E. coli by ultracentrifugation, gel filtration, anion and cation exchange chromatography allowed the enzyme to be purified to homogeneity with an overall yield of 87%. The properties of the recombinant enzyme were identical to those of the B. cereus enzyme; e.g., the electrophoretic mobilities of the purified recombinant enzyme and the purified B. cereus enzyme were identical in both native and SDS gel electrophoresis As with the B. cereus enzyme, K{sub m} and V{sub max} for the recombinant enzyme are 0.39 mM and 1,333 units/mg protein, respectively. Likewise, the Co(II)-reconstituted recombinant enzyme has electronic spectra with maxima at 347, 551, 617 and 646 nm and extinction coefficients of 900, 250, 173 and 150 M{sup {minus}1} cm{sup {minus}1}, respectively. This heterologous construct and purification scheme will be used to produce and purify site-directed mutant proteins for use in exploring the reaction mechanisms of B. cereus metallo-{beta}-lactamases.

  10. A Simple and Accurate Method for Measuring Enzyme Activity.

    Science.gov (United States)

    Yip, Din-Yan

    1997-01-01

    Presents methods commonly used for investigating enzyme activity using catalase and presents a new method for measuring catalase activity that is more reliable and accurate. Provides results that are readily reproduced and quantified. Can also be used for investigations of enzyme properties such as the effects of temperature, pH, inhibitors,…

  11. A Simple and Accurate Method for Measuring Enzyme Activity.

    Science.gov (United States)

    Yip, Din-Yan

    1997-01-01

    Presents methods commonly used for investigating enzyme activity using catalase and presents a new method for measuring catalase activity that is more reliable and accurate. Provides results that are readily reproduced and quantified. Can also be used for investigations of enzyme properties such as the effects of temperature, pH, inhibitors,…

  12. Methods for Isolation, Purification, and Propagation of Bacteriophages of Campylobacter jejuni

    DEFF Research Database (Denmark)

    Gencay, Yilmaz Emre; Birk, Tina; Sørensen, Martine Camilla Holst

    2017-01-01

    Here, we describe the methods for isolation, purification, and propagation of Campylobacter jejuni bacteriophages from samples expected to contain high number of phages such as chicken feces. The overall steps are (1) liberation of phages from the sample material; (2) observation of plaque...

  13. Growth of Acinetobacter gerneri P7 on polyurethane and the purification and characterization of a polyurethanase enzyme.

    Science.gov (United States)

    Howard, Gary T; Norton, William N; Burks, Timothy

    2012-07-01

    A soil microorganism, designated as P7, was characterized and investigated for its ability to degrade polyurethane (PU). This bacterial isolate was identified as Acinetobacter gerneri on the basis of 16 s rRNA sequencing and biochemical phenotype analysis. The ability of this organism to degrade polyurethane was characterized by the measurement of growth, SEM observation, measurement of electrophoretic mobility and the purification and characterization of a polyurethane degrading enzyme. The purified protein has a molecular weight of approximately 66 kDa as determined by SDS-PAGE. Substrate specificity was examined using p-nitrophenyl substrates with varying carbon lengths. The highest substrate specificity was observed using p-nitrophenyl-propanate with an activity of 37.58 ± 0.21 U mg(-1). Additionally, the enzyme is inhibited by phenylmethylsulfonylfluoride and by ethylenediamine-tetra acetic acid. When grown on Impranil DLN(™) YES medium, a lag phase was noted for the first 3 h which was followed by logarithmic growth for 5 h. For the linear portion of growth between 5 and 9 h, a μ value of 0.413 doublings h(-1) was calculated. After 9 h of incubation the cell number dramatically decreased resulting in a chalky precipitate. Measurements of electrophoretic mobility indicated the formation of a complex between the PU and A. gerneri P7 cells. A hybrid zeta potential had been generated between the cells and polyurethane. Further evidence for a complex was provided by SEM observation where cells appeared to cluster along the surface of polyurethane particles and along edges of polyurethane films. Occasionally, the cells established an anchor-like structure that connected the cells to polyurethane particles.

  14. Lipolytic enzymes in bovine thyroid tissue. I. Subcellular localization, purification and characterization of acid phospholipase A1.

    Science.gov (United States)

    De Wolf, M; Lagrou, A; Hilderson, H J; Dierick, W

    1978-12-01

    In mammalian cells the catabolism of membrane phosphoglycerides proceeds probably entirely through a deacylation pathway catalysed by phospholipase A and lysophospholipase (Wise & Elwyn, 1965). In the initial attack of diacylphosphoglycerides by phospholipase A two enzymatic activities with different positional specificities have been distinguished: phospholipase A1 (phosphatidate 1-acyl hydrolase EN 3.1.1.32) and phospholipase A2 (phosphatidate 2-acyl hydrolase EN 3.1.1.4) (Van Deenen & De Haas, 1966). Studies on these intracellular phospholipases were mainly concerned with their subcellular localization. Only occasionally more detailed enzymatic investigations have been conducted on them, in contrast to export phospholipases e.g. from snake venom, bee venom and porcine pancreas, which have been extensively investigated (Brockerhoff & Jensen 1974a). In a previous paper (De Wolf et al., 1976a), the presence of phospholipase A1 and phospholipase A2 activities in bovine thyroid was demonstrated, using 1-[9, 10-3H] stearoyl-2-[1-14C] linoleyl-sn-glycero-3-phosphocholine as a substrate. Optimal activity was observed in both instances at pH 4. Addition of the anionic detergent sodium taurocholate increased the A2 type activity and decreased the A1 type activity suggesting the presence of different enzymes. The lack of influence of Ca2+-ions and EDTA and the acid pH optima could suggest lysosomal localization. In this paper the subcellular distribution of both acid phospholipase activities is described as well as a purification scheme for phospholipase A1. Some characteristics of the purified enzyme preparation are discussed.

  15. Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

    CERN Document Server

    Foulon, V; Croes, K; Waelkens, E

    1999-01-01

    Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

  16. RESEARCH METHODS OF WATER PURIFICATION FROM POLLUTION WITH PETROLEUM AND PETROLEUM PRODUCTS

    OpenAIRE

    Privalova N. M.; Dvadnenko M. V.; Nekrasova A. A.; Popova O. S.; Privalov D. M.

    2015-01-01

    This article provides an overview of the currently existing methods of purification of waters from pollution with petroleum and petroleum products. The most popular cleaning ways and new emerging technologies are considered. For each method of combating with petroleum pollution the circumstances and the factors are given, under which the application of this method is the most efficient and cost-effective. The article briefly describes the technology of each method, and its main strengths and ...

  17. Combustion water purification techniques influence on OBT analysing using liquid scintillation counting method

    Energy Technology Data Exchange (ETDEWEB)

    Varlam, C.; Vagner, I.; Faurescu, I.; Faurescu, D. [National Institute for Cryogenics and Isotopic Technologies, Valcea (Romania)

    2015-03-15

    In order to determine organically bound tritium (OBT) from environmental samples, these must be converted into water, measurable by liquid scintillation counting (LSC). For this purpose we conducted some experiments to determine OBT level of a grass sample collected from an uncontaminated area. The studied grass sample was combusted in a Parr bomb. However usual interfering phenomena were identified: color or chemical quench, chemiluminescence, overlap over tritium spectrum because of other radionuclides presence as impurities ({sup 14}C from organically compounds, {sup 36}Cl as chloride and free chlorine, {sup 40}K as potassium cations) and emulsion separation. So the purification of the combustion water before scintillation counting appeared to be essential. 5 purification methods were tested: distillation with chemical treatment (Na{sub 2}O{sub 2} and KMnO{sub 4}), lyophilization, chemical treatment (Na{sub 2}O{sub 2} and KMnO{sub 4}) followed by lyophilization, azeotropic distillation with toluene and treatment with a volcanic tuff followed by lyophilization. After the purification step each sample was measured and the OBT measured concentration, together with physico-chemical analysis of the water analyzed, revealed that the most efficient method applied for purification of the combustion water was the method using chemical treatment followed by lyophilization.

  18. Comparison of Methods for the Purification of Alpha-1 Acid Glycoprotein from Human Plasma

    Directory of Open Access Journals (Sweden)

    Teresa R. McCurdy

    2011-01-01

    Full Text Available Alpha-1 acid glycoprotein (AGP is a highly glycosylated, negatively charged plasma protein suggested to have anti-inflammatory and/or immunomodulatory activities. Purification of AGP could be simplified if methods that exploit its high solubility under chemically harsh conditions could be demonstrated to leave the protein in its native conformation. Procedures involving exposure of AGP to hot phenol or sulphosalicylic acid (SSA were compared to solely chromatographic methods. Hot phenol-purified AGP was more rapidly cleared from mice in vivo following intravenous injection than chromatographically purified AGP. In contrast, SSA-purified AGP demonstrated an identical in vivo clearance profile and circular dichroism spectrum to chromatographically purified AGP. Similarly, no differences in susceptibility to enzymatic deglycosylation or reactivity with Sambucus nigra lectin were detected between AGP purified via the two methods. Incorporation of the SSA step in the purification scheme for AGP eliminated the need for a large (4 mL resin/mL of plasma initial chromatographic step and simplified its purification without causing any detectable distortion in the conformation of the protein. Confirmation that this procedure is nondenaturing will simplify AGP purification and investigation of its possible biological roles in laboratory animals.

  19. RAPID AND EFFICIENT METHOD FOR ENVIRONMENTAL DNA EXTRACTION AND PURIFICATION FROM SOIL

    Directory of Open Access Journals (Sweden)

    J. Hamedi

    2016-06-01

    Full Text Available Large proportion of microbial population in the world is unculturable. Extraction of total DNA from soil is usually a crucial step considering to the difficulties of study the uncultivable microorganisms. Humic acid is considered as the main inhibitory agent in the environmental DNA studies. Here, we introduced a rapid and efficient method for DNA extraction and purification from soil. Yield of DNA extraction by the presented method was 130 ng/µl. Three conventional methods of DNA extraction including liquid nitrogen incursion, bead beating and sonication were performed as control methods. Yield of DNA extraction by these methods were 110, 90 and 50 ng/µl, respectively. A rapid and efficient one step DNA purification method was introduced instead of hazardous conventional phenol-chloroform methods. Humic acid removal percentage by the introduced method was 95.8 % that is comparable with 97 % gained by the conventional gel extraction method and yield of DNA after purification was 84 % and 73 %, respectively. This study could be useful in molecular ecology and metagenomics study as a fast and reliable method.

  20. Purification and characterization of hatching enzyme from brine shrimp Artemia salina.

    Science.gov (United States)

    Fan, Tingjun; Wang, Jing; Yuan, Wenpeng; Zhong, Qiwang; Shi, Ying; Cong, Rishan

    2010-02-01

    By using Artemia chorion as a specific substrate, the hatching enzyme from Artemia salina (AHE) was purified by gel-filtration and ion-exchange chromatography, and characterized biochemically and enzymatically in this study. It was found that the AHE had a molecular weight of 82.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and often contained 73.3 kDa molecules in preparation. The AHE had obvious choriolytic activity, which was optimal at pH 7.0 and a temperature of 408C. The Km value of the AHE for dimethyl casein was 8.20 mg/ml. The AHE activity was almost completely inhibited by soybean trypsin inhibitor and p-amidinophenyl methane sulfonyl fluoride hydrochloride, greatly inhibited by N-tosyl-L-lysyl chloromethyl ketone, phenylmethanesulfonyl fluoride, and lima bean trypsin inhibitor, slightly inhibited by pepstatin, N-tosyl-L-phenylalanyl chloromethyl ketone, leupeptin, N-ethylmaleimide, and iodoacetamide, and not inhibited by chymostatin and bestatin. All these results imply that AHE is most probably a trypsin-type serine protease. Besides of these, AHE was also sensitive to EDTA and Zn21. Combined with the results that the EDTA-pre-treated HE activity could be perfectly recovered by Zn21, it is indicated that AHE might be also a kind of Zn-metalloprotease.

  1. Cyclomaltodextrin glucanotransferase from Bacillus circulans E 192. I. Purification and characterization of the enzyme.

    Science.gov (United States)

    Bovetto, L J; Backer, D P; Villette, J R; Sicard, P J; Bouquelet, S J

    1992-02-01

    The cyclomaltrodextrin glucanotransferase (CGTase) [1,4-alpha-D-glucan:4-alpha-D-(1,4-alpha-D-glucano)-transferase (cyclizing), EC 2.4.1.19] from Bacillus circulans E 192 has been purified to homogeneity by Cetavlon treatment, ammonium sulfate precipitation, DEAE Trisacryl M chromatography, Q Fast Flow chromatography, and affinity on beta-cyclodextrin-Sepharose 4B. Two isoenzymes were separated by FPLC on a Mono Q column. Their isoelectric points were estimated as 6.7 and 6.9 and they represented 13 and 87%, respectively, of the initial activity. Their molecular weight, pH, and temperature optima were estimated as 78,000, 5.5, and 60 degrees C, respectively. Kinetic parameters indicated that both enzymes had the same properties; they preferentially modified high-molecular-weight substrates to produce cyclodextrins. The apparent Vmax and Km values for soluble starch were 43 mumol of beta-cyclodextrin/min/mg of protein and 0.57% (w/v), respectively. Although this CGTase is not markedly thermostable, it is protected against heat denaturation by substrate, product, and/or calcium ions. The ratios of alpha-, beta-, and gamma-cyclodextrins produced have been determined as 1/7/2 in the initial phase of the reaction and 3/3/1 at equilibrium.

  2. Method of preparing cross-linked enzyme particles

    NARCIS (Netherlands)

    Mateo, C.; Van Langen, L.M.; Van Rantwijk, F.

    2004-01-01

    The invention relates to a method of preparing cross-linked enzyme particles using a cross-linking agent. According to the invention, the enzyme particles are formed and subsequently cross-linked using a cross-linking agent having at least n reactive groups where N>=3 and a molecular weight of

  3. Method of preparing cross-linked enzyme particles

    NARCIS (Netherlands)

    Mateo, C.; Van Langen, L.M.; Van Rantwijk, F.

    2004-01-01

    The invention relates to a method of preparing cross-linked enzyme particles using a cross-linking agent. According to the invention, the enzyme particles are formed and subsequently cross-linked using a cross-linking agent having at least n reactive groups where N>=3 and a molecular weight of >2,00

  4. Rapid Purification and Procoagulant and Platelet Aggregating Activities of Rhombeobin: A Thrombin-Like/Gyroxin-Like Enzyme from Lachesis muta rhombeata Snake Venom

    Directory of Open Access Journals (Sweden)

    Frank Denis Torres-Huaco

    2013-01-01

    Full Text Available We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47 from Lachesis muta rhombeata venom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C8 Discovery BIO Wide Pore, showing high degree of molecular homogeneity with molecular mass of 47035.49 Da. Rhombeobin showed amidolytic activity upon BAρNA, with a broad optimum pH (7–10 and was stable in solution up to 60°C. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents, but not chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with other SVSPs, especially with LM-TL (L. m. muta and Gyroxin (C. d. terrificus. Rhombeobin acts, in vitro, as a strong procoagulant enzyme on mice citrated plasma, shortening the APTT and PT tests in adose-dependent manner. The protein showed, “ex vivo”, a strong defibrinogenating effect with 1 µg/animal. Lower doses activated the intrinsic and extrinsic coagulation pathways and impaired the platelet aggregation induced by ADP. Thus, this is the first report of a venom component that produces a venom-induced consumptive coagulopathy (VICC.

  5. Identification, purification and characterization of furfural transforming enzymes from Clostridium beijerinckii NCIMB 8052.

    Science.gov (United States)

    Zhang, Yan; Ujor, Victor; Wick, Macdonald; Ezeji, Thaddeus Chukwuemeka

    2015-06-01

    Generation of microbial inhibitory compounds such as furfural and 5-hydroxymethylfurfural (HMF) is a formidable roadblock to fermentation of lignocellulose-derived sugars to butanol. Bioabatement offers a cost effective strategy to circumvent this challenge. Although Clostridium beijerinckii NCIMB 8052 can transform 2-3 g/L of furfural and HMF to their less toxic alcohols, higher concentrations present in biomass hydrolysates are intractable to microbial transformation. To delineate the mechanism by which C. beijerinckii detoxifies furfural and HMF, an aldo/keto reductase (AKR) and a short-chain dehydrogenase/reductase (SDR) found to be over-expressed in furfural-challenged cultures of C. beijerinckii were cloned and over-expressed in Escherichia coli Rosetta-gami™ B(DE3)pLysS, and purified by histidine tag-assisted immobilized metal affinity chromatography. Protein gel analysis showed that the molecular weights of purified AKR and SDR are close to the predicted values of 37 kDa and 27 kDa, respectively. While AKR has apparent Km and Vmax values of 32.4 mM and 254.2 mM s(-1) respectively, using furfural as substrate, SDR showed lower Km (26.4 mM) and Vmax (22.6 mM s(-1)) values on the same substrate. However, AKR showed 7.1-fold higher specific activity on furfural than SDR. Further, both AKR and SDR were found to be active on HMF, benzaldehyde, and butyraldehyde. Both enzymes require NADPH as a cofactor for aldehydes reduction. Based on these results, it is proposed that AKR and SDR are involved in the biotransformation of furfural and HMF by C. beijerinckii.

  6. Purification and some physico-chemical and enzymic properties of a calcium ion-activated neutral proteinase from rabbit skeletal muscle

    Science.gov (United States)

    Azanza, Jean-Louis; Raymond, Jacques; Robin, Jean-Michel; Cottin, Patrick; Ducastaing, André

    1979-01-01

    Ca2+-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial–Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulphate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only one polypeptide chain of mol.wt. 73000. The purification procedure used allowed us to eliminate a contaminant containing two components of mol.wt. about 30000 each. Whole casein or α1-casein were hydrolysed with a maximum rate at 30°C, pH7.5, and with 5mm-CaCl2, but myofibrils were found to be a very susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30000-dalton component as well as in various other higher- and lower-molecular-weight peptide fragments. Troponin T, troponin I, α-tropomyosin, some high-molecular-weight proteins (M protein, heavy chain of myosin) and three unidentified proteins are degraded. Thus the number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton, Goll, Zeece, Robson & Reville [(1976) Biochemistry 15, 2150–2158]. The Ca2+-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. Thus the proteinase was proved to have an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca2+-activated neutral proteinase. ImagesFig. 1.Fig. 2.Fig. 3. PMID:534501

  7. A Simple and Efficient Method for Purification of Egg White Major Proteins Using Ion Exchange Chromatography

    OpenAIRE

    Sh. Veisi; A. Mostafaie; Z. Mohammad Hasan

    2008-01-01

    Introduction & Objective: Egg white contains four high-quantity proteins which have numerous applications. In this research, a simple and efficient method for the purification of those proteins was designed and performed based on ion exchange chromatography.Materials & Methods: In this experimental study egg white was initially separated from insoluble substances by acidic pH. The resulting extract was isolated after two steps of ion exchange chromatography using CM-Sepharose and DEAE-Sepharo...

  8. Rescue of cystathionine beta-synthase (CBS) mutants with chemical chaperones: purification and characterization of eight CBS mutant enzymes.

    Science.gov (United States)

    Majtan, Tomas; Liu, Lu; Carpenter, John F; Kraus, Jan P

    2010-05-21

    Missense mutations represent the most common cause of many genetic diseases including cystathionine beta-synthase (CBS) deficiency. Many of these mutations result in misfolded proteins, which lack biological function. The presence of chemical chaperones can sometimes alleviate or even restore protein folding and activity of mutant proteins. We present the purification and characterization of eight CBS mutants expressed in the presence of chemical chaperones such as ethanol, dimethyl sulfoxide, or trimethylamine-N-oxide. Preliminary screening in Escherichia coli crude extracts showed that their presence during protein expression had a significant impact on the amount of recovered CBS protein, formation of tetramers, and catalytic activity. Subsequently, we purified eight CBS mutants to homogeneity (P49L, P78R, A114V, R125Q, E176K, P422L, I435T, and S466L). The tetrameric mutant enzymes fully saturated with heme had the same or higher specific activities than wild type CBS. Thermal stability measurements demonstrated that the purified mutants are equally or more thermostable than wild type CBS. The response to S-adenosyl-L-methionine stimulation or thermal activation varied. The lack of response of R125Q and E176K to both stimuli indicated that their specific conformations were unable to reach the activated state. Increased levels of molecular chaperones in crude extracts, particularly DnaJ, indicated a rather indirect effect of the chemical chaperones on folding of CBS mutants. In conclusion, the chemical chaperones present in the expression medium were able to fully restore the activity of eight CBS mutants by improving their protein folding. This finding could have direct implications for the development of a therapeutical approach to pyridoxine unresponsive homocystinuria.

  9. QS-21 Adjuvant: Laboratory-Scale Purification Method and Formulation Into Liposomes.

    Science.gov (United States)

    Brunner, Livia; Barnier-Quer, Christophe; Collin, Nicolas

    2017-01-01

    QS-21, a saponin extracted from the tree Quillaja saponaria Molina, is a vaccine adjuvant which has been shown to elicit robust antibody and cell-mediated immune responses in a variety of preclinical and clinical studies [1]. Its purification from the natural source is a lengthy and difficult process. The commercially available saponin mixture Quil-A(®) is a fraction of the bark extract containing a variety of saponins, including QS-21. In order to facilitate access to QS-21 at laboratory-scale amounts, we propose here a method of purification of QS-21 starting from Quil-A(®). In addition, we describe a protocol to appropriately formulate QS-21 into cholesterol-containing, neutral liposomes which are known to decrease QS-21's hemolytic activity while retaining the adjuvant effect. Methods for the physicochemical characterization of purified QS-21 and of the QS-21/liposome formulations are also described.

  10. A simplified method for purification of recombinant soluble DnaA proteins.

    Science.gov (United States)

    Zawilak-Pawlik, Anna M; Kois, Agnieszka; Zakrzewska-Czerwinska, Jolanta

    2006-07-01

    An improved, simplified method for the purification of recombinant, tagged DnaA proteins is described. The presented protocol allowed us to purify soluble DnaA proteins from two different bacterial species: Helicobacter pylori and Streptomyces coelicolor, but it can most likely also be used for the isolation of DnaA proteins from other bacteria, as it was adapted for Mycobacterium tuberculosis DnaA. The isolation procedure consists of protein precipitation with ammonium sulphate followed by affinity chromatography. The composition of the buffers used at each purification step is crucial for the successful isolation of the recombinant DnaA proteins. The universality of the method in terms of its application to differently tagged proteins (His-tagged or GST-tagged) as well as different properties of purified proteins (e.g., highly aggregating truncated forms) makes the protocol highly useful for all studies requiring purified and active DnaA proteins.

  11. Effect of Water Volume and Biogas Volumetric Flowrate in Biogas Purification Through Water Scrubbing Method

    Directory of Open Access Journals (Sweden)

    Hendry Sakke Tira

    2014-10-01

    Full Text Available Energy supply is a crucial issue in the world in the last few years. The increase in energy demand caused by population growth and resource depletion of world oil reserves provides determination to produce and to use renewable energies. One of the them is biogas. However, until now the use of biogas has not yet been maximized because of its poor purity. According to the above problem, the research has been carried out using the method of water absorption. Under this method it is expected that the rural community is able to apply it. Therefore, their economy and productivity can be increased. This study includes variations of absorbing water volume (V and input biogas volume flow rate (Q. Raw biogas which is flowed into the absorbent will be analyzed according to the determined absorbing water volume and input biogas volume rate. Improvement on biogas composition through the biogas purification method was obtained. The level of CO2 and H2S was reduced significantly specifically in the early minutes of purification process. On the other hand, the level of CH4 was increased improving the quality of raw biogas. However, by the time of biogas purification the composition of purified biogas was nearly similar to the raw biogas. The main reason for this result was an increasing in pH of absorbent. It was shown that higher water volume and slower biogas volume rate obtained better results in reducing the CO2 and H2S and increasing CH4 compared to those of lower water volume and higher biogas volume rate respectively. The purification method has a good promising in improving the quality of raw biogas and has advantages as it is cheap and easy to be operated.

  12. Effect of Water Volume and Biogas Volumetric Flowrate in Biogas Purification Through Water Scrubbing Method

    Directory of Open Access Journals (Sweden)

    Hendry Sakke Tira

    2016-05-01

    Full Text Available Energy supply is a crucial issue in the world in the last few years. The increase in energy demand caused by population growth and resource depletion of world oil reserves provides determination to produce and to use renewable energies. One of the them is biogas. However, until now the use of biogas has not yet been maximized because of its poor purity. According to the above problem, the research has been carried out using the method of water absorption. Under this method it is expected that the rural community is able to apply it. Therefore, their economy and productivity can be increased. This study includes variations of absorbing water volume (V and input biogas volume flow rate (Q. Raw biogas which is flowed into the absorbent will be analyzed according to the determined absorbing water volume and input biogas volume rate. Improvement on biogas composition through the biogas purification method was obtained. The level of CO2 and H2S was reduced significantly specifically in the early minutes of purification process. On the other hand, the level of CH4 was increased improving the quality of raw biogas. However, by the time of biogas purification the composition of purified biogas was nearly similar to the raw biogas. The main reason for this result was an increasing in pH of absorbent. It was shown that higher water volume and slower biogas volume rate obtained better results in reducing the CO2 and H2S and increasing CH4 compared to those of lower water volume and higher biogas volume rate respectively. The purification method has a good promising in improving the quality of raw biogas and has advantages as it is cheap and easy to be operated.

  13. Sample displacement chromatography as a method for purification of proteins and peptides from complex mixtures

    OpenAIRE

    Gajdosik, Martina Srajer; Clifton, James; Josic, Djuro

    2012-01-01

    Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method takes advantage of relative binding affinities of components in a sample mixture. During loading, there is a competition among different sample components for the sorption on the surface of the stationary phase. SDC was first used for the preparative purification of proteins. Later, it was demonstrated that this kind of chromatography can also be perform...

  14. Purification of a chymotrypsin-like enzyme present on adult Schistosoma mansoni worms from infected mice and its characterization as a host carboxylesterase.

    Science.gov (United States)

    Igetei, Joseph E; Liddell, Susan; El-Faham, Marwa; Doenhoff, Michael J

    2016-04-01

    A serine protease-like enzyme found in detergent extracts of Schistosoma mansoni adult worms perfused from infected mice has been purified from mouse blood and further characterized. The enzyme is approximately 85 kDa and hydrolyses N-acetyl-DL-phenylalanine β-naphthyl-ester, a chromogenic substrate for chymotrypsin-like enzymes. The enzyme from S. mansoni worms appears to be antigenically and enzymatically similar to a molecule that is present in normal mouse blood and so is seemingly host-derived. The enzyme was partially purified by depleting normal mouse serum of albumin using sodium chloride and cold ethanol, followed by repeated rounds of purification by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified material was subjected to tandem mass spectrometry and its derived peptides found to belong to mouse carboxylesterase 1C. Its ability to hydrolyse α- or β-naphthyl acetates, which are general esterase substrates, has been confirmed. A similar carboxylesterase was purified and characterized from rat blood. Additional evidence to support identification of the enzyme as a carboxylesterase has been provided. Possible roles of the enzyme in the mouse host-parasite relationship could be to ease the passage of worms through the host's blood vessels and/or in immune evasion.

  15. Purification and Partial Characterization of a Collagenolytic Enzyme Produced by Pseudomonas aeraginosa SCU Screened from Rotten Hides

    Institute of Scientific and Technical Information of China (English)

    Yang Guangyao(杨光垚); Zhang Yizheng

    2004-01-01

    Strain Pseudomonas Aeraginosa SCU isolated from rotten hides is shown to produce various gelatinolytic enzymes with molecular masses ranging from ~50 to ~200 kD. A gelatinolytic enzyme called PAC exhibiting collagenolytic activity is purified by SP sepharose fast flow, Sephadex G-200 gel filtration and native PAGE cutting method. The purified enzyme has an apparent molecular weight of about 110 kD by SDS PAGE without β-mercaptoethanol. Treatment withβ-Me suggests that PAC is dissociated into three subunits approximately 33 kD,25 kD and 20 kD with a ratio of 2∶1∶1, named sub A, sub B and sub C repectively. EDTA and EGTA display a significant inhibitory effect on the enzyme activity while PMSF, leupeptin and pepstain do not appreciably inhibit it. The first 15 amino acid residues of the major subunit (subA) are determined and the sequence is Ala-Glu-Ala-Gly-Gly-Pro-Gly-Gly-Asn-Gln-Lys-Ile-Gly -Lys-Tyr. This sequence is identical to that of elastase of P.aeruginosa. The fragment of encoding mature sub A is cloned and its sequence is determined, which has a high homology with the gene of elastase. These results indicate that PAC is a novel collagenolytic metalloprotease composed of three kinds of subunits, of which elastase is the major one.

  16. An efficient method for the purification of proteins from four distinct toxin-antitoxin modules.

    Science.gov (United States)

    Sterckx, Yann G-J; De Gieter, Steven; Zorzini, Valentina; Hadži, San; Haesaerts, Sarah; Loris, Remy; Garcia-Pino, Abel

    2015-04-01

    Toxin-antitoxin (TA) modules are stress response elements that are ubiquitous in the genomes of bacteria and archaea. Production and subsequent purification of individual TA proteins is anything but straightforward as over-expression of the toxin gene is lethal to bacterial and eukaryotic cells and over-production of the antitoxin leads to its proteolytic degradation because of its inherently unstructured nature. Here we describe an effective production and purification strategy centered on an on-column denaturant-induced dissociation of the toxin-antitoxin complex. The success of the method is demonstrated by its application on four different TA families, encoding proteins with distinct activities and folds. A series of biophysical and in vitro activity tests show that the purified proteins are of high quality and suitable for structural studies.

  17. Method and apparatus for capacitive deionization and electrochemical purification and regeneration of electrodes

    Science.gov (United States)

    Tran, Tri D.; Farmer, Joseph C.; Murguia, Laura

    2001-01-01

    An electrically regeneratable electrochemical cell (30) for capacitive deionization and electrochemical purification and regeneration of electrodes includes two end plates (31, 32), one at each end of the cell (30). A new regeneration method is applied to the cell (30) which includes slowing or stopping the purification cycle, electrically desorbing contaminants and removing the desorbed contaminants. The cell (30) further includes a plurality of generally identical double-sided intermediate electrodes (37-43) that are equidistally separated from each other, between the two end electrodes (35, 36). As the electrolyte enters the cell, it flows through a continuous open serpentine channel (65-71) defined by the electrodes, substantially parallel to the surfaces of the electrodes. By polarizing the cell (30), ions are removed from the electrolyte and are held in the electric double layers formed at the carbon aerogel surfaces of the electrodes. The cell (30) is regenerated electrically to desorb such previously removed ions.

  18. Enzyme

    Science.gov (United States)

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  19. Soils and waste water purification from oil products using combined methods under the North conditions.

    Science.gov (United States)

    Evdokimova, Galina A; Gershenkop, Alexander Sh; Mozgova, Natalia P; Myazin, Vladimir A; Fokina, Nadejda V

    2012-01-01

    Oil and gas production and transportation in Russia is increasingly moving to the north regions. Such regions are characterized by relatively low self-purification capacity of the natural environments from the contaminants due to slow character of the energy exchange and mass transfer processes. Off-shore field development in the Barents Sea and oil product transportation can result in contamination, as confirmed by the national and international practice of the developed oil and gas regions. The research aims at development of the soil bioremediation methods and industrial waste water purification contaminated by oil products in the north-western region of Russia. The dynamics of oil products carry-over have been investigated under the field model experiments in podzolic soils: gas condensate, diesel fuel and mazut from oil and the plants were selected for phyto-remediation of contaminated soils under high north latitudes. It is shown that soil purification from light hydrocarbons takes place during one vegetation period. In three months of the vegetation period the gas condensate was completely removed from the soil, diesel fuel - almost completely (more than 90%). Residual amounts of heavy hydrocarbons were traced, even 1.5 later. The following plants that were highly resistant to the oil product contamination were recommended for bioremediation: Phalaroides arundinacea, Festuca pratensis, Phleum pratense, Leymus arenarius. There has been developed and patented the combined method of treatment of waste water contaminated with hydrocarbons based on inorganic coagulants and local oil-oxidizing bacteria.

  20. Purification of d-a-tocopheryl polyethylene glycol 1000 succinate (TPGS) by a temperature-modulated silica gel column chromatography: use of Taguchi method to optimize purification conditions.

    Science.gov (United States)

    Chang, Yinzi; Cao, Yucheng; Zhang, Jin; Wen, Yangyi; Ren, Qilong

    2011-12-05

    The demand for high purity d-a-tocopheryl polyethylene glycol 1000 succinate (TPGS) is increasing with the exploitation of TPGS-related products. Previously, we synthesized a TPGS mixture by esterifying vitamin E succinate with polyethyleneglycol 1000. In this study, a temperature-modulated silica gel chromatographic column was used to purify the synthesized TPGS. Taguchi method was used to optimize purification conditions associated with column temperature, loading amount, feedstock concentration and flow rate of mobile phases. Purification efficacy under the Taguchi optimized conditions was predicted theoretically and the predicted results were verified experimentally. High-performance liquid chromatography was used to quantify the unpurified and purified TPGS. The Taguchi-based analysis separately produced an optimum combination of purification conditions for TPGS purity and recovery. Under the optimized conditions, both the theoretical prediction and the confirmatory experiment yielded TPGS purity and recovery approximating to 98% each. Impressively, the study also found that column temperature had a considerable effect on purification efficacy, in particular on TPGS purity, although it was a less influential factor compared to loading amount and feedstock concentration.

  1. Purification and characterization of protease enzyme from actinomycetes and its cytotoxic effect on cancer cell line (A549)

    Institute of Scientific and Technical Information of China (English)

    C Balachandran; V Duraipandiyan; S Ignacimuthu

    2012-01-01

    Objective: To isolate active actinomycetes from soil samples of Northern Himalayas and study their culture characterization, protease production and cytotoxic effects on cancer cell line (A549). Methods: Forty six strains of actinomycetes were isolated from the soil collected from Northern Himalayas, India. Isolation of actinomycetes was performed by serial dilution plate technique. Forty six isolated actinomycetes cultures were grown in ISP 2 medium to study the morphology and biochemical characteristics. Isolated strains were studied for protease enzyme production in skim milk agar medium with solubilising capacity. Seven isolates were studied for melanin pigmentation and different NaCl concentration. Effects of environmental conditions influencing protease enzyme production of seven isolated strains were also studied at different pH, temperature and metal ions (β-mercaptoethanol, dithiothreitol, iodoacetamide, MgSO4, CaCl2 and EDTA). The seven isolates were also studied for lytic enzyme activity using different bacteria and yeast such as Pseudomonas aeruginosa (P. aeruginosa), Enterococcus feacalis (E. feacalis), Escherishia coli (E. coli), Candida albicans (C. albicans), Bacillus subtilis (B. subtilis), Klebsiella pneumonia (K. pneumonia) and Staphylococcus aureus (S. aureus). Results: Isolates ERIA-31 and ERIA-33 produced more protease enzyme activity in modified nutrient agar media compared to other actinomycetes cultures. ERIA-31 and ERIA-33 were tested for cytotoxic effect in human adenocarcinoma cancer cell line (A549). IC50 for ERIA-31 was 57.04 μg/mL and IC50 for ERIA-33 was 55.07 μg/mL. Conclusion: Actinomycete being a protease producing bacteria has the potential for use in industrial purpose, pharmaceuticals, cytotoxic agent and its proteolytic activity. Isolates of ERIA-31 and ERIA-33 produced significant amount of protease enzymes.

  2. Two-phase aqueous micellar systems: an alternative method for protein purification

    Directory of Open Access Journals (Sweden)

    Rangel-Yagui C. O.

    2004-01-01

    Full Text Available Two-phase aqueous micellar systems can be exploited in separation science for the extraction/purification of desired biomolecules. This article reviews recent experimental and theoretical work by Blankschtein and co-workers on the use of two-phase aqueous micellar systems for the separation of hydrophilic proteins. The experimental partitioning behavior of the enzyme glucose-6-phosphate dehydrogenase (G6PD in two-phase aqueous micellar systems is also reviewed and new results are presented. Specifically, we discuss very recent work on the purification of G6PD using: i a two-phase aqueous micellar system composed of the nonionic surfactant n-decyl tetra(ethylene oxide (C10E4, and (ii a two-phase aqueous mixed micellar system composed of C10E4 and the cationic surfactant decyltrimethylammonium bromide (C10TAB. Our results indicate that the two-phase aqueous mixed (C10E4/C10TAB micellar system can improve significantly the partitioning behavior of G6PD relative to that observed in the two-phase aqueous C10E4 micellar system.

  3. Evaluation of an enzymic method for starch purity determination

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, J.F. (Research Lab. for the Chemistry of Bioactive Carbohydrates and Proteins, Birmingham Univ. (United Kingdom)); Cabalda, V.M. (Inst. of Research and Development, Chembiotech Ltd., Birmingham (United Kingdom))

    1993-02-01

    Independent evaluation of an enzyme method (AFNOR) on a wider than original array of starch types showed incomplete conversion of some starch components (up to 2.5%) to glucose, as assayed per gel permeation chromatography. The AFNOR method uses only a single enzyme regime consisting of amyloglucosidase from Aspergillus niger. Negligible amounts of starch were found in the precipitates/residues obtained after starch hydrolysis of the starch hydrolysates (<0.1%). Standard deviation of the method was <1% absolute for all starches studied. (orig.).

  4. Improved methods of isolation and purification of myxobacteria and development of fruiting body formation of two strains.

    Science.gov (United States)

    Zhang, LiPing; Wang, HaiYing; Fang, XiaoMei; Stackebrandt, Erko; Ding, YanBo

    2003-07-01

    By using baiting techniques and different purification methods, a high number of myxobacterial strains have been isolated as pure cultures from soil of different regions of China. Because myxobacterial cells do not disperse easily in liquid media, a medium containing an enzymatic hydrolysate of casein (CEH) medium have been used for purification and purity tests combined in a single step. The key method, in which isolates are reintroduced to sterile rabbit dung to induce fruiting bodies formation, facilitates purification of myxobacteria. Sterile rabbit dung pellets are used to mimic the natural growth substance of these organisms which has the advantage that characteristic fruiting bodies emerge, which is a key characteristics in the taxonomy of myxobacteria. In this study, the optimum program of isolation and purification of some myxobacteria strains has been established which will facilitate screening programs. Moreover, the development of fruiting body formation of strain BD20 (Chondromyces) and strain BD54 (Cystobacter) have been recorded in this study.

  5. Evaluation method for the drying performance of enzyme containing formulations

    DEFF Research Database (Denmark)

    Sloth, Jakob; Bach, P.; Jensen, Anker Degn;

    2008-01-01

    A method is presented for fast and cheap evaluation of the performance of enzyme containing formulations in terms of preserving the highest enzyme activity during spray drying. The method is based on modeling the kinetics of the thermal inactivation reaction which occurs during the drying process....... Relevant kinetic parameters are determined from differential scanning calorimeter (DSC) experiments and the model is used to simulate the severity of the inactivation reaction for temperatures and moisture levels relevant for spray drying. After conducting experiments and subsequent simulations...... for a number of different formulations it may be deduced which formulation performs best. This is illustrated by a formulation design study where 4 different enzyme containing formulations are evaluated. The method is validated by comparison to pilot scale spray dryer experiments....

  6. Purification, crystallization and preliminary X-ray diffraction analysis of saxthrombin, a thrombin-like enzyme from Gloydius saxatilis venom

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Wenqing; Zhao, Wei [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Wang, Xiaoping [National Conservation of Snake Island and Laotieshan Mountain, Dalian, Liaoning, 116041 (China); Teng, Maikun, E-mail: mkteng@ustc.edu.cn; Niu, Liwen, E-mail: mkteng@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027 (China)

    2007-08-01

    The thrombin-like enzyme saxthrombin has been purified from G. saxatilis snake venom. Crystallization conditions were found and a data set was obtained to 1.43 Å. The snake-venom thrombin-like enzymes (SVTLEs) are a class of serine proteinases that show fibrinogen-clotting and esterolytic activities. Most TLEs convert fibrinogen to fibrin by releasing either fibrinopeptide A or fibrinopeptide B and cannot activate factor XIII. The enzymes hydrolyze fibrinogen to produce non-cross-linked fibrins, which are susceptible to the lytic action of plasmin. Because of these physiological properties, TLEs have important medical applications in myocardial infarction, ischaemic stroke and thrombotic diseases. Here, a three-step chromatography procedure was used to purify saxthrombin (AAP20638) from Gloydius saxatilis venom to homogeneity. Its molecular weight is about 30 kDa as estimated by SDS–PAGE. A saxthrombin crystal was obtained using the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 1.43 Å. The crystal belongs to space group C2, with unit-cell parameters a = 97.23, b = 52.21, c = 50.10 Å, β = 96.72°, and the Matthews coefficient (V{sub M}) was calculated to be 2.13 Å{sup 3} Da{sup −1} with one molecule in the asymmetric unit.

  7. An improved method for purification of recombinant truncated heme oxygenase-1 by expanded bed adsorption and gel filtration.

    Science.gov (United States)

    Hu, Hong-Bo; Wang, Wei; Han, Ling; Zhou, Wen-Pu; Zhang, Xue-Hong

    2007-03-01

    Recombinant truncated human heme oxygenase-1 (hHO-1) expressed in Escherichia coli was efficiently separated and purified from feedstock by DEAE-ion exchange expanded bed adsorption. Protocol optimization of hHO-1 on DEAE adsorbent resulted in adsorption in 0 M NaCl and elution in 150 mM NaCl at a pH of 8.5. The active enzyme fractions separated from the expanded bed column were further purified by a Superdex 75 gel filtration step. The specific hHO-1 activity increased from 0.82 +/- 0.05 to 24.8 +/- 1.8 U/mg during the whole purification steps. The recovery and purification factor of truncated hHO-1 of the whole purification were 72.7 +/- 4.7 and 30.2 +/- 2.3%, respectively. This purification process can decrease the demand on the preparation of feedstock and simplify the purification process.

  8. Alternative purification method for recombinant measles viral nucleoprotein expressed in insect cells by ion-exchange chromatography.

    Science.gov (United States)

    Lee, Han Saem; Kim, You-Jin; Yang, Jeongsun; Yoon, Hee Sook; Kim, Seung Tae; Kim, Kisoon

    2014-03-01

    Recombinant measles virus nucleoproteins (rMeV N) and fusion (F) proteins were characterized as major antigenic proteins expressed in insect cells mediated by recombinant baculoviruses (rBVs). Band intensities were analyzed by Western blotting to recognize IgG and IgM antibodies against the rMeV N and F proteins in human sera and cerebrospinal fluids (CSFs) from patients with measles infections. Positive results from the blots using the rMeV N were consistent with the results of enzyme-linked immunosorbent assays (ELISAs) in which whole viral proteins were used as antigens. Human sera and CSFs reacted more strongly with the rMeV N than with the rMeV F proteins prepared in an identical expression system. For efficient and reliable purification, ion-exchange chromatography using Source Q anion resin was applied, and high-purity rMeV N protein was harvested. To characterize the similarity with the native viral protein to purified N protein, structural mimicry of purified recombinant proteins with intact rMeV N was shown through transmission electron microscopy, and the truncation and the phosphorylation status of the expressed protein were analyzed. These results suggest that the rMeV N purified by ion-exchange chromatography has features similar to those of naïve N including a self-assembled structure, phosphorylation and antigenic function. Thus, these expression and purification methods can be applied to the large-scale production of the rMeV N, which is essential for the development of new diagnostic tools and vaccines for acute and chronic MeV infections.

  9. Method and apparatus for efficient photodetachment and purification of negative ion beams

    Science.gov (United States)

    Beene, James R.; Liu, Yuan; Havener, Charles C.

    2008-02-26

    Methods and apparatus are described for efficient photodetachment and purification of negative ion beams. A method of purifying an ion beam includes: inputting the ion beam into a gas-filled multipole ion guide, the ion beam including a plurality of ions; increasing a laser-ion interaction time by collisional cooling the plurality of ions using the gas-filled multipole ion guide, the plurality of ions including at least one contaminant; and suppressing the at least one contaminant by selectively removing the at least one contaminant from the ion beam by electron photodetaching at least a portion of the at least one contaminant using a laser beam.

  10. A new method for the purification of the different stages of carrot embryoids.

    Science.gov (United States)

    Giuliano, G; Rosellini, D; Terzi, M

    1983-08-01

    An easy method is presented for the purification of the different stages of carrot embryoids. This is based on a synchronization of the regenerating culture and on a filtration through filters of various pore sizes. A differential sedimentation was used for removing undifferentiated cells. At the end of the process, the different stages: globular, heart- and torpedo-shaped were obtained with a degree of purity that always exceeded 90%. This method can be used for the separation of relatively large numbers of embryoids (from thousands to a million) of haploid and diploid carrot lines and is very gentle on embryoids in that it does not affect their viability or further development.

  11. Computer Aided Methods & Tools for Separation & Purification of Fine Chemical & Pharmaceutical Products

    DEFF Research Database (Denmark)

    Afonso, Maria B.C.; Soni, Vipasha; Mitkowski, Piotr Tomasz

    2006-01-01

    aided system. The methods and tools are linked through the problems they are able to solve and the associated data-flow. The integrated computer aided system has been used to solve a number of industrial problems and summarized results from a selection, involving separation and purification issues......An integrated approach that is particularly suitable for solving problems related to product-process design from the fine chemicals, agrochemicals, food and pharmaceutical industries is presented together with the corresponding methods and tools, which forms the basis for an integrated computer...

  12. Purification and Stability of an Antithrombus Enzyme from Bacillus subtilis%一种溶解酶(ATE)的分离纯化及其稳定性

    Institute of Scientific and Technical Information of China (English)

    关怡新; 姚善泾; 俞丽华; 梅乐和

    2004-01-01

    An antithrombus enzyme (ATE) was precipitated by (NH4)2SO4 or ethanol from supernatant of Bacillus subtilis culture broth then purified using ion exchange chromatography on CM-sepharose fast flow. The effects of ionic strength and pH value on protein adsorption, the gradient elution at different flow rates and step elution were examined respectively. The recovery yield of the optimised process was 74.5% with a purification factor 8.1. The ATE molecular weight was estimated as 30ku by SDS-PAGE. The experimental results showed that the enzyme was stable in the range of pH 7 to pH11, and temperature 25℃ to 37℃.

  13. Method of water purification from chromium (VI with the presence of microorganisms

    Directory of Open Access Journals (Sweden)

    Олена Георгіївна Горшкова

    2015-09-01

    Full Text Available The high efficiency of water purification from chromium (VI by the polyfunctional bacterial suspension consisted of the association of non-pathogenic bacteria strains of the genus Pseudomonas: P. fluorescens ONU328, P. maltophilia ONU329, P. cepacia ONU327 in a volume ratio of 1:1:1 is experimentally confirmed. The method allows in the presence of hydrogen peroxide and calcium chloride to purify contaminated water from chromium (VI with concentration up to 70 mg/dm3 to values of concentration smaller than the maximum allowable concentration

  14. A rapid and scalable density gradient purification method for Plasmodium sporozoites

    Directory of Open Access Journals (Sweden)

    Kennedy Mark

    2012-12-01

    Full Text Available Abstract Background Malaria remains a major human health problem, with no licensed vaccine currently available. Malaria infections initiate when infectious Plasmodium sporozoites are transmitted by Anopheline mosquitoes during their blood meal. Investigations of the malaria sporozoite are, therefore, of clear medical importance. However, sporozoites can only be produced in and isolated from mosquitoes, and their isolation results in large amounts of accompanying mosquito debris and contaminating microbes. Methods Here is described a discontinuous density gradient purification method for Plasmodium sporozoites that maintains parasite infectivity in vitro and in vivo and greatly reduces mosquito and microbial contaminants. Results This method provides clear advantages over previous approaches: it is rapid, requires no serum components, and can be scaled to purify >107 sporozoites with minimal operator involvement. Moreover, it can be effectively applied to both human (Plasmodium falciparum, Plasmodium vivax and rodent (Plasmodium yoelii infective species with excellent recovery rates. Conclusions This novel method effectively purifies viable malaria sporozoites by greatly reducing contaminating mosquito debris and microbial burdens associated with parasite isolation. Large-scale preparations of purified sporozoites will allow for enhanced in vitro infections, proteomics, and biochemical characterizations. In conjunction with aseptic mosquito rearing techniques, this purification technique will also support production of live attenuated sporozoites for vaccination.

  15. Optimization of Expression and Purification of Recombinant Archeoglobus fulgidus F420H2:NADP+ Oxidoreductase, an F420 Cofactor Dependent Enzyme.

    Science.gov (United States)

    Le, Cuong Quang; Joseph, Ebenezer; Nguyen, Toan; Johnson-Winters, Kayunta

    2015-12-01

    Methanogens play a critical role in carbon cycling and contain a number of intriguing biosynthetic pathways. One unusual cofactor found in methanogenic and sulfate reducing archaea is Factor 420 (F420), which can be interconverted between its reduced and oxidized forms by the F420H2:NADP(+) oxidoreductase (Fno) through hydride transfer mechanisms. Here, we report an optimized expression and purification method for recombinant Fno derived from the extreme thermophile Archeoglobus fulgidus. An expression vector that is codon-optimized for heterologous expression in Escherichia coli, modified growth conditions, and a modified purification protocol involving a key polyethyleneimine precipitation step results in a highly purified, homogeneous preparation of Fno that displays high catalytic activity with a truncated F420 analog. This method should accelerate studies on how Fno uses the unusual F420 cofactor during catalysis.

  16. Optimization of the Purification Methods for Recovery of Recombinant Growth Hormone from Paralichthys olivaceus

    Institute of Scientific and Technical Information of China (English)

    ZANG Xiaonan; ZHANG Xuecheng; MU Xiaosheng; LIU Bin

    2013-01-01

    This study aimed to optimize the purification of recombinant growth hormone from Paralichthys olivaceus.Recombinant flounder growth hormone (r-fGH) was expressed by Escherichia coli in form of inclusion body or as soluble protein under different inducing conditions.The inclusion body was renatured using two recovery methods,i.e.,dilution and dialysis.Thereafter,the refolded protein was purified by Glutathione Sepharase 4B affinity chromatography and r-fGH was obtained by cleavage of thrombin.For soluble products,r-fGH was directly purified from the lysates by Glutathione Sepharase 4B affinity chromatography.ELISA-receptor assay demonstrated that despite its low receptor binding activity,the r-fGH purified from refolded inclusion body had a higher yield (2.605 mg L-1) than that from soluble protein (1.964 mg L-1).Of the tested recovery methods,addition of renaturing buffer (pH 8.5) into denatured inclusion body yielded the best recovery rate (17.9%).This work provided an optimized purification method for high recovery of r-fGH,thus contributing to the application of r-fGH to aquaculture.

  17. Optimization of purification method and characterization of recombinant human Centrin-1.

    Science.gov (United States)

    Phanindranath, Regur; Sudhakar, Digumarthi V S; Sharma, Anand Kumar; Thangaraj, Kumarasamy; Sharma, Yogendra

    2016-08-01

    Centrins are acidic proteins, present in all eukaryotes to perform imperative roles in centrosome positioning and segregation. Existing methods for the purification of centrins for biophysical studies involves either multiple steps or yields protein with an affinity tag, which pins additional tag-cleavage step. Therefore, we have made an attempt to develop a simple and single step method for protein purification. We have performed categorical evaluation of existing methods, and describe a one-step procedure based on cleavable Intein-tag, which can be utilized for routine preparation of any isoform of centrins. Since human Centrin-1 and Centrin-2 are devoid of Trp, we exploit this feature to assess the purity of the protein using Tyr fluorescence; an essential point ignored generally. In addition, we report important spectral and hydrodynamic characteristics of human Centrin-1, accounting that HsCentrin-1 has moderate affinity for Ca(2+). Centrin-1 does not gain structure as seen by far- and near-UV circular dichroism, rather there is a loss of ellipticity, though inconsiderable upon binding Ca(2+). Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Optimization of the purification methods for recovery of recombinant growth hormone from Paralichthys olivaceus

    Science.gov (United States)

    Zang, Xiaonan; Zhang, Xuecheng; Mu, Xiaosheng; Liu, Bin

    2013-03-01

    This study aimed to optimize the purification of recombinant growth hormone from Paralichthys olivaceus. Recombinant flounder growth hormone (r-fGH) was expressed by Escherichia coli in form of inclusion body or as soluble protein under different inducing conditions. The inclusion body was renatured using two recovery methods, i.e., dilution and dialysis. Thereafter, the refolded protein was purified by Glutathione Sepharase 4B affinity chromatography and r-fGH was obtained by cleavage of thrombin. For soluble products, r-fGH was directly purified from the lysates by Glutathione Sepharase 4B affinity chromatography. ELISA-receptor assay demonstrated that despite its low receptor binding activity, the r-fGH purified from refolded inclusion body had a higher yield (2.605 mg L-1) than that from soluble protein (1.964 mg L-1). Of the tested recovery methods, addition of renaturing buffer (pH 8.5) into denatured inclusion body yielded the best recovery rate (17.9%). This work provided an optimized purification method for high recovery of r-fGH, thus contributing to the application of r-fGH to aquaculture.

  19. Metodologia de seleção de cepas para produção etodologia da ciclodextrina glicosiltransferase e para purificação da enzima = Strains selection methodology for cyclodextrin glycosyltransferase production and enzyme purification

    Directory of Open Access Journals (Sweden)

    Glauciane de Lara Costa

    2007-01-01

    Full Text Available As ciclodextrinas (CDs são maltooligossacarídeos, produzidas a partir do amido, pela enzima ciclodextrina glicosiltransferase (CGTase. Esta pesquisa teve por objetivo estabelecer metodologias de seleção de cepas para produção de CGTase e para purificação da enzima. Os microrganismos foram selecionados a partir de 53 análises de solos de cultura de amido, em placas contendo meio de cultivo específico, para seleção de cepas produtoras de CGTase. As enzimas foram obtidas com cultivo destes microrganismos em meio líquido. As atividades enzimáticas das CGTases foram determinadas pelos métodos espectrofotométricos e precipitação com tricloroetileno. A cepa isolada do solo de aveia foi a que apresentou maior atividade [0,1864 mmol de b-CD (min mL-1]. Esta cepa foi utilizada para a produção da enzima em escala laboratorial e purificação em cromatografia deafinidade bioespecífica. A cepa selecionada nesta pesquisa abre novas perspectivas para produção de enzima e CDs em escala industrial.Cyclodextrins (CDs are maltooligosaccharides produced from starch by cyclodextrin glycosyltransferase (CGTase enzyme. This researchaimed at establishing method for strains selection for CGTase production and enzyme purification. The microorganisms were selected from 53 analyses of starch cultures soils on plates containing specific culture medium for strains selection that produce CGTase. Theenzymes were obtained by culturing these microorganisms in liquid medium. The enzyme activity was determined with photospectrometric methods and precipitation with trichloroethylene. The strain isolated from oat soil was the one that showed the highest activity [0.1864 mmol of b-CD (min mL-1]. This strain was used for enzyme production in laboratory scale and purification by biospecific affinity chromatography. The strain selected in this research opens new perspectives for enzymes production and CDs in industrial scale.

  20. Purification of a Recombinant Glutathione Transferase from the Causative Agent of Hydatidosis, "Echinococcus granulosus"

    Science.gov (United States)

    Fleitas, Andrea L.; Randall, Lía M.; Möller, Matías N.; Denicola, Ana

    2016-01-01

    This practical class activity was designed to introduce students to recombinant protein expression and purification. The principal goal is to shed light on basic aspects concerning recombinant protein production, in particular protein expression, chromatography methods for protein purification, and enzyme activity as a tool to evaluate purity and…

  1. Protein purification-free method of binding affinity determination by microscale thermophoresis.

    Science.gov (United States)

    Khavrutskii, Lyuba; Yeh, Joanna; Timofeeva, Olga; Tarasov, Sergey G; Pritt, Samuel; Stefanisko, Karen; Tarasova, Nadya

    2013-08-15

    Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

  2. Analysis of exosome purification methods using a model liposome system and tunable-resistive pulse sensing

    Science.gov (United States)

    Lane, Rebecca E.; Korbie, Darren; Anderson, Will; Vaidyanathan, Ramanathan; Trau, Matt

    2015-01-01

    Exosomes are vesicles which have garnered interest due to their diagnostic and therapeutic potential. Isolation of pure yields of exosomes from complex biological fluids whilst preserving their physical characteristics is critical for downstream applications. In this study, we use 100 nm-liposomes from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol as a model system as a model system to assess the effect of exosome isolation protocols on vesicle recovery and size distribution using a single-particle analysis method. We demonstrate that liposome size distribution and ζ-potential are comparable to extracted exosomes, making them an ideal model for comparison studies. Four different purification protocols were evaluated, with liposomes robustly isolated by three of them. Recovered yields varied and liposome size distribution was unaltered during processing, suggesting that these protocols do not induce particle aggregation. This leads us to conclude that the size distribution profile and characteristics of vesicles are stably maintained during processing and purification, suggesting that reports detailing how exosomes derived from tumour cells differ in size to those from normal cells are reporting a real phenomenon. However, we hypothesize that larger particles present in most purified exosome samples represent co-purified contaminating non-exosome debris. These isolation techniques are therefore likely nonspecific and may co-isolate non-exosome material of similar physical properties.

  3. Two-stage method for purification of ceruloplasmin based on its interaction with neomycin.

    Science.gov (United States)

    Sokolov, A V; Kostevich, V A; Romanico, D N; Zakharova, E T; Vasilyev, V B

    2012-06-01

    A two-stage chromatography that yields highly purified ceruloplasmin (CP) from human plasma and from rat and rabbit serum is described. The isolation procedure is based on the interaction of CP with neomycin, and it provides a high yield of CP. Constants of inhibition by gentamycin, kanamycin, and neomycin of oxidase activity of CP in its reaction with p-phenylenediamine were assayed. The lowest K(i) for neomycin (11 µM) corresponded to the highest specific adsorption of CP on neomycin-agarose (10 mg CP/ml of resin). Isolation of CP from 1.4 liters of human plasma using ion-exchange chromatography on UNO-Sphere Q and affinity chromatography on neomycin-agarose yields 348 mg of CP with 412-fold purification degree. Human CP preparation obtained with A(610)/A(280) ~ 0.052 contained neither immunoreactive prothrombin nor active thrombin. Upon storage at 37°C under sterile conditions, the preparation remained stable for two months. Efficient preparation of highly purified CP from rat and rabbit sera treated according to a similar protocol suggests the suitability of our method for isolation of CP from plasma and serum of other animals. The yield of CP in three separate purifications was no less than 78%.

  4. Statistical evaluation and optimization of zinc electrolyte hot purification process by Taguchi method

    Institute of Scientific and Technical Information of China (English)

    Bahram Behnajady; Javad Moghaddam

    2015-01-01

    The neutral zinc sulfate solution obtained from hydrometallurgical process of Angouran zinc concentrate has cadmium, nickel and cobalt impurities, that must be purified before electrowinning. Therefore, cadmium and nickel are usually cemented out by addition of zinc dust and remained nickel and cobalt cemented out at second stage with zinc powder and arsenic trioxide. In this research, a new approach is described for determination of effective parameters and optimization of zinc electrolyte hot purification process using statistical design of experiments. The Taguchi method based on orthogonal array design (OAD) has been used to arrange the experimental runs. The experimental conditions involved in the work are as follows: the temperature range of 70−90°C for reaction temperature (T), 30−90 min for reaction time (t), 2−4 g/L for zinc powder mass concentration (M), one to five series for zinc dust particle size distributions (S1−S5), and 0.1−0.5 g/L (C) for arsenic trioxide mass concentration. Optimum conditions for hot purification obtained in this work areT4 (85 °C),t4=75 min,M4=3.5 g/L,S4 (Serie 4), andC2=0.2 g/L.

  5. FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

    Directory of Open Access Journals (Sweden)

    Lu Jia

    2011-10-01

    Full Text Available Abstract Background Although a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process. Results Here we report a highly simplified, reliable, and efficient PCR-based cloning technique to insert any DNA fragment into a plasmid vector or into a gene (cDNA in a vector at any desired position. With this method, the vector and insert are PCR amplified separately, with only 18 cycles, using a high fidelity DNA polymerase. The amplified insert has the ends with ~16-base overlapping with the ends of the amplified vector. After DpnI digestion of the mixture of the amplified vector and insert to eliminate the DNA templates used in PCR reactions, the mixture is directly transformed into competent E. coli cells to obtain the desired clones. This technique has many advantages over other cloning methods. First, it does not need gel purification of the PCR product or linearized vector. Second, there is no need of any cloning kit or specialized enzyme for cloning. Furthermore, with reduced number of PCR cycles, it also decreases the chance of random mutations. In addition, this method is highly effective and reproducible. Finally, since this cloning method is also sequence independent, we demonstrated that it can be used for chimera construction, insertion, and multiple mutations spanning a stretch of DNA up to 120 bp. Conclusion Our FastCloning technique provides a very simple, effective, reliable, and versatile tool for molecular cloning, chimera construction, insertion of any DNA sequences of interest and also for multiple mutations in a short stretch of a cDNA.

  6. Purification and characterization of alkaline-thermostable protease enzyme from Pitaya (Hylocereus polyrhizus) waste: a potential low cost of the enzyme.

    Science.gov (United States)

    Amid, Mehrnoush; Manap, Mohd Yazid A B D; Zohdi, Nor Khanani

    2014-01-01

    The thermoalkaline protease enzyme from pitaya (Hylocereus polyrhizus) waste was purified by a factor of 221.2 with 71.3% recovery using ammonium sulphate precipitation, gel filtration, and cation exchange chromatography. Gel filtration chromatography together with sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) revealed that the enzyme is monomeric with a molecular weight of 26.7 kDa. The apparent K m and V max of the protease were 2.8 mg/mL and 31.20 u/min, respectively. The optimum pH and temperature were 8.0 and 70°C. The enzyme was highly active and stable over a wide pH range (from pH 3.0 to pH 11.0 with the optimum activity at pH 8.0). The protease has broad specificity toward azocasein, casein, hemoglobin, and gelatine. Activity of the enzyme was inhibited by Fe(2+) and Zn(2+), while protease activity was increased in the presence of Ca(2+) and Mg(2+) and Cu(2+) by factors of 125%, 110%, and 105%, respectively. The alkaline protease showed extreme stability toward surfactants and oxidizing agent. The purified protease exhibited extreme stability in the presence of organic solvents and inhibitors. In addition, the enzyme was relativity stable toward organic solvents and chelating agents, such as ethylenediaminetetraacetic acid (EDTA). The enzyme, derived from pitaya peel, possesses unique characteristics and could be used in various industrial and biotechnological applications.

  7. Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

    NARCIS (Netherlands)

    Wang, C.T.; Ji, B.P.; Li, B.; Nout, M.J.R.; Li, P.L.; Ji, H.; Chen, L.F.

    2006-01-01

    Bacillus subtilis DC33 producing a novel fibrinolytic enzyme was isolated from Ba-bao Douchi, a traditional soybean-fermented food in China. The strong fibrin-specific enzyme subtilisin FS33 was purified to electrophoretic homogeneity using the combination of various chromatographic steps. The

  8. Method and system for purification of gas/liquid streams for fuel cells or electrolysis cells

    DEFF Research Database (Denmark)

    2013-01-01

    The present invention provides in embodiments a method for purification of inlet gas/liquid streams in a fuel cell or electrolysis cell, the fuel cell or electrolysis cell comprising at least a first electrode, an electrolyte and a second electrode, the method comprising the steps of: - providing...... at least one scrubber in the gas/liquid stream at the inlet side of the first electrode of the fuel cell or electrolysis cell; and/or providing at least one scrubber in the gas/liquid stream at the inlet side of the second electrode of the fuel cell or electrolysis cell; and - purifying the gas...... with the at least one scrubber, with the proviso that the fuel cell or electrolysis cell is not a solid oxide cell....

  9. Evaluation of immunoglobulin purification methods and their impact on quality and yield of antigen-specific antibodies

    Directory of Open Access Journals (Sweden)

    Khan Farhat

    2008-07-01

    Full Text Available Abstract Background Antibodies are the main effectors against malaria blood-stage parasites. Evaluation of functional activities in immune sera from Phase 2a/b vaccine trials may provide invaluable information in the search for immune correlates of protection. However, the presence of anti-malarial-drugs, improper collection/storage conditions or concomitant immune responses against other pathogens can contribute to non-specific anti-parasite activities when the sera/plasma are tested in vitro. Purification of immunoglobulin is a standard approach for reducing such non-specific background activities, but the purification method itself can alter the quality and yield of recovered Ag-specific antibodies. Methods To address this concern, various immunoglobulin (Ig purification methods (protein G Sepharose, protein A/G Sepharose, polyethylene glycol and caprylic acid-ammonium sulphate precipitation were evaluated for their impact on the quality, quantity and functional activity of purified rabbit and human Igs. The recovered Igs were analysed for yield and purity by SDS-PAGE, for quality by Ag-specific ELISAs (determining changes in titer, avidity and isotype distribution and for functional activity by in vitro parasite growth inhibition assay (GIA. Results This comparison demonstrated that overall polyethylene glycol purification of human serum/plasma samples and protein G Sepharose purification of rabbit sera are optimal for recovering functional Ag-specific antibodies. Conclusion Consequently, critical consideration of the purification method is required to avoid selecting non-representative populations of recovered Ig, which could influence interpretations of vaccine efficacy, or affect the search for immune correlates of protection.

  10. PEG precipitation coupled with chromatography is a new and sufficient method for the purification of botulinum neurotoxin type B [corrected].

    Directory of Open Access Journals (Sweden)

    Yao Zhao

    Full Text Available Clostridium botulinum neurotoxins are used to treat a variety of neuro-muscular disorders, as well as in cosmetology. The increased demand requires efficient methods for the production and purification of these toxins. In this study, a new purification process was developed for purifying type B neurotoxin. The kinetics of C.botulinum strain growth and neurotoxin production were determined for maximum yield of toxin. The neurotoxin was purified by polyethylene glycol (PEG precipitation and chromatography. Based on design of full factorial experiment, 20% (w/v PEG-6000, 4 °C, pH 5.0 and 0.3 M NaCl were optimal conditions to obtain a high recovery rate of 87% for the type B neurotoxin complex, as indicated by a purification factor of 61.5 fold. Furthermore, residual bacterial cells, impurity proteins and some nucleic acids were removed by PEG precipitation. The following purification of neurotoxin was accomplished by two chromatography techniques using Sephacryl™ S-100 and phenyl HP columns. The neurotoxin was recovered with an overall yield of 21.5% and the purification factor increased to 216.7 fold. In addition, a mouse bioassay determined the purified neurotoxin complex possessed a specific toxicity (LD(50 of 4.095 ng/kg.

  11. High-yield expression in Escherichia coli and purification of mouse ubiquitin-activating enzyme E1.

    Science.gov (United States)

    Carvalho, Andreia F; Pinto, Manuel P; Grou, Cláudia P; Vitorino, Rui; Domingues, Pedro; Yamao, Fumiaki; Sá-Miranda, Clara; Azevedo, Jorge E

    2012-07-01

    Research in the ubiquitin field requires large amounts of ubiquitin-activating enzyme (E1) for in vitro ubiquitination assays. Typically, the mammalian enzyme is either isolated from natural sources or produced recombinantly using baculovirus/insect cell protein expression systems. Escherichia coli is seldom used to produce mammalian E1 probably due to the instability and insolubility of this high-molecular mass protein. In this report, we show that 5-10 mg of histidine-tagged mouse E1 can be easily obtained from a 1 l E. coli culture. A low temperature during the protein induction step was found to be critical to obtain an active enzyme.

  12. Purification and characterisation of trypsin-like enzyme from the pyloric caeca of cod (Gadus morhua) II

    OpenAIRE

    Luiz Henrique Beirão; Ian Mckintoch Mackie; Evanilda Teixeira; César Damian

    2001-01-01

    A trypsin -like enzyme from the pyloric caeca of cod (Gadus morhua) was purified by affinity chromatography on CHOM Sepharose 4B. Some characteristics were established by its catalytic activity on T.A.M.E., typical enzyme substrate, and serine protease inhibitors. The enzyme had an isoelectric point of 5.30 and 5.89 and was very similar in amino acid composition to bovine trypsin, but differed in having a higher relative amount of acidic amino acids and a lower amount of basic amino acids. Th...

  13. Enhanced molten salt purification by electrochemical methods: feasibility experiments with flibe

    Energy Technology Data Exchange (ETDEWEB)

    Alan K Wertsching; Brandon S Grover; Pattrick Calderoni

    2010-09-01

    Molten salts are considered within the Very High Temperature Reactor program as heat transfer media because of their intrinsically favorable thermo-physical properties at temperatures starting from 300 C and extending up to 1200 C. In this context two main applications of molten salt are considered, both involving fluoride-based materials: as primary coolants for a heterogeneous fuel reactor core and as secondary heat transport medium to a helium power cycle for electricity generation or other processing plants, such as hydrogen production. The reference design concept here considered is the Advanced High Temperature Reactor (AHTR), which is a large passively safe reactor that uses solid graphite-matrix coated-particle fuel (similar to that used in gas-cooled reactors) and a molten salt primary and secondary coolant with peak temperatures between 700 and 1000 C, depending upon the application. However, the considerations included in this report apply to any high temperature system employing fluoride salts as heat transfer fluid, including intermediate heat exchangers for gas-cooled reactor concepts and homogenous molten salt concepts, and extending also to fast reactors, accelerator-driven systems and fusion energy systems. The most important initial requirement for heat transfer test of molten salt systems is the establishment of reference coolant materials to use in the experiments. An earlier report produced within the same project (INL/EXT-10-18297) highlighted how thermo-physical properties of the materials that directly impact the heat transfer behavior are strongly correlated to the of composition and impurities concentration of the melt. It is therefore essential to establish laboratory techniques that can measure the melt composition, and to develop purification methods that would allow the production of large quantities of coolant with the desired purity. A companion report titled ‘An experimental test plan for the characterization of molten salt thermo

  14. Purification and Characterization of Alkaline-Thermostable Protease Enzyme from Pitaya (Hylocereus polyrhizus Waste: A Potential Low Cost of the Enzyme

    Directory of Open Access Journals (Sweden)

    Mehrnoush Amid

    2014-01-01

    Full Text Available The thermoalkaline protease enzyme from pitaya (Hylocereus polyrhizus waste was purified by a factor of 221.2 with 71.3% recovery using ammonium sulphate precipitation, gel filtration, and cation exchange chromatography. Gel filtration chromatography together with sodium dodecyl sulphate gel electrophoresis (SDS-PAGE revealed that the enzyme is monomeric with a molecular weight of 26.7 kDa. The apparent Km and Vmax of the protease were 2.8 mg/mL and 31.20 u/min, respectively. The optimum pH and temperature were 8.0 and 70°C. The enzyme was highly active and stable over a wide pH range (from pH 3.0 to pH 11.0 with the optimum activity at pH 8.0. The protease has broad specificity toward azocasein, casein, hemoglobin, and gelatine. Activity of the enzyme was inhibited by Fe2+ and Zn2+, while protease activity was increased in the presence of Ca2+ and Mg2+ and Cu2+ by factors of 125%, 110%, and 105%, respectively. The alkaline protease showed extreme stability toward surfactants and oxidizing agent. The purified protease exhibited extreme stability in the presence of organic solvents and inhibitors. In addition, the enzyme was relativity stable toward organic solvents and chelating agents, such as ethylenediaminetetraacetic acid (EDTA. The enzyme, derived from pitaya peel, possesses unique characteristics and could be used in various industrial and biotechnological applications.

  15. NO.sub.x catalyst and method of suppressing sulfate formation in an exhaust purification system

    Science.gov (United States)

    Balmer-Millar, Mari Lou; Park, Paul W.; Panov, Alexander G.

    2007-06-26

    The activity and durability of a zeolite lean-burn NOx catalyst can be increased by loading metal cations on the outer surface of the zeolite. However, the metal loadings can also oxidize sulfur dioxide to cause sulfate formation in the exhaust. The present invention is a method of suppressing sulfate formation in an exhaust purification system including a NO.sub.x catalyst. The NO.sub.x catalyst includes a zeolite loaded with at least one metal. The metal is selected from among an alkali metal, an alkaline earth metal, a lanthanide metal, a noble metal, and a transition metal. In order to suppress sulfate formation, at least a portion of the loaded metal is complexed with at least one of sulfate, phosphate, and carbonate.

  16. Chromatofocusing: a new method for purification of staphylococcal enterotoxins B and C1.

    Science.gov (United States)

    Ende, I A; Terplan, G; Kickhöfen, B; Hammer, D K

    1983-12-01

    A new chromatographic procedure was developed which obtained highly purified preparations of staphylococcal enterotoxins B and C1 in yields of 60% from cultures of Staphylococcus aureus and which is faster than any of the separation methods used previously. The procedure involves chromatography on carboxymethylcellulose, removal of alpha-toxin by adsorption to rabbit erythrocyte membranes, and finally, chromatofocusing as the fundamental new step. Enterotoxins were obtained in highly purified form and behaved in a homogeneous manner as determined by ultracentrifugation and electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, with molecular weights of 34,000 for staphylococcal enterotoxin B and 30,000 for staphylococcal enterotoxin C1. Using chromatofocusing as the final purification step, we isolated three B and six C1 distinct but immunologically identical enterotoxin fractions, which were found to be devoid of any impurities and to possess a marked degree of toxicity in monkeys.

  17. Chromatofocusing: a new method for purification of staphylococcal enterotoxins B and C1.

    Science.gov (United States)

    Ende, I A; Terplan, G; Kickhöfen, B; Hammer, D K

    1983-01-01

    A new chromatographic procedure was developed which obtained highly purified preparations of staphylococcal enterotoxins B and C1 in yields of 60% from cultures of Staphylococcus aureus and which is faster than any of the separation methods used previously. The procedure involves chromatography on carboxymethylcellulose, removal of alpha-toxin by adsorption to rabbit erythrocyte membranes, and finally, chromatofocusing as the fundamental new step. Enterotoxins were obtained in highly purified form and behaved in a homogeneous manner as determined by ultracentrifugation and electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, with molecular weights of 34,000 for staphylococcal enterotoxin B and 30,000 for staphylococcal enterotoxin C1. Using chromatofocusing as the final purification step, we isolated three B and six C1 distinct but immunologically identical enterotoxin fractions, which were found to be devoid of any impurities and to possess a marked degree of toxicity in monkeys. Images PMID:6660872

  18. Purification of a derepressible arylsulfatase from Chlamydomonas reinhardti. Properties of the enzyme in intact cells and in purified state.

    Science.gov (United States)

    Lien, T; Schreiner, O; Steine, M

    1975-03-28

    Arylsulfatase (aryl-sulfate sulfohdydrolase, EC 3.1.6.1) has been purified from SO4-2-minus-starved cells of Chlamydomonas reinhardti. The enzyme was isolated from acetone-powder extract by (NH4)2SO4 precipitation, Sephadex G-200 filtration and ion-exchange chromatography. Only one fraction of aryl-sulfatase was found. The final preparation was homogenous by the criteria of sedimentation, diffusion and polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of about 150 000, estimated by ultracentrifugation and gel filtration, and an isoelectric point of 9.0. The properties of the enzyme as investigated in intact cells and in the purified state were found to be very similar except for the temperature optimum. Imidazole strongly increased the enzyme by increasing the V, but reduced the affinity for the substrate. The enzyme activity was competitively inhibited by borate with a greater affinity for borate than for the substrate. The Chlamydomonas enzyme is a Type I arylsulfatase since it was inhibited by CN-minus, but not SO4-2-minus and phosphate.

  19. Identification of protein partners in mycobacteria using a single-step affinity purification method.

    Directory of Open Access Journals (Sweden)

    Przemysław Płociński

    Full Text Available Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis, is thus vital. We have implemented improved screening methods for protein-protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium- and high-throughput studies aiming to characterize protein-protein interaction networks of tubercle bacilli. Of the 4 tested epitopes FLAG, enhanced green fluorescent protein (eGFP, protein A and haemagglutinin, the eGFP tag was found to be most useful on account of its easily monitored expression and its ability to function as a simultaneous tool for subcellular localization studies. It presents a relatively low background with cost-effective purification. RNA polymerase subunit A (RpoA was used as a model for investigation of a large protein complex. When used as bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH, making it applicable for semi-quantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells treated with UV light were used to co-purify UvrA interaction partners.

  20. Identification of protein partners in mycobacteria using a single-step affinity purification method.

    Science.gov (United States)

    Płociński, Przemysław; Laubitz, Daniel; Cysewski, Dominik; Stoduś, Krystian; Kowalska, Katarzyna; Dziembowski, Andrzej

    2014-01-01

    Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein-protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium- and high-throughput studies aiming to characterize protein-protein interaction networks of tubercle bacilli. Of the 4 tested epitopes FLAG, enhanced green fluorescent protein (eGFP), protein A and haemagglutinin, the eGFP tag was found to be most useful on account of its easily monitored expression and its ability to function as a simultaneous tool for subcellular localization studies. It presents a relatively low background with cost-effective purification. RNA polymerase subunit A (RpoA) was used as a model for investigation of a large protein complex. When used as bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH), making it applicable for semi-quantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells treated with UV light were used to co-purify UvrA interaction partners.

  1. Purification and Characterization of Tryptophan Hydroxylase

    DEFF Research Database (Denmark)

    Haahr, Lærke Tvedebrink

    This thesis deals with the purification and characterization of the iron-containing enzyme tryptophan hydroxylase (TPH). TPH exists in two isoforms, called TPH1 and TPH2. Each isoform consists of threestructural distinct domains: the regulatory, the catalytic and the tetramerization domain. TPH...... of this project was to developpurification methods for full-length TPH1 and TPH2 as well as to characterize purified TPH variants. A successful purification method for full-length human TPH1 (hTPH1) was developed, which resulted in pure, active and stable protein. The method includes affinity-purification using....... The crystallization procedure for the catalytic domain of gallus gallus TPH1 (cgTPH1) was optimized to faster crystal growth by addition of tryptophan and incubation at room temperature. Crystals without imidazole in the crystallization conditions could be obtained. The solved structures were however of poor quality...

  2. Purification and Characterization of Jararassin-I,A Thrombin-like Enzyme from Bothrops jararaca Snake Venom

    Institute of Scientific and Technical Information of China (English)

    Débora F. VIEIRA; Leandra WATANABE; Carolina D. SANT'ANA; Silvana MARCUSSI; Suely V. SAMPAIO; Andreimar M. SOARES; Raghuvir K. ARNI

    2004-01-01

    A thrombin-like serine protease, jararassin-I, was isolated from the venom of Bothrops jararaca. The protein was obtained in high yield and purity by a single chromatographic step using the affinity resin Benzamidine-Sepharose CL-6B. SDS-PAGE and dynamic light scattering analyses indicated that the molecular mass of the enzyme was about 30 kD. The enzyme possessed fibrinogenolytic and coagulant activities. The jararassin-I degraded the Bβ chain of fibrinogen while the Aα chain and γ chain were unchanged.Proteases inhibitors, PMSF and benzamidine inhibited the coagulant activity. These results showed jararassinI is a serine protease similar to coagulating thrombin-like snake venom proteases, but it specifically cleaves Bβ chain of bovine fibrinogen. Single crystals of enzyme were obtained (0.2 mm×0.2 mm×0.2 mm) and used for X-ray diffraction experiments.

  3. Purification and Characterization of a Dimethoate-Degrading Enzyme of Aspergillus niger ZHY256, Isolated from Sewage

    Science.gov (United States)

    Liu, Yu-Huan; Chung, Ying-Cheng; Xiong, Ya

    2001-01-01

    A dimethoate-degrading enzyme from Aspergillus niger ZHY256 was purified to homogeneity with a specific activity of 227.6 U/mg of protein. The molecular mass of the purified enzyme was estimated to be 66 kDa by gel filtration and 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.4, and the enzyme activity was optimal at 50°C and pH 7.0. The activity was inhibited by most of the metal ions and reagents, while it was induced by Cu2+. The Michaelis constant (Km) and Vmax for dimethoate were 1.25 mM and 292 μmol min−1 mg of protein−1, respectively. PMID:11472959

  4. Extracellular cellulolytic enzyme system of Aspergillus japonicus: Pt. 2. Purification and characterization of an inducible extracellular. beta. -glucosidase

    Energy Technology Data Exchange (ETDEWEB)

    Sanyal, Arunik; Kundu, R.K.; Dube, S.; Dube, D.K.

    1988-02-01

    A high molecular weight ..beta..-glucosidase (mol. wt. > 240 000 daltons) was isolated from the culture filtrate of Aspergillus japonicus and was finally purified to 86-fold by alcohol precipitation, gel filtration and ion exchange chromatography on Whatman DE-52. An apparently homogeneous form of the enzyme appeared in the polyacrylamide gel electrophoresis. It is capable of utilizing cellobiose, salicin, o-nitrophenyl-..beta..-D-glucoside (ONPG), methyl-..beta..-D-glucoside and amygdalin effectively as substrates but not arbutin, esculin hydrate and phloridzin. No metal ion is required for its catalytic activity. Hg/sup ++/ and p-chloromercuricbenzoate (PCMB) are strong inhibitors for the enzyme. Nojirimycin and glucono-delta-lactone are two competitive inhibitors of the same enzyme, and nojirimycin is the more potent of the two.

  5. Serine transhydroxymethylase: a simplified radioactive assay; purification and stabilization of enzyme activity employing Affi-Gel Blue.

    Science.gov (United States)

    Braman, J C; Black, M J; Mangum, J H

    1981-01-01

    An improved radioactive assay has been developed for serine transhydroxymethylase. This assay involves the direct measurement of the [14C]HCHO which is generated when [3- 14C]-serine is employed as the substrate. The new assay eliminates the need for a solvent extraction of a [14C]HCHO-dimedon adduct which is the basis of the assay devised by Taylor and Weissbach. The enzyme has been purified employing Affi-Gel Blue. The purified enzyme retains full activity when bound to this affinity chromatography matrix and can be stored in this state at 4 degrees indefinitely.

  6. Comparison of Influenza Virus Particle Purification Using Magnetic Sulfated Cellulose Particles with an Established Centrifugation Method for Analytics.

    Science.gov (United States)

    Serve, Anja; Pieler, Michael Martin; Benndorf, Dirk; Rapp, Erdmann; Wolff, Michael Werner; Reichl, Udo

    2015-11-03

    A method for the purification of influenza virus particles using novel magnetic sulfated cellulose particles is presented and compared to an established centrifugation method for analytics. Therefore, purified influenza A virus particles from adherent and suspension MDCK host cell lines were characterized on the protein level with mass spectrometry to compare the viral and residual host cell proteins. Both methods allowed one to identify all 10 influenza A virus proteins, including low-abundance proteins like the matrix protein 2 and nonstructural protein 1, with a similar impurity level of host cell proteins. Compared to the centrifugation method, use of the novel magnetic sulfated cellulose particles reduced the influenza A virus particle purification time from 3.5 h to 30 min before mass spectrometry analysis.

  7. Assay Methods for H2S Biogenesis and Catabolism Enzymes

    Science.gov (United States)

    Banerjee, Ruma; Chiku, Taurai; Kabil, Omer; Libiad, Marouane; Motl, Nicole; Yadav, Pramod K.

    2015-01-01

    H2S is produced from sulfur-containing amino acids, cysteine and homocysteine, or a catabolite, 3-mercaptopyruvate, by three known enzymes: cystathionine β-synthase, γ-cystathionase, and 3-mercaptopyruvate sulfurtransferase. Of these, the first two enzymes reside in the cytoplasm and comprise the transsulfuration pathway, while the third enzyme is found both in the cytoplasm and in the mitochondrion. The following mitochondrial enzymes oxidize H2S: sulfide quinone oxidoreductase, sulfur dioxygenase, rhodanese, and sulfite oxidase. The products of the sulfide oxidation pathway are thiosulfate and sulfate. Assays for enzymes involved in the production and oxidative clearance of sulfide to thiosulfate are described in this chapter. PMID:25725523

  8. A purification method for a molecular complex in which a scaffold molecule is fully loaded with heterogeneous molecules.

    Directory of Open Access Journals (Sweden)

    Shoji J Ohuchi

    Full Text Available An affinity resin-based pull-down method is convenient for the purification of biochemical materials. However, its use is difficult for the isolation of a molecular complex fully loaded with multiple components from a reaction mixture containing the starting materials and intermediate products. To overcome this problem, we have developed a new purification procedure that depends on sequential elimination of the residues. In practice, two affinity resins were used for purifying a triangular-shaped RNP (RNA-protein complex consisting of three ribosomal proteins (L7Ae bound to an RNA scaffold. First, a resin with immobilized L7Ae protein captured the incomplete RNP complexes and the free RNA scaffold. Next, another resin with an immobilized chemically modified RNA of a derivative of Box C/D motif, the binding partner of L7Ae, was used to capture free protein. The complete triangular RNP was successfully purified from the mixture by these two steps. Obviously, the purified triangular RNP displaying three protein-binding peptides exhibited an improved performance when compared with the unrefined product. Conceptually, this purification procedure should be applicable for the purification of a variety of complexes consisting of multiple components other than RNP.

  9. Purification and characterization of a collagenolytic enzyme produced by Rathayibacter sp. strains isolated from cultures of Clavibacter michiganensis subsp. michiganensis.

    Science.gov (United States)

    Labadie, J; Hébraud, M

    1997-02-01

    We show in this work that collagenolytic Rathayibacter sp. are isolated with phytopathogenic Clavibacter michiganensis subsp. michiganensis strains. The Rathayibacter strains isolated all produced collagenases. One of these collagenases (from the strain 1715) was purified by ammonium sulphate precipitation, DEAE cellulose and Sephadex G 200 chromatography. Characterization of the enzyme showed that it is a true collagenase which is able to degrade both native collagen, gelatin and probably other proteins from plants sharing sequence homologies with collagen.

  10. Purification and characterization of 3-dehydroshikimate dehydratase, an enzyme in the inducible quinic acid catabolic pathway of Neurospora crassa.

    Science.gov (United States)

    Strøman, P; Reinert, W R; Giles, N H

    1978-07-10

    3-Dehydroshikimate dehydratase catalyzes the third reaction in the inducible quinic acid catabolic pathway of Neurospora crassa and is encoded in the qa-4 gene of the qa gene cluster. As part of continuing genetic and biochemical studies concerning the organization and regulation of this gene cluster, 3-dehydroshikimate dehydratase has been purified and characterized biochemically. The enzyme was purified 1650-fold using the following techniques: 1) (NH4)2SO4 fractionation; 2) ion exchange chromatography on DEAE-cellulose; 3) gel filtration on Sephadex G-100; 4) ion exchange chromatography on Cellex QAE (quaternary aminoethyl); and 5) hydroxylapatite chromatography. 3-Dehydroshikimate dehydratase is a monomer with a molecular weight of about 37,000 and a sedimentation coefficient of 3.27 S. It has a Km value of 5.9 X 10(-4) and an average isoelectric point of 4.92. The purified enzyme is extremely sensitive to thermal denaturation but can be significantly stabilized by Mg2+ ions. The purified enzyme also exhibits maximal catalytic activity only when assayed in the presence of certain divalent cations, e.g. magnesium. The NH2-terminal residue of 3-dehydroshikimate dehydratase is proline, and its alpha-amino group is unblocked.

  11. Purification of Single-walled Carbon Nanotubes Grown by a Chemical Vapour Deposition (CVD) Method

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A procedure for purification of single-walled carbon nanotubes(SWNTs) grown by the chemical vapour deposition (CVD) of carbon monooxide has been developed. Based on the result from TGA/DTA of as-prepared sample, the oxidation temperature was determined. The process included sonication, oxidation and acid washing steps. The purity and yield after purification were determined and estimated by TEM. Moreover, for the first time, a loop structure for CVD SWNTs has been observed.

  12. Method and system for purification of gas/liquid streams for fuel cells or electrolysis cells

    DEFF Research Database (Denmark)

    2013-01-01

    The present invention provides in embodiments a method for purification of inlet gas/liquid streams in a fuel cell or electrolysis cell, the fuel cell or electrolysis cell comprising at least a first electrode, an electrolyte and a second electrode, the method comprising the steps of: - providing...... at least one scrubber in the gas/liquid stream at the inlet side of the first electrode of the fuel cell or electrolysis cell; and/or providing at least one scrubber in the gas/liquid stream at the inlet side of the second electrode of the fuel cell or electrolysis cell; and - purifying the gas/liquid...... streams towards the first and second electrode; wherein the at least one scrubber in the gas/liquid stream at the inlet side of the first electrode and/or the at least one scrubber in the gas/liquid stream at the inlet side of the second electrode comprises a material suitable as an electrolyte material...

  13. High oleic sunflower bio diesel: quality control and different purification methods

    Energy Technology Data Exchange (ETDEWEB)

    Pighlinelli, A. L. M. T.; Ferrari, R. A.; Miguel, A. M. R. O.; Park, K. J.

    2011-07-01

    The objective of the present work is to evaluate the production of bio diesel using ethanol and sunflower oil. The extraction of the sunflower oil was evaluated first. An experimental design was used to estimate the influence of the independent variables grain temperature (25 degree centigrade to 110 degree centigrade) and expelled rotation (85 to 119rpm) on the crude oil. The best result obtained was 68.38%, achieved with a rotation from 100 to 115rpm, grain temperature ranging from 25 degree centigrade to 30 degree centigrade and moisture content of around 7%. The next study consisted of transesterification, evaluating the influence of the ethanol, oil molar ratio and the catalyst concentration (sodium methylate) on the ester-rich phase yield. The highest yield was 98.39% obtained with a molar ratio of 9:1 and 3% catalyst. An experiment was then carried out on a small reactor and the bio diesel produced was purified by three different methods: acidified water, silica and distillation. The quality aspects of the purified bio diesel samples were evaluated according to the Brazilian specifications for bio diesel, and distillation was shown to be the best method of purification. (Author) 28 refs.

  14. 4-Dihydromethyltrisporate dehydrogenase from Mucor mucedo, an enzyme of the sexual hormone pathway: purification, and cloning of the corresponding gene.

    Science.gov (United States)

    Czempinski, K; Kruft, V; Wöstemeyer, J; Burmester, A

    1996-09-01

    We have purified the NADP-dependent 4-dihydromethyltrisporate dehydrogenase from the zygomycete Mucor mucedo. The enzyme is involved in the biosynthesis of trisporic acid, the sexual hormone of zygomycetes, which induces the first steps of zygophore development. Protein was obtained from the (-) mating type of M. mucedo after induction with trisporic acid, and purified by gel filtration and affinity chromatography steps. On SDS-PAGE a band with an apparent molecular mass of 33 kDa was ascribed to the enzyme. After transferring onto PVDF membranes the protein was digested with endoprotease Lys-C, and several peptides were sequenced. Oligonucleotides derived from protein sequence data were used for PCR amplification of genomic M. mucedo DNA. The PCR fragment was used as probe for isolation of the corresponding cDNA and complete genomic DNA clones. Comparison of protein and DNA sequence data showed that the cloned fragment corresponded to the purified protein. Search for similarity with protein sequences of the Swiss-Prot database revealed a relationship to enzymes belonging to the aldo/keto reductase superfamily. Southern-blot analysis of genomic DNA with the labelled cloned fragment detected a single-copy gene in both mating types of M. mucedo. PCR with genomic DNA from other zygomycetes gave rise to several fragments. Hybridization analysis with the cloned M. mucedo fragment showed that a fragment of similar length cross-hybridized in Blakeslea trispora (Choanephoraceae) as well as in Parasitella parasitica and Absidia glauca (Mucoraceae). The promoter region of the gene contains DNA elements with similarity to a cAMP-regulated gene of Dictyostelium discoideum.

  15. Insights into the Hypertensive Effects of Tityus serrulatus Scorpion Venom: Purification of an Angiotensin-Converting Enzyme-Like Peptidase

    Directory of Open Access Journals (Sweden)

    Daniela Cajado-Carvalho

    2016-11-01

    Full Text Available The number of cases of envenomation by scorpions has grown significantly in Brazil since 2007, with the most severe cases being caused by the Tityus serrulatus scorpion. Although envenomed patients mostly suffer neurotoxic manifestations, other symptoms, such as hypertension, cannot be exclusively attributed to neurotoxins. Omics analyses have detected plentiful amounts of metalloproteases in T. serrulatus venom. However, the roles played by these enzymes in envenomation are still unclear. Endeavoring to investigate the functions of scorpion venom proteases, we describe here for the first time an Angiotensin I-Converting Enzyme-like peptidase (ACE-like purified from T. serrulatus venom. The crude venom cleaved natural and fluorescent substrates and these activities were inhibited by captopril. Regarding the serum neutralization, the scorpion antivenom was more effective at blocking the ACE-like activity than arachnid antivenom, although neither completely inhibited the venom cleavage action, even at higher doses. ACE-like was purified from the venom after three chromatographic steps and its identity was confirmed by mass spectrometric and transcriptomic analyses. Bioinformatics analysis showed homology between the ACE-like transcript sequences from Tityus spp. and human testis ACE. These findings advance our understanding of T. serrulatus venom components and may improve treatment of envenomation victims, as ACE-like may contribute to envenomation symptoms, especially the resulting hypertension.

  16. Insights into the Hypertensive Effects of Tityus serrulatus Scorpion Venom: Purification of an Angiotensin-Converting Enzyme-Like Peptidase

    Science.gov (United States)

    Cajado-Carvalho, Daniela; Kuniyoshi, Alexandre Kazuo; Duzzi, Bruno; Iwai, Leo Kei; de Oliveira, Úrsula Castro; Junqueira de Azevedo, Inácio de Loiola Meirelles; Kodama, Roberto Tadashi; Portaro, Fernanda Vieira

    2016-01-01

    The number of cases of envenomation by scorpions has grown significantly in Brazil since 2007, with the most severe cases being caused by the Tityus serrulatus scorpion. Although envenomed patients mostly suffer neurotoxic manifestations, other symptoms, such as hypertension, cannot be exclusively attributed to neurotoxins. Omics analyses have detected plentiful amounts of metalloproteases in T. serrulatus venom. However, the roles played by these enzymes in envenomation are still unclear. Endeavoring to investigate the functions of scorpion venom proteases, we describe here for the first time an Angiotensin I-Converting Enzyme-like peptidase (ACE-like) purified from T. serrulatus venom. The crude venom cleaved natural and fluorescent substrates and these activities were inhibited by captopril. Regarding the serum neutralization, the scorpion antivenom was more effective at blocking the ACE-like activity than arachnid antivenom, although neither completely inhibited the venom cleavage action, even at higher doses. ACE-like was purified from the venom after three chromatographic steps and its identity was confirmed by mass spectrometric and transcriptomic analyses. Bioinformatics analysis showed homology between the ACE-like transcript sequences from Tityus spp. and human testis ACE. These findings advance our understanding of T. serrulatus venom components and may improve treatment of envenomation victims, as ACE-like may contribute to envenomation symptoms, especially the resulting hypertension. PMID:27886129

  17. Complex Enzyme-Assisted Extraction, Purification, and Antioxidant Activity of Polysaccharides from the Button Mushroom, Agaricus bisporus (Higher Basidiomycetes).

    Science.gov (United States)

    Yin, Xiulian; You, Qinghong; Zhou, Xinghai

    2015-01-01

    Agaricus bisporus polysaccharides (ABP) were extracted by complex enzyme-assisted extraction methodology. The following were optimal conditions for the extraction of crude ABP: complex enzyme amount, 2.2%; temperature, 62°C; time, 3 h; and pH, 4. Under these conditions, the experimental yield of crude ABP was 6.87%. The crude ABP was purified by diethylaminoethyl-cellulose 52 chromatography and Sephadex G-100 chromatography, and one fraction-namely, ABP-1-was produced. The ABP-1 contained 93.67% carbohydrate, 1.46% protein, and 0.62% uronic acid. The constituent monosaccharides were predominantly glucose, galactose, mannose, and xylose. The antioxidant activities of ABP-1 were investigated by measuring its scavenging ability on 2,2-diphenyl-1-picrylhydrazyl and hydroxyl radicals, its ferric-reducing activity power, and the reducing power assay. At a concentration of 1.2 mg/mL, ABP-1 seemed to possess good free radical scavenging activity, with a scavenging value of about 56%. The results indicate that ABP-1 has good antioxidant activity.

  18. Methods for isolation, purification and structural elucidation of bioactive secondary metabolites from marine invertebrates.

    Science.gov (United States)

    Ebada, Sherif S; Edrada, Ru Angelie; Lin, Wenhan; Proksch, Peter

    2008-01-01

    In the past few decades, marine natural products bioprospecting has yielded a considerable number of drug candidates. Two marine natural products have recently been admitted as new drugs: Prialt (also known as ziconotide) as a potent analgesic for severe chronic pain and Yondelis (known also as trabectedin or E-743) as antitumor agent for the treatment of advanced soft tissue sarcoma. In this protocol, methods for bioactivity-guided isolation, purification and identification of secondary metabolites from marine invertebrates such as sponges, tunicates, soft corals and crinoids are discussed. To achieve this goal, solvent extraction of usually freeze-dried sample of marine organisms is performed. Next, the extract obtained is fractionated by liquid-liquid partitioning followed by various chromatographic separation techniques including thin layer chromatography, vacuum liquid chromatography, column chromatography (CC) and preparative high-performance reversed-phase liquid chromatography. Isolation of bioactive secondary metabolites is usually monitored by bioactivity assays, e.g., antioxidant (2,2-diphenyl-1-picryl hydrazyl) and cytotoxicity (microculture tetrazolium) activities that ultimately yield the active principles. Special care should be taken when performing isolation procedures adapted to the physical and chemical characteristics of the compounds isolated, particularly their lipo- or hydrophilic characters. Examples of isolation of compounds of different polarities from extracts of various marine invertebrates will be presented in this protocol. Structure elucidation is achieved using recent spectroscopic techniques, especially 2D NMR and mass spectrometry analysis.

  19. Preparation of recombinant firefly luciferase by a simple and rapid expression and purification method and its application in bacterial detection

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A simple and rapid expression and purification method of recombinant firefly luciferase was developed for bacteria detection. A modified luciferase gene from North American firefly Photinus pyralis was cloned into pET28a expression vector and the recombinant protein was produced in Escherichia coli BL21. The recombinant luciferase,equipped with a polyhistidine affinity tag,was purified by immobilized metal ion affinity chromatography (IMAC). The approach generated an abundant expression and an efficient pur...

  20. Proteinases from buckwheat (Fagopyrum esculentum moench seeds: Purification and properties of the 47 kDa enzyme

    Directory of Open Access Journals (Sweden)

    Timotijević Gordana S.

    2006-01-01

    Full Text Available Aspartic proteinases from buckwheat seeds are analyzed. Three forms of 47 kDa, 40 kDa and 28 kDa, were purified from mature buckwheat seeds, while two forms of 47 kDa and 28 kDa were detected in developing buckwheat seeds using pepstatin A affinity chromatography. A form of 47 kDa was selectively precipitated from other forms by ammonium sulfate precipitation. This enzyme resembles the chymosin-like pattern of proteolytic activity, as it was shown using BSA and k-casein as substrates, clarifying its ability for milk-clotting. The 47 kDa aspartic proteinase form is localized in the membrane fraction. .

  1. Arabinoxylan-degrading enzyme system of the fungus Aspergillus awamori: purification and properties of an alpha-L-arabinofuranosidase.

    Science.gov (United States)

    Wood, T M; McCrae, S I

    1996-05-01

    An alpha-L-arabinofuranosidase produced by the fungus Aspergillus awamori had a molecular mass of approximately 64 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) and was optimally active at pH 4.6 and 50 degrees C. The enzyme, which chromatographed as a single component on SDS-PAGE, appeared to consist of two isoenzymes of pI 3.6 and 3.2. Acting in isolation, the alpha-L-arabinofuranosidase had only a very limited capacity to release L-arabinose (less than 11%) directly from arabinoxylans that had been extracted from a number of plant cell wall preparations using 18% alkali, but a much higher proportion of the L-arabinose (46%) was released from a wheat straw arabinoxylan that had been isolated by steam treatment. There was a marked synergistic effect between the alpha-L-arabinofuranosidase and an endo-(1 --> 4)-beta-D-xylanase produced by A. awamori in both the rate and extent of the release of L-arabinose from both oat straw and wheat straw arabinoxylans, suggesting that L-arabinose-substituted oligosaccharides generated by the endoxylanase action were better substrates for enzyme action. A novel property of the alpha-L-arabinofuranosidase was its capacity to release a substantial proportion (42%) of feruloyl L-arabinose from intact wheat straw arabinoxylan. The concerted action of the alpha-L-arabinofuranosidase and endoxylanase released 71% of the feruloyl L-arabinose and 69% of the p-coumaroyl L-arabinose substituents from wheat straw arabinoxylan.

  2. Purification and biochemical characterization of a novel fibrinolytic enzyme, PSLTro01, from a medicinal animal Porcellio scaber Latreille.

    Science.gov (United States)

    Tian, Zhou; Li, Bo; Guo, Liwei; Wu, Mianhua; Fu, Tingming; Cheng, Haibo; Zhu, Huaxu

    2015-09-01

    A novel protease, named PSLTro01, with fibrinolytic and anticoagulant activity was isolated from Porcellio scaber Latreille and was purified by a combination of hollow fibre membrane molecular weight cut-off (MWCO), ammonium sulfate fractionation, gel filtration and ion-exchange chromatography. PSLTro01 is a single-chain protein with a molecular mass of 38,497 Da as estimated by non-reduced SDS-PAGE and MALDI-TOF MS spectrometry, and its N-terminal 15 amino acid sequence was determined as DINGGGATLPQPLYQ. PSLTro01 is stable in the range of 20-40 °C and pH 6.0-10.0, with a maximum fibrinolytic activity at 40 °C and pH 7.0. The PSLTro01-induced fibrinolytic activity was not influenced by K(+) or Na(+) but was slightly increased by Mg(2+) and completely inhibited by aprotinin and pepstatin A. Fibrin plate assays revealed that PSLTro01 could not directly degrade fibrin but was a plasminogen activator. PSLTro01 exhibited high specificity for the substrate S-2251 for plasmin, followed by S-2238 for thrombin and S-2444 for urokinase. Moreover, the fibrinogenolysis pattern of PSLTro01 was Aα-chains>Bβ-chains>γ-chain. Tail-thrombus of the enzyme treated group was significantly shorter than the physiological saline treated group and the thrombus decrement was correlated with the enzyme dose. PSLTro01 prolongs both thrombin time (TT) and activated partial thromboplastin time (APTT). These results indicate that PSLTro01 may have potential applications in the prevention and treatment of thrombosis.

  3. Heterodimeric l-amino acid oxidase enzymes from Egyptian Cerastes cerastes venom: Purification, biochemical characterization and partial amino acid sequencing

    Directory of Open Access Journals (Sweden)

    A.E. El Hakim

    2015-12-01

    Full Text Available Two l-amino acid oxidase enzyme isoforms, Cc-LAAOI and Cc-LAAOII were purified to apparent homogeneity from Cerastes cerastes venom in a sequential two-step chromatographic protocol including; gel filtration and anion exchange chromatography. The native molecular weights of the isoforms were 115 kDa as determined by gel filtration on calibrated Sephacryl S-200 column, while the monomeric molecular weights of the enzymes were, 60, 56 kDa and 60, 53 kDa for LAAOI and LAAOII, respectively. The tryptic peptides of the two isoforms share high sequence homology with other snake venom l-amino acid oxidases. The optimal pH and temperature values of Cc-LAAOI and Cc-LAAOII were 7.8, 50 °C and 7, 60 °C, respectively. The two isoenzymes were thermally stable up to 70 °C. The Km and Vmax values were 0.67 mM, 0.135 μmol/min for LAAOI and 0.82 mM, 0.087 μmol/min for LAAOII. Both isoenzymes displayed high catalytic preference to long-chain, hydrophobic and aromatic amino acids. The Mn2+ ion markedly increased the LAAO activity for both purified isoforms, while Na+, K+, Ca2+, Mg2+ and Ba2+ ions showed a non-significant increase in the enzymatic activity of both isoforms. Furthermore, Zn2+, Ni2+, Co2+, Cu2+ and AL3+ ions markedly inhibited the LAAOI and LAAOII activities. l-Cysteine and reduced glutathione completely inhibited the LAAO activity of both isoenzymes, whereas, β-mercaptoethanol, O-phenanthroline and PMSF completely inhibited the enzymatic activity of LAAOII. Furthermore, iodoacitic acid inhibited the enzymatic activity of LAAOII by 46% and had no effect on the LAAOI activity.

  4. Nanolipoprotein particles comprising a natural rubber biosynthetic enzyme complex and related products, methods and systems

    Energy Technology Data Exchange (ETDEWEB)

    Hoeprich, Paul D.; Whalen, Maureen

    2016-04-05

    Provided herein are nanolipoprotein particles that comprise a biosynthetic enzyme more particularly an enzyme capable of catalyzing rubber or other rubbers polymerization, and related assemblies, devices, methods and systems.

  5. Two-step Purification Method for Aging Pigments A2E and Iso-A2E Using Medium Pressure Liquid Chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Park, Sang Il; Park, Sang Cheol [Kyunghee Univ., Seoul (Korea, Republic of); Kim, So Ra; Jang, Young Pyo [Seoul National Univ., Seoul (Korea, Republic of)

    2016-09-15

    A newly modified method for the efficient purification of A2E and iso-A2E using reverse phase silica gel resin with medium pressure liquid chromatography (MPLC) was suggested. MPLC is one of the various preparative column chromatography techniques and separation under pressure renders the use of smaller particle size resins possible and increases the diversity of available stationary phases. A simplified two-step purification method was developed for the purification of aging pigments in eye, A2E and iso-A2E. With simple two-step elution of mobile phase in reverse phase MPLC, A2E and iso-A2E were successfully purified with great efficiency compare with previous column chromatography and HPLC method. This method provides more simple, convenient, cost-effective, and less time-consuming procedure for mass purification of aging pigments A2E and iso-A2E.

  6. Development of an aptamer-based affinity purification method for vascular endothelial growth factor

    Directory of Open Access Journals (Sweden)

    Maren Lönne

    2015-12-01

    Full Text Available Since aptamers bind their targets with high affinity and specificity, they are promising alternative ligands in protein affinity purification. As aptamers are chemically synthesized oligonucleotides, they can be easily produced in large quantities regarding GMP conditions allowing their application in protein production for therapeutic purposes. Several advantages of aptamers compared to antibodies are described in general within this paper. Here, an aptamer directed against the human Vascular Endothelial Growth Factor (VEGF was used as affinity ligand for establishing a purification platform for VEGF in small scale. The aptamer was covalently immobilized on magnetic beads in a controlled orientation resulting in a functional active affinity matrix. Target binding was optimized by introduction of spacer molecules and variation of aptamer density. Further, salt-induced target elution was demonstrated as well as VEGF purification from a complex protein mixture proving the specificity of protein-aptamer binding.

  7. Produção, purificação, clonagem e aplicação de enzimas líticas Production, purification, cloning and application of lytic enzymes

    Directory of Open Access Journals (Sweden)

    Luciana Francisco Fleuri

    2005-10-01

    Full Text Available Lytic enzymes such as beta-1,3 glucanases, proteases and chitinases are able to hydrolyse, respectively, beta-1,3 glucans, mannoproteins and chitin, as well as the cell walls of many yeast species. Lytic enzymes are useful in a great variety of applications including the preparation of protoplasts; the extraction of proteins, enzymes, pigments and functional carbohydrates; pre-treatment for the mechanical rupture of cells; degradation of residual yeast cell mass for the preparation of animal feed; analysis of the yeast cell wall structure and composition; study of the yeast cell wall synthesis and the control of pathogenic fungi. This review presents the most important aspects with respect to lytic enzymes, especially their production, purification, cloning and application.

  8. THERMOSTABLE ALGINATE DEGRADING ENZYMES AND THEIR METHODS OF USE

    NARCIS (Netherlands)

    Hreggvidsson, Gudmundur Oli; Jonsson, Oskar W.J.; Bjornsdottir, Bryndis; Fridjonsson, Hedinn O; Altenbuchner, Josef; Watzlawick, Hildegard; Dobruchowska, Justyna; Kamerling, Johannis

    2015-01-01

    The present invention relates to the identification, production and use of thermostable alginate lyase enzymes that can be used to partially degrade alginate to yield oligosaccharides or to give complete degradation of alginate to yield (unsaturated) mono-uronates.

  9. A User-Friendly Method for Teaching Restriction Enzyme Mapping.

    Science.gov (United States)

    Ehrman, Patrick

    1990-01-01

    Presented is a teaching progression that enhances learning through low-cost, manipulative transparencies. Discussed is instruction about restriction enzymes, plasmids, cutting plasmids, plasmid maps, recording data, and mapping restriction sites. Mapping wheels for student use is included. (CW)

  10. Thermostable Alginate degrading enzymes and their methods of use

    NARCIS (Netherlands)

    Hreggvidsson, Gudmundur Oli; Jonsson, Oskar W.J.; Bjornsdottir, Bryndis; Fridjonsson, Hedinn O; Altenbuchner, Josef; Watzlawick, Hildegard; Dobruchowska, Justyna; Kamerling, Johannis

    2015-01-01

    The present invention relates to the identification, production and use of thermostable alginate lyase enzymes that can be used to partially degrade alginate to yield oligosaccharides or to give complete degradation of alginate to yield (unsaturated) mono-uronates.

  11. Biochemistry in an Industrial Context: Methods of Protein Purification and Downstream Processing.

    Science.gov (United States)

    Weathers, Pamela J.

    1988-01-01

    Explores a graduate level bioprocess engineering course in protein purification and downstream processing. Designed to provide students with hands-on training in the design and implementation of product processing for the biotechnology industry. Includes syllabus and plan of study. (MVL)

  12. A Robust and Fully-Automated Chromatographic Method for the Quantitative Purification of Ca and Sr for Isotopic Analysis

    Science.gov (United States)

    Smith, H. B.; Kim, H.; Romaniello, S. J.; Field, P.; Anbar, A. D.

    2014-12-01

    High throughput methods for sample purification are required to effectively exploit new opportunities in the study of non-traditional stable isotopes. Many geochemical isotopic studies would benefit from larger data sets, but these are often impractical with manual drip chromatography techniques, which can be time-consuming and demand the attention of skilled laboratory staff. Here we present a new, fully-automated single-column method suitable for the purification of both Ca and Sr for stable and radiogenic isotopic analysis. The method can accommodate a wide variety of sample types, including carbonates, bones, and teeth; silicate rocks and sediments; fresh and marine waters; and biological samples such as blood and urine. Protocols for these isotopic analyses are being developed for use on the new prepFAST-MCTM system from Elemental Scientific (ESI). The system is highly adaptable and processes up to 24-60 samples per day by reusing a single chromatographic column. Efficient column cleaning between samples and an all Teflon flow path ensures that sample carryover is maintained at the level of background laboratory blanks typical for manual drip chromatography. This method is part of a family of new fully-automated chromatographic methods being developed to address many different isotopic systems including B, Ca, Fe, Cu, Zn, Sr, Cd, Pb, and U. These methods are designed to be rugged and transferrable, and to allow the preparation of large, diverse sample sets via a highly repeatable process with minimal effort.

  13. Optimizing the Performance of Porous Electrochemical Cells for Flue Gas Purification using the DOE method

    DEFF Research Database (Denmark)

    Andersen, Kjeld Bøhm; Nygaard, Frederik Berg; He, Zeming;

    2011-01-01

    The DOE model was used to improve the performance of cells for electrochemical gas purification. Three factors were chosen: the amount of graphite, the Lanthanum Strontium Manganate/Gadolinium-doped Cerium oxide weight % ratio, and the Lanthanum Strontium Manganate pre-calcination temperature (with......, that a change in a factor not only changes the performance property that one would expect, but also influence other properties....

  14. High-yield fermentation and a novel heat-precipitation purification method for hydrophobin HGFI from Grifola frondosa in Pichia pastoris.

    Science.gov (United States)

    Song, Dongmin; Gao, Zhendong; Zhao, Liqiang; Wang, Xiangxiang; Xu, Haijin; Bai, Yanling; Zhang, Xiuming; Linder, Markus B; Feng, Hui; Qiao, Mingqiang

    2016-12-01

    Hydrophobins are proteins produced by filamentous fungi with high natural-surfactant activities and that can self-assemble in interfaces of air-water or solid-water to form amphiphilic membranes. Here, we reported a high-yield fermentation method for hydrophobin HGFI from Grifola frondosa in Pichia pastoris, attaining production of 300 mg/L by keeping the dissolved oxygen level at 15%-25% by turning the methanol-feeding speed. We also developed a novel HGFI-purification method enabling large-scare purification of HGFI, with >90% recovery. Additionally, we observed that hydrophobin HGFI in fermentation broth precipitated at pH 90 °C. We also identified the structure and properties of proteins purified by this method through atomic force microscopy, circular dichroism, X-ray photoelectron spectroscopy, and water-contact angle measurement, which is similar to protein purification by ultrafiltration without heating treatment that enables our method to maintain native HGFI structure and properties. Furthermore, the purification method presented here can be applied to large-scale purification of other type I hydrophobins. Copyright © 2016. Published by Elsevier Inc.

  15. SMET: systematic multiple enzyme targeting - a method to rationally design optimal strains for target chemical overproduction.

    Science.gov (United States)

    Flowers, David; Thompson, R Adam; Birdwell, Douglas; Wang, Tsewei; Trinh, Cong T

    2013-05-01

    Identifying multiple enzyme targets for metabolic engineering is very critical for redirecting cellular metabolism to achieve desirable phenotypes, e.g., overproduction of a target chemical. The challenge is to determine which enzymes and how much of these enzymes should be manipulated by adding, deleting, under-, and/or over-expressing associated genes. In this study, we report the development of a systematic multiple enzyme targeting method (SMET), to rationally design optimal strains for target chemical overproduction. The SMET method combines both elementary mode analysis and ensemble metabolic modeling to derive SMET metrics including l-values and c-values that can identify rate-limiting reaction steps and suggest which enzymes and how much of these enzymes to manipulate to enhance product yields, titers, and productivities. We illustrated, tested, and validated the SMET method by analyzing two networks, a simple network for concept demonstration and an Escherichia coli metabolic network for aromatic amino acid overproduction. The SMET method could systematically predict simultaneous multiple enzyme targets and their optimized expression levels, consistent with experimental data from the literature, without performing an iterative sequence of single-enzyme perturbation. The SMET method was much more efficient and effective than single-enzyme perturbation in terms of computation time and finding improved solutions.

  16. Effects of Extraction and Purification Methods on Degradation Kinetics and Stability of Lycopene from Watermelon under Storage Conditions.

    Science.gov (United States)

    Saeid, Abu; Eun, Jong Bang; Sagor, Md Shafiul Azam; Rahman, Atikur; Akter, Mst Sorifa; Ahmed, Maruf

    2016-09-28

    Lycopene was extraction, isolation and purification using recrystallization, column chromatography, and preparative thin layer chromatography (TLC) methods as well as degradation kinetics of lycopene were studied at refrigerated temperature and room temperature for 3 wk from watermelon. Higher lycopene degradation was observed at refrigerated temperature as compared to ambient temperature throughout the storage periods. The highest amount of lycopene retained in recrystallization (101.69 μg/g) followed by column chromatography (18.20 μg/g) and preparative TLC (15.57 μg/g). Color parameters, half-life time (t1/2 ), and color retention (%R) were dependent on extraction, isolation, and purification methods and storage life. Recrystallization and preparative TLC were followed by first order reaction model. Preparative TLC exhibited higher activation energy than did the recrystallization and column chromatography. Therefore, the result shows that recrystallization method could apply to extract and purify lycopene from watermelon that would also be used as a natural colorant as well as value-added product.

  17. 偏析法净化原铝的研究%Purification of Aluminium Ingot by Segregation Method

    Institute of Scientific and Technical Information of China (English)

    符岩; 张晓明; 孙中祺; 翟秀静

    2001-01-01

    考察了原铝熔体偏析净化过程中冷却速度、搅拌方式、温度梯度等因素对偏析净化的影响,并对净化得到的杂质富集相和纯化相进行了结构研究.结果表明,冷却速度、搅拌方式和温度梯度在偏析净化过程中起着重要的作用.当冷却速度缓慢,搅拌充分,温度梯度较大时,偏析净化效果比较好.杂质Si,Fe主要分布在杂质富集相中,并以Al-Si-Fe和Al-Si化合物的形式存在.%The factors such as distribution of temperature field,coolingrate and thermal gradient were examined during purification of aluminium melt by the segregation method. The structure of two phases (enriching phase of impurities and purifying phase) were researched. The results show that the important factors are cooling rate, agitation condition and thermal gradient in the course of segregation purification. When the melt is cooled slowly, agitated fully and under large thermal gradient, the purification results were in good satisfactory. The impurities of Fe and Si are distributed in the enriching phase existed in the formation of Al-Si-Fe and Al-Si compounds.

  18. Expression and purification of splicing proteins from mammalian cells.

    Science.gov (United States)

    Allemand, Eric; Hastings, Michelle L

    2014-01-01

    Pre-mRNA splicing is a complex process that is carried out by a large ribonucleoprotein enzyme, termed the spliceosome, which comprises up to 200 proteins. Despite this complexity, the role of individual spliceosomal proteins in the splicing reaction has been successfully investigated using cell-free assays. In many cases, the splicing factor of interest must be expressed and purified in order to study its function in vitro. Posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination of splicing factors are important for activity. Thus, their purification from mammalian cells presents numerous advantages. Here, we describe a method for expression and purification of splicing proteins from mammalian cells.

  19. RECOVERY OF MORE THAN 10 YEARS-DRYING m o N ascus CULTURES AND ITS PURIFICATION METHODS FROM FUNGAL AND BACTERIAL CONTAMINATION

    Directory of Open Access Journals (Sweden)

    NANDANG SUHARNA

    2008-01-01

    Full Text Available This study was carried out to understand the recovery capability of more than 10 years- drying Monascus cultures. A new simple purification technique from fungal contamination using ethanol-soaking treatment was also reported as a part of this study. The result showed that all drying cultures were recovered well and retained their characters such as good growth, pigmen-tation and production of fruit bodies (ascomata, sexual spores (ascospores and asexual spores. Several cultures showed its good growth in 20% ethanol medium. This study also reported suc-cessful purification of cultures from fungal contamination using ethanol-soaking treatment. This self-drying method, therefore, could be suggested as a good long-term preservation method for Monascus cultures. Moreover, purification method from fungal contamination soaked in ethanol 70% or 95% was successfully effective.

  20. Comparing Russian and Finnish standards of water purification

    OpenAIRE

    Maria, Pupkova

    2012-01-01

    The subject of this thesis is water purification. The first aim of this thesis is to consider different ways of water purification. The second aim is to compare Finnish and Russian standards of water purification. The third one is to show water purification methods on the pattern of Mikkeli water purification plan. Water purification methods of water intended for human consumption will be described.Combined tables will be done according to the quality requirement of drinking water of both,...

  1. A new versatile microarray-based method for high throughput screening of carbohydrate-active enzymes.

    Science.gov (United States)

    Vidal-Melgosa, Silvia; Pedersen, Henriette L; Schückel, Julia; Arnal, Grégory; Dumon, Claire; Amby, Daniel B; Monrad, Rune Nygaard; Westereng, Bjørge; Willats, William G T

    2015-04-03

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths.

  2. Metodologia de seleção de cepas para produção etodologia da ciclodextrina glicosiltransferase e para purificação da enzima - DOI: 10.4025/actascihealthsci.v29i1.133 Strains selection methodology for cyclodextrin glycosyltransferase production and enzyme purification - DOI: 10.4025/actascihealthsci.v29i1.133

    Directory of Open Access Journals (Sweden)

    Graciette Matioli

    2007-12-01

    Full Text Available As ciclodextrinas (CDs são maltooligossacarídeos, produzidas a partir do amido, pela enzima ciclodextrina glicosiltransferase (CGTase. Esta pesquisa teve por objetivo estabelecer metodologias de seleção de cepas para produção de CGTase e para purificação da enzima. Os microrganismos foram selecionados a partir de 53 análises de solos de cultura de amido, em placas contendo meio de cultivo específico, para seleção de cepas produtoras de CGTase. As enzimas foram obtidas com cultivo destes microrganismos em meio líquido. As atividades enzimáticas das CGTases foram determinadas pelos métodos espectrofotométricos e precipitação com tricloroetileno. A cepa isolada do solo de aveia foi a que apresentou maior atividade [0,1864 mol de -CD (min mL-1]. Esta cepa foi utilizada para a produção da enzima em escala laboratorial e purificação em cromatografia de afinidade bioespecífica. A cepa selecionada nesta pesquisa abre novas perspectivas para produção de enzima e CDs em escala industrial. Palavras-chave: ciclodextrina, glicosiltransferase, CGTase.Cyclodextrins (CDs are maltooligosaccharides produced from starch by cyclodextrin glycosyltransferase (CGTase enzyme. This research aimed at establishing method for strains selection for CGTase production and enzyme purification. The microorganisms were selected from 53 analyses of starch cultures soils on plates containing specific culture medium for strains selection that produce CGTase. The enzymes were obtained by culturing these microorganisms in liquid medium. The enzyme activity was determined with photospectrometric methods and precipitation with trichloroethylene. The strain isolated from oat soil was the one that showed the highest activity [0.1864 µmol of ß-CD (min mL-1]. This strain was used for enzyme production in laboratory scale and purification by biospecific affinity chromatography. The strain selected in this research opens new perspectives for enzymes

  3. PDZ affinity chromatography: a general method for affinity purification of proteins based on PDZ domains and their ligands.

    Science.gov (United States)

    Walkup, Ward G; Kennedy, Mary B

    2014-06-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins.

  4. Extraction, isolation and purification of exopolysaccharide from lactic acid bacteria using ethanol precipitation method

    Directory of Open Access Journals (Sweden)

    Vivek K. Bajpai

    2016-09-01

    Full Text Available Lactic acid bacteria are classified ‘Generally Recognized As Safe’ (GRAS with most effective potential to divert significant amount of fermentable sugars towards the biosynthesis of functional exopolysaccharide. Exopolysaccharides from lactic acid bacteria are receiving a renewed interest due to the claims of human health benefits, such as modulation of immune response system and more importantly in food and pharma industries as a texturizer, viscosifer, emulsifier and syneresis-lowering agent. Its purification methodology involves: a Extraction of cell-free supernatant from lactic acid bacteria; b Denature of protein using trichloroacetic acid; c Ethanol precipitation; d Dialysis; and e Freeze drying. However, depending on nature of research, compounds can be further purified using scanning electron microscopy (SEM, infrared spectrum (IR; and nuclear magnetic resonance (NMR spectral analyses.

  5. Keto-isovalerate decarboxylase enzymes and methods of use thereof

    Science.gov (United States)

    McElvain, Jessica; O'Keefe, Daniel P.; Paul, Brian James; Payne, Mark S.; Rothman, Steven Cary; He, Hongxian

    2016-01-19

    Provided herein are polypeptides and polynucleotides encoding such polypeptides which have ketoisovalerate decarboxylase activity. Also provided are recombinant host cells comprising such polypeptides and polynucleotides and methods of use thereof.

  6. Centrifugal gas-phase transition magnetophoresis (GTM)--a generic method for automation of magnetic bead based assays on the centrifugal microfluidic platform and application to DNA purification.

    Science.gov (United States)

    Strohmeier, Oliver; Emperle, Alexander; Roth, Günter; Mark, Daniel; Zengerle, Roland; von Stetten, Felix

    2013-01-07

    Transportation of magnetic beads between different reagents plays a crucial role in many biological assays e.g. for purification of biomolecules or cells where the beads act as a mobile solid support. Therefore, usually a complex set-up either for fluidic processing or for manipulation of magnetic beads is required. To circumvent these drawbacks, we present a facile and automated method for the transportation of magnetic beads between multiple microfluidic chambers on a centrifugal microfluidic cartridge "LabDisk". The method excels by requiring only one stack of stationary permanent magnets, a specific microfluidic layout without actively controlled valves and a predefined frequency protocol for rotation of the LabDisk. Magnetic beads were transported through three fluidically separated chambers with a yield of 82.6% ± 3.6%. Bead based DNA purification from a dilution series of a Listeria innocua lysate and from a lambda phage DNA standard was demonstrated where the three chambers were used for binding, washing and elution of DNA. Recovery of L. innocua DNA was up to 68% ± 24% and for lambda phage DNA 43% ± 10% compared to manual reference purification in test tubes. Complete purification was conducted automatically within 12.5 min. Since all reagents can be preloaded onto the LabDisk prior to purification, no further hands-on steps are required during processing. Due to its modular and generic character, the presented method could also be adapted to other magnetic bead based assays e.g. to immunoassays or protein affinity purification, solely requiring the adjustment of number and volumes of the fluidic chambers.

  7. Method of reduction of nitroaromatics by enzymatic reaction with redox enzymes

    Science.gov (United States)

    Shah, Manish M.

    2000-01-01

    A method for the controlled reduction of nitroaromatic compounds such as nitrobenzene and 2,4,6-trinitrotoluene by enzymatic reaction with redox enzymes, such as Oxyrase (Trademark of Oxyrase, Inc., Mansfield, Ohio).

  8. Method of controlled reduction of nitroaromatics by enzymatic reaction with oxygen sensitive nitroreductase enzymes

    Science.gov (United States)

    Shah, Manish M.; Campbell, James A.

    1998-01-01

    A method for the controlled reduction of nitroaromatic compounds such as nitrobenzene and 2,4,6-trinitrotoluene by enzymatic reaction with oxygen sensitive nitroreductase enzymes, such as ferredoxin NADP oxidoreductase.

  9. Simple method for preparation of nanostructure on microchannel surface and its usage for enzyme-immobilization.

    Science.gov (United States)

    Miyazaki, Masaya; Kaneno, Jun; Uehara, Masato; Fujii, Masayuki; Shimizu, Hazime; Maeda, Hideaki

    2003-03-07

    We developed a novel preparation method of nanostructure on the microchannel surface formed by sol-gel like simple treatment with 3-aminopropyltriethoxysilane, which is suitable for a highly efficient enzyme-immobilized microchannel reactor.

  10. Influence of different purification and drying methods on rheological properties and viscoelastic behaviour of durian seed gum.

    Science.gov (United States)

    Amid, Bahareh Tabatabaee; Mirhosseini, Hamed

    2012-09-01

    The aim of the present study was to investigate the effects of different purification and drying methods on the viscoelastic behaviour and rheological properties of durian seed gum. The results indicated that the purified gum A (using isopropanol and ethanol) and D (using hydrochloric acid and ethanol) showed the highest and lowest viscosity, respectively. Four drying techniques included oven drying (105 °C), freeze drying, spray drying and vacuum oven drying. In the present work, all purified gums exhibited more elastic (gel-like) behaviour than the viscous (liquid-like) behaviour (G″gum. The freeze-dried gum and oven-dried (105 °C) gum exhibited the highest and lowest viscous modulus (G″), respectively.

  11. Preparation of SiO2 nanowires from rice husks by hydrothermal method and the RNA purification performance

    Science.gov (United States)

    Huang, Meiyan; Cao, Jianping; Meng, Xing; Liu, Yangsi; Ke, Wei; Wang, Jialiang; Sun, Ling

    2016-10-01

    In this study, SiO2 nanowires were prepared by using rice husks as silicon source via a hydrothermal method. The microstructure, thermal stability and morphology of SiO2 nanowires were characterized by X-ray diffraction, infrared spectroscopy, thermal gravimetric analysis and scanning electron microscope. SiO2 nanowires with a diameter of 30-100 nm were obtained and the formation mechanism of SiO2 nanowires during the hydrothermal reaction was proposed. The SiO2 nanowires were introduced into membrane spin columns to isolate RNA and the values of A260/280 and A260/230 were 2.0-2.1 and 1.8-2.0, respectively, which shows the SiO2 nanowires were effective for RNA purification.

  12. Partial Purification and Characterization of Extracellular Protease ...

    African Journals Online (AJOL)

    USER

    Purification of the enzyme by gel filtration chromatography on Sephadex G75 following ammonium sulphate precipitation gave 2.26 fold increase in purification with specific activity of 46.13 .... minutes in a water bath and were allowed to cool.

  13. An efficient sample preparation method for high-throughput analysis of 15(S)-8-iso-PGF2α in plasma and urine by enzyme immunoassay.

    Science.gov (United States)

    Bielecki, A; Saravanabhavan, G; Blais, E; Vincent, R; Kumarathasan, P

    2012-01-01

    Although several methods have been reported on the analysis of the oxidative stress marker 15(S)-8-iso-prostaglandin-F2alpha (8-iso-PGF2α) in biological fluids, they either involve extensive sample preparation and costly technology or require high sample volume. This study presents a sample preparation method that utilizes low sample volume for 8-iso-PGF2α analysis in plasma and urine by an enzyme immunoassay (EIA). In brief, 8-iso-PGF2α in deproteinized plasma or native urine sample is complexed with an antibody and then captured by molecular weight cut-off filtration. This method was compared with two other sample preparation methods that are typically used in the analysis of 8-iso-PGF2α by EIA: Cayman's affinity column purification method and solid-phase extraction on C-18. The immunoaffinity purification method described here was superior to the other two sample preparation methods and yielded recovery values of 99.8 and 54.1% for 8-iso-PGF2α in plasma and urine, respectively. Analytical precision (relative standard deviation) was ±5% for plasma and ±15% for urine. The analysis of healthy human plasma and urine resulted in basal 8-iso-PGF2α levels of 31.8 ± 5.5 pg/mL and 2.9 ± 2.0 ng/mg creatinine, respectively. The robustness and analytical performance of this method makes it a promising tool for high-throughput screening of biological samples for 8-iso-PGF2α.

  14. 碳纳米管纯化处理的研究进展%Research Progress on Purification Methods of Carbon Nanotubes

    Institute of Scientific and Technical Information of China (English)

    张翼; 齐暑华; 王洲; 段国晨

    2011-01-01

    Carbon nanotubes have been prepared by many methods at present. However, the as-prepared pro ducts almost have impurities, such as amorphous carbon, carbon nanopartieles and catalyst partieles, which directly affect the properties and the applications of carbon nanotubes. So the purification process is necessary. Recent research in this area and characteristics of physical and chemical purification methods are introduced simply. The physical puri fication methods, the chemical purification methods and the comprehensive purification methods are described,in de tail. The good prospects of purification methods are forecasted.%目前制备碳纳米管的方法有很多,但这些方法制备的碳纳米管样品中大部分含有无定形碳、碳纳米颗粒和催化剂等杂质,这些杂质的存在直接影响其性能并限制其应用,因此碳纳米管的纯化研究很有必要.结合该领域的研究前沿概述了碳纳米管纯化处理的研究现状,简单介绍了物理纯化方法和化学纯化方法的特点,详细介绍了物理纯化法、化学纯化法和综合纯化法,并展望了纯化方法的趋势.

  15. Development and application of a method for the purification of free shigatoxigenic bacteriophage from environmental samples.

    Science.gov (United States)

    Rooks, David J; Libberton, Ben; Woodward, Martin J; Allison, Heather E; McCarthy, Alan J

    2012-11-01

    Shiga toxin producing Escherichia coli (STEC) strains are foodborne pathogens whose ability to produce Shiga toxin (Stx) is due to the integration of Stx-encoding lambdoid bacteriophage (Stx phage). Circulating, infective Stx phages are very difficult to isolate, purify and propagate such that there is no information on their genetic composition and properties. Here we describe a novel approach that exploits the phage's ability to infect their host and form a lysogen, thus enabling purification of Stx phages by a series of sequential lysogen isolation and induction steps. A total of 15 Stx phages were rigorously purified from water samples in this way, classified by TEM and genotyped using a PCR-based multi-loci characterisation system. Each phage possessed only one variant of each target gene type, thus confirming its purity, with 9 of the 15 phages possessing a short tail-spike gene and identified by TEM as Podoviridae. The remaining 6 phages possessed long tails, four of which appeared to be contractile in nature (Myoviridae) and two of which were morphologically very similar to bacteriophage lambda (Siphoviridae). Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Overview of methods in RNA nanotechnology: synthesis, purification, and characterization of RNA nanoparticles.

    Science.gov (United States)

    Haque, Farzin; Guo, Peixuan

    2015-01-01

    RNA nanotechnology encompasses the use of RNA as a construction material to build homogeneous nanostructures by bottom-up self-assembly with defined size, structure, and stoichiometry; this pioneering concept demonstrated in 1998 (Guo et al., Molecular Cell 2:149-155, 1998; featured in Cell) has emerged as a new field that also involves materials engineering and synthetic structural biology (Guo, Nature Nanotechnology 5:833-842, 2010). The field of RNA nanotechnology has skyrocketed over the last few years, as evidenced by the burst of publications in prominent journals on RNA nanostructures and their applications in nanomedicine and nanotechnology. Rapid advances in RNA chemistry, RNA biophysics, and RNA biology have created new opportunities for translating basic science into clinical practice. RNA nanotechnology holds considerable promise in this regard. Increased evidence also suggests that substantial part of the 98.5 % of human genome (Lander et al. Nature 409:860-921, 2001) that used to be called "junk DNA" actually codes for noncoding RNA. As we understand more on how RNA structures are related to function, we can fabricate synthetic RNA nanoparticles for the diagnosis and treatment of diseases. This chapter provides a brief overview of the field regarding the design, construction, purification, and characterization of RNA nanoparticles for diverse applications in nanotechnology and nanomedicince.

  17. Accurate prediction of enzyme subfamily class using an adaptive fuzzy k-nearest neighbor method.

    Science.gov (United States)

    Huang, Wen-Lin; Chen, Hung-Ming; Hwang, Shiow-Fen; Ho, Shinn-Ying

    2007-01-01

    Amphiphilic pseudo-amino acid composition (Am-Pse-AAC) with extra sequence-order information is a useful feature for representing enzymes. This study first utilizes the k-nearest neighbor (k-NN) rule to analyze the distribution of enzymes in the Am-Pse-AAC feature space. This analysis indicates the distributions of multiple classes of enzymes are highly overlapped. To cope with the overlap problem, this study proposes an efficient non-parametric classifier for predicting enzyme subfamily class using an adaptive fuzzy r-nearest neighbor (AFK-NN) method, where k and a fuzzy strength parameter m are adaptively specified. The fuzzy membership values of a query sample Q are dynamically determined according to the position of Q and its weighted distances to the k nearest neighbors. Using the same enzymes of the oxidoreductases family for comparisons, the prediction accuracy of AFK-NN is 76.6%, which is better than those of Support Vector Machine (73.6%), the decision tree method C5.0 (75.4%) and the existing covariant-discriminate algorithm (70.6%) using a jackknife test. To evaluate the generalization ability of AFK-NN, the datasets for all six families of entirely sequenced enzymes are established from the newly updated SWISS-PROT and ENZYME database. The accuracy of AFK-NN on the new large-scale dataset of oxidoreductases family is 83.3%, and the mean accuracy of the six families is 92.1%.

  18. Measurement of peroxisomal enzyme activities in the liver of brown trout (Salmo trutta, using spectrophotometric methods

    Directory of Open Access Journals (Sweden)

    Resende Albina D

    2003-03-01

    Full Text Available Abstract Background This study was aimed primarily at testing in the liver of brown trout (Salmo trutta spectrophotometric methods previously used to measure the activities of catalase and hydrogen peroxide producing oxidases in mammals. To evaluate the influence of temperature on the activities of those peroxisomal enzymes was the second objective. A third goal of this work was the study of enzyme distribution in crude cell fractions of brown trout liver. Results The assays revealed a linear increase in the activity of all peroxisomal enzymes as the temperature rose from 10° to 37°C. However, while the activities of hydrogen peroxide producing oxidases were strongly influenced by temperature, catalase activity was only slightly affected. A crude fraction enriched with peroxisomes was obtained by differential centrifugation of liver homogenates, and the contamination by other organelles was evaluated by the activities of marker enzymes for mitochondria (succinate dehydrogenase, lysosomes (aryl sulphatase and microsomes (NADPH cytochrome c reductase. For peroxisomal enzymes, the activities per mg of protein (specific activity in liver homogenates were strongly correlated with the activities per g of liver and with the total activities per liver. These correlations were not obtained with crude peroxisomal fractions. Conclusions The spectrophotometric protocols originally used to quantify the activity of mammalian peroxisomal enzymes can be successfully applied to the study of those enzymes in brown trout. Because the activity of all studied peroxisomal enzymes rose in a linear mode with temperature, their activities can be correctly measured between 10° and 37°C. Probably due to contamination by other organelles and losses of soluble matrix enzymes during homogenisation, enzyme activities in crude peroxisomal fractions do not correlate with the activities in liver homogenates. Thus, total homogenates will be used in future seasonal and

  19. Aqueous enzyme assisted oil extraction from oilseeds and emulsion de-emulsifying methods: a review

    OpenAIRE

    Mat Yusoff, Masni; Gordon, Mike; Niranjan, Keshavan

    2014-01-01

    Regulatory, safety, and environmental issues have prompted the development of aqueousenzymatic extraction (AEE) for extracting components from oil-bearing materials. The emulsion resulting from AEE requires de-emulsification to separate the oil; when enzymes are used for this purpose, the method is known as aqueous enzymatic emulsion de-emulsification (AEED). In general, enzyme assisted oil extraction is known to yield oil having highly favourable characteristics. This review covers techno...

  20. [Progress in quantitative methods based on liquid chromatography-mass spectrometry for drug metabolizing enzymes in human liver microsomes].

    Science.gov (United States)

    Wang, Huanhuan; Lu, Yayao; Peng, Bo; Qian, Xiaohong; Zhang, Yangjun

    2015-06-01

    Cytochrome P450 (CYP) enzymes and uridine 5-diphospho-glucuronosyltransferase (UGT) enzymes are critical enzymes for drug metabolism. Both chemical drugs and traditional Chinese medicines are converted to more readily excreted compounds by drug metabolizing enzymes in human livers. Because of the disparate expression of CYP and UGT enzymes among different individuals, accurate quantification of these enzymes is essential for drug pharmacology, drug-drug interactions and drug clinical applications. The research progress in quantitative methods based on liquid chromatography-mass spectrometry for drug metabolizing enzymes in human liver microsomes in the recent decade is reviewed.

  1. Simplified method to obtain enhanced expression of tau protein from E. coli and one-step purification by direct boiling.

    Science.gov (United States)

    KrishnaKumar, V Guru; Gupta, Sharad

    2017-05-28

    Tau is an intrinsically disordered protein responsible for maintaining the structure and stability of axonal microtubules. However, in certain disease conditions including Alzheimer's disease, tau protein may undergo biochemical and structural changes to form intracellular aggregates. Since tau is a proline- and arginine-rich eukaryotic protein, heterologous expression in Escherichia coli often results in poor yield and has been a major technical challenge. In the current work, we have improved the expressed yield of tau by overcoming codon bias problem and established a simplified protocol for efficient extraction. The reported method has two distinct features: (i) enhanced tau expression (upto eightfold) by supplementing deficient tRNAs that aid in rapid translation and (ii) direct boiling of expressed E. coli cells to extract tau with no separate cell lysis step. We further demonstrate that tau extracted by the direct boiling method is similar to tau purified by size-exclusion chromatography exhibiting similar structural and biophysical characteristics including aggregation propensity. Since morphologies and in vitro toxicity of fibrillar tau aggregates were also similar, tau extracted by the one-step direct boiling method can be used for tau aggregation assays without any additional purification.

  2. Combined electron-beam and coagulation purification of molasses distillery slops. Features of the method, technical and economic evaluation of large-scale facility

    Energy Technology Data Exchange (ETDEWEB)

    Pikaev, A.K. E-mail: pikaev@ipc.rssi.ru; Ponomarev, A.V.; Bludenko, A.V.; Minin, V.N.; Elizar' eva, L.M

    2001-04-01

    The paper summarizes the results obtained from the study on combined electron-beam and coagulation method for purification of molasses distillery slops from distillery produced ethyl alcohol by fermentation of grain, potato, beet and some other plant materials. The method consists in preliminary mixing of industrial wastewater with municipal wastewater, electron-beam treatment of the mixture and subsequent coagulation. Technical and economic evaluation of large-scale facility (output of 7000 m{sup 3} day{sup -1}) with two powerful cascade electron accelerators (total maximum beam power of 400 kW) for treatment of the wastewater by the above method was carried out. It was calculated that the cost of purification of the wastes is equal to 0.25 US$ m{sup -3} that is noticeably less than in the case of the existing method.

  3. Combined electron-beam and coagulation purification of molasses distillery slops. Features of the method, technical and economic evaluation of large-scale facility

    Science.gov (United States)

    Pikaev, A. K.; Ponomarev, A. V.; Bludenko, A. V.; Minin, V. N.; Elizar'eva, L. M.

    2001-04-01

    The paper summarizes the results obtained from the study on combined electron-beam and coagulation method for purification of molasses distillery slops from distillery produced ethyl alcohol by fermentation of grain, potato, beet and some other plant materials. The method consists in preliminary mixing of industrial wastewater with municipal wastewater, electron-beam treatment of the mixture and subsequent coagulation. Technical and economic evaluation of large-scale facility (output of 7000 m 3 day -1) with two powerful cascade electron accelerators (total maximum beam power of 400 kW) for treatment of the wastewater by the above method was carried out. It was calculated that the cost of purification of the wastes is equal to 0.25 US$ m -3 that is noticeably less than in the case of the existing method.

  4. New effective method for analysis of the component composition of enzyme complexes from Trichoderma reesei.

    Science.gov (United States)

    Markov, A V; Gusakov, A V; Kondratyeva, E G; Okunev, O N; Bekkarevich, A O; Sinitsyn, A P

    2005-06-01

    A method for analysis of the component composition of multienzyme complexes secreted by the filamentous fungus Trichoderma reesei was developed. The method is based on chromatofocusing followed by further identification of protein fractions according to their substrate specificity and molecular characteristics of the proteins. The method allows identifying practically all known cellulases and hemicellulases of T. reesei: endoglucanase I (EG I), EG II, EG III, cellobiohydrolase I (CBH I), CBH II, xylanase I (XYL I), XYL II, beta-xylosidase, alpha-L-arabinofuranosidase, acetyl xylan esterase, mannanase, alpha-galactosidase, xyloglucanase, polygalacturonase, and exo-beta-1,3-glucosidase. The component composition of several laboratory and commercial T. reesei preparations was studied and the content of the individual enzymes in these preparations was quantified. The influence of fermentation conditions on the component composition of secreted enzyme complexes was revealed. The characteristic features of enzyme preparations obtained in "cellulase" and "xylanase" fermentation conditions are shown.

  5. Application of modified enzyme digestion method in rapid primary culture of human glioma cells

    Directory of Open Access Journals (Sweden)

    Wei XIANG

    2016-06-01

    Full Text Available Objective  To explore the applied value of modified enzyme digestion method in primary culture of human glioma cells. Methods  A traditional enzyme digestion method was modified based on literatures and our work experience. The glioma cells from 32 glioma patients with different grades were primarily cultured by the modified enzyme digestion method. The morphological features of these cells were observed under an inverted phase contrast microscope. The primary cells were purified by differential adhesion during passage. The primary cells were identified by immunofluorescence technique, and the growth curves were drawn by cell proliferation assays (CCK-8 method for investigating the proliferation of the cells cultured in vitro. Results  The primary human glioma cells were successfully cultured and transferred by the new method, with a success rate of 87.5%. The cells cultured successfully in vitro showed good adherent growth, stable morphologies, thus can be passaged. Fluoroimmunoassay showed positive expression of glial fibrillary acidic protein, which confirms the cultured cells were glioma cells. Cell proliferation assays revealed active cell proliferation in vitro, the higher the tumor grade, the higher the proliferative capacity. Conclusion  The modified enzyme digestion method is simpler and more efficient for primary culture of human glioma cells, and the success rate is also higher, thus being able to provide a good guarantee for fundamental research of glioma. DOI: 10.11855/j.issn.0577-7402.2016.06.06

  6. A rapid and versatile method for the isolation, purification and cryogenic storage of Schwann cells from adult rodent nerves.

    Science.gov (United States)

    Andersen, Natalia D; Srinivas, Shruthi; Piñero, Gonzalo; Monje, Paula V

    2016-08-23

    We herein developed a protocol for the rapid procurement of adult nerve-derived Schwann cells (SCs) that was optimized to implement an immediate enzymatic dissociation of fresh nerve tissue while maintaining high cell viability, improving yields and minimizing fibroblast and myelin contamination. This protocol introduces: (1) an efficient method for enzymatic cell release immediately after removal of the epineurium and extensive teasing of the nerve fibers; (2) an adaptable drop-plating method for selective cell attachment, removal of myelin debris, and expansion of the initial SC population in chemically defined medium; (3) a magnetic-activated cell sorting purification protocol for rapid and effective fibroblast elimination; and (4) an optional step of cryopreservation for the storage of the excess of cells. Highly proliferative SC cultures devoid of myelin and fibroblast growth were obtained within three days of nerve processing. Characterization of the initial, expanded, and cryopreserved cell products confirmed maintenance of SC identity, viability and growth rates throughout the process. Most importantly, SCs retained their sensitivity to mitogens and potential for differentiation even after cryopreservation. To conclude, this easy-to-implement and clinically relevant protocol allows for the preparation of expandable homogeneous SC cultures while minimizing time, manipulation of the cells, and exposure to culture variables.

  7. Development of supercritical fluid extraction and supercritical fluid chromatography purification methods using rapid solubility screening with multiple solubility chambers.

    Science.gov (United States)

    Gahm, Kyung H; Huang, Ke; Barnhart, Wesley W; Goetzinger, Wolfgang

    2011-01-01

    Rapid solubility screening in diverse supercritical fluids (SCFs) was carried out via multiple solubility chambers with a trapping device and online ultraviolet (UV) detection. With this device, it was possible to rapidly study the solubility variations of multiple components in a mixture. Results from solubility studies have been used to develop efficient supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC) methods. After the investigation of solubilities of theophylline and caffeine in several neat organic solvents and SCFs, advantages of SFE over conventional organic solvent extraction were demonstrated with a model mixture of theophylline and caffeine. The highest solubility ratio of 1:40 (theophylline:caffeine) was observed in the SCF with 20% acetonitrile (MeCN), where a ratio of 1:11 was the highest in the neat organic solvents. A model mixture of theophylline:caffeine (85:15 w/w, caffeine as an impurity) was successfully purified by SFE by leveraging the highest solubility difference. The SCF with 20% MeCN selectively removed caffeine and left theophylline largely intact. Rapid SCF solubility screening was applied to development of SFE and SFC methods in a drug discovery environment. Two successful applications were demonstrated with proprietary Amgen compounds to either remove an achiral impurity before chiral purification or enhance chiral chromatographic throughput.

  8. RNA integrity as a quality indicator during the first steps of RNP purifications : A comparison of yeast lysis methods

    Directory of Open Access Journals (Sweden)

    Jansen Ralf-Peter

    2004-10-01

    Full Text Available Abstract Background The completion of several genome-sequencing projects has increased our need to assign functions to newly identified genes. The presence of a specific protein domain has been used as the determinant for suggesting a function for these new genes. In the case of proteins that are predicted to interact with mRNA, most RNAs bound by these proteins are still unknown. In yeast, several protocols for the identification of protein-protein interactions in high-throughput analyses have been developed during the last years leading to an increased understanding of cellular proteomics. If any of these protocols or similar approaches shall be used for the identification of mRNA-protein complexes, the integrity of mRNA is a critical factor. Results We compared the effect of different lysis protocols on RNA integrity. We report dramatic differences in RNA stability depending on the method used for yeast cell lysis. Glass bead milling and French Press lead to degraded mRNAs even in the presence of RNase inhibitors. Thus, they are not suitable to purify intact mRNP complexes or to identify specific mRNAs bound to proteins. Conclusion We suggest a novel protocol, grinding deep-frozen cells, for the preparation of protein extracts that contain intact RNAs, as lysis method for the purification of mRNA-protein complexes from yeast cells.

  9. [Separation and purification of cellulase using affinity membrane].

    Science.gov (United States)

    Shi, Xiang-zhu; Guo, Chun-teng; Zhou, Jian-wu; Wang, Zhong-lai; Rao, Ping-fan

    2002-07-01

    The importance of cellulase as a means for the efficient utilization of abundant cellulose resources in the world has been well recognized. Many researchers devote themselves to studying the mechanism of the action of cellulase to cellulose so that such expensive enzyme can be used much more widely. The first step is to obtain cellulase of high purity. So purification of cellulase is the key point in this field. However, the major problem in isolation is that cellulase is a complicated enzyme system and needs too many steps for separation, and that every cellulase needs special purification processing which cannot be used for the others. A novel method for the separation of the cellulase from crude extraction of Aspergillus niger with normal qualitative filter paper processed by 5 mol/L sodium hydroxide without precipitation and desalting steps was developed. Further purification of the cellulase was achieved by using an anion-exchange column of POROS 20HQ. The cellulase purified was identified as a new endoglucanase that had relatively high endurance to pH and temperature. Its relative molecular mass was estimated to be 60,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme exhibited very high activity towards carboxymethyl cellulose (CMC) with specific activity of 350 U.mg-1 and the recovery of activity of 9.7%. Its optimum pH and temperature were 4.0 and 70 degrees C, respectively. This is a simple, rapid and efficient method for purifying cellulase with high activity.

  10. Oxidation of protoporphyrinogen to protoporphyrin, a step in chlorophyll and haem biosynthesis. Purification and partial characterization of the enzyme from barley organelles.

    OpenAIRE

    Jacobs, J. M.; Jacobs, N J

    1987-01-01

    The protoporphyrinogen-oxidizing enzyme from Triton X-100 extracts of the mitochondrial and etioplast fractions of etiolated barley was purified by using ion-exchange and hydroxyapatite chromatography. The purified enzyme from both organelle fractions exhibited a Km of 5 microM and was labile to mild heat and acidification. The pH optimum (5-6) and the substrate-specificity (mesoporphyrinogen was oxidized as rapidly as protoporphyrinogen) revealed properties very different from the protoporph...

  11. Hamiltonian purification

    Energy Technology Data Exchange (ETDEWEB)

    Orsucci, Davide [Scuola Normale Superiore, I-56126 Pisa (Italy); Burgarth, Daniel [Department of Mathematics, Aberystwyth University, Aberystwyth SY23 3BZ (United Kingdom); Facchi, Paolo; Pascazio, Saverio [Dipartimento di Fisica and MECENAS, Università di Bari, I-70126 Bari (Italy); INFN, Sezione di Bari, I-70126 Bari (Italy); Nakazato, Hiromichi; Yuasa, Kazuya [Department of Physics, Waseda University, Tokyo 169-8555 (Japan); Giovannetti, Vittorio [NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, I-56126 Pisa (Italy)

    2015-12-15

    The problem of Hamiltonian purification introduced by Burgarth et al. [Nat. Commun. 5, 5173 (2014)] is formalized and discussed. Specifically, given a set of non-commuting Hamiltonians (h{sub 1}, …, h{sub m}) operating on a d-dimensional quantum system ℋ{sub d}, the problem consists in identifying a set of commuting Hamiltonians (H{sub 1}, …, H{sub m}) operating on a larger d{sub E}-dimensional system ℋ{sub d{sub E}} which embeds ℋ{sub d} as a proper subspace, such that h{sub j} = PH{sub j}P with P being the projection which allows one to recover ℋ{sub d} from ℋ{sub d{sub E}}. The notions of spanning-set purification and generator purification of an algebra are also introduced and optimal solutions for u(d) are provided.

  12. A novel Method for the selective recovery and purification of gamma-polyglutamic acid from Bacillus licheniformis fermentation broth.

    Science.gov (United States)

    Manocha, Bhavik; Margaritis, Argyrios

    2010-01-01

    Microbially produced gamma-polyglutamic acid (gamma-PGA) is a commercially important biopolymer with many applications in biopharmaceutical, food, cosmetic and waste-water treatment industries. Owing to its increasing demand in various industries, production of gamma-PGA is well documented in the literature, however very few methods have been reported for its recovery. In this paper, we report a novel method for the selective recovery and purification of gamma-PGA from cell-free fermentation broth of Bacillus licheniformis. The cell-free fermentation broth was treated with divalent copper ions, resulting in the precipitation of gamma-PGA, which was collected as a pellet by centrifugation. The pellet was resolubilized and dialyzed against de-ionized water to obtain the purified gamma-PGA biopolymer. The efficiency and selectivity of gamma-PGA recovery was compared with ethanol precipitation method. We found that 85% of the original gamma-PGA content in the broth was recovered by copper sulfate-induced precipitation, compared to 82% recovery by ethanol precipitation method. Since ethanol is a commonly used solvent for protein precipitation, the purity of gamma-PGA precipitate was analyzed by measuring proteins that co-precipitated with gamma-PGA. Of the total proteins present in the broth, 48% proteins were found to be co-precipitated with gamma-PGA by ethanol precipitation, whereas in copper sulfate-induced precipitation, only 3% of proteins were detected in the final purified gamma-PGA, suggesting that copper sulfate-induced precipitation offers better selectivity than ethanol precipitation method. Total metal content analysis of the purified gamma-PGA revealed the undetectable amount of copper ions, whereas other metal ions detected were in low concentration range. The purified gamma-PGA was characterized using infrared spectroscopy. Copyright 2010 American Institute of Chemical Engineers

  13. Purification of glucose-6-phosphate dehydrogenase and glutathione reductase enzymes from the gill tissue of Lake Van fish and analyzing the effects of some chalcone derivatives on enzyme activities.

    Science.gov (United States)

    Kuzu, Muslum; Aslan, Abdulselam; Ahmed, Ishtiaq; Comakli, Veysel; Demirdag, Ramazan; Uzun, Naim

    2016-04-01

    Glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR) are metabolically quite important enzymes. Within this study, these two enzymes were purified for the first time from the gills of Lake Van fish. In the purifying process, ammonium sulfate precipitation and 2',5'-ADP Sepharose 4B affinity column chromatography techniques for glucose-6-phosphate dehydrogenase, temperature degradation and 2',5'-ADP Sepharose 4B affinity column chromatography for glutathione reductase enzyme were used. The control of the enzyme purity and determination of molecular weight were done with sodium dodecyl sulfate polyacrylamide gel electrophoresis. K(M) and V(max) values were determined with Lineweaver-Burk plot. Besides, the effects of some chalcone derivatives on the purified enzymes were analyzed. For the ones showing inhibition effect, % activity-[I] figures were drawn and IC50 values were determined. K(i) value was calculated by using Cheng-Prusoff equation.

  14. Production, partial purification and characterization of feruloyl esterase by Aspergillus awamori in submerged fermentation.

    Science.gov (United States)

    Fazary, Ahmed Eid; Ju, Yi-Hsu

    2008-10-01

    Microbial feruloyl esterases acting on plant cell wall polymers represent key tools for the degradation of plant cell wall. In this paper, we describe in detail the microbial production, partial purification and characterization of feruloyl esterase from a culture medium of Aspergillus awamori strain IFO4033 obtained from a crude hemicellulose preparation of wheat straw, corncobs and wheat germ. Feruloyl esterase was extracted using centrifugation and dialysis, and then purified by ion exchange chromatography and microfiltration to homogeneity, which was checked by SDSPAGE and isoelectric focusing-PAGE. Protein content and activity of the enzyme were measured in each step of extraction and purification. Biomass was determined by the dry weight method. pH and temperature optima of feruloyl esterase enzyme were also determined. The effects of culturing time, and carbon and nitrogen sources on enzyme production were systematically investigated. Finally, enzyme activities under different storage conditions were examined.

  15. H sup + -ATP synthase from rat liver mitochondria. A simple, rapid purification method of the functional complex and its characterization

    Energy Technology Data Exchange (ETDEWEB)

    Yoshihara, Yutaka; Nagase, Hideki; Yamane, Takeshi; Oka, Hideki; Tani, Isamu; Higuti, Tomihiko (Univ. of Tokushima (Japan))

    1991-07-16

    A novel, simple, and rapid preparative method for purification of rat liver H{sup +}-ATP synthase by anion-exchange HPLC was developed. The H{sup +}-ATP synthase purified had higher ATPase activity in the absence of added phospholipids than any preparation reported previously, and this activity was completely inhibited by oligomycin. When reconstituted into proteoliposomes, the H{sup +}-ATP synthase showed an ATP-dependent 8-anilinonaphthalene-1-sulfonate response and ATP-P{sub i} exchange activity, both of which were also completely inhibited by oligomycin and an uncoupler, indicating the intactness of the H{sup +}-ATP synthase. An immunochemical study and a labeling experiment with N,N{prime}-({sup 14}C)dicyclohexylcarbondiimide (({sup 14}C)DCCD) demonstrated the presence of chargerin II (a product of mitochondrial A6L DNA) and DCCD-binding protein (subunit c) in the complex. The subunits of the complex were separated into 11 main fractions by reverse-phase HPLC, and 3 of them and the {sigma} subunit in F{sub 1} were partially sequenced. A search for sequence homologies indicated that these components were subunit b, coupling factor 6, subunit {sigma}, and subunit e. This is the first report of the existence of subunit b, factor 6, and chargerin II in K{sup +}-ATP synthase purified from rat liver mitochondria.

  16. Defeating numts: semi-pure mitochondrial DNA from eggs and simple purification methods for field-collected wildlife tissues.

    Science.gov (United States)

    Ibarguchi, Gabriela; Friesen, Vicki L; Lougheed, Stephen C

    2006-11-01

    Mitochondrial DNA (mtDNA) continues to play a pivotal role in phylogeographic, phylogenetic, and population genetic studies. PCR amplification with mitochondrial primers often yields ambiguous sequences, in part because of the co-amplification of nuclear copies of mitochondrial genes (numts) and true mitochondrial heteroplasmy arising from mutations, hybridization with paternal leakage, gene duplications, and recombination. Failing to detect numts or to distinguish the origin of such homologous sequences results in the incorrect interpretation of data. However, few studies obtain purified mtDNA to confirm the mitochondrial origin of the first reference sequences for a species. Here, we demonstrate the importance and ease of obtaining semi-pure mtDNA from wildlife tissues, preserved under various typical field conditions, and investigate the success of 3 commercial extraction kits, cesium-chloride gradient mtDNA purification, long-template PCR amplification, cloning, and more species-specific degenerate primers. Using more detailed avian examples, we illustrate that unfertilized or undeveloped eggs provide the purest sources of mtDNA; that kits provide an alternative to cesium-chloride gradient methods; and that long-template PCR, cloning, and degenerate primers cannot be used to produce reliable mitochondrial reference sequences, but can be powerful tools when used in conjunction with purified mtDNA stocks to distinguish numts from true heteroplasmy.

  17. Development and validation of a method for purification of mallein for the diagnosis of glanders in equines

    Directory of Open Access Journals (Sweden)

    de Carvalho Filho Maurício

    2012-09-01

    Full Text Available Abstract Background The allergic test of mallein is one of the most frequently used tests, together with the Complement Fixation Test (CFT, for the diagnosis of glanders in endemic areas. Mallein, a purified protein derivative (PPD, is produced similarly to PPD tuberculin and the end product is a primarily proteic antigen, which is only poorly purified. The immuno-allergic activity of mallein is believed to be due to a high molecular weight group of proteins present in the antigen. To improve the quality of the antigen, in terms of sensitivity and specificity, a new method of mallein production was developed, in which purification was accomplished by ultrafiltration in a Tangential Flow Filtration system (TFF. Results The TFF methodology efficiently separated the high and low molecular weight protein groups of mallein. The five TFF-purified malleins, produced from Burkholderia mallei strains isolated from clinical cases of glanders in Brazil, proved to be more potent than standard mallein in the induction of an allergic reaction in sensitized animals. Regarding specificity, two of the purified malleins were equivalent to the standard and three were less specific. Conclusion Some of the TFF-purified malleins showed considerable potential to be used as an auxiliary test in the diagnosis of glanders.

  18. Advances on Aquaculture Water Purification Method%水产养殖水体处理方法研究进展

    Institute of Scientific and Technical Information of China (English)

    黄蔷; 刘松; 杨立群

    2014-01-01

    Purifying aquaculture water of ponds to improve water of breeding environment has become a research focus of breeding ecology and environmental.The research progress of the pond water quality purification methods were described, the handle principles and efficiency of a variety of water purification methods were focused on, and several recommendations of water treatment for aquaculture development direction were prospected.%如何净化养殖池塘水质、改善养殖水体环境质量已成为渔业养殖环境和生态研究的重点。阐述了池塘水质净化方法研究的进展,着重讨论了各种水质净化方法的处理原理和处理效率,并就水产养殖水体处理发展方向提出建议。

  19. 碳纳米管的纯化——电化学氧化法%Purification of Carbon Nanotubes Oxidation Method by Using Electrochemistry

    Institute of Scientific and Technical Information of China (English)

    杨占红; 吴浩青; 李晶; 李新海

    2001-01-01

    用电化学氧化法对碳纳米管进行纯化, 从稳态极化曲线出发, 对反应的可行性进行了分析, 考察了支持电解质、 电流密度、 时间等因素对反应的影响, 确定了最佳实验条件, 同时对纯化机理进行了解释.%Carbon nanotubes were purified by use of electrical chemistry oxidation method. In the view of polarization curves it was discussed that electrochemistry method could be used in the purification of carbon nanotubus. The influence of current density, sulfuric acid concentration and reaction time on the reaction was studied, the optimum experimental condition was obtained and the mechanism of purification was also discussed.

  20. Superparamagnetic poly(methyl methacrylate) beads for nattokinase purification from fermentation broth.

    Science.gov (United States)

    Yang, Chengli; Xing, Jianmin; Guan, Yueping; Liu, Huizhou

    2006-09-01

    An effective method for purification of nattokinase from fermentation broth using magnetic poly(methyl methacrylate) (PMMA) beads immobilized with p-aminobenzamidine was proposed in this study. Firstly, magnetic PMMA beads with a narrow size distribution were prepared by spraying suspension polymerization. Then, they were highly functionalized via transesterification reaction with polyethylene glycol. The surface hydroxyl-modified magnetic beads obtained were further modified with chloroethylamine to transfer the surface amino-modified magnetic functional beads. The morphology and surface functionality of the magnetic beads were examined by scanning electron microscopy and Fourier transform infrared. An affinity ligand, p-aminobenzamidine was covalently immobilized to the amino-modified magnetic beads by the glutaraldehyde method for nattokinase purification directly from the fermentation broth. The purification factor and the recovery of the enzyme activity were found to be 8.7 and 85%, respectively. The purification of nattokinase from fermentation broth by magnetic beads only took 40 min, which shows a very fast purification of nattokinase compared to traditional purification methods.

  1. Estimation of the overall kinetic parameters of enzyme inactivation using an isoconversional method

    OpenAIRE

    2008-01-01

    Estimation of the overall kinetic parameters of enzyme inactivation using an isoconversional method correspondance: Corresponding author. Tel./fax: +40 213275116, +40 722624585 (Mobile). (Raducan, Adina) (Raducan, Adina) Department of Physical Chemistry--> , Faculty of Chemistry--> , University of Bucharest--> , Bd. Elisabeta 4-12--> , 030018--> , Bucharest--> - RO...

  2. A simple enzymic method for the synthesis of [32P]phosphoenolpyruvate.

    Science.gov (United States)

    Parra, F

    1982-09-01

    A rapid and simple enzymic method is described for the synthesis of [(32)P]phosphoenolpyruvate from [(32)P]P(i), with a reproducible yield of 74%. The final product was shown to be a good substrate for pyruvate kinase (EC 2.7.1.40).

  3. Simple enzymic method for the synthesis of (/sup 32/P)phosphoenolpyruvate

    Energy Technology Data Exchange (ETDEWEB)

    Parra, F. (Cambridge Univ. (UK). Dept. of Biochemistry)

    1982-09-01

    A rapid and simple enzymic method is described for the synthesis of (/sup 32/P)phosphoenolpyruvate from (/sup 32/P)Psub(i), with a reproducible yield of 74%. The final product was shown to be a good substrate for pyruvate kinase (EC 2.7.1.40).

  4. A method for selection of restriction enzymes for sdCAPS marker construction

    Science.gov (United States)

    Development of PCR-based markers for SNP detection is prerequisite for various genetic analyses. The use of restriction enzymes following PCR amplification is a common and relatively low cost method for SNP detection. Simple and cost-effective methodologies for SNP marker development that would en...

  5. An enzyme-based in situ hybridisation method for the identification of Streptococcus suis - Brief report

    DEFF Research Database (Denmark)

    Madsen, L.W.; Boye, Mette; Jensen, Henrik E

    2001-01-01

    A method for enzyme-based in situ hybridisation of Streptococcus suis was developed. It enables the light microscopic localization of bacterial ribosomal RNA (rRNA) in formalin-fixed paraffin-embedded tissues. A unique sequence in the 16S rRNA of S. suis was targeted. Different pretreatment...

  6. Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Yacoby, I.; Tegler, L. T.; Pochekailov, S.; Zhang, S.; King, P. W.

    2012-04-01

    Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L{sup -1} of culture from E. coli with specific activities of 1000 U (U = 1 {micro}mol hydrogen evolved mg{sup -1} min{sup -1}). The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.

  7. Partial purification, characterization, and kinetic studies of a low-molecular-weight, alkali-tolerant chitinase enzyme from Bacillus subtilis JN032305, A potential biocontrol strain.

    Science.gov (United States)

    Shivakumar, Srividya; Karmali, Anika Nayak; Ruhimbana, Charles

    2014-01-01

    A new alkalophilic low-molecular-mass chitinase of 14 kD from the potent biocontrol agent Bacillus subtilis JN032305 was partially purified and enzymology of the chitinase was studied. The enzyme showed optimal pH of 9.0 and temperature of 50°C. The enzyme was found stable during the 60-min incubation at 50 °C. The chitinase was inhibited by group specific agents like IAA, DAN, TLCK, and SDS and metal ions Mg(2+), Ca(2+), Fe(2+), Mn(2+), Ba(2+), and Hg(2+), whereas Zn(2+) did not show significant inhibitory effect against the chitinase. PMSF partially inhibited the enzyme. Substrates specificity tests indicated that the enzyme showed 75% of relative activity on glycol chitin, 58% on carboxymethylcellulose (CMC), 33% on chitin flakes, and 166% laminarin compared to that on colloidal chitin. The enzyme also hydrolyzed 4-methylumbelliferyl-N-acetyl-D-glucosaminide, indicating its chitobiase activity. The chitinase of this study has broad specificity, which could hydrolyze not only the glycosidic bond in GlcNAc-GlcNAc but also that of related carbohydrates with glycosidic linkages. The partially purified chitinase not only showed antifungal activity against Rhizoctonia solani and Colletotrichum gloeosporioides, two potent phytopathogens of chilli, but also increased the germination of chilli seeds when infected with the two potent phytopathogenic fungi.

  8. Cysteine sulfinate desulfinase, a NIFS-like protein of Escherichia coli with selenocysteine lyase and cysteine desulfurase activities. Gene cloning, purification, and characterization of a novel pyridoxal enzyme.

    Science.gov (United States)

    Mihara, H; Kurihara, T; Yoshimura, T; Soda, K; Esaki, N

    1997-09-05

    Selenocysteine lyase (EC 4.4.1.16) exclusively decomposes selenocysteine to alanine and elemental selenium, whereas cysteine desulfurase (NIFS protein) of Azotobacter vinelandii acts indiscriminately on both cysteine and selenocysteine to produce elemental sulfur and selenium respectively, and alanine. These proteins exhibit some sequence homology. The Escherichia coli genome contains three genes with sequence homology to nifS. We have cloned the gene mapped at 63.4 min in the chromosome and have expressed, purified to homogeneity, and characterized the gene product. The enzyme comprises two identical subunits with 401 amino acid residues (Mr 43,238) and contains pyridoxal 5'-phosphate as a coenzyme. The enzyme catalyzes the removal of elemental sulfur and selenium atoms from L-cysteine, L-cystine, L-selenocysteine, and L-selenocystine to produce L-alanine. Because L-cysteine sulfinic acid was desulfinated to form L-alanine as the preferred substrate, we have named this new enzyme cysteine sulfinate desulfinase. Mutant enzymes having alanine substituted for each of the four cysteinyl residues (Cys-100, Cys-176, Cys-323, and Cys-358) were all active. Cys-358 corresponds to Cys-325 of A. vinelandii NIFS, which is conserved among all NIFS-like proteins and catalytically essential (Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714-4720), is not required for cysteine sulfinate desulfinase. Thus, the enzyme is distinct from A. vinelandii NIFS in this respect.

  9. A high volume extraction and purification method for recovering DNA from human bone.

    Science.gov (United States)

    Marshall, Pamela L; Stoljarova, Monika; Schmedes, Sarah E; King, Jonathan L; Budowle, Bruce

    2014-09-01

    DNA recovery, purity and overall extraction efficiency of a protocol employing a novel silica-based column, Hi-Flow(®) (Generon Ltd., Maidenhead, UK), were compared with that of a standard organic DNA extraction methodology. The quantities of DNA recovered by each method were compared by real-time PCR and quality of DNA by STR typing using the PowerPlex(®) ESI 17 Pro System (Promega Corporation, Madison, WI) on DNA from 10 human bone samples. Overall, the Hi-Flow method recovered comparable quantities of DNA ranging from 0.8ng±1 to 900ng±159 of DNA compared with the organic method ranging from 0.5ng±0.9 to 855ng±156 of DNA. Complete profiles (17/17 loci tested) were obtained for at least one of three replicates for 3/10 samples using the Hi-Flow method and from 2/10 samples with the organic method. All remaining bone samples yielded partial profiles for all replicates with both methods. Compared with a standard organic DNA isolation method, the results indicated that the Hi-Flow method provided equal or improved recovery and quality of DNA without the harmful effects of organic extraction. Moreover, larger extraction volumes (up to 20mL) can be employed with the Hi-Flow method which enabled more bone sample to be extracted at one time. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  10. Purification of flavonoids from licorice using an off-line preparative two-dimensional normal-phase liquid chromatography/reversed-phase liquid chromatography method.

    Science.gov (United States)

    Fan, Yunpeng; Fu, Yanhui; Fu, Qing; Cai, Jianfeng; Xin, Huaxia; Dai, Mei; Jin, Yu

    2016-07-01

    An orthogonal (71.9%) off-line preparative two-dimensional normal-phase liquid chromatography/reversed-phase liquid chromatography method coupled with effective sample pretreatment was developed for separation and purification of flavonoids from licorice. Most of the nonflavonoids were firstly removed using a self-made Click TE-Cys (60 μm) solid-phase extraction. In the first dimension, an industrial grade preparative chromatography was employed to purify the crude flavonoids. Click TE-Cys (10 μm) was selected as the stationary phase that provided an excellent separation with high reproducibility. Ethyl acetate/ethanol was selected as the mobile phase owing to their excellent solubility for flavonoids. Flavonoids co-eluted in the first dimension were selected for further purification using reversed-phase liquid chromatography. Multiple compounds could be isolated from one normal-phase fraction and some compounds with bad resolution in one-dimensional liquid chromatography could be prepared in this two-dimensional system owing to the orthogonal separation. Moreover, this two-dimensional liquid chromatography method was beneficial for the preparation of relatively trace flavonoid compounds, which were enriched in the first dimension and further purified in the second dimension. Totally, 24 flavonoid compounds with high purity were obtained. The results demonstrated that the off-line two-dimensional liquid chromatography method was effective for the preparative separation and purification of flavonoids from licorice.

  11. Preparation of monoclonal antibody of anti-feline calicivirus and establishment of double-antibody sandwich enzyme-linked immunosorbent assay detecting method.

    Science.gov (United States)

    Yuan, B; Ai, C-X; Yuan, L; Gao, W; Hu, J-P; Chen, J; Ren, W-Z

    2014-09-12

    This study aimed to prepare monoclonal antibody of feline calicivirus (FCV) and identify its basic biological characteristics. Saturated ammonium sulfate precipitation, combined differential centrifugation, and cesium chloride density gradient centrifugation were used for the purification of FCV. The purified FCV was used as antigen to immunize BALB/c mice. The hybridoma lines of anti-FCV monoclonal antibodies were established using cell fusion and hybridoma screening techniques. The subtypes of the monoclonal antibody were identified. The results showed that 3 strains of hybridoma cell lines stably secreted anti-FCV monoclonal antibody; they were named as D8, E5, and H4. The D8 and E5 were IgM subtype antibodies, and H4 was IgG2b subtype antibody. The monoclonal antibody obtained shared no cross-reactivity with feline parvovirus, canine parvovirus, and canine distemper virus. According to the different recognition sites of 2 monoclonal antibodies E5 and H4 to the FCV, they were used to coat microtiter plates and prepare 2 enzyme-labeled secondary antibodies to establish double-antibody sandwich enzyme-linked immunosorbent assay detecting method.

  12. Blood purification and hemo- perfusion

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The method of blood purification is a new overlapping frontierdiscipline which develops quickly in recent years. It helps overcoming many serious and complicated diseases, even including some incurable illnesses.

  13. Hydrodynamic Voltammetry as a Rapid and Simple Method for Evaluating Soil Enzyme Activities

    Directory of Open Access Journals (Sweden)

    Kazuto Sazawa

    2015-03-01

    Full Text Available Soil enzymes play essential roles in catalyzing reactions necessary for nutrient cycling in the biosphere. They are also sensitive indicators of ecosystem stress, therefore their evaluation is very important in assessing soil health and quality. The standard soil enzyme assay method based on spectroscopic detection is a complicated operation that requires the removal of soil particles. The purpose of this study was to develop a new soil enzyme assay based on hydrodynamic electrochemical detection using a rotating disk electrode in a microliter droplet. The activities of enzymes were determined by measuring the electrochemical oxidation of p-aminophenol (PAP, following the enzymatic conversion of substrate-conjugated PAP. The calibration curves of β-galactosidase (β-gal, β-glucosidase (β-glu and acid phosphatase (AcP showed good linear correlation after being spiked in soils using chronoamperometry. We also performed electrochemical detection using real soils. Hydrodynamic chronoamperometry can be used to assess the AcP in soils, with a detection time of only 90 s. Linear sweep voltammetry was used to measure the amount of PAP released from β-gal and β-glu by enzymatic reaction after 60 min. For the assessment of soil enzymes, the results of hydrodynamic voltammetry assay compared favorably to those using a standard assay procedure, but this new procedure is more user-friendly, rapid and simple.

  14. Hydrodynamic voltammetry as a rapid and simple method for evaluating soil enzyme activities.

    Science.gov (United States)

    Sazawa, Kazuto; Kuramitz, Hideki

    2015-03-04

    Soil enzymes play essential roles in catalyzing reactions necessary for nutrient cycling in the biosphere. They are also sensitive indicators of ecosystem stress, therefore their evaluation is very important in assessing soil health and quality. The standard soil enzyme assay method based on spectroscopic detection is a complicated operation that requires the removal of soil particles. The purpose of this study was to develop a new soil enzyme assay based on hydrodynamic electrochemical detection using a rotating disk electrode in a microliter droplet. The activities of enzymes were determined by measuring the electrochemical oxidation of p-aminophenol (PAP), following the enzymatic conversion of substrate-conjugated PAP. The calibration curves of β-galactosidase (β-gal), β-glucosidase (β-glu) and acid phosphatase (AcP) showed good linear correlation after being spiked in soils using chronoamperometry. We also performed electrochemical detection using real soils. Hydrodynamic chronoamperometry can be used to assess the AcP in soils, with a detection time of only 90 s. Linear sweep voltammetry was used to measure the amount of PAP released from β-gal and β-glu by enzymatic reaction after 60 min. For the assessment of soil enzymes, the results of hydrodynamic voltammetry assay compared favorably to those using a standard assay procedure, but this new procedure is more user-friendly, rapid and simple.

  15. An Approximate Analytical Method for the Evaluation of the Concentrations and Current for Hybrid Enzyme Biosensor

    OpenAIRE

    2013-01-01

    Mathematical modeling of amperometric biosensor with cyclic reaction is discussed. Analytical expressions pertaining to the concentration of substrate, cosubstrate, reducing agent and medial product and current for hybrid enzyme biosensor are obtained in terms of Thiele module and saturation parameters. In this paper, a powerful analytical method, called homotopy analysis method (HAM) is used to solve the system of nonlinear differential equations. Furthermore, in this work the numerical simu...

  16. Purification, characterization, and cloning of the gene for a biodegradable plastic-degrading enzyme from Paraphoma-related fungal strain B47-9.

    Science.gov (United States)

    Suzuki, Ken; Noguchi, Masako Tsujimoto; Shinozaki, Yukiko; Koitabashi, Motoo; Sameshima-Yamashita, Yuka; Yoshida, Shigenobu; Fujii, Takeshi; Kitamoto, Hiroko K

    2014-05-01

    Paraphoma-related fungal strain B47-9 secreted a biodegradable plastic (BP)-degrading enzyme which amounted to 68 % (w/w) of the total secreted proteins in a culture medium containing emulsified poly(butylene succinate-co-adipate) (PBSA) as sole carbon source. The gene for this enzyme was found to be composed of an open reading frame consisting of 681 nucleotides encoding 227 amino acids and two introns. Southern blot analysis showed that this gene exists as a single copy. The deduced amino acid sequence suggested that this enzyme belongs to the cutinase (E.C.3.1.1.74) family; thus, it was named P araphoma-related fungus cutinase-like enzyme (PCLE). It degraded various types of BP films, such as poly(butylene succinate), PBSA, poly(butylene adipate-co-terephthalate), poly(ε-caprolactone), and poly(DL-lactic acid). It has a molecular mass of 19.7 kDa, and an optimum pH and temperature for degradation of emulsified PBSA of 7.2 and 45 °C, respectively. Ca(2+) ion at a concentration of about 1.0 mM markedly enhanced the degradation of emulsified PBSA.

  17. The purification, crystallization and preliminary structural characterization of FAD-dependent monooxygenase PhzS, a phenazine-modifying enzyme from Pseudomonas aeruginosa

    Science.gov (United States)

    The blue chloroform-soluble bacterial metabolite pyocyanin (1-hydroxy-5-methyl-phenazine) contributes to the survival and virulence of Pseudomonas aeruginosa, an important Gram-negative opportunistic pathogen of humans and animals. Little is known about the two enzymes, designated PhzM and PhzS, tha...

  18. Purification of a new manganese peroxidase of the white-rot fungus Irpex lacteus, and degradation of polycyclic aromatic hydrocarbons by the enzyme.

    Science.gov (United States)

    Baborová, Petra; Möder, Monika; Baldrian, Petr; Cajthamlová, Kamila; Cajthaml, Tomás

    2006-04-01

    The white-rot fungus Irpex lacteus has been reported to be an efficient degrader of polycyclic aromatic hydrocarbons, polychlorinated biphenyls and pentachlorophenol. The fungus produces ligninolytic enzymes laccase, lignin peroxidase and manganese peroxidase (MnP), the latter being the major one produced. MnP was purified using anion exchange and size exclusion chromatography. SDS-PAGE showed the purified MnP to be a monomeric protein of 37 kDa (37.5 kDa using MALDI-TOF) with an isoelectric point at 3.55. The pH optimum was relatively broad, from 4.0 to 7.0 with a peak at pH 5.5. Kinetic constants K(m) were 8 microM for H(2)O(2) and 12 or 31 microM for Mn(2+) depending on the substrate. The enzyme did not perform oxidation in the absence of H(2)O(2) or Mn(2+). MnP was active at 5-70 degrees C with an optimum between 50-60 degrees C. At temperatures above 65 degrees C the enzyme rapidly lost activity. Degradation of four representatives of PAHs (phenanthrene, anthracene, fluoranthene, and pyrene) was tested and the enzyme showed the ability to degrade them in vitro. Major degradation products of anthracene were identified. The results confirm the role of MnP in PAH degradation by I. lacteus, including cleavage of the aromatic ring.

  19. Purification and characterization of 2-keto-3-deoxy-6-phosphogluconate aldolase from Azotobacter vinelandii: evidence that the enzyme is bifunctional towards 2-keto-4-hydroxy glutarate cleavage.

    Science.gov (United States)

    Taha, T S; Deits, T L

    1994-04-15

    2-keto-3-deoxy-6-phosphogluconate aldolase (E.C. 4.1.2.14) has been purified in two chromatographic steps to 99% purity in 73% overall yield from Azotobacter vinelandii. The pure enzyme is a 70 kD trimeric Class I aldolase, inhibitable by bromopyruvate or pyruvate plus sodium borohydride, with a specific activity of 625 mumol per min per mg protein and a Km of 38 microM for 2-keto-3-deoxy-6-phosphogluconate. The enzyme also has 2-keto-4-hydroxy glutarate aldolase (E.C. 4.1.3.16) activity, with a specific activity of 4.8 mumol per min per mg protein and a Km of 39 microM. 2-keto-4-hydroxy glutarate inhibits the 2-keto-3-deoxy-6-phosphogluconate aldolase activity of the enzyme with an apparent Ki of 0.17 mM. Slow steps following formation of the Schiff base intermediate between KHG and the enzyme are responsible for both the slower turnover of this substrate and for its inhibitory effect.

  20. A Method of Purification of Bovine Colostrum sIgA

    Institute of Scientific and Technical Information of China (English)

    LIU Xiaofei; GAO Xuejun; YAO Yonghao; ZHOU Shenghua

    2008-01-01

    sIgA in bovine colostrum was purified by ultrafiltration and enzymolysis methods in this experiment, and the prepared substance was detected by Western Blot. The purity and yield were up to 73.6% and 65.2%, respectively. This convenient technique offers helpful exploration for production of bovine colostrum sIgA.

  1. Evaluation of methods for the extraction and purification of DNA from the human microbiome.

    Directory of Open Access Journals (Sweden)

    Sanqing Yuan

    Full Text Available BACKGROUND: DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used. CONCLUSIONS/SIGNIFICANCE: Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.

  2. Purification of human albumin by the combination of the method of Cohn with liquid chromatography

    Directory of Open Access Journals (Sweden)

    Tanaka K.

    1998-01-01

    Full Text Available Large volumes of plasma can be fractionated by the method of Cohn at low cost. However, liquid chromatography is superior in terms of the quality of the product obtained. In order to combine the advantages of each method, we developed an integrated method for the production of human albumin and immunoglobulin G (IgG. The cryoprecipitate was first removed from plasma for the production of factor VIII and the supernatant of the cryoprecipitate was fractionated by the method of Cohn. The first precipitate, containing fractions (F-I + II + III, was used for the production of IgG by the chromatographic method (see Tanaka K et al. (1998 Brazilian Journal of Medical and Biological Research, 31: 1375-1381. The supernatant of F-I + II + III was submitted to a second precipitation and F-IV was obtained and discarded. Albumin was obtained from the supernatant of the precipitate F-IV by liquid chromatography, ion-exchange on DEAE-Sepharose FF, filtration through Sephacryl S-200 HR and introduction of heat treatment for fatty acid precipitation. Viral inactivation was performed by pasteurization at 60ºC for 10 h. The albumin product obtained by the proposed procedure was more than 99% pure for the 15 lots of albumin produced, with a mean yield of 25.0 ± 0.5 g/l plasma, containing 99.0 to 99.3% monomer, 0.7 to 1.0% dimers, and no polymers. Prekallikrein activator levels were <=5 IU/ml. This product satisfies the requirements of the 1997 Pharmacopée Européenne.

  3. Use of an enzyme-assisted method to improve protein extraction from olive leaves.

    Science.gov (United States)

    Vergara-Barberán, M; Lerma-García, M J; Herrero-Martínez, J M; Simó-Alfonso, E F

    2015-02-15

    The improvement of protein extraction from olive leaves using an enzyme-assisted protocol has been investigated. Using a cellulase enzyme (Celluclast® 1.5L), different parameters that affect the extraction process, such as the influence and amount of organic solvent, enzyme amount, pH and extraction temperature and time, were optimised. The influence of these factors was examined using the standard Bradford assay and the extracted proteins were characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum extraction parameters were: 30% acetonitrile, 5% (v/v) Celluclast® 1.5L at pH 5.0 and 55°C for 15min. Under these conditions, several protein extracts from olive leaves of different genetic variety (with a total protein amount comprised between 1.87 and 6.64mgg(-1)) were analysed and compared by SDS-PAGE, showing differences in their electrophoretic protein profiles. The developed enzyme-assisted extraction method has shown a faster extraction, higher recovery and reduced solvent usage with respect to the use of the non-enzymatic methods described in literature.

  4. A robust and efficient method for estimating enzyme complex abundance and metabolic flux from expression data.

    Science.gov (United States)

    Barker, Brandon E; Sadagopan, Narayanan; Wang, Yiping; Smallbone, Kieran; Myers, Christopher R; Xi, Hongwei; Locasale, Jason W; Gu, Zhenglong

    2015-12-01

    A major theme in constraint-based modeling is unifying experimental data, such as biochemical information about the reactions that can occur in a system or the composition and localization of enzyme complexes, with high-throughput data including expression data, metabolomics, or DNA sequencing. The desired result is to increase predictive capability and improve our understanding of metabolism. The approach typically employed when only gene (or protein) intensities are available is the creation of tissue-specific models, which reduces the available reactions in an organism model, and does not provide an objective function for the estimation of fluxes. We develop a method, flux assignment with LAD (least absolute deviation) convex objectives and normalization (FALCON), that employs metabolic network reconstructions along with expression data to estimate fluxes. In order to use such a method, accurate measures of enzyme complex abundance are needed, so we first present an algorithm that addresses quantification of complex abundance. Our extensions to prior techniques include the capability to work with large models and significantly improved run-time performance even for smaller models, an improved analysis of enzyme complex formation, the ability to handle large enzyme complex rules that may incorporate multiple isoforms, and either maintained or significantly improved correlation with experimentally measured fluxes. FALCON has been implemented in MATLAB and ATS, and can be downloaded from: https://github.com/bbarker/FALCON. ATS is not required to compile the software, as intermediate C source code is available. FALCON requires use of the COBRA Toolbox, also implemented in MATLAB.

  5. A new multi-wavelength model-based method for determination of enzyme kinetic parameters

    Indian Academy of Sciences (India)

    Mohammad-Hossein Sorouraddin; Kaveh Amini; Abdolhossein Naseri; Javad Vallipour; Jalal Hanaee; Mohammad-Reza Rashidi

    2010-09-01

    Lineweaver–Burk plot analysis is the most widely used method to determine enzyme kinetic parameters. In the spectrophotometric determination of enzyme activity using the Lineweaver–Burk plot, it is necessary to find a wavelength at which only the substrate or the product has absorbance without any spectroscopic interference of the other reaction components. Moreover, in this method, different initial concentrations of the substrate should be used to obtain the initial velocities required for Lineweaver–Burk plot analysis. In the present work, a multi-wavelength model-based method has been developed and validated to determine Michaelis–Menten constants for some enzyme reactions. In this method, a selective wavelength region and several experiments with different initial concentrations of the substrate are not required. The absorbance data of the kinetic assays are fitted by non-linear regression coupled to the numeric integration of the related differential equation. To indicate the applicability of the proposed method, the Michaelis–Menten constants for the oxidation of phenanthridine, 6-deoxypenciclovir and xanthine by molybdenum hydroxylases were determined using only a single initial concentration of the substrate, regardless of any spectral overlap.

  6. A new multi-wavelength model-based method for determination of enzyme kinetic parameters.

    Science.gov (United States)

    Sorouraddin, Mohammad-Hossein; Amini, Kaveh; Naseri, Abdolhossein; Vallipour, Javad; Hanaee, Jalal; Rashidi, Mohammad-Reza

    2010-09-01

    Lineweaver-Burk plot analysis is the most widely used method to determine enzyme kinetic parameters. In the spectrophotometric determination of enzyme activity using the Lineweaver-Burk plot, it is necessary to find a wavelength at which only the substrate or the product has absorbance without any spectroscopic interference of the other reaction components. Moreover, in this method, different initial concentrations of the substrate should be used to obtain the initial velocities required for Lineweaver-Burk plot analysis. In the present work, a multi-wavelength model-based method has been developed and validated to determine Michaelis-Menten constants for some enzyme reactions. In this method, a selective wavelength region and several experiments with different initial concentrations of the substrate are not required. The absorbance data of the kinetic assays are fitted by non-linear regression coupled to the numeric integration of the related differential equation. To indicate the applicability of the proposed method, the Michaelis-Menten constants for the oxidation of phenanthridine, 6-deoxypenciclovir and xanthine by molybdenum hydroxylases were determined using only a single initial concentration of the substrate, regardless of any spectral overlap.

  7. A rapid, cost-effective method of assembly and purification of synthetic DNA probes >100 bp.

    Directory of Open Access Journals (Sweden)

    Michael A Jensen

    Full Text Available Here we introduce a rapid, cost-effective method of generating molecular DNA probes in just under 15 minutes without the need for expensive, time-consuming gel-extraction steps. As an example, we enzymatically concatenated six variable strands (50 bp with a common strand sequence (51 bp in a single pool using Fast-Link DNA ligase to produce 101 bp targets (10 min. Unincorporated species were then filtered out by passing the crude reaction through a size-exclusion column (12 could be achieved with further optimization. Moreover, for large-scale assays, we envision this method to be fully automated with the use of robotics such as the Biomek FX; here, potentially thousands of samples could be pooled, ligated and purified in either a 96, 384 or 1536-well platform in just minutes.

  8. Synthesis of Cell Adhesive Motif RGD Tripeptide by a Novel Chemical Method and Its Purification

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The cell adhesive motif RGD tripeptide was synthesized by using a novel chemical method. First, Gly-Asp(GD)was synthesized in two steps including the chloroacetylation of free L-aspartic acid and the ammonolysis of the chloroacetylated L-aspartic acid. The yield of chloroacetylated L-aspartic acid was 83.0%. For the ammonolysis of chloroacetylated L-aspartic acid, the yield of the ammonolyzed product was 92.3%. Second, the coupling between Arg and Gly-Asp was carried out by using the NCA method. The maximum yield of RGD was about 50% at 0℃ and pH =9. 5. The prepared RGD tripeptide was confirmed by using amino acid component analysis and mass spectrographic analysis.

  9. Development of a nucleotide sugar purification method using a mixed mode column & mass spectrometry detection.

    Science.gov (United States)

    Eastwood, Heather; Xia, Fang; Lo, Mei-Chu; Zhou, Jing; Jordan, John B; McCarter, John; Barnhart, Wesley W; Gahm, Kyung-Hyun

    2015-11-10

    Analysis of nucleotide sugars, nucleoside di- and triphosphates and sugar-phosphates is an essential step in the process of understanding enzymatic pathways. A facile and rapid separation method was developed to analyze these compounds present in an enzymatic reaction mixture utilized to produce nucleotide sugars. The Primesep SB column explored in this study utilizes hydrophobic interactions as well as electrostatic interactions with the phosphoric portion of the nucleotide sugars. Ammonium formate buffer was selected due to its compatibility with mass spectrometry. Negative ion mode mass spectrometry was adopted for detection of the sugar phosphate (fucose-1-phophate), as the compound is not amenable to UV detection. Various mobile phase conditions such as pH, buffer concentration and organic modifier were explored. The semi-preparative separation method was developed to prepare 30mg of the nucleotide sugar. (19)F NMR was utilized to determine purity of the purified fluorinated nucleotide sugar. The collected nucleotide sugar was found to be 99% pure.

  10. Human papillomavirus DNA from warts for typing by endonuclease restriction patterns: purification by alkaline plasmid methods.

    Science.gov (United States)

    Chinami, M; Tanikawa, E; Hachisuka, H; Sasai, Y; Shingu, M

    1990-01-01

    The alkaline plasmid DNA extraction method of Birnboim and Doly was applied for the isolation of human papillomavirus (HPV) from warts. Tissue from common and plantar warts was digested with proteinase K, and the extrachromosomal circular covalently-closed form of HPV-DNA was rapidly extracted by alkaline sodium dodecyl sulphate and phenol-chloroform treatment. Recovery of HPV-DNA from the tissue was sufficient for determination of endonuclease restriction patterns by agarose gel electrophoresis.

  11. Chromatofocusing: a new method for purification of staphylococcal enterotoxins B and C1.

    OpenAIRE

    Ende, I A; Terplan, G; Kickhöfen, B.; Hammer, D K

    1983-01-01

    A new chromatographic procedure was developed which obtained highly purified preparations of staphylococcal enterotoxins B and C1 in yields of 60% from cultures of Staphylococcus aureus and which is faster than any of the separation methods used previously. The procedure involves chromatography on carboxymethylcellulose, removal of alpha-toxin by adsorption to rabbit erythrocyte membranes, and finally, chromatofocusing as the fundamental new step. Enterotoxins were obtained in highly purified...

  12. Nanolipoprotein particles and related methods and systems for protein capture, solubilization, and/or purification

    Energy Technology Data Exchange (ETDEWEB)

    Chromy, Brett A.; Henderson, Paul; Hoeprich, Jr, Paul D.

    2016-10-04

    Provided herein are methods and systems for assembling, solubilizing and/or purifying a membrane associated protein in a nanolipoprotein particle, which comprise a temperature transition cycle performed in presence of a detergent, wherein during the temperature transition cycle the nanolipoprotein components are brought to a temperature above and below the gel to liquid crystalling transition temperature of the membrane forming lipid of the nanolipoprotein particle.

  13. Standardization of water purification in the central dialysis fluid delivery system: validation and parametric method.

    Science.gov (United States)

    Tomo, Tadashi; Shinoda, Tosiho

    2009-01-01

    The central dialysis fluid delivery system (CDDS) has been mainly used for hemodialysis therapy in Japan. Validation and a parametric method are necessary for the quality control of dialysis fluid in CDDS. Validation is a concept for the assurance of system compatibility and product quality, and is defined as follows: the manufacturing and quality control methods including the system design and equipment of the manufacturing facility, manufacturing procedure and processes. Confirmed results must be kept within acceptable limits and they must be documented in a record. Important parameters for validating CDDS include: (1) setting the sterilized area; (2) decision of sterilization level; (3) confirmation of the maximum bio-burden; (4) performance of endotoxin retentive filter and reverse osmosis (RO) module, and (5) checkpoints of purity of dialysis water in the system. Taking the concept of validation and a parametric method in the management of CDDS into consideration enables the supply the purified dialysis fluid or the online prepared substitution fluid that meet the 2008 standards of the Japanese Society for Dialysis Therapy.

  14. A new versatile microarray-based method for high-throughput screening of carbohydrate-active enzymes

    DEFF Research Database (Denmark)

    Vidal Melgosa, Silvia; Pedersen, Henriette Lodberg; Schückel, Julia;

    2015-01-01

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing, together with associated bioinformatic tools have identified vast numbers of putative carbohydrate degrading and modifying enzymes including glycoside hydrolases...... and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high-throughput and versatile...... semi-quantitative enzyme-screening technique which requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme cocktails and crude culture broths against single substrates, substrate mixtures and biomass samples. Moreover, we show...

  15. Benzylidenemalononitrile derivatives as substrates and inhibitors of a new NAD(P)H dehydrogenase of erythrocytes. Purification and crystallisation of two forms of the enzyme.

    Science.gov (United States)

    Ueberschär, K H; Kille, S; Laule, G; Maurer, P; Wallenfels, K

    1979-10-01

    Using the powerful lachrymator (2-chlorobenzylidene)malononitrile as electron acceptor, two types of NAD(P)H dehydrogenases have been isolated from human blood. Crystallisation of the homogenous enzymes was performed in 50% polyethylene glycol solution. The enzymes (average molecular weight 18 000) are composed of only one polypeptide chain and have a very similar amino acid composition. B-side stereospecificity was determined with respect to the cofactor by gas chromatography-mass spectrometry for the reductase. Besides (2-chlorobenzylidene)malononitrile, 2,6-dichloroindophenol, methylene blue, 4-benzoquinone, FMN and FAD are also reduced using NADH or NADPH as hydrogen donor with the rates decreasing in the given order. Reduction of methemoglobin is observed only upon addition of methylene blue, FMN or FAD as carriers. (2-Chlorobenzylidene)malononitrile reduction is inhibited by most of the compounds known to be decouplers of oxidative phosphorylation.

  16. 4-dihydrotrisporin-dehydrogenase, an enzyme of the sex hormone pathway of Mucor mucedo: purification, cloning of the corresponding gene, and developmental expression.

    Science.gov (United States)

    Wetzel, Jana; Scheibner, Olaf; Burmester, Anke; Schimek, Christine; Wöstemeyer, Johannes

    2009-01-01

    The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (-) mating-type-specific enzyme in the pathway from beta-carotene to trisporic acid. This substance and its isomers and derivatives represent the general system of sexual communication in zygomycetes. The (-) mating type of Mucor mucedo was stimulated by trisporic acid and the enzyme was purified by ion exchange and affinity chromatography. Several peptides of the 26-kDa protein, digested with trypsin, were sequenced by mass spectrometry. Oligonucleotides based on protein sequence data were used for PCR amplification of genomic DNA. The primary PCR fragment was sequenced and the complete gene, TSP2, was isolated. A labeled TSP2 hybridization probe detects a single-copy gene in the genome of M. mucedo. Northern blot analysis with RNAs from different growth stages reveals that the expression of the gene depends on the developmental stage of the mycelium in both mating types of M. mucedo. At the enzyme level, activity is found exclusively in the (-) mating type. However, renaturation of proteins in sodium dodecyl sulfate-containing gels revealed the TSP2 gene product in both mating types. Analyzing the protein sequence places the enzyme in the short chain dehydrogenase superfamily. Thus, it has an evolutionary origin distinct from that of the previously isolated 4-dihydromethyltrisporate dehydrogenase, which belongs to the aldo/keto reductase superfamily. Apart from the TSP2 genes in the three sequenced zygomycetous genomes (Phycomyces blakesleeanus, Rhizopus oryzae, and Mucor circinelloides), the closest relative is the Myxococcus xanthus CsgA gene product, which is also a short chain dehydrogenase, involved in C signaling and fruiting body formation.

  17. 4-Dihydrotrisporin-Dehydrogenase, an Enzyme of the Sex Hormone Pathway of Mucor mucedo: Purification, Cloning of the Corresponding Gene, and Developmental Expression▿

    Science.gov (United States)

    Wetzel, Jana; Scheibner, Olaf; Burmester, Anke; Schimek, Christine; Wöstemeyer, Johannes

    2009-01-01

    The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (−) mating-type-specific enzyme in the pathway from β-carotene to trisporic acid. This substance and its isomers and derivatives represent the general system of sexual communication in zygomycetes. The (−) mating type of Mucor mucedo was stimulated by trisporic acid and the enzyme was purified by ion exchange and affinity chromatography. Several peptides of the 26-kDa protein, digested with trypsin, were sequenced by mass spectrometry. Oligonucleotides based on protein sequence data were used for PCR amplification of genomic DNA. The primary PCR fragment was sequenced and the complete gene, TSP2, was isolated. A labeled TSP2 hybridization probe detects a single-copy gene in the genome of M. mucedo. Northern blot analysis with RNAs from different growth stages reveals that the expression of the gene depends on the developmental stage of the mycelium in both mating types of M. mucedo. At the enzyme level, activity is found exclusively in the (−) mating type. However, renaturation of proteins in sodium dodecyl sulfate-containing gels revealed the TSP2 gene product in both mating types. Analyzing the protein sequence places the enzyme in the short chain dehydrogenase superfamily. Thus, it has an evolutionary origin distinct from that of the previously isolated 4-dihydromethyltrisporate dehydrogenase, which belongs to the aldo/keto reductase superfamily. Apart from the TSP2 genes in the three sequenced zygomycetous genomes (Phycomyces blakesleeanus, Rhizopus oryzae, and Mucor circinelloides), the closest relative is the Myxococcus xanthus CsgA gene product, which is also a short chain dehydrogenase, involved in C signaling and fruiting body formation. PMID:18931040

  18. PEGylated Human Serum Albumin: Review of PEGylation, Purification and Characterization Methods

    Directory of Open Access Journals (Sweden)

    Parvin Akbarzadehlaleh

    2016-09-01

    Full Text Available Human serum albumin (HSA is a non-glycosylated, negatively charged protein (Mw: about 65-kDa that has one free cystein residue (Cys 34, and 17 disulfide bridges that these bridges have main role in its stability and longer biological life-time (15 to 19 days. As HSA is a multifunctional protein, it can also bind to other molecules and ions in addition to its role in maintaining colloidal osmotic pressure (COP in various diseases. In critical illnesses changes in the level of albumin between the intravascular and extravascular compartments and the decrease in its serum concentration need to be compensated using exogenous albumin; but as the size of HSA is an important parameter in retention within the circulation, therefore increasing its molecular size and hydrodynamic radius of HSA by covalent attachment of poly ethylene glycol (PEG, that is known as PEGylation, provides HSA as a superior volume expander that not only can prevent the interstitial edema but also can reduce the infusion frequency. This review focuses on various PEGylation methods of HSA (solid phase and liquid phase, and compares various methods to purifiy and characterize the pegylated form.

  19. PEGylated Human Serum Albumin: Review of PEGylation, Purification and Characterization Methods

    Science.gov (United States)

    Akbarzadehlaleh, Parvin; Mirzaei, Mona; Mashahdi-Keshtiban, Mahdiyeh; Shamsasenjan, Karim; Heydari, Hamidreza

    2016-01-01

    Human serum albumin (HSA) is a non-glycosylated, negatively charged protein (Mw: about 65-kDa) that has one free cystein residue (Cys 34), and 17 disulfide bridges that these bridges have main role in its stability and longer biological life-time (15 to 19 days). As HSA is a multifunctional protein, it can also bind to other molecules and ions in addition to its role in maintaining colloidal osmotic pressure (COP) in various diseases. In critical illnesses changes in the level of albumin between the intravascular and extravascular compartments and the decrease in its serum concentration need to be compensated using exogenous albumin; but as the size of HSA is an important parameter in retention within the circulation, therefore increasing its molecular size and hydrodynamic radius of HSA by covalent attachment of poly ethylene glycol (PEG), that is known as PEGylation, provides HSA as a superior volume expander that not only can prevent the interstitial edema but also can reduce the infusion frequency. This review focuses on various PEGylation methods of HSA (solid phase and liquid phase), and compares various methods to purifiy and characterize the pegylated form. PMID:27766215

  20. Methods for Efficiently and Accurately Computing Quantum Mechanical Free Energies for Enzyme Catalysis.

    Science.gov (United States)

    Kearns, F L; Hudson, P S; Boresch, S; Woodcock, H L

    2016-01-01

    Enzyme activity is inherently linked to free energies of transition states, ligand binding, protonation/deprotonation, etc.; these free energies, and thus enzyme function, can be affected by residue mutations, allosterically induced conformational changes, and much more. Therefore, being able to predict free energies associated with enzymatic processes is critical to understanding and predicting their function. Free energy simulation (FES) has historically been a computational challenge as it requires both the accurate description of inter- and intramolecular interactions and adequate sampling of all relevant conformational degrees of freedom. The hybrid quantum mechanical molecular mechanical (QM/MM) framework is the current tool of choice when accurate computations of macromolecular systems are essential. Unfortunately, robust and efficient approaches that employ the high levels of computational theory needed to accurately describe many reactive processes (ie, ab initio, DFT), while also including explicit solvation effects and accounting for extensive conformational sampling are essentially nonexistent. In this chapter, we will give a brief overview of two recently developed methods that mitigate several major challenges associated with QM/MM FES: the QM non-Boltzmann Bennett's acceptance ratio method and the QM nonequilibrium work method. We will also describe usage of these methods to calculate free energies associated with (1) relative properties and (2) along reaction paths, using simple test cases with relevance to enzymes examples. © 2016 Elsevier Inc. All rights reserved.

  1. 天然气净化业务能效对标方法探索%Energy efficiency benchmarking methods in natural gas purification business

    Institute of Scientific and Technical Information of China (English)

    苟小静; 黄朝齐; 龚毅然; 陈世明; 王灵军

    2014-01-01

    According to the requirements of PetroChina Exploration and Production Branch , Southwest Oil & Gasfield Company carried out energy efficiency benchmarking for gas purifica-tion business .The method of “subdivision unit ,and ratings contrast”was used to basically over-come the differences between purification devices ,and eliminate the impact level of non-compara-ble factors ,w hich provides a feasible idea for energy efficiency benchmarking of oil and gas field upstream business .The technology difficulties and solution ideas of energy efficiency benchmark-ing in gas purification business were summarized .%按照中国石油勘探与生产分公司要求,西南油气田公司开展了天然气净化业务能效对标试点,采用“细分单元、分级对比”的方法,基本实现了克服天然气净化装置间的差异性、消除不可比因素的影响程度,为油气田上游业务能效对标工作的开展提供了一种可行的思路,并总结了天然气净化业务能效对标技术难点与解决思路。

  2. Distinguishing enzymes using metabolome data for the hybrid dynamic/static method

    Directory of Open Access Journals (Sweden)

    Nakayama Yoichi

    2007-05-01

    Full Text Available Abstract Background In the process of constructing a dynamic model of a metabolic pathway, a large number of parameters such as kinetic constants and initial metabolite concentrations are required. However, in many cases, experimental determination of these parameters is time-consuming. Therefore, for large-scale modelling, it is essential to develop a method that requires few experimental parameters. The hybrid dynamic/static (HDS method is a combination of the conventional kinetic representation and metabolic flux analysis (MFA. Since no kinetic information is required in the static module, which consists of MFA, the HDS method may dramatically reduce the number of required parameters. However, no adequate method for developing a hybrid model from experimental data has been proposed. Results In this study, we develop a method for constructing hybrid models based on metabolome data. The method discriminates enzymes into static modules and dynamic modules using metabolite concentration time series data. Enzyme reaction rate time series were estimated from the metabolite concentration time series data and used to distinguish enzymes optimally for the dynamic and static modules. The method was applied to build hybrid models of two microbial central-carbon metabolism systems using simulation results from their dynamic models. Conclusion A protocol to build a hybrid model using metabolome data and a minimal number of kinetic parameters has been developed. The proposed method was successfully applied to the strictly regulated central-carbon metabolism system, demonstrating the practical use of the HDS method, which is designed for computer modelling of metabolic systems.

  3. Purification and immobilization of L-arginase from thermotolerant Penicillium chrysogenum KJ185377.1; with unique kinetic properties as thermostable anticancer enzyme.

    Science.gov (United States)

    El-Sayed, Ashraf S; Shindia, Ahmed A; Diab, Ayman A; Rady, Amgad M

    2014-10-18

    L-Arginase, hydrolyzing L-arginine to L-ornithine and urea, is a powerful anticancer, L-arginine-depleting agent, against argininosuccinate synthase expressing tumors. Otherwise, the higher antigenicity and lower thermal stability of this enzyme was the main biochemical hurdles. Since, the intrinsic thermal stability of enzymes follow the physiological temperature of their producer, thus, characterization of L-arginase from thermotolerant Penicillium chrysogenum was the objective of this study. L-Arginase (Arg) was purified to its homogeneity from P. chrysogenum by 10.1-fold, with 37.0 kDa under denaturing PAGE, optimum reaction at 50 °C, pH stability (6.8-7.9), with highest molar ratio of constitutional arginine, glutamic acid, lysine and aspartic acid. The purified enzyme was PEGylated and immobilized on chitosan, with 41.9 and 22.1 % yield of immobilization. At 40 °C, the T1/2 value of free-Arg, PEG-Arg and Chit-Arg was 10.4, 15.6, 20.5 h, respectively. The free-Arg and Chit-Arg have a higher affinity to L-arginine (K m 4.8 mM), while, PEG-Arg affinity was decreased by about 3 fold (K m 15.2 mM). The inhibitory constants to the free and PEG-Arg were relatively similar towards HA and PPG. The IC50 for the free enzyme against HEPG-2 and A549 tumor cells was 0.136 and 0.165 U/ml, comparing to 0.232 and 0.496 U/ml for PEG-Arg, respectively. The in vivo T1/2 to the free Arg and PEG-Arg was 16.4 and 20.4 h, respectively as holo-enzyme. The residual L-arginine level upon using free Arg was 156.9 and 144.5 µM, after 6 and 8 h, respectively, regarding to initials at 253.6 µM, while for Peg-Arg the level of L-arginine was nil till 7 h of initial dosing. The titer of IgG was induced by 10-15 % in response to free-Arg after 28 days comparing to IgG titer for PEG-Arg.

  4. Preparation of 5N high purified indium by the method of chemical purification-electrolysis

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The application of indium requires high purity indium as material. 5N high purity indium had been prepared by the method of a combination of chemically smelting and electrolysis. Smelting time was 10 min, the abstraction rate of cadmium was 80%-90% when used solution of I2-KI and glycerine to smelt indium. 4N metal indium was used as anode, high purity indium as cathode, In2(SO4)3-H2SO4 system as electrolyte, and In content is 100 g/L, pH 2-3 and current density 80-100 A/m2. The thallium was removed by smelting indium using 15% NH4Cl-glycerine solution for 20 min and tin by smelting indium using NaOH and NaNO3 for 20 min. The removed rate of tin was 60%.The product quality of indium reached national standard of 5N high purity indium.

  5. Development of a method for the purification and culture of rodent astrocytes.

    Science.gov (United States)

    Foo, Lynette C; Allen, Nicola J; Bushong, Eric A; Ventura, P Britten; Chung, Won-Suk; Zhou, Lu; Cahoy, John D; Daneman, Richard; Zong, Hui; Ellisman, Mark H; Barres, Ben A

    2011-09-08

    The inability to purify and culture astrocytes has long hindered studies of their function. Whereas astrocyte progenitor cells can be cultured from neonatal brain, culture of mature astrocytes from postnatal brain has not been possible. Here, we report a new method to prospectively purify astrocytes by immunopanning. These astrocytes undergo apoptosis in culture, but vascular cells and HBEGF promote their survival in serum-free culture. We found that some developing astrocytes normally undergo apoptosis in vivo and that the vast majority of astrocytes contact blood vessels, suggesting that astrocytes are matched to blood vessels by competing for vascular-derived trophic factors such as HBEGF. Compared to traditional astrocyte cultures, the gene profiles of the cultured purified postnatal astrocytes much more closely resemble those of in vivo astrocytes. Although these astrocytes strongly promote synapse formation and function, they do not secrete glutamate in response to stimulation.

  6. [Potential of nitrification and denitrification in water purification system with hydroponic bio-filter method].

    Science.gov (United States)

    Li, Xian-ing; Lu, Xi-wu; Song, Hai-liang; Osamu, Nishimura; Yuhei, Inamori

    2005-03-01

    The potential of nitrification and denitrification of sediment and the density of ammonium-oxidizing bacteria and nitrite-oxidizing bacteria in sediment in water quality purifying system with hydroponic bio-filter method (HBFM) were measured. The variation of nitrification and denitrification potential of the sediment along the stream way was quantitatively studied. The results show that among the sediments from front, middle and retral part of the stream way, the sediment from middle part reached a maximum nitrification potential . nitrification potential of 4.76 x 10(-6) g/(g x h), while the sediment from front part reached a maximum denitrification potential of 8 .1 x 10(-7) g/(g x h). The distribution of nitrification potential accords with the ammonium-oxidizing bacteria density. The key for improving nitrogen removal efficiency of HBFM system consists in changing nitrification & denitrification region distributing and accordingly enhances denitrification process.

  7. Improvised purification methods for obtaining individual drinking water supply under war and extreme shortage conditions.

    Science.gov (United States)

    Kozlicic, A; Hadzic, A; Bevanda, H

    1994-01-01

    Supplying an adequate amount of drinking water to a population is a complex problem that becomes an extremely difficult task in war conditions. In this paper, several simple methods for obtaining individual supplies of drinking water by filtration of atmospheric water with common household items are reported. Samples of atmospheric water (rain and snow) were collected, filtered, and analyzed for bacteriological and chemical content. The ability of commonly available household materials (newspaper, filter paper, gauze, cotton, and white cotton cloth) to filter water from the environmental sources was compared. According to chemical and biological analysis, the best results were obtained by filtering melted snow from the ground through white cotton cloth. Atmospheric water collected during war or in extreme shortage conditions can be purified with simple improvised filtering techniques and, if chlorinated, used as an emergency potable water source.

  8. Polonium purification

    Energy Technology Data Exchange (ETDEWEB)

    Baker, J.D.

    1996-09-01

    Three processes for the purification of {sup 210}Po from irradiated bismuth targets are described. Safety equipment includes shielded hotcells for the initial separation from other activation products, gloveboxes for handling the volatile and highly toxic materials, and provisions for ventilation. All chemical separations must be performed under vacuum or in inerted systems. Two of the processes require large amounts of electricity; the third requires vessels made from exotic materials.

  9. Production of microbial enzymes by new method of cultivation of microorganisms

    Directory of Open Access Journals (Sweden)

    Blieva Raushan

    2014-10-01

    Full Text Available We have developed efficient methods for long-term culturing and selection of highly active versions of the original cultures of micromycetes – producers of enzymes. We theoretically substantiated and experimentally confirmed an advantage of growing micromycetes in a new filament-spongy immobilized growth structure on the substrate relative to the traditional method of deep cultivation of free cells in the form of pellets. When comparing a traditional with our innovative method of cultivation, many advantages of the latter are revealed, above all being the possibility of the formation of new highly selective cultures in the long process of their growth with modified culturally - morphological properties.

  10. Elaboration of Method of Long-Term Culturing and Selection of Enzyme Producers

    Directory of Open Access Journals (Sweden)

    Blieva Raushan

    2014-06-01

    Full Text Available On the basis of the conducted researches on pectin lyase and proteolytic enzyme biosynthesis by Penicillium and Asperaillus micromycetes we have developed efficient methods for their cultivation and selection. We theoretically substantiated and experimentally confirmed an advantage of growing micromycetes in a new filament-spongy immobilized growth structure on the substrate relative to the traditional method of deep cultivation of free cells in the form of pellets. When comparing a traditional with our innovative method of cultivation, many advantages of the latter are revealed, above all being the possibility of the formation of new highly selective cultures in the long process of their growth with modified culturally - morphological properties.

  11. Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them

    Science.gov (United States)

    Gray, Kevin A [San Diego, CA; Zhao, Lishan [Emeryville, CA; Cayouette, Michelle H [San Diego, CA

    2012-01-24

    The invention provides polypeptides having any cellulolytic activity, e.g., a cellulase activity, a endoglucanase, a cellobiohydrolase, a beta-glucosidase, a xylanase, a mannanse, a .beta.-xylosidase, an arabinofuranosidase, and/or an oligomerase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. In one aspect, the invention provides polypeptides having an oligomerase activity, e.g., enzymes that convert recalcitrant soluble oligomers to fermentable sugars in the saccharification of biomass. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

  12. Enzymic sulfation of bile salts. Partial purification and characterization of an enzyme from the liver of the shark Heterodontus portusjacksoni that catalyses the sulfation of the shark bile steroid 5 beta-scymnol.

    Science.gov (United States)

    Macrides, T A; Faktor, D A; Kalafatis, N; Amiet, R G

    1994-03-01

    An enzyme system which catalyses the transfer of the sulfate group from 3'-phosphoadenosine-5'-phosphosulfate to the bile steroid 5 beta-scymnol has been isolated and characterized from the liver of the shark Heterodontus portusjacksoni (Meyer 1793). The enzyme is present in the cytosol fraction of liver cells. It was partially purified by hydroxylapatite chromatography, molecular sizing by G100-Sephadex and isoelectrofocusing electrophoresis. The apparent Km value for 3'-phosphoadenosine-5'-phosphosulfate was 4 microM and that for 5 beta-scymnol, 14 microM. The enzyme activity is inhibited by iodoacetate and p-chloromercuribenzoate indicating the possible requirement of a sulfhydryl group for activity. The molecular weight of the enzyme was estimated to be 40 kDa by gel filtration. This was verified by running the partially purified material on a native gel and electrophoretically separating two major bands corresponding to molecular weights of 40 and 45 kDa, respectively. Isoelectric focusing of the purified material resulted in two major bands with pI values of 5.0 and 5.85. Enzymatic activity was found to be optimal at a pH of 6.5 with little activity recorded at pH 5.0 and 8.0.

  13. Culturing Schwann Cells from Neonatal Rats by Improved Enzyme Digestion Combined with Explants-culture Method.

    Science.gov (United States)

    Liu, Di; Liang, Xiao-Chun; Zhang, Hong

    2016-08-01

    Objective To develop an improved method for culturing Schwann cells(SCs) by using both enzyme digestion and explants-culture approaches and compared with traditional explants-culture method and general hemi-explants-culture method. Methods Bilaterally sciatic nerves and brachial plexus nerves were dissected from 3 to 5-day-old neonatal SD rats and explants-culture method,general hemi-explants-culture method,and improved enzyme digestion combined with explants-culture method were adopted to culture SCs,respectively. SCs were digested and passaged after 7 days in culture and counted under the microscope. The purity of SCs was identified by S-100 immunofluorescence staining. Results The SCs of improved method group grew fastest and the total number of cells obtained was(1.85±0.13)×10(6);the SCs of the hemi-explants-culture method group grew slower than the improved method group and the total number of cells obtained was (1.10±0.10)×10(6);the SCs of the explants-culture method group grew slowest and the total number of cells obtained was (0.77±0.03)×10(6).The total number of cells obtained showed significant difference among the three groups(Pculture method group,and (74.50±4.23)% in the explants-culture method group(Pculture method can obtain sufficient amount of high-purity SCs in a short time and thus may be applied in further research on peripheral nerve regeneration.

  14. Simple method for Shiga toxin 2e purification by affinity chromatography via binding to the divinyl sulfone group.

    Directory of Open Access Journals (Sweden)

    Hideyuki Arimitsu

    Full Text Available Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e, a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease.

  15. Comparison of expression, purification and characterization of a new pectate lyase from Phytophthora capsici using two different methods

    Directory of Open Access Journals (Sweden)

    Zhang Xiuguo

    2011-04-01

    Full Text Available Abstract Background Pectate lyases (PELs play an important role in the infection process of plant pathogens and also have a commercial significance in industrial applications. Most of the PELs were expressed as soluble recombinant proteins, while a few recombinant proteins were insoluble. The production of a large-scale soluble recombinant PEL would allow not only a more detailed structural and functional characterization of this enzyme but also may have important applications in the food industry. Results We cloned a new pectate lyase gene (Pcpel2 from Phytophthora capsici. Pcpel2 was constructed by pET system and pMAL system, and both constructs were used to express the PCPEL2 in Escherichia coli BL21 (DE3 pLysS. The expressed products were purified using affinity chromatography and gel filtration chromatography. The purity, specific activity and pathogenicity of the purified PCPEL2 expressed by the pMAL system were higher than the purified PCPEL2 expressed by the pET system. In addition, some other characteristics of the purified PCPEL2 differed from the two systems, such as crystallographic features. Purified PCPEL2 expressed by the pMAL system was crystallized by the hanging-drop vapour-diffusion method at 289 K, and initial crystals were grown. Conclusion The two different methods and comparison presented here would be highly valuable in obtaining an ideal enzyme for the downstream experiments, and supply an useful alternative to purify some insoluble recombinant proteins.

  16. Purification and biochemical characterization of a new alkali-stable laccase from Trametes sp. isolated in Tunisia: role of the enzyme in olive mill waste water treatment.

    Science.gov (United States)

    Daâssi, Dalel; Zouari-Mechichi, Héla; Prieto, Alicia; Martínez, María Jesús; Nasri, Moncef; Mechichi, Tahar

    2013-11-01

    A white-rot basidiomycete, isolated from decayed acacia wood (from Northwest of Tunisia) and identified as Trametes sp, was selected in a broad plate screening because of its ability to decolorize and dephenolize olive oil mill wastewater (OMW) efficiently. The major laccase was purified and characterized as a monomeric protein with apparent molecular mass of 61 kDa (SDS-PAGE). It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 4.0 and a temperature of 60 °C. The purified laccase is stable at alkaline pH values. The enzyme retained 50 % of its activity after 90 min of incubation at 55 °C. Using ABTS, this laccase presented K m and V max values of 0.05 mM and 212.73 μmoL min(-1) mg(-1), respectively. It has shown a degrading activity towards a variety of phenolic compounds. The purified laccase was partially inhibited by Fe(2+), Zn(2+), Cd(2+) and Mn(2+), while Cu(2+) acted as inducer. EDTA (10 mM) and NaN3 (10 mM) were found to completely inhibit its activity. 73 % OMW was dephenolized after 315 min incubation at 30 °C with 2 U mL(-1) of laccase and 2 mM HBT.

  17. An enzyme-based DNA preparation method for application to forensic biological samples and degraded stains.

    Science.gov (United States)

    Lounsbury, Jenny A; Coult, Natalie; Miranian, Daniel C; Cronk, Stephen M; Haverstick, Doris M; Kinnon, Paul; Saul, David J; Landers, James P

    2012-09-01

    Extraction of DNA from forensic samples typically uses either an organic extraction protocol or solid phase extraction (SPE) and these methods generally involve numerous sample transfer, wash and centrifugation steps. Although SPE has been successfully adapted to the microdevice, it can be problematic because of lengthy load times and uneven packing of the solid phase. A closed-tube enzyme-based DNA preparation method has recently been developed which uses a neutral proteinase to lyse cells and degrade proteins and nucleases [14]. Following a 20 min incubation of the buccal or whole blood sample with this proteinase, DNA is polymerase chain reaction (PCR)-ready. This paper describes the optimization and quantitation of DNA yield using this method, and application to forensic biological samples, including UV- and heat-degraded whole blood samples on cotton or blue denim substrates. Results demonstrate that DNA yield can be increased from 1.42 (±0.21)ng/μL to 7.78 (±1.40)ng/μL by increasing the quantity of enzyme per reaction by 3-fold. Additionally, there is a linear relationship between the amount of starting cellular material added and the concentration of DNA in the solution, thereby allowing DNA yield estimations to be made. In addition, short tandem repeat (STR) profile results obtained using DNA prepared with the enzyme method were comparable to those obtained with a conventional SPE method, resulting in full STR profiles (16 of 16 loci) from liquid samples (buccal swab eluate and whole blood), dried buccal swabs and bloodstains and partial profiles from UV or heat-degraded bloodstains on cotton or blue denim substrates. Finally, the DNA preparation method is shown to be adaptable to glass or poly(methyl methacrylate) (PMMA) microdevices with little impact on STR peak height but providing a 20-fold reduction in incubation time (as little as 60 s), leading to a ≥1 h reduction in DNA preparation time.

  18. Efficient extraction of olive pulp and stone proteins by using an enzyme-assisted method.

    Science.gov (United States)

    Vergara-Barberán, María; Lerma-García, María Jesús; Herrero-Martínez, José Manuel; Simó-Alfonso, Ernesto Francisco

    2014-07-01

    An efficient protein extraction protocol for proteins from olive pulp and stone by using enzymes was developed. For this purpose, different parameters that affect the extraction process, such as enzyme type and content, pH, and extraction temperature and time, were tested. The influence of these factors on protein recovery was examined using the standard Bradford assay, while the extracted proteins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The best extraction conditions were achieved at pH 7.0 and 5% (v/v) Palatase® 20000 L (lipase) for pulp and Lecitase® Ultra (phospholipase) for stone proteins. The optimal extraction temperature and time were 30 and 40 °C for 15 min for pulp and stone tissues, respectively. Under these conditions, several protein extracts coming from olive fruits of different genetic variety were analyzed, their profiles being compared by SDS-PAGE. The developed enzyme-assisted extraction method showed faster extraction, higher recovery, and reduced solvent usage than the nonenzymatic methods previously described in the literature. In the case of stone proteins, different electrophoretic profiles and band intensities were obtained that could be helpful to distinguish samples according to their genetic variety.

  19. Purification and characterization of an anti-cancer enzyme produced by marine Vibrio Costicola under a novel solid state fermentation process

    Directory of Open Access Journals (Sweden)

    G. Nagendra Prabhu

    1999-01-01

    Full Text Available L - Glutaminase, a therapeutically and industrially important enzyme, was produced from marine Vibrio costicola by a novel solid state fermentation process using polystyrene beads as inert support. The new fermentation system offered several advantages over the conventional systems, such as the yield of leachate with minimum viscosity and high specific activity for the target product besides facilitating the easy estimation of biomass. The enzyme thus produced was purified and characterised. It was active at physiological pH, showed high substrate specificity towards L - glutamine and had a Km value of 7.4 x 10-2 M. It also exhibited high salt and temperature tolerance indicating good scope for its industrial and therapeutic applications.O L - Glutaminase, uma enzima terapêutica industrialmente importante foi produzida a partir do Vibrio costicola marinho. Por um processo de fermentação no estado sólido, em particular contas de poliestireno foram utilizadas como suporte inerte. O novo sistema de fermentação ofereceu várias vantagens sobre os sistemas convencionais, como rendimento de "leachate" com viscosidade mínima e atividade específica alta para o produto; facilidade a estimação da biomassa. A enzima assim produzida foi purificada e caracterizada. A enzima apresentou atividade elevada em pH fisiológico e alta especificidade ao substrato em direção a L - glutamina com um valor Km de 7.4 x 10-2 M. A enzima também exibiu alta tolerância ao sal e temperatura demonstrando ser um bom indicador para aplicações terapêuticas e industriais.

  20. Enzyme-Based Logic Gates and Networks with Output Signals Analyzed by Various Methods.

    Science.gov (United States)

    Katz, Evgeny

    2017-07-05

    The paper overviews various methods that are used for the analysis of output signals generated by enzyme-based logic systems. The considered methods include optical techniques (optical absorbance, fluorescence spectroscopy, surface plasmon resonance), electrochemical techniques (cyclic voltammetry, potentiometry, impedance spectroscopy, conductivity measurements, use of field effect transistor devices, pH measurements), and various mechanoelectronic methods (using atomic force microscope, quartz crystal microbalance). Although each of the methods is well known for various bioanalytical applications, their use in combination with the biomolecular logic systems is rather new and sometimes not trivial. Many of the discussed methods have been combined with the use of signal-responsive materials to transduce and amplify biomolecular signals generated by the logic operations. Interfacing of biocomputing logic systems with electronics and "smart" signal-responsive materials allows logic operations be extended to actuation functions; for example, stimulating molecular release and switchable features of bioelectronic devices, such as biofuel cells. The purpose of this review article is to emphasize the broad variability of the bioanalytical systems applied for signal transduction in biocomputing processes. All bioanalytical systems discussed in the article are exemplified with specific logic gates and multi-gate networks realized with enzyme-based biocatalytic cascades. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. An improved method for the cost-effective expression and purification of large quantities of KcsA.

    Science.gov (United States)

    Tilegenova, Cholpon; Vemulapally, Spandana; Cortes, Doris M; Cuello, Luis G

    2016-11-01

    KcsA, the bacterial K(+) channel from Streptomyces lividans, is the prototypical model system to study the functional and structural correlations of the pore domain of eukaryotic voltage-gated K(+) channels (Kv channels). It contains all the molecular elements responsible for ion conduction, activation, deactivation and inactivation gating [1]. KcsA's structural simplicity makes it highly amenable for structural studies. Therefore, it is methodological advantageous to produce large amounts of functional and properly folded KcsA in a cost-effective manner. In the present study, we show an optimized protocol for the over-expression and purification of large amounts of high-quality, fully functional and crystallizable KcsA using inexpensive detergents, which significantly lowered the cost of the purification process. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Faster, safer, and better DNA purification by ultracentrifugation using GelRed stain and development of mismatch oligo DNA for genome walking.

    Science.gov (United States)

    Kasajima, Ichiro; Ohtsubo, Norihiro; Sasaki, Katsutomo

    2014-01-01

    Purification of plant DNA involves lengthy ultracentrifugation using ethidium bromide. Here, ultracentrifugation method is improved by staining with GelRed. The resulting method is faster, safer and of higher sensitivity. Purified DNA quality was confirmed by treatment with restriction enzymes and isolation of gene promoters. New type of long adaptor with mismatch sequence was also developed for promoter isolation.

  3. THE PURIFICATION OF GLUCOSE SYRUP FROM TAPIOCA BY USING ABSORPTION METHOD AND THE CONCENTRATION PROCESS BY VACUUM EVAPORATOR

    OpenAIRE

    Zainal; Laga, Amran; Bastian, Februadi

    2013-01-01

    The glucose syrupe production from tapioca needs to remove dirt and the colour. The water content should also be reduced. The aobjectives of this research were to identify the efffectiveness of glucose syrup purification by using the combination of activated charcoal and zeolit, and to determine the optimal evaporation time on the concentration process of glucose syrup to produce high glucose syrup. The materials were tapioca, activated charcoal, and zeolit. The research was started with conv...

  4. Purification of RNA by SDS solubilization and phenol extraction.

    Science.gov (United States)

    Rio, Donald C; Ares, Manuel; Hannon, Gregory J; Nilsen, Timothy W

    2010-06-01

    This protocol describes a method for RNA purification by sodium dodecyl sulfate (SDS) solubilization and phenol extraction. It is of wide utility and is used routinely to deproteinize RNAs in biological material that has been solubilized in SDS, an ionic detergent that dissolves membranes, disrupts protein-nucleic acid interactions, and inactivates ribonucleases. Once solubilized, addition of phenol or phenol:chloroform:isoamyl alcohol (PCA) completely denatures the protein, and it becomes insoluble in aqueous solution. PCA extraction is the method of choice for preparing cytoplasmic RNA from tissue culture cells or in any other situation (e.g., enzyme reactions) where solubilization in SDS is easily achievable.

  5. Inorganic Materials as Supports for Covalent Enzyme Immobilization: Methods and Mechanisms

    Directory of Open Access Journals (Sweden)

    Paolo Zucca

    2014-09-01

    Full Text Available Several inorganic materials are potentially suitable for enzymatic covalent immobilization, by means of several different techniques. Such materials must meet stringent criteria to be suitable as solid matrices: complete insolubility in water, reasonable mechanical strength and chemical resistance under the operational conditions, the capability to form manageable particles with high surface area, reactivity towards derivatizing/functionalizing agents. Non-specific protein adsorption should be always considered when planning covalent immobilization on inorganic solids. A huge mass of experimental work has shown that silica, silicates, borosilicates and aluminosilicates, alumina, titania, and other oxides, are the materials of choice when attempting enzyme immobilizations on inorganic supports. More recently, some forms of elemental carbon, silicon, and certain metals have been also proposed for certain applications. With regard to the derivatization/functionalization techniques, the use of organosilanes through silanization is undoubtedly the most studied and the most applied, although inorganic bridge formation and acylation with selected acyl halides have been deeply studied. In the present article, the most common inorganic supports for covalent immobilization of the enzymes are reviewed, with particular focus on their advantages and disadvantages in terms of enzyme loadings, operational stability, undesired adsorption, and costs. Mechanisms and methods for covalent immobilization are also discussed, focusing on the most widespread activating approaches (such as glutaraldehyde, cyanogen bromide, divinylsulfone, carbodiimides, carbonyldiimidazole, sulfonyl chlorides, chlorocarbonates, N-hydroxysuccinimides.

  6. Polydopamine microcapsules with different wall structures prepared by a template-mediated method for enzyme immobilization.

    Science.gov (United States)

    Shi, Jiafu; Yang, Chen; Zhang, Shaohua; Wang, Xiaoli; Jiang, Zhongyi; Zhang, Wenyan; Song, Xiaokai; Ai, Qinghong; Tian, Chunyong

    2013-10-23

    Microcapsules with diverse wall structures may exhibit different performance in specific applications. In the present study, three kinds of mussel-inspired polydopamine (PDA) microcapsules with different wall structures have been prepared by a template-mediated method. More specifically, three types of CaCO3 microspheres (poly(allylamine hydrochloride), (PAH)-doped CaCO3; pure-CaCO3; and poly(styrene sulfonate sodium), (PSS)-doped CaCO3) were synthesized as sacrificial templates, which were then treated by dopamine to obtain the corresponding PDA-CaCO3 microspheres. Through treating these microspheres with disodium ethylene diamine tetraacetic acid (EDTA-2Na) to remove CaCO3, three types of PDA microcapsules were acquired: that was (1) PAH-PDA microcapsule with a thick (∼600 nm) and highly porous capsule wall composed of interconnected networks, (2) pure-PDA microcapsule with a thick (∼600 nm) and less porous capsule wall, (3) PSS-PDA microcapsule with a thin (∼70 nm) and dense capsule wall. Several characterizations confirmed that a higher degree in porosity and interconnectivity of the capsule wall would lead to a higher mass transfer coefficient. When serving as the carrier for catalase (CAT) immobilization, these enzyme-encapsulated PDA microcapsules showed distinct structure-related activity and stability. In particular, PAH-PDA microcapsules with a wall of highly interconnected networks displayed several significant advantages, including increases in enzyme encapsulation efficiency and enzyme activity/stability and a decrease in enzyme leaching in comparison with other two types of PDA microcapsules. Besides, this hierarchically structured PAH-PDA microcapsule may find other promising applications in biocatalysis, biosensors, drug delivery, etc.

  7. The purification, crystallization and preliminary structural characterization of FAD-dependent monooxygenase PhzS, a phenazine-modifying enzyme from Pseudomonas aeruginosa

    Energy Technology Data Exchange (ETDEWEB)

    Gohain, Neelakshi [Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund (Germany); Thomashow, Linda S. [Department of Plant Pathology, Washington State University, Pullman, Washington 99164-6430 (United States); USDA Agricultural Research Service, Root Disease and Biological Control Research Unit, Pullman, Washington 99164-6430 (United States); Mavrodi, Dmitri V. [Department of Plant Pathology, Washington State University, Pullman, Washington 99164-6430 (United States); Blankenfeldt, Wulf, E-mail: wulf.blankenfeldt@mpi-dortmund.mpg.de [Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)

    2006-10-01

    PhzS, an FAD-dependent monooxygenase that catalyzes a reaction involved in the biosynthesis of the virulence factor pyocyanin in P. aeruginosa, was cloned, overexpressed and crystallized. Data collection from native and seleno-l-methionine-labelled crystals is reported. The blue chloroform-soluble bacterial metabolite pyocyanin (1-hydroxy-5-methyl-phenazine) contributes to the survival and virulence of Pseudomonas aeruginosa, an important Gram-negative opportunistic pathogen of humans and animals. Little is known about the two enzymes, designated PhzM and PhzS, that function in the synthesis of pyocyanin from phenazine-1-carboxylic acid. In this study, the FAD-dependent monooxygenase PhzS was purified and crystallized from lithium sulfate/ammonium sulfate/sodium citrate pH 5.5. Native crystals belong to space group C2, with unit-cell parameters a = 144.2, b = 96.2, c = 71.7 Å, α = γ = 90, β = 110.5°. They contain two monomers of PhzS in the asymmetric unit and diffract to a resolution of 2.4 Å. Seleno-l-methionine-labelled PhzS also crystallizes in space group C2, but the unit-cell parameters change to a = 70.6, b = 76.2, c = 80.2 Å, α = γ = 90, β = 110.5° and the diffraction limit is 2.7 Å.

  8. Comparison of four methods for the purification and refolding of human interleukin-2-mouse granulocyte/macrophage colony-stimulating factor fusion protein.

    Science.gov (United States)

    Wen, Qian; Ma, Li; Luo, Wei; Zhou, Ming-Qian; He, Dong; Lin, Ying; Wu, Zhen-Qiang; He, Xiao-Wei; Wang, Ju-Fang; Wang, Xiao-Ning

    2008-05-01

    The combination of IL-2 (interleukin-2) and GM-CSF (granulocyte/macrophage colony-stimulating factor) has been broadly studied in antitumour immune therapy, but its efficacy is uncertain. To better exert the activities of the two cytokines and study them in a mouse model, we have constructed a bifunctional protein, hIL-2-mGM-CSF (human IL-2-mouse GM-CSF), fused to a C-terminal tag of six histidine residues (His(6)). The fusion protein was expressed in Escherichia coli as IBs (inclusion bodies). After extracting and clarifying the IBs, four methods of protein purification and refolding were compared in order to optimize the preparation technique. Of these methods, the best result was obtained with a four-step process consisting of (1) purification with denaturing affinity chromatography, (2) followed by fully denaturing the protein with system conversion, (3) then refolding by isovolumetric ultrafiltration and (4) finally, purification by anion-exchange chromatography. The purity of the hIL-2-mGM-CSF was approx. 95%, yielding approx. 20 mg of protein/l of culture. The fusion protein retained the natural activities of IL-2 and GM-CSF, with specific activities of 8.7 x 10(6) and 1.1 x 10(7) i.u./mg respectively. Flow-cytometric analysis indicated that hIL-2-mGM-CSF could specifically bind to the corresponding receptor-positive cells. The present study provides important preliminary information for studying the antitumour activity of hIL-2-mGM-CSF in vivo, which will facilitate future clinical research into the use of hIL-2/hGM-CSF in immune therapy.

  9. Inhibition of Re Du Ning Injection on Enzyme Activities of Rat Liver Microsomes Using Cocktail Method

    Institute of Scientific and Technical Information of China (English)

    Xiao-qian Xu; Ting Geng; She-bing Zhang; Dan-yu Kang; Yan-jing Li; Gang Ding; Wen-zhe Huang; Zhen-zhong Wang; Wei Xiao

    2016-01-01

    Objective Re Du Ning Injection(RDN), a Chinese materia medica injection, is made from the extracts of Lonicerae Japonicae Flos, Gardeniae Fructus, and Artemisiae Annuae Herba. Since last decade, RDN has been widely used in China for the treatment of viral infection, fever, and inflammation. To assess the potential interacting of RDN with co-administered drugs, the inhibitory effects of RDN on the enzyme activities(CYP1A1, CYP1A2, CYP2C11, CYP2D1, and CYP3A1/2) of rat liver microsomes were investigated by a cocktail method. Methods A sensitive and specific LC-MS method capable of simultaneous quantification of five metabolites in rat liver microsomes was developed and validated. Then RDN(0.625%–1.0%) was incubated with rat liver microsomes and specific substrates. The enzyme activities were expressed as the formation rate of the specific metabolites of the substrates(pmol·mg·protein-1·min-1). Results RDN competitively inhibited the activities of CYP1A2 and CYP2C11, with inhibition constant(Ki) values determined to be 0.18% and 0.63%, respectively. RDN exhibited the mixed inhibition on the activity of CYP2D1, with a Ki value of 0.15%. The activities of CYP1A1 and CYP3A1/2 were not markedly inhibited even by 1.0% RDN. Conclusion RDN could inhibit the rat enzyme activities of CYP1A2, 2C11, and 2D1 in vitro with different inhibition modes, which is worthy of promoting safety and efficacy of RDN.

  10. [Effects of different tillage methods on phospholipid fatty acids and enzyme activities in calcareous cinnamon soil].

    Science.gov (United States)

    Pei, Xue-Xia; Dang, Jian-You; Zhang, Ding-Yi; Wang, Jiao-Ai; Zhang, Jing

    2014-08-01

    In order to study changes of physical and chemical characteristics and microbial activities in soil under different tillage methods, effects of four tillage methods, rotary tillage (RT), subsoil tillage (ST), conventional tillage (CT) with corn straw returned to soil, and rotary tillage with no corn straw returned to soil (CK), on phospholipid fatty acids (PLFA) characteristics and hydrolase enzymes activities in calcareous cinnamon soil were investigated. The results showed that soil hydrolase enzymes activities, nutrient contents, microbial diversity varied greatly with the different tillage methods. Returning corn straw to soil increased the kinds, amount of soil total PLFAs, bacteria PLFAs and actonomycetes PLFAs, while decreased the fungi PLFAs, indicating that fungi was more adaptable than bacteria to an infertile environment. ST and CT resulted in higher amounts of total PLFAs, which were 74.7% and 53.3% higher than that of CK, indicating they were more beneficial to the growth of plants. They could also improve soil physical and chemical properties, increase alk-phosphatase, protease and urease activities, which would provide a favorable soil condition for high and stable crop yields.

  11. Expression and purification of a single-chain Type IV restriction enzyme Eco94GmrSD and determination of its substrate preference.

    Science.gov (United States)

    He, Xinyi; Hull, Victoria; Thomas, Julie A; Fu, Xiaoqing; Gidwani, Sonal; Gupta, Yogesh K; Black, Lindsay W; Xu, Shuang-yong

    2015-05-19

    The first reported Type IV restriction endonuclease (REase) GmrSD consists of GmrS and GmrD subunits. In most bacteria, however, the gmrS and gmrD genes are fused together to encode a single-chain protein. The fused coding sequence for ECSTEC94C_1402 from E. coli strain STEC_94C was expressed in T7 Express. The protein designated as Eco94GmrSD displays modification-dependent ATP-stimulated REase activity on T4 DNA with glucosyl-5-hydroxymethyl-cytosines (glc-5hmC) and T4gt DNA with 5-hydroxymethyl-cytosines (5hmC). A C-terminal 6xHis-tagged protein was purified by two-column chromatography. The enzyme is active in Mg(2+) and Mn(2+) buffer. It prefers to cleave large glc-5hmC- or 5hmC-modified DNA. In phage restriction assays, Eco94GmrSD weakly restricted T4 and T4gt, whereas T4 IPI*-deficient phage (Δip1) were restricted more than 10(6)-fold, consistent with IPI* protection of E. coli DH10B from lethal expression of the closely homologous E. coli CT596 GmrSD. Eco94GmrSD is proposed to belong to the His-Asn-His (HNH)-nuclease family by the identification of a putative C-terminal REase catalytic site D507-H508-N522. Supporting this, GmrSD variants D507A, H508A, and N522A displayed no endonuclease activity. The presence of a large number of fused GmrSD homologs suggests that GmrSD is an effective phage exclusion protein that provides a mechanism to thwart T-even phage infection.

  12. Evaluation of enzyme immobilization methods for paper-based devices--A glucose oxidase study.

    Science.gov (United States)

    Nery, Emilia Witkowska; Kubota, Lauro T

    2016-01-01

    Paper-based sensors gained almost explosive attention during the last few years. A large number of systems, often destined to resource limited settings is based on enzymatic reactions. Choice of an adequate immobilization method could significantly prolong the shelf-life of such sensors, especially in applications, where exposure to high temperatures during storage and transport is more than a threat. We are seeking to compare a variety of immobilization methods based on different phenomena (adsorption, entrapment in gel, microencapsulation, covalent linkage), with total of 33 methods tested. Glucose oxidase was used as a model enzyme. Enzymatic activity of immobilized samples was accompanied for a period of 24 weeks considering two sets of samples, one stored in 4 °C and other in ambient temperature.

  13. Purification of 6-phosphogluconate dehydrogenase from parsley (Petroselinum hortense) leaves and investigation of some kinetic properties.

    Science.gov (United States)

    Demir, Hülya; Ciftçi, Mehmet; Küfrevioğlu, O Irfan

    2003-02-01

    In this study, 6-phosphogluconate dehydrogenase (E.C.1.1.44; 6PGD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps that are preparation of homogenate ammonium sulfate fractionation and on DEAE-Sephadex A50 ion exchange. The enzyme was obtained with a yield of 49% and had a specific activity of 18.3 U (mg proteins)(-1) (Lehninger, A.L.; Nelson, D.L.; Cox, M.M. Principles of Biochemistry, 2nd Ed.; Worth Publishers Inc.: N.Y., 2000, 558-560). The overall purification was about 339-fold. A temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method at 340 mn. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 97.5 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a subunit molecular weight of 24.1 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found as 8.0, 8.0, and 50 degrees C, respectively. In addition, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk plots.

  14. Comparative analysis of antibiotic residue in milk using enzyme and microbiological methods

    Directory of Open Access Journals (Sweden)

    Petrović Jelena

    2003-01-01

    Full Text Available Antibiotic residue can have a harmful effect on human health and can disrupt the processing of milk and milk products. In order to prevent these unwanted effects of residue, different screening methods are used today. The basic goal of this paper is to compare screening methods performed during the testing of milk from different points of the production chain. In this paper we have comparatively analyzed three screening methods: microbiological methods - the Delvo SP test and the diffusion method with B. stearothermophilus as the test microorganism, and an enzyme method - the Penzym S test. Twenty samples of farm milk from collective tanks were analyzed, as well as 20 samples of milk from transport cisterns, 10 samples of pasteurized milk and 10 samples of sterilized market milk. Based on the comparative analysis of the diffusion method, Delvo SP test and Panzym S test, we conclude that all three methods are in hgih mutual accordance (the kappa value oscillates from nearly ideal to ideal coinciding and thus meet one of the criteria for being included in the systematic control of milk for the presence of antibiotic residue.

  15. Study on Method of Purification of GST-tagged Proteins from Inclusion Bodies%GST融合蛋白包涵体纯化方法的探索

    Institute of Scientific and Technical Information of China (English)

    季林; 刘佩娟; 王毅; 赵勇; 毛峰峰; 赵善民; 尚淑琴; 师长宏

    2012-01-01

    The aim was to study the method of purification of GST-tagged proteins from inclusion bodies. The vector PGEX-4T1-Rpfd expressed Rpfd -GST fusion protein was transformed into E. Coli DH5a and protein expression was induced with IPTG. The effect of these factors is observed on the impact of protein purification by comparing lysis with guanidine hydrochloride and pretreatment with Novagen Protein Refolding Kit, and adjusting the different types of protein refolding solution. The active GST fusion protein can be efficiently purified, through removing soluble protein after ultrasonic treatment, and then using the reaction mixture containing 20 mmol Tris-HCl and 0.1 mmol DTT for rehabilitation. To remove soluble protein expression and change the formula of protein refolding solutions can, effectively improve protein purification of GST-tagged proteins from inclusion bodies.%探讨GST融合蛋白包涵体纯化的方法.将表达Rpfd与GST融合蛋白的质粒PGEX-4T1 -Rpfd转入大肠杆菌DH5a,IPTG诱导表达目的蛋白.通过比较盐酸胍裂解和Novagen蛋白折叠试剂盒预处理,及调整不同类型蛋白复性液,观察上述因素对GST融合蛋白包涵体纯化效果的影响.结果:超声后先去除可溶性表达,再使用20 mmolris-HC1和0.1 mmol DTT进行复性,可获得高效纯化,具有活性的GST融合蛋白.对于GST包涵体蛋白进行纯化时,去除可溶性表达蛋白,改变蛋白复性液的成分,可有效提高蛋白纯化效果.

  16. Enzyme catalysis enhanced dark-field imaging as a novel immunohistochemical method

    Science.gov (United States)

    Fan, Lin; Tian, Yanyan; Yin, Rong; Lou, Doudou; Zhang, Xizhi; Wang, Meng; Ma, Ming; Luo, Shouhua; Li, Suyi; Gu, Ning; Zhang, Yu

    2016-04-01

    Conventional immunohistochemistry is limited to subjective judgment based on human experience and thus it is clinically required to develop a quantitative immunohistochemical detection. 3,3'-Diaminobenzidin (DAB) aggregates, a type of staining product formed by conventional immunohistochemistry, were found to have a special optical property of dark-field imaging for the first time, and the mechanism was explored. On this basis, a novel immunohistochemical method based on dark-field imaging for detecting HER2 overexpressed in breast cancer was established, and the quantitative analysis standard and relevant software for measuring the scattering intensity was developed. In order to achieve a more sensitive detection, the HRP (horseradish peroxidase)-labeled secondary antibodies conjugated gold nanoparticles were constructed as nanoprobes to load more HRP enzymes, resulting in an enhanced DAB deposition as a dark-field label. Simultaneously, gold nanoparticles also act as a synergistically enhanced agent due to their mimicry of enzyme catalysis and dark-field scattering properties.Conventional immunohistochemistry is limited to subjective judgment based on human experience and thus it is clinically required to develop a quantitative immunohistochemical detection. 3,3'-Diaminobenzidin (DAB) aggregates, a type of staining product formed by conventional immunohistochemistry, were found to have a special optical property of dark-field imaging for the first time, and the mechanism was explored. On this basis, a novel immunohistochemical method based on dark-field imaging for detecting HER2 overexpressed in breast cancer was established, and the quantitative analysis standard and relevant software for measuring the scattering intensity was developed. In order to achieve a more sensitive detection, the HRP (horseradish peroxidase)-labeled secondary antibodies conjugated gold nanoparticles were constructed as nanoprobes to load more HRP enzymes, resulting in an enhanced DAB

  17. Biogas Purification up to Final Product

    Directory of Open Access Journals (Sweden)

    Yu. Losiuk

    2012-01-01

    Full Text Available The paper considers main technological methods for biogas purification from impurities that permit to increase energy value of the product and decrease its corrosion activity.  While evaluating economic efficiency due to introduction of the corresponding purification technology, in addition, it is necessary to take into account an ecological factor.

  18. Purification and characterization of a thermostable glucoamylase ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-06-07

    Jun 7, 2010 ... tryptophan and serine residues in the catalytic process. Raw corn .... selected for amylase production, purification and characterization. .... chromatogram was developed with solvent system of butanol/ ... starch affinity and acetone precipitation method for ..... Optimization of extraction and purification of.

  19. HOUSEHOLD PURIFICATION OF FLUORIDE CONTAMINATED MAGADI (TRONA)

    DEFF Research Database (Denmark)

    1997-01-01

    Purification of fluoride contaminated magadi is studied using bone char sorption and calcium precipitation. The bone char treatment is found to be workable both in columns and in batches where the magadi is dissolved in water prior to treatment. The concentrations in the solutions were 89 g magadi...... treatment method. A procedure for purification of fluoride contaminated magadi at household level is described....

  20. Purification method for recombinant proteins based on a fusion between the target protein and the C-terminus of calmodulin

    Science.gov (United States)

    Schauer-Vukasinovic, Vesna; Deo, Sapna K.; Daunert, Sylvia

    2002-01-01

    Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein. The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins. A CaM-GFP fusion protein was expressed in E. coli and purified on a phenothiazine-derivatized silica column. CaM binds to the phenothiazine on the column in a Ca(2+)-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP. The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa. Pure GFP was eluted with a Ca(2+)-containing buffer, while CaM was eluted later with a buffer containing the Ca(2+)-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.

  1. X-ray fluorescence as a method of monitoring metal catalyst content during the purification of carbon nanotubes

    Science.gov (United States)

    Cavness, Brandon; Heimbecker, Joshua; Velasquez, Joe; Williams, S.

    2012-02-01

    There have been several studies that suggest that catalyst metals in carbon nanotubes (CNTs) may pose a health threat. As there are many potential applications of CNTs in medicine, it is important to be able to quantitatively determine the amount of metal catalyst contained in a CNT sample. The relative catalyst content of carbon nanotube samples synthesized via arc-discharge has been determined at various stages of the purification process using X-ray fluorescence (XRF) analysis. Purification was achieved by immersing samples in heated nitric acid. The intensities of the nickel K α X-rays were studied to determine the relative catalyst content in the samples. Scanning electron microscopy (SEM) images of purified nanotubes have been compared to the images of a sample that has been irradiated by 0-15 keV bremsstrahlung in order to determine if the XRF analysis of the nanotubes is in any way destructive. No obvious structural defects were observed as the result of irradiation.

  2. Cathepsin S from bovine spleen. Purification, distribution, intracellular localization and action on proteins.

    OpenAIRE

    Kirschke, H; Wiederanders, B; Brömme, D.; Rinne, A

    1989-01-01

    Cathepsin S was detected in bovine kidney, spleen, lymph nodes and lung by immunochemical methods. The immunostaining of cathepsin S in kidney was concentrated to the cells of the proximal tubule, where the enzyme was present in cytoplasmic granules. The purification method for cathepsin S from bovine spleen involved (NH4)2SO4 fractionation, chromatography on CM-Sephadex C-50, gel filtration on Sephacryl S-200 and chromatofocusing (pH 8.0-6.0). The enzyme was partially destroyed by autolysis ...

  3. Purification and characterization of a branched-chain amino acid aminotransferase from Lactobacillus paracasei subsp paracasei CHCC 2115

    DEFF Research Database (Denmark)

    Thage, B.V.; Rattray, F.P.; Laustsen, M.W.;

    2004-01-01

    Purification and characterization of an aminotransferase (AT) specific for the degradation of branched-chain amino acids from Lactobacillus paracasei subsp. paracasei CHCC 2115. Methods and Results: The purification protocol consisted of anion exchange chromatography, affinity chromatography...... of other metal ions, thiol- and carbonyl-binding agents. The N-terminal sequence of the enzyme was SVNIDWNNLGFDYMQLPYRYVAHXKDGVXD, and had at the amino acid level, 60 and 53% identity to a branched-chain amino acid AT of Lact. plantarum and Lactococcus lactis, respectively. Conclusions: The results suggest...

  4. A Bayesian method for identifying missing enzymes in predicted metabolic pathway databases

    Directory of Open Access Journals (Sweden)

    Karp Peter D

    2004-06-01

    Full Text Available Abstract Background The PathoLogic program constructs Pathway/Genome databases by using a genome's annotation to predict the set of metabolic pathways present in an organism. PathoLogic determines the set of reactions composing those pathways from the enzymes annotated in the organism's genome. Most annotation efforts fail to assign function to 40–60% of sequences. In addition, large numbers of sequences may have non-specific annotations (e.g., thiolase family protein. Pathway holes occur when a genome appears to lack the enzymes needed to catalyze reactions in a pathway. If a protein has not been assigned a specific function during the annotation process, any reaction catalyzed by that protein will appear as a missing enzyme or pathway hole in a Pathway/Genome database. Results We have developed a method that efficiently combines homology and pathway-based evidence to identify candidates for filling pathway holes in Pathway/Genome databases. Our program not only identifies potential candidate sequences for pathway holes, but combines data from multiple, heterogeneous sources to assess the likelihood that a candidate has the required function. Our algorithm emulates the manual sequence annotation process, considering not only evidence from homology searches, but also considering evidence from genomic context (i.e., is the gene part of an operon? and functional context (e.g., are there functionally-related genes nearby in the genome? to determine the posterior belief that a candidate has the required function. The method can be applied across an entire metabolic pathway network and is generally applicable to any pathway database. The program uses a set of sequences encoding the required activity in other genomes to identify candidate proteins in the genome of interest, and then evaluates each candidate by using a simple Bayes classifier to determine the probability that the candidate has the desired function. We achieved 71% precision at a

  5. Rapid purification of recombinant histones.

    Directory of Open Access Journals (Sweden)

    Henrike Klinker

    Full Text Available The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method "rapid histone purification" (RHP as it circumvents isolation of inclusion bodies and thereby cuts out the most time-consuming step of traditional purification protocols. Instead of inclusion body isolation, whole cell extracts are prepared under strongly denaturing conditions that directly solubilize inclusion bodies. By ion exchange chromatography, the histones are purified from the extracts. The protocol has been successfully applied to all four canonical Drosophila and human histones. RHP histones and histones that were purified from isolated inclusion bodies had similar purities. The different purification strategies also did not impact the quality of octamers reconstituted from these histones. We expect that the RHP protocol can be readily applied to the purification of canonical histones from other species as well as the numerous histone variants.

  6. 植物几丁质酶纯化、测定及应用研究进展%Review on purification, enzyme assay and application of plant chitinases

    Institute of Scientific and Technical Information of China (English)

    杨海霞; 邓建军; 张建; 赵广华

    2011-01-01

    植物几丁质酶是植物体中能够水解几丁质多聚体的一种致病性相关蛋白(Pathogenesis-related proteins).近年来对于几丁质酶的研究报道中,大量新型的植物几丁质酶被分离纯化,并建立了不同的酶活测定方法,在几丁质酶的结构及分类方面也逐步有了系统的研究.从几丁质酶的结构及分类,分离纯化以及已建立的酶活测定方法等方面取得的新进展进行了综述,并展望了几丁质酶在农业、食品生产及药用领域的应用前景.%Plant chitinases which hydrolyze the chitin is one of pathogenesis-related proteins and can be induced in resistance of plants to fungal pathogens. Thus, plant chitinases has a wide range of application as a antisepticise material. Recently, chitinases from different plants have been purified and their enzymatic activities have been assayed with varied methods. The structures and classifications, different methods on enzymatic activity assay and purification were summarized. Moreover, applications of chitinases such as in food and medicine field were prospected.

  7. Purificação parcial, por dois diferentes métodos cromatográficos, da lipase produzida por Rhizopus sp. Partial purification of the lipase from Rhizopus sp by two different chromatographic methods

    Directory of Open Access Journals (Sweden)

    Maria Gabriela Bello Koblitz

    2004-06-01

    Full Text Available Lipases, especialmente as de origem microbiana, são largamente utilizadas em processos e na obtenção de produtos para as indústrias química, cosmética, farmacêutica e alimentícia. A produção de enzimas de elevada pureza é importante, principalmente, do ponto de vista do controle dos processos (ausência de interferentes, porém as etapas necessárias à purificação, em geral, provocam perdas na atividade das enzimas e aumentam seu custo final. O objetivo deste trabalho foi propor a melhor metodologia de purificação para a lipase de Rhizopus sp. através do teste de dois diferentes métodos cromatográficos (troca iônica e interação hidrofóbica e, ainda verificar o melhor planejamento estatístico para caracterização bioquímica da mesma. Foi possível purificar parcialmente a lipase de Rhizopus sp. com o uso de coluna de DEAE Sepharose (troca aniônica e de FENIL Sepharose (interação hidrofóbica. A primeira, embora mais seletiva para a enzima em questão, parece provocar redução de sua atividade. A presença de maiores concentrações de íons Na+1 na fração purificada por FENIL Sepharose parece contribuir para o aumento de atividade da lipase. Embora os resultados obtidos por análise multivariável para determinação das características bioquímicas da lipase sejam compatíveis com a análise univariável, aquele planejamento não foi considerado indicado no presente caso.Lipases, especially of microbial origin, are widely applied in processes and in the production of insumesfor the chemical, cosmetic, pharmaceutical and food industries. To produce highly pure enzyme is important to process control (absence of interferentes. However the necessary stages to the purification, in general, cause losses of activity and increases enzyme final cost. The aim of this work was to consider the best methodology of purification for the Rhizopus sp. lipase through the test of two different chromatographic methods (ion exchange

  8. Monitoring the process of purification of crude glycerol derived from biodiesel production: a method based on fluorescence spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Magalhaes, Keurison F.; Caires, Anderson R.L. [Universidade Federal da Grande Dourados, MS (Brazil). Grupo de Optica Aplicada; Oliveira, Samuel L. [Universidade Federal de Mato Grosso do Sul (UFMS), MS (Brazil). Grupo de Optica e Fotonica

    2011-07-01

    Full text. The use of biodiesel has increased worldwide. The biodiesel production on an industrial scale has been based on the transesterification of vegetable oils and fats with methanol in the presence of an alkaline catalyst. During the transesterification, one molecule of triglyceride reacts with three molecules of alcohol to produce glycerol and molecules of alkyl esters (biodiesel). As a result, an increase in biodiesel production also enhances the availability of glycerol on the market. However, crude glycerin has about 30% of impurities which are inherent to biodiesel production such as catalyst, alcohol and fatty acids. The present study evaluated the usefulness of the fluorescence spectroscopy as a tool to monitor the glycerol purification process. Glycerol samples were obtained from transesterification of soybean, canola, and sunflower oils in the presence of NaOH. After stirring time, the solutions were let to stand in separating funnels, then two phases were observed: one containing mainly biodiesel and other consisting of glycol. Then, the respective glycerol samples were collected, henceforth called G1. After that, it was added H2SO4 (20%) in the crude glycerol samples to reduce their pH to 4 in order to remove fatty acids. The solutions were stored for 24 hours in separating funnels. The glycerol (heavy phase), hereafter named G2, was then separated and filtered. To remove other impurities from G2 samples by means of ionic exchange columns, the samples were neutralized and diluted using Milli-Q water (G3 samples). Aliquots of 20 mL were then passed through cationic and anionic resins (G4 and G5 samples, respectively). Emission and excitation spectra of the G1-G5 samples as well as of the glycerol PA-ACS (reference) were recorded at room temperature using a spectrofluorimeter. The emission spectra were obtained setting the excitation at 325nm and monitoring the emission in the 330-800nm range. Fluorimetric maps were also achieved by pumping the

  9. Purification and characterization of Ocimum basilicum L. polyphenol oxidase.

    Science.gov (United States)

    Dogan, Serap; Turan, Pinar; Dogan, Mehmet; Arslan, Oktay; Alkan, Mahir

    2005-12-28

    A partial characterization of polyphenol oxidase (PPO) activity in Ocimum basilicum L. is described. PPO in O. basilicum L. was extracted and purified through (NH4)2SO4 precipitation, dialysis, and a Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity column. The samples obtained from (NH4)2SO4 precipitation and dialysis were used for the characterization of PPO. At the end of purification by affinity chromatography, 11.5-fold purification was achived. The purified enzyme exhibited a clear single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the enzyme was estimated to be approximately 54 kDa. The contents of total phenolic and protein of O. basilicum L. extracts were determined. The total phenolic content of O. basilicum L. was determined spectrophotometrically according to the Folin-Ciocalteu procedure and was found to be 280 mg 100 g(-1) on a fresh weight basis. The protein content was determined according to the Bradford method. The enzyme showed activity to 4-methylcatechol, catechol, and pyrogallol substrates, but not to tyrosine. Therefore, of these three substrates, 4-methylcatecol was the best substrate due to the highest V(max)/K(m) value, followed by pyrogallol and catechol. The optimum pH was at 6, 8, and 9 for 4-methylcatechol, catechol, and pyrogallol, respectively. The enzyme had an optimum temperature of 20, 40, and 50 degrees C for 4-methylcatechol, catechol, and pyrogallol, respectively. It was found that optimum temperature and pH were dependent on the substrates studied. The enzyme activity with increasing temperature and inactivation time for 4-methylcatechol, catechol, and pyrogallol substrates decreased due to heat denaturation of the enzyme.

  10. Purification and properties of thioether methyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Mozier, N.M.

    1988-01-01

    A method to assay activity was developed which measures acceptance of methyl groups from (methyl-{sup 3}H)-S-adenosylmethionine by dimethyl selenide. The product, ({sup 3}H)trimethylselenonium ion, is separated by HPLC and quantitated by scintillation counting. Thioether methyltransferase from mouse liver and lung resides primarily in the cytosol. In terms of specific activity the enzyme is most active in the lung and liver. Purification from lung cytosol requires a three-step process of DEAE and gel filtration column chromatographies followed by chromatofocusing. SDS-Polyacrylamide gel electrophoresis shows a single homogeneous band with a molecular mass of 28,000 daltons. Vmax and Km values for dimethyl selenide as a substrate are 15. 7 pmol/min and 0.44 {mu}M, respectively. Our studies have also shown that this purified enzyme is capable of methylating a wide range of compounds. To further test the enzyme's role in detoxification, in vivo studies were performed by injecting mice with substrate and (methyl-{sup 3}H)methionine and analyzing tissue extracts and urine for (methyl-{sup 3}H)sulfonium.

  11. Development of a novel baculovirus titration method using the Enzyme-linked immunosorbent spot (ELISPOT) assay.

    Science.gov (United States)

    Wang, Wei; Cheng, Tong; Ma, Ke; Xia, Dezhen; Wang, Yongmei; Liu, Jian; Du, Hailian; Shih, James Wai Kuo; Zhang, Jun; Zhao, Qinjian; Xia, Ningshao

    2013-03-01

    The baculovirus expression vector system (BEVS) is one of the most powerful methods for production of recombinant proteins for research or commercial purposes. Titration of viable virus in insect cell culture is often required when BEVS is used for basic research or bioprocessing. An enzyme-linked immunosorbent spot (ELISPOT) assay using monoclonal antibodies against the major capsid protein VP39 of both Autographa californica nuclear polyhedrosis virus (AcMNPV) and Bombyx mori nuclear polyhedrosis virus (BmNPV) was developed for baculovirus quantitation at 48h post-infection. The titer was determined by visualizing infected insect cells as blue spots and automated spot counting was achieved with ELISPOT hardware and software. Log-scale comparison of the results between the ELISPOT assay and a conventional end point dilution assay using a fluorescent marker showed a good correlation for both AcMNPV (R(2)=0.9980, pspot counting.

  12. Enzyme sequence similarity improves the reaction alignment method for cross-species pathway comparison

    Energy Technology Data Exchange (ETDEWEB)

    Ovacik, Meric A. [Chemical and Biochemical Engineering Department, Rutgers University, Piscataway, NJ 08854 (United States); Androulakis, Ioannis P., E-mail: yannis@rci.rutgers.edu [Chemical and Biochemical Engineering Department, Rutgers University, Piscataway, NJ 08854 (United States); Biomedical Engineering Department, Rutgers University, Piscataway, NJ 08854 (United States)

    2013-09-15

    Pathway-based information has become an important source of information for both establishing evolutionary relationships and understanding the mode of action of a chemical or pharmaceutical among species. Cross-species comparison of pathways can address two broad questions: comparison in order to inform evolutionary relationships and to extrapolate species differences used in a number of different applications including drug and toxicity testing. Cross-species comparison of metabolic pathways is complex as there are multiple features of a pathway that can be modeled and compared. Among the various methods that have been proposed, reaction alignment has emerged as the most successful at predicting phylogenetic relationships based on NCBI taxonomy. We propose an improvement of the reaction alignment method by accounting for sequence similarity in addition to reaction alignment method. Using nine species, including human and some model organisms and test species, we evaluate the standard and improved comparison methods by analyzing glycolysis and citrate cycle pathways conservation. In addition, we demonstrate how organism comparison can be conducted by accounting for the cumulative information retrieved from nine pathways in central metabolism as well as a more complete study involving 36 pathways common in all nine species. Our results indicate that reaction alignment with enzyme sequence similarity results in a more accurate representation of pathway specific cross-species similarities and differences based on NCBI taxonomy.

  13. 岩栖蝮蛇类凝血酶纯化、cDNA克隆和序列分析%Purification, cDNA cloning and sequence analysis of thrombin-like enzyme from Gloydius saxatilis

    Institute of Scientific and Technical Information of China (English)

    孙德军; 杨春伟; 杨同书; 颜炜群; 王伟

    2003-01-01

    Thrombin-like enzyme has great medical application in treating thrombus. A thrombin-like enzyme from Gloydius saxatilis snake venom was isolated and purified to homogeneity by a rapid and effective method using ion-exchange chromatography on DEAE-Sepharose and affinity chromatography on heparin-sepharose.SDS-polyacrylamide electrophoresis under reducing condition revealed that the purified enzyme had a single protein band and its molecular weight was 32000 dalton.Total RNAs were extracted from the venom gland of the G.saxatilis snake.Using degenerate primers,we amplified the cDNA of the thrombin-like enzyme gene in the venom gland of G.saxatilis using the reverse transcription-polymerase chainreaction (RT-PCR) method.The cDNA fragment was inserted into pGEMT vector,cloned and its nucleotide sequence was determined.Its open reading frame is composed of 774 nucleotides and codes a protein prezymogen of 258 amino acids,including a putative secretory signal peptide of 18 amino acids and a proposed pro-peptide of 6 amino acid residues.It contains 12 cysteine residues.The sequence analysis indicates that the deduced amino acid sequence of the cDNA fragment shares high identity with the thrombin-like enzyme genes of other snakes in the gene bank.The query sequence exhibits strong amino acid sequence homology of 88%,88% and 86% to the serine proteas of T.gramineus,thrombin-like defibrase Ⅰ of D.acutus and serine protease catroxase Ⅱ of C.atrox respectively.Based on the amino acid sequences of other thrombin-like enzymes,the catalytic residues and disulfide bridges of this thrombin-like enzyme are deduced as follows:catalytic residues,His65,Asp110,Ser%204;and six disulfide bridges Cys31-Cys163,Cys50-Cys66,Cys98-Cys256,Cys142-Cys210,Cys174-Cys189 and Cys200-Cys225.According to the possible linked glycosylation sites N-X-T (Asn-X-Thr) or N-X-S (Asn-X-Ser),its possible glycosylation sites are N44-S45-T46 and N251-T252-T253 residues [Acta Zoologica Sinica 49(6):878-882,2003].

  14. Nanospines incorporation into the structure of the hydrophobic cryogels via novel cryogelation method: an alternative sorbent for plasmid DNA purification.

    Science.gov (United States)

    Üzek, Recep; Uzun, Lokman; Şenel, Serap; Denizli, Adil

    2013-02-01

    In this study, it was aimed to prepare hydrophobic cryogels for plasmid DNA (pDNA) purification from Escherichia coli lysate. The hydrophobicity was achieved by incorporating a hydrophobic ligand, N-methacryloyl-(L)-phenylalanine (MAPA), into the cryogel backbone. In addition to the conventional cryogelation process, freeze-drying step was included to create nanospines. Three different cryogels {poly(2-hydoxyethyl methacrylate-N-methacryloyl-L-phenylalanine)-freeze dried, [P(HEMA-MAPA)-FD]; poly(2-hydoxyethyl methacrylate-N-methacryloyl-L-phenylalanine, [P(HEMA-MAPA)] and poly(2-hydoxyethyl methacrylate)-freeze dried, [P(HEMA)-FD]} were prepared, characterized, and used for DNA (salmon sperm DNA) adsorption studies from aqueous solution. The specific surface areas of cryogels were determined to be 21.4 m(2)/g for P(HEMA)-FD, 17.65 m(2)/g for P(HEMA-MAPA) and 36.0 m(2)/g for P(HEMA-MAPA)-FD. The parameters affecting adsorption such as temperature, initial DNA concentration, salt type and concentration were examined in continuous mode. The maximum adsorption capacities were observed as 45.31 mg DNA/g, 27.08 mg DNA/g and 1.81 mg DNA/g for P(HEMA-MAPA)-FD, P(HEMA-MAPA) and P(HEMA)-FD, respectively. Desorption process was performed using acetate buffer (pH 5.50) without salt. First, pDNA was isolated from E. coli lysate and the purity of pDNA was then determined by agarose gel electrophoresis. Finally, the chromatographic performance of P(HEMA-MAPA)-FD cryogel for pDNA purification was tested in FPLC. The resolution (R(s)) was 2.84, and the specific selectivity for pDNA was 237.5-folds greater than all impurities.

  15. 猪肺血管紧张素转化酶分离纯化的工艺优化%Process optimization for separation and purification of angiotensin converting enzyme from pig lung

    Institute of Scientific and Technical Information of China (English)

    涂振兴; 陈锡鸿; 廖丹葵; 兰雄雕; 孙丽霞; 和筱宇; 卢姗姗; 王燕兰

    2014-01-01

    Angiotensin converting enzyme ( ACE ) is one kind of protease existed in the biological tissue and blood, which plays a very important role in blood pressure regulation. How to purify ACE efficiently is significant for the study of ACE structure and function. Fresh pig lung was homogenized and digested with the trypsin. The influence factors including concentration of homogenization and initial concentration of protein were optimized. High activity ACE was obtained after salting out, di-alysis and ion exchange chromatograph. The enzyme activity was increased by 27. 06% when the pig lung was homogenized at ratio of 1:3 and digested by the trypsin for 1 h. The homogenarate was cen-trifuged and the protein concentration of the supernatant was diluted to 3% before two steps salting-out, while the primary waste deposition was recycled. The ACE was purified to 3. 94 fold and activi-ty recovery 73. 73% which was improved more than 24. 33% in the comparision with traditional treatment. After ion exchange chromatography, the specific activity of ACE was 0. 130 3 U/mg and the activity recovery reached to 59. 14%. The anzymatic characterization of ACE was investigated. The results showed that the optimized catalytic temperature was 42 ℃ and pH of ACE was 8. 3 . The Michaelis constant and the maximum reaction rate of pig lung ACE were 1. 521 mmol/L, 17. 301 nmol/min, respectively. ACE activity recovery rate could be improved significantly by the new purification process.%血管紧张素转化酶(angiotensin converting enzyme, ACE)存在于生物体组织和血液中,并在血压调控方面发挥着重要作用,高效分离纯化ACE对研究其结构和功能具有重要意义。文中将猪肺匀浆液进行酶解处理,优化浆液比、盐析初始蛋白浓度等条件,并经盐析、透析和离子交换层析等步骤,得到高活性的ACE。当猪肺以1:3浆液比匀浆并经胰蛋白酶处理1 h后,其总酶活可增加27.06%;匀浆后初始蛋白浓度为3%时

  16. Identification of Functionally Related Enzymes by Learning-to-Rank Methods.

    Science.gov (United States)

    Stock, Michiel; Fober, Thomas; Hüllermeier, Eyke; Glinca, Serghei; Klebe, Gerhard; Pahikkala, Tapio; Airola, Antti; De Baets, Bernard; Waegeman, Willem

    2014-01-01

    Enzyme sequences and structures are routinely used in the biological sciences as queries to search for functionally related enzymes in online databases. To this end, one usually departs from some notion of similarity, comparing two enzymes by looking for correspondences in their sequences, structures or surfaces. For a given query, the search operation results in a ranking of the enzymes in the database, from very similar to dissimilar enzymes, while information about the biological function of annotated database enzymes is ignored. In this work, we show that rankings of that kind can be substantially improved by applying kernel-based learning algorithms. This approach enables the detection of statistical dependencies between similarities of the active cleft and the biological function of annotated enzymes. This is in contrast to search-based approaches, which do not take annotated training data into account. Similarity measures based on the active cleft are known to outperform sequence-based or structure-based measures under certain conditions. We consider the Enzyme Commission (EC) classification hierarchy for obtaining annotated enzymes during the training phase. The results of a set of sizeable experiments indicate a consistent and significant improvement for a set of similarity measures that exploit information about small cavities in the surface of enzymes.

  17. Nanoarmored Enzymes for Organic Enzymology: Synthesis and Characterization of Poly(2-Alkyloxazoline)-Enzyme Conjugates.

    Science.gov (United States)

    Leurs, Melanie; Tiller, Joerg C

    2017-01-01

    The properties of enzymes can be altered significantly by modification with polymers. Numerous different methods are known to obtain such polymer-enzyme conjugates (PECs). However, there is no universal method to render enzymes into PECs that are fully soluble in organic solvents. Here, we present a method, which achieves such high degree of modification of proteins that the majority of modified enzymes will be soluble in organic solvents. This is achieved by preparing poly(2-alkyloxazoline)s (POx) with an NH2 end group and coupling this functional polymer via pyromellitic acid dianhydride onto the amino groups of the respective protein. The resulting PECs are capable of serving as surfactants for unmodified proteins, rendering the whole mixture organosoluble. Depending on the nature of the POx and the molecular weight and the nature of the enzyme, the PECs are soluble in chloroform or even toluene. Another advantage of this method is that the poly(2-alkyloxazoline) can be activated with the coupling agent and used for the enzyme conjugation without further purification. The POx-enzyme conjugates generated by this modification strategy show modulated catalytic activity in both, aqueous and organic, systems. © 2017 Elsevier Inc. All rights reserved.

  18. Robust methods for purification of histones from cultured mammalian cells with the preservation of their native modifications.

    Science.gov (United States)

    Rodriguez-Collazo, Pedro; Leuba, Sanford H; Zlatanova, Jordanka

    2009-06-01

    Post-translational modifications (PTMs) of histones play a role in modifying chromatin structure for DNA-templated processes in the eukaryotic nucleus, such as transcription, replication, recombination and repair; thus, histone PTMs are considered major players in the epigenetic control of these processes. Linking specific histone PTMs to gene expression is an arduous task requiring large amounts of highly purified and natively modified histones to be analyzed by various techniques. We have developed robust and complementary procedures, which use strong protein denaturing conditions and yield highly purified core and linker histones from unsynchronized proliferating, M-phase arrested and butyrate-treated cells, fully preserving their native PTMs without using enzyme inhibitors. Cell hypotonic swelling and lysis, nuclei isolation/washing and chromatin solubilization under mild conditions are bypassed to avoid compromising the integrity of histone native PTMs. As controls for our procedures, we tested the most widely used conventional methodologies and demonstrated that they indeed lead to drastic histone dephosphorylation. Additionally, we have developed methods for preserving acid-labile histone modifications by performing non-acid extractions to obtain highly purified H3 and H4. Importantly, isolation of histones H3, H4 and H2A/H2B is achieved without the use of HPLC. Functional supercoiling assays reveal that both hyper- and hypo-phosphorylated histones can be efficiently assembled into polynucleosomes. Notably, the preservation of fully phosphorylated mitotic histones and their assembly into polynucleosomes should open new avenues to investigate an important but overlooked question: the impact of mitotic phosphorylation in chromatin structure and function.

  19. Effect of Different Purification Techniques on the Characteristics of Heteropolysaccharide-Protein Biopolymer from Durian (Durio zibethinus Seed

    Directory of Open Access Journals (Sweden)

    Hamed Mirhosseini

    2012-09-01

    Full Text Available Natural biopolymers from plant sources contain many impurities (e.g., fat, protein, fiber, natural pigment and endogenous enzymes, therefore, an efficient purification process is recommended to minimize these impurities and consequently improve the functional properties of the biopolymer. The main objective of the present study was to investigate the effect of different purification techniques on the yield, protein content, solubility, water- and oil-holding capacity of a heteropolysaccharide-protein biopolymer obtained from durian seed. Four different purification methods using different chemicals and solvents (i.e., A (isopropanol and ethanol, B (isopropanol and acetone, C (saturated barium hydroxide, and D (Fehling solution] to liberate the purified biopolymer from its crude form were compared. In most cases, the purification process significantly (p < 0.05 improved the physicochemical properties of heteropolysaccharide-protein biopolymer from durian fruit seed. The present work showed that the precipitation using isopropanol and acetone (Method B resulted in the highest purification yield among all the tested purification techniques. The precipitation using saturated barium hydroxide (Method C led to induce the highest solubility and relatively high capacity of water absorption. The current study reveals that the precipitation using Fehling solution (Method D most efficiently eliminates the protein fraction, thus providing more pure biopolymer suitable for biological applications.

  20. The Establishment and Appraisal of the Methods for the Extraction, Separation and Purification of Rat Tail Collagen%鼠尾胶原蛋白提取、分离、纯化方法的建立及鉴定

    Institute of Scientific and Technical Information of China (English)

    任海涛; 钟志勇; 郑佳琳; 饶子亮; 邝少松; 王刚; 唐小江

    2012-01-01

    Objective To establish a highly efficient method for the extraction , separation and purification of rat tail collagen. Methods The rat tail tendon has been obtained through stripping the rat tail ; the rat tail collagen stoste has been developed through processing with Tris -HC1 buffer, pepsin; sodium chloride solution has been repeatedly used for graded salting-out; acetic acid has been used for the purification of collagen . To get purified rat tail collagen , inorganic salt has to be removed with ultra-pure water dialysis. Appraisal is to be made through such technical means such as SDS -PAGE protein electrophoresis, amino acid content analysis. Results Rat tail collagen of high purity can be developed with the method established in this study , whose purity can reach electrophoresis level . When compared with those commercialized rat tail collagen products imported from overseas there is no difference . The effects the parameters for the extraction , separation and purification have exerted on yield and purity has been deliberated , established The optimal conditions for the extraction of rat tail collagen has been established, pepsin usage; l:500, enzyme solution time : 72 h, salting-out concentration; 2 mol/L, the acid solution used for extraction : 0.05 mol/L acetum. Conclusion For rat tail collagen enlargement production to provide the proper processing parameters have been provided for the enlargement of production of rat tail collagen, for a large gain rat tail collagen and deeper efficacy research provides the theoretical support and practical basis have also been provided for the obtaining of large quantities of rat tail collagen so as to carry out deeper research on its efficacy.%目的 建立一种高效提取、分离、纯化鼠尾胶原蛋白的方法.方法 通过对鼠尾进行剥离获得鼠尾腱,用Tris-HCl缓冲液、胃蛋白酶处理获得鼠尾胶原蛋白原液、反复使用氯化钠溶液进行分级盐析、醋酸溶液复溶进行

  1. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    Science.gov (United States)

    Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

    2013-07-23

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  2. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Ward, Thomas E.

    2016-03-22

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  3. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

    2014-04-08

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  4. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Ward, Thomas E.

    2017-08-15

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  5. Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods

    Directory of Open Access Journals (Sweden)

    Ken Motohashi

    2017-03-01

    Full Text Available Simple and low-cost recombinant enzyme-free seamless DNA cloning methods have recently become available. In vivo Escherichia coli cloning (iVEC can directly transform a mixture of insert and vector DNA fragments into E. coli, which are ligated by endogenous homologous recombination activity in the cells. Seamless ligation cloning extract (SLiCE cloning uses the endogenous recombination activity of E. coli cellular extracts in vitro to ligate insert and vector DNA fragments. An evaluation of the efficiency and utility of these methods is important in deciding the adoption of a seamless cloning method as a useful tool. In this study, both seamless cloning methods incorporated inserting DNA fragments into linearized DNA vectors through short (15–39 bp end homology regions. However, colony formation was 30–60-fold higher with SLiCE cloning in end homology regions between 15 and 29 bp than with the iVEC method using DH5α competent cells. E. coli AQ3625 strains, which harbor a sbcA gene mutation that activates the RecE homologous recombination pathway, can be used to efficiently ligate insert and vector DNA fragments with short-end homology regions in vivo. Using AQ3625 competent cells in the iVEC method improved the rate of colony formation, but the efficiency and accuracy of SLiCE cloning were still higher. In addition, the efficiency of seamless cloning methods depends on the intrinsic competency of E. coli cells. The competency of chemically competent AQ3625 cells was lower than that of competent DH5α cells, in all cases of chemically competent cell preparations using the three different methods. Moreover, SLiCE cloning permits the use of both homemade and commercially available competent cells because it can use general E. coli recA− strains such as DH5α as host cells for transformation. Therefore, between the two methods, SLiCE cloning provides both higher efficiency and better utility than the iVEC method for seamless DNA plasmid

  6. Development of a Spectrophotometric Method for Monitoring Angiotensin-Converting Enzyme in Dairy Products

    Directory of Open Access Journals (Sweden)

    Julijana Tomovska*, S. Presilski, N. Gjorgievski, N. Tomovska1, M. S. Qureshi2 and N. P. Bozinovska3

    2013-01-01

    Full Text Available The angiotensin-converting enzyme (ACE regulates the levels of blood pressure through generation of angiotensin-II from angiotensin-I. It is of great importance to have a reliable and yet simple method for a quantitative determination ACE inhibitory peptides in whey of milk products. A rapid, simple, sensitive and accurate spectrophotometric kinetic method has been developed for determination of ACE inhibitory peptides, using competitive inhibition. Samples of dairy product from the market were used for the determination of ACE inhibitory peptides in whey. Holmquist’s kinetic method was used for determining ACE inhibitory activity in blood serum and Ronca-Testoni method was used for the determination of ACE inhibitory activity in whey. Enzymatic inhibition activity was determined using 0.8 mmol/L FAPGG (N-[3-(Furyl –Acryloyl]-L-Phenylalanyl Glycyl Glycyne as the substrate in 50 mmol/L Tris buffer at pH 8.2 at 37°C and a standard serum containing ACE. First, a solution of whey was mixed in a 1 to 10 ratio with serum (elevation containing high ACE activity. The enzymatic activity was determined by monitoring the decrease in absorbance at 340 nm as result of hydrolysis of the substrate. The concentration of ACE inhibitory peptides was determined from a standard curve of inhibitor concentration versus percent of ACE inhibition. The study suggests that the method possesses good reproducibility and accuracy. The linear range enabled determination of high enzymatic activity of ACE and all ACE inhibitory peptides from dairy products act as competitive inhibitors.

  7. An effective high-speed countercurrent chromatographic method for preparative isolation and purification of mollugin directly from the ethanol extract of the Chinese medicinal plant Rubia cordifolia.

    Science.gov (United States)

    Lu, Yanbin; Liu, Rui; Sun, Cuirong; Pan, Yuanjiang

    2007-06-01

    The medicinal plant Rubia cordifolia has been used widely in traditional Chinese medicine (TCM) for its antibacterial, antioxidant and anti-inflammatory activities. In this study, a preparative high-speed countercurrent chromatography (HSCCC) method for isolation and purification of the bioactive component mollugin directly from the ethanol extract of R. cordifolia was successfully established by using light petroleum (bp 60-90 degrees C)/ethanol/diethyl ether/water as the two-phase solvent system. The upper phase of light petroleum/ethanol/diethyl ether/water (5:4:3:1 v/v) was used as the stationary phase of HSCCC. Under the optimum conditions, 46 mg of mollugin at 98.5% purity, as determined by HPLC, could be yielded from 500 mg of the crude extract in a single HSCCC separation. The peak fraction of HSCCC was identified by 1H NMR and 13C NMR.

  8. Structure-Based Design and Synthesis of a New Phenylboronic-Modified Affinity Medium for Metalloprotease Purification

    Science.gov (United States)

    Li, Shangyong; Wang, Linna; Xu, Ximing; Lin, Shengxiang; Wang, Yuejun; Hao, Jianhua; Sun, Mi

    2016-01-01

    Metalloproteases are emerging as useful agents in the treatment of many diseases including arthritis, cancer, cardiovascular diseases, and fibrosis. Studies that could shed light on the metalloprotease pharmaceutical applications require the pure enzyme. Here, we reported the structure-based design and synthesis of the affinity medium for the efficient purification of metalloprotease using the 4-aminophenylboronic acid (4-APBA) as affinity ligand, which was coupled with Sepharose 6B via cyanuric chloride as spacer. The molecular docking analysis showed that the boron atom was interacting with the hydroxyl group of Ser176 residue, whereas the hydroxyl group of the boronic moiety is oriented toward Leu175 and His177 residues. In addition to the covalent bond between the boron atom and hydroxyl group of Ser176, the spacer between boronic acid derivatives and medium beads contributes to the formation of an enzyme-medium complex. With this synthesized medium, we developed and optimized a one-step purification procedure and applied it for the affinity purification of metalloproteases from three commercial enzyme products. The native metalloproteases were purified to high homogeneity with more than 95% purity. The novel purification method developed in this work provides new opportunities for scientific, industrial and pharmaceutical projects. PMID:28036010

  9. Structure-Based Design and Synthesis of a New Phenylboronic-Modified Affinity Medium for Metalloprotease Purification

    Directory of Open Access Journals (Sweden)

    Shangyong Li

    2016-12-01

    Full Text Available Metalloproteases are emerging as useful agents in the treatment of many diseases including arthritis, cancer, cardiovascular diseases, and fibrosis. Studies that could shed light on the metalloprotease pharmaceutical applications require the pure enzyme. Here, we reported the structure-based design and synthesis of the affinity medium for the efficient purification of metalloprotease using the 4-aminophenylboronic acid (4-APBA as affinity ligand, which was coupled with Sepharose 6B via cyanuric chloride as spacer. The molecular docking analysis showed that the boron atom was interacting with the hydroxyl group of Ser176 residue, whereas the hydroxyl group of the boronic moiety is oriented toward Leu175 and His177 residues. In addition to the covalent bond between the boron atom and hydroxyl group of Ser176, the spacer between boronic acid derivatives and medium beads contributes to the formation of an enzyme-medium complex. With this synthesized medium, we developed and optimized a one-step purification procedure and applied it for the affinity purification of metalloproteases from three commercial enzyme products. The native metalloproteases were purified to high homogeneity with more than 95% purity. The novel purification method developed in this work provides new opportunities for scientific, industrial and pharmaceutical projects.

  10. Intein-mediated purification system: mechanism and applications

    Institute of Scientific and Technical Information of China (English)

    Sarra setrerrahmane; Shuhua Tan

    2013-01-01

    The incorporation of self-cleaving protein elements into a variety of fusion-based purification systems; has been an important development in the area of recombinant protein purification. The self-cleaving capability of these tags has recently been combined with additional purification tags to generate novel and convenient protein purification methods. This review elucidates the properties of intein, the mechanism of the intein-based protein splicing and the progress of intein-based protein purification procedures, and recent advances in the applications of intein.

  11. Teaching Enzymes to Pre-Service Science Teachers through POE (Predict, Observe, Explain) Method: The Case of Catalase

    Science.gov (United States)

    Güngör, Sema Nur; Özkan, Muhlis

    2016-01-01

    The aim of this study is to teach enzymes, which are one of the biology subjects in understanding which students have a big difficulty, to pre-service teachers through POE method in the case of catalase, which is an oxidoreductase. Descriptive analysis method was employed in this study in which 38 second grade pre-service teachers attending Uludag…

  12. Enzyme-polysaccharide interaction: a method for improved stability of horseradish peroxidase.

    Science.gov (United States)

    Kagliwal, Lalit D; Singhal, Rekha S

    2014-08-01

    With the advent of green technology, use of enzymes as biocatalyst has become increasingly popular. However, in doing so, enzymes can lose their structure and catalytic activity under conditions that might be necessary for other components of processes. Compared to other strategies, chemical modification is a simple and effective technique for generating stable enzyme. Horseradish peroxidase (HRP; EC 1.11.1.7) was chemically modified by conjugating with 10 different polysaccharides. All polysaccharides were found to increase the thermal and pH stability of HRP with starch being most promising. Further, different parameters were evaluated for effective conjugation and thus stability of HRP conjugate. The degradation kinetics and storage stability of HRP proved the conjugate to be 6.4 times more stable than free enzyme. The starch conjugated HRP and free HRP were further evaluated for its application in decolorization of bromophenol blue dye. Both the enzymes were able to efficiently (>90%) decolorize the dye within minutes.

  13. Enzyme detection by microfluidics

    DEFF Research Database (Denmark)

    2013-01-01

    Microfluidic-implemented methods of detecting an enzyme, in particular a DNA-modifying enzyme, are provided, as well as methods for detecting a cell, or a microorganism expressing said enzyme. The enzyme is detected by providing a nucleic acid substrate, which is specifically targeted...... by that enzyme...

  14. 两种豆制品厂煎炸油净化方法效果的比较研究%The comparative analysis of two kinds of purification methods of frying oil in processing factory of bean products

    Institute of Scientific and Technical Information of China (English)

    谢志镭; 任莉萍; 杨琦

    2015-01-01

    This paper has conducted the research of the effection of purification methods on quality in two different bean products factory of frying oil. It choosed two companies using the method of natural sedimentation purification and water purification,then choosed two similar products to study the index of acid value,carbonyl value and polar compounds in frying oil which under factory conditions. The results showed that the two kind of frying oil purification methods has significant effect on the quality of frying oil.%本文对两种不同的豆制品厂煎炸油净化方法对煎炸油品质的影响进行了研究,选择了两家分别采用自然沉降净化法和加水净化法的企业,再选择两种相同的产品,在工厂条件下对煎炸油的酸价、羰基价和极性物质指标进行研究。研究结果显示两种煎炸油净化方法对煎炸油品质具有显著影响。

  15. Accelerated purification of colloidal silica sols

    Science.gov (United States)

    Bahnsen, E. B.; Garofalini, S.; Pechman, A.

    1979-01-01

    Accelerated purification process for colloidal sols using heat/deionization scheme, sharply reduces waiting time between deionization cycles from several months to a few days. Process produces same high purity silica sols as conventional methods.

  16. A selective molecularly imprinted polymer for immobilization of acetylcholinesterase (AChE): an active enzyme targeted and efficient method.

    Science.gov (United States)

    Demirci, Gökhan; Doğaç, Yasemin İspirli; Teke, Mustafa

    2015-11-01

    In the present study, we immobilized acetylcholinesterase (AChE) enzyme onto acetylcholine removed imprinted polymer and acetylcholine containing polymer. First, the polymers were produced with acetylcholine, substrate of AChE, by dispersion polymerization. Then, the enzyme was immobilized onto the polymers by using two different methods: In the first method (method A), acetylcholine was removed from the polymer, and then AChE was immobilized onto this polymer (acetylcholine removed imprinted polymer). In the second method (method B), AChE was immobilized onto acetylcholine containing polymer by affinity. In method A, enzyme-specific species (binding sites) occurred by removing acetylcholine from the polymer. The immobilized AChE reached 240% relative specific activity comparison with free AChE because the active enzyme molecules bounded onto the polymer. Transmission electron microscopy results were taken before and after immobilization of AChE for the assessment of morphological structure of polymer. Also, the experiments, which include optimum temperature (25-65 °C), optimum pH (3-10), thermal stability (4-70 °C), kinetic parameters, operational stability and reusability, were performed to determine the characteristic of the immobilized AChE.

  17. Optimization of crude enzyme preparation methods for analysis of glutamine synthetase activity in phytoplankton and field samples

    Institute of Scientific and Technical Information of China (English)

    WANG Yujue; WANG Dazhi; HONG Huasheng

    2009-01-01

    Glutamine synthetase (GS) is an important enzyme involved in nitrogen assimilation and metabolism in marine phytoplankton. However, little work has been done in situ due to the limitation of crude enzyme preparation methods. In this study, three enzyme preparation methods, high-speed centrifugation (HC, <10 000 g), ultracentrifugation (UC, 70 000 g), and ultrafiltration (UF) with 100 kμ, molecular weight cutoff, were compared using two diatom species (Asterionellopsis glacialis and Thalassiosira weissflogii), and two dinoflagellate species (Alexandrium catenella and Prorocentrum donghaiense) as experimental materials together with field samples collected from Xiamen Harbor, China. The results showed that HC is the best method to prepare crude enzymes for glutamine synthetase activity (GSA) in diatom species and diatom-dominant samples, while UF is the best method to extract GS from dinoflagellate species and dinoflagellate-dominant samples. For the HC method, the optimal centrifugal speed and time were 10 000 g and 35 min, respectively, and under these conditions, the highest GSA was obtained in all samples. This study indicates that both methods (HC and UF) overcome the limitation of centrifugal speed and could be applied to in situ GSA analysis, especially at sea.

  18. Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

    Science.gov (United States)

    Gray, Kevin A.; Zhao, Lishan; Cayouette, Michelle H.

    2015-09-08

    The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

  19. Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

    Energy Technology Data Exchange (ETDEWEB)

    Gray, Kevin A; Zhao, Lishan; Cayouette, Michelle H

    2015-11-04

    The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

  20. Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes

    Science.gov (United States)

    Greenough, Lucia; Schermerhorn, Kelly M.; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Slatko, Barton E.; Gardner, Andrew F.

    2016-01-01

    Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3′-5′ exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner. PMID:26365239

  1. Simultaneous Measurement of the Galacturonate and Neutral Sugar Contents of Pectic Substances by an Enzymic-HPLC Method.

    Science.gov (United States)

    Matsuhashi, S; Inoue, S; Hatanaka, C

    1992-01-01

    An enzymic-HPLC method was successfully applied to the simultaneous analysis of the galacturonate and neutral sugar contents of pectic substances. A mixture of seven neutral sugars that are present in oridnary pectins was eluted as one peak on a Shodex SUGAR SH1821 column, using 0.001 N sulfuric acid as the eluent; the peak was completely separated from that of galacturonate. No difference in the peak-area ratios of individual neutral sugars to glycerol (internal standard) was found among the seven; the relationship between the peak response and concentration was strictly linear throughout the entire concentration range studied. Seventeen pectic samples, including pectins, pectates, and rhamnogalacturonan fragments, were completely prehydrolyzed by Driselase, an industrial enzyme product from Irpex lacteus, and then analyzed for their constituent sugar contents. The enzymic-HPLC method was simple, accurate, and particularly useful for routine pectin analyses.

  2. Development and Application of a New Microarray- Based Method for High-Throughput Screening of Carbohydrate Active Enzymes

    DEFF Research Database (Denmark)

    Vidal Melgosa, Silvia

    biological roles in plants and in addition to biofuel production they are extensively used in other industrial processes including in detergents, textiles, paper and the food industry. A vast repertoire of CAZymes exists in nature but there is a growing disparity between our ability to putatively identify....... The applicability of the method to identify the substrate specificities of purified uncharacterised enzymes as well as for screening CAZyme activities in complex enzyme mixtures, such as crude culture broths and plant extracts, is shown by examples presented in this thesis. We envisage that the method......The effective and sustainable use of plant biomass for second generation biofuels is of vital importance for reducing dependence on fossil fuels. Carbohydrate-active enzymes (CAZymes) that degrade lignocellulosic plant cell wall material are an important part of this effort. CAZymes have multiple...

  3. Isolation and purification of fungal pathogen (Macrophomina phaseolina induced chitinase from moth beans (Phaseolus aconitifolius

    Directory of Open Access Journals (Sweden)

    Neelima Garg

    2010-01-01

    Full Text Available Objective : Chitinase (EC 3.2.1.14 is one of the major pathogenesis-related proteins, which is a polypeptide that accumulates extracellularly in infected plant tissue. An attempt was made to isolate and purify the chitanase enzyme using moth beans as an enzyme source. Materials and Method : The enzyme was isolated and purified from moth beans against the fungal pathogen Macrophomina phaseolina strain 2165. The isolation and purification was done in both in vitro and in vivo conditions. Purification of chitinase was carried out to obtain three fractions, viz. 50°C heated, ammonium sulfate precipitated and sephadex G-25 column-eluted fractions. The molecular mass of Chitinase was directly estimated by sodium dodecyl sulfate-polyacryamide gel electroresis (SDS-PAGE. Result : The yield is sufficient for initial characterization studies of the enzyme. The molecular study of the enzyme shows the possibility of generating the defense mechanism in plants in which it cannot occur. Chitinase was purified by gel filtration chromatography with 20.75-fold and 32.78-fold purification in the in vitro and in vivo conditions, respectively. The enzyme shows a maximum activity after 90 min with 0.1 ml of colloidal chitin as a substrate and 0.4 ml of crude chitinase extract. The optimum pH of 5.0 and an optimum temperature of 40°C was found for maximal activity. The molecular weight of purified chitinase was estimated to be 30 kDa by SDS-PAGE. Conclusion : The chitinase isolated in both in vitro and in vivo conditions is stable andactive.

  4. A mass spectrometric method to determine activities of enzymes involved in polyamine catabolism

    Energy Technology Data Exchange (ETDEWEB)

    Moriya, Shunsuke; Iwasaki, Kaori [Department of Molecular Medicine, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kami-kitazawa, Setagaya-ku, Tokyo 156-8506 (Japan); Samejima, Keijiro, E-mail: samejima-kj@igakuken.or.jp [Department of Molecular Medicine, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kami-kitazawa, Setagaya-ku, Tokyo 156-8506 (Japan); Takao, Koichi [Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295 (Japan); Kohda, Kohfuku [Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo, Tokyo 202-8585 (Japan); Hiramatsu, Kyoko; Kawakita, Masao [Department of Molecular Medicine, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kami-kitazawa, Setagaya-ku, Tokyo 156-8506 (Japan)

    2012-10-20

    Highlights: Black-Right-Pointing-Pointer Compounds in polyamine catabolic pathway were determined by a column-free ESI-TOF MS. Black-Right-Pointing-Pointer N{sup 1}- and N{sup 8}-acetylspermidine were determined by a column-free ESI-MS/MS. Black-Right-Pointing-Pointer The method was applied to determine activities of APAO, SMO, and SSAT in the pathway. Black-Right-Pointing-Pointer The assay method contained stable isotope-labeled natural substrates. Black-Right-Pointing-Pointer It is applicable to biological samples containing natural substrate and product. - Abstract: An analytical method for the determination of three polyamines (putrescine, spermidine, and spermine) and five acetylpolyamines [N{sup 1}-acetylspermidine (N{sup 1}AcSpd), N{sup 8}-acetylspermidine (N{sup 8}AcSpd), N{sup 1}-acetylspermine, N{sup 1},N{sup 8}-diacetylspermidine, and N{sup 1},N{sup 12}-diacetylspermine] involved in the polyamine catabolic pathway has been developed using a hybrid tandem mass spectrometer. Heptafluorobutyryl (HFB) derivatives of these compounds and respective internal standards labeled with stable isotopes were analyzed simultaneously by TOF MS, based on peak areas appearing at appropriate m/z values. The isomers, N{sup 1}AcSpd and N{sup 8}AcSpd were determined from their fragment ions, the acetylamidopropyl and acetylamidobutyl groups, respectively, using MS/MS with {sup 13}C{sub 2}-N{sup 1}AcSpd and {sup 13}C{sub 2}-N{sup 8}AcSpd which have the {sup 13}C{sub 2}-acetyl group as an internal standard. The TOF MS method was successfully applied to measure the activity of enzymes involved in polyamine catabolic pathways, namely N{sup 1}-acetylpolyamine oxidase (APAO), spermine oxidase (SMO), and spermidine/spermine N{sup 1}-acetyltransferase (SSAT). The following natural substrates and products labeled with stable isotopes considering the application to biological samples were identified; for APAO, [4,9,12-{sup 15}N{sub 3}]-N{sup 1}-acetylspermine and [1,4,8-{sup 15}N{sub 3

  5. Toward Radiocarbon Measurement of Individual Amino Acids in Marine Dissolved Organic Matter (DOM): Δ14C Blank Quantification for an HPLC Purification Method.

    Science.gov (United States)

    Bour, A. L.; Broek, T.; Walker, B. D.; Mccarthy, M. D.

    2014-12-01

    The presence of much of the marine dissolved organic nitrogen (DON) pool as uncharacterized, biologically recalcitrant molecules is a central mystery in the marine nitrogen cycle. Radiocarbon (Δ14C) isotopic measurements have been perhaps the most important data constraining the cycling of dissolved organic matter (DOM), but little Δ14C data specific to DON is available. Amino acids (AAs) are the major component of DON that can be isolated on a molecular level. Δ14C measurements for the operational "protein-like" fraction of DOM in the deep ocean indicate that this compound class has radiocarbon ages greater than several ocean mixing cycles, suggesting remarkable preservation of labile AAs exported from the surface. However, it is possible that the previously defined operational "protein-like" fraction may also contain non-AA material. Radiocarbon measurement of purified individual AAs would provide a more direct and reliable proxy for DON Δ14C age and cycling rate. We present here Δ14C blank characterization of an AA purification method based on HPLC, with on-line fraction collection. This method allows the recovery of unmodified AAs, but accurate measurement of small AA samples that can be extracted from DOM requires a system with extremely low Δ 14C blanks. Here we assess the impact of HPLC purification on the Δ14C age of known amino acids standards. Individual AA standards with contrasting (modern vs. dead) and well- characterized Δ14C ages were processed in a range of sample sizes. The eluted peaks were collected and dried, and measurement of their post-chromatography Δ14C content allowed for determination of the Δ14C blank by method of additions. The same protocol was applied to a mixture of six AA standards, to evaluate tailing effects in consecutive AA peaks of contrasting Δ14C age. AA standards were selected to include both Δ14C modern and dead AAs that elute both early and late in the chromatographic solvent program. We discuss implications

  6. Encapsulation of enzyme via one-step template-free formation of stable organic-inorganic capsules: A simple and efficient method for immobilizing enzyme with high activity and recyclability.

    Science.gov (United States)

    Huang, Renliang; Wu, Mengyun; Goldman, Mark J; Li, Zhi

    2015-06-01

    Enzyme encapsulation is a simple, gentle, and general method for immobilizing enzyme, but it often suffers from one or more problems regarding enzyme loading efficiency, enzyme leakage, mechanical stability, and recyclability. Here we report a novel, simple, and efficient method for enzyme encapsulation to overcome these problems by forming stable organic-inorganic hybrid capsules. A new, facile, one-step, and template-free synthesis of organic-inorganic capsules in aqueous phase were developed based on PEI-induced simultaneous interfacial self-assembly of Fmoc-FF and polycondensation of silicate. Addition of an aqueous solution of Fmoc-FF and sodium silicate into an aqueous solution of PEI gave a new class of organic-inorganic hybrid capsules (FPSi) with multi-layered structure in high yield. The capsules are mechanically stable due to the incorporation of inorganic silica. Direct encapsulation of enzyme such as epoxide hydrolase SpEH and BSA along with the formation of the organic-inorganic capsules gave high yield of enzyme-containing capsules (∼1.2 mm in diameter), >90% enzyme loading efficiency, high specific enzyme loading (158 mg protein g(-1) carrier), and low enzyme leakage (capsules catalyzed the hydrolysis of cyclohexene oxide to give (1R, 2R)-cyclohexane-1,2-diol in high yield and concentration, with high specific activity (6.94 U mg(-1) protein) and the same high enantioselectivity as the free enzyme. The immobilized SpEH demonstrated also excellent operational stability and recyclability: retaining 87% productivity after 20 cycles with a total reaction time of 80 h. The new enzyme encapsulation method is efficient, practical, and also better than other reported encapsulation methods. © 2015 Wiley Periodicals, Inc.

  7. Purification and characterisation of a metallopeptidase of Candida albicans.

    Science.gov (United States)

    el Moudni, B; Rodier, M H; Barrault, C; Ghazali, M; Jacquemin, J L

    1995-10-01

    A novel aminopeptidase was purified by high performance liquid chromatography from a cytosoluble 100,000 g extract of Candida albicans on the basis of its ability to cleave L-arginine 7-amino-4-methylcoumarin. The purification factor was 36 and the yield was 20%. The native enzyme had a mol. wt of 52 kDa as demonstrated by SDS-PAGE in the presence or absence of reducing conditions and exhibited an iso-electric point of 4.3. The aminopeptidase showed optimum activity at pH 7.2, a Michaelis constant of c. 50 microM and a Vmax at 19 mM AMC released/min/mg of protein for L-Arg-AMC. This enzyme was shown to cleave at low affinity L-leucine-7-amino-4-methylcoumarin as demonstrated by the spectrofluorimetric method. The enzyme was strongly inhibited by specific metallo-enzyme inhibitors-EDTA and o-phenanthroline. Furthermore, there is evidence that a similar or identical enzyme occurs in other C. albicans clinical isolates and other Candida spp.

  8. Designing and constructing an 100 bp DNA Ladder by combining PCR and enzyme digestion methods

    Directory of Open Access Journals (Sweden)

    Saidijam M

    2011-05-01

    Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Molecular DNA markers are one of the most important tools in molecular biology labs. The size of DNA molecules is determined by comparing them with known bands of markers during gel electrophoresis. There are many different protocols to produce these kinds of molecular markers. In this study we have suggested an efficient strategy to produce molecular weight markers in industrial proportions."n"nMethods : To achieve the desired sizes of DNA fragments, a combination of two previously known methods, restriction enzyme digestion and polymerase chain reaction (PCR, were used. The enzymatic digestion process was based on designing and constructing plasmids which equaled in size with the desired length of DNA fragments and produced the desired DNA fragment upon linearization. In the PCR method, the desired length of DNA fragments were cloned in multiple cloning sites of pTZ57R plasmid and in a PCR reaction, the new constructed plasmid was used as a template to produce the final fragment."n"nResults : Upon application of this strategy, 2000 and 3000 bp DNA fragments were produced by enzymatic digestion of plasmids of the same size. Moreover, 100 to 1500 bp fragments were produced during PCR using only a set of

  9. Identification and differentiation of Fasciola hepatica and Fasciola gigantica using a simple PCR-restriction enzyme method.

    Science.gov (United States)

    Rokni, Mohammad Bagher; Mirhendi, Hossein; Mizani, Azadeh; Mohebali, Mehdi; Sharbatkhori, Mitra; Kia, Eshrat Beigom; Abdoli, Hamid; Izadi, Shahrokh

    2010-02-01

    Accurate morphological differentiation between the liver fluke species Fasciola hepatica and Fasciola gigantica is difficult. We evaluated PCR-restriction enzyme profiles of internal transcribed spacer 1 (ITS1) that could aid in their identification. Fifty F. hepatica and 30 F. gigantica specimens were collected from different hosts in three provinces of Iran. For DNA extraction, we crushed fragments of the worms between two glass slides as a new method to break down the cells. DNA from the crushed materials was then extracted with a conventional phenol-chloroform method and with the newly developed technique, commercial FTA cards. A primer pair was selected to amplify a 463-bp region of the ITS1 sequence. After sequencing 14 samples and in silico analysis, cutting sites of all known enzymes were predicted and TasI was selected as the enzyme that yielded the most informative profile. Crushing produced enough DNA for PCR amplification with both the phenol-chloroform and commercial FTA card method. The DNA extracted from all samples was successfully amplified and yielded a single sharp band of the expected size. Digestion of PCR products with TasI allowed us to distinguish the two species. In all samples, molecular identification was consistent with morphological identification. Our PCR-restriction enzyme profile is a simple, rapid and reliable method for differentiating F. hepatica and F. gigantica, and can be used for diagnostic and epidemiological purposes.

  10. 浮空器氦气纯化方法应用研究%Research on application of aerostat helium purification method

    Institute of Scientific and Technical Information of China (English)

    万小刚; 傅剑; 赵林华

    2011-01-01

    在浮空器使用过程中,外界空气的混入将导致其内部氦气纯度降低,从而影响到浮空器正常使用.文中通过对常用气体分离方法的分析研究,提出了以高压低温冷凝和低温吸附相结合的方式作为浮空器氦气提纯方式的思路,并详细给出了实现工艺流程和控制方案.换热计算结果表明:该纯化方法液氮消耗量小、氦气提纯成本低.%Aerostat’s working capability is influenced when atmosphere mixed into its envelope and debased the inner helium purity. Through analysis of general gas separation methods, this paper presented a kind of high - pressure condensation and adsorption method as aerostat’s heiium purification way, detailed working flow and control scheme were also presented. Heat exchange calculation shows that, this method costs low liquid nitrogen and low expense.

  11. An improved method for specificity annotation shows a distinct evolutionary divergence among the microbial enzymes of the cholylglycine hydrolase family.

    Science.gov (United States)

    Panigrahi, Priyabrata; Sule, Manas; Sharma, Ranu; Ramasamy, Sureshkumar; Suresh, C G

    2014-06-01

    Bile salt hydrolases (BSHs) are gut microbial enzymes that play a significant role in the bile acid modification pathway. Penicillin V acylases (PVAs) are enzymes produced by environmental microbes, having a possible role in pathogenesis or scavenging of phenolic compounds in their microbial habitats. The correct annotation of such physiologically and industrially important enzymes is thus vital. The current methods relying solely on sequence homology do not always provide accurate annotations for these two members of the cholylglycine hydrolase (CGH) family as BSH/PVA enzymes. Here, we present an improved method [binding site similarity (BSS)-based scoring system] for the correct annotation of the CGH family members as BSH/PVA enzymes, which along with the phylogenetic information incorporates the substrate specificity as well as the binding site information. The BSS scoring system was developed through the analysis of the binding sites and binding modes of the available BSH/PVA structures with substrates glycocholic acid and penicillin V. The 198 sequences in the dataset were then annotated accurately using BSS scores as BSH/PVA enzymes. The dataset presented contained sequences from Gram-positive bacteria, Gram-negative bacteria and archaea. The clustering obtained for the dataset using the method described above showed a clear distinction in annotation of Gram-positive bacteria and Gram-negative bacteria. Based on this clustering and a detailed analysis of the sequences of the CGH family in the dataset, we could infer that the CGH genes might have evolved in accordance with the hypothesis stating the evolution of diderms and archaea from the monoderms.

  12. Methods for demonstration of enzyme activity in muscle fibres at the muscle/bone interface in demineralized tissue

    DEFF Research Database (Denmark)

    Kirkeby, S; Vilmann, H

    1981-01-01

    A method for demonstration of activity for ATPase and various oxidative enzymes (succinic dehydrogenase, alpha-glycerophosphate dehydrogenase, and lactic dehydrogenase) in muscle/bone sections of fixed and demineralized tissue has been developed. It was found that it is possible to preserve...... considerable amounts of the above mentioned enzymes in the muscle fibres at the muscle/bone interfaces. The best results were obtained after 20 min fixation, and 2-3 weeks of storage in MgNa2EDTA containing media. As the same technique previously has been used to describe patterns of resorption and deposition...

  13. Partial purification and characterization of an endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242.

    OpenAIRE

    Ishii-Karakasa, I; Iwase, H.; Hotta, K; Tanaka, Y.; Omura, S

    1992-01-01

    For the purification of a new type of endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 (endo-GalNAc-ase-S) [Iwase, Ishii, Ishihara, Tanaka, Omura & Hotta (1988) Biochem. Biophys. Res. Commun. 151, 422-428], a method for assaying enzyme activity was established. Using purified pig gastric mucus glycoprotein (PGM) as the substrate, oligosaccharides liberated from PGM were pyridylaminated, and the reducing terminal sugars of oligosaccharides larger than G...

  14. Purification and concentration of alphavirus.

    Science.gov (United States)

    Lundstrom, Kenneth

    2012-07-01

    The alphaviruses Semliki Forest virus and Sindbis virus have been used frequently as expression vectors in vitro and in vivo. Usually, these systems consist of replication-deficient vectors that require a helper vector for packaging of recombinant particles. Replication-proficient vectors have also been engineered. Alphaviral vectors can be used as nucleic-acid-based vectors (DNA and RNA) or infectious particles. High-titer viral production is achieved in alphaviruses facilitates studies in mammalian and nonmammalian cell lines, primary cells in culture, and in vivo. The strong preference for expression in neuronal cells has made alphaviruses particularly useful in neurobiological studies. Unfortunately, their strong cytotoxic effect on host cells, relatively short-term transient expression patterns, and the reasonably high cost of viral production remain drawbacks. However, novel mutant alphaviruses have showed reduced cytotoxicity and prolonged expression. Membrane proteins (which are generally difficult to express at high levels in recombinant systems) have generated high yields and facilitate applications in structural biology. Alphaviruses have also been applied in vaccine development and gene therapy. Generally, purification or concentration of alphaviruses is not necessary. However, for instance, the medium derived from baby hamster kidney cells is toxic to primary neurons in culture. Including a purification step substantially improves the survival of the transduced neurons. Viral concentration and purification may also be advantageous for in vivo studies in animal models and are mandatory for clinical applications. This protocol describes three methods for purification and concentration of alphavirus.

  15. Biobased monoliths for adenovirus purification.

    Science.gov (United States)

    Fernandes, Cláudia S M; Gonçalves, Bianca; Sousa, Margarida; Martins, Duarte L; Barroso, Telma; Pina, Ana Sofia; Peixoto, Cristina; Aguiar-Ricardo, Ana; Roque, A Cecília A

    2015-04-01

    Adenoviruses are important platforms for vaccine development and vectors for gene therapy, increasing the demand for high titers of purified viral preparations. Monoliths are macroporous supports regarded as ideal for the purification of macromolecular complexes, including viral particles. Although common monoliths are based on synthetic polymers as methacrylates, we explored the potential of biopolymers processed by clean technologies to produce monoliths for adenovirus purification. Such an approach enables the development of disposable and biodegradable matrices for bioprocessing. A total of 20 monoliths were produced from different biopolymers (chitosan, agarose, and dextran), employing two distinct temperatures during the freezing process (-20 °C and -80 °C). The morphological and physical properties of the structures were thoroughly characterized. The monoliths presenting higher robustness and permeability rates were further analyzed for the nonspecific binding of Adenovirus serotype 5 (Ad5) preparations. The matrices presenting lower nonspecific Ad5 binding were further functionalized with quaternary amine anion-exchange ligand glycidyltrimethylammonium chloride hydrochloride by two distinct methods, and their performance toward Ad5 purification was assessed. The monolith composed of chitosan and poly(vinyl) alcohol (50:50) prepared at -80 °C allowed 100% recovery of Ad5 particles bound to the support. This is the first report of the successful purification of adenovirus using monoliths obtained from biopolymers processed by clean technologies.

  16. Optimization of a method for isolation and purification of early pregnancy factor%早孕因子分离纯化方法的优化

    Institute of Scientific and Technical Information of China (English)

    温志锋; 练玉银; 王家骥; 聂湘辉; 林燕棉

    2012-01-01

    This study aimed to optimize the method for isolation and purification of early pregnancy factor (EPF) in serum from the normal early pregnancy women so as to improve the access rate of EPF. EPF was purified from the mixed serum of normal early pregnancy women with 12 gestational weeks by using the DEAE Fast Flow ion exchange chromatography and Heparin Fast Flow affinity chromatography. The biological activity of each extraction was detected by erythrocty active rosette inhibition test (Ea-RIT) and the purified product was identified by sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE) to determinate its molecular weight. And its specificity was analyzed by Western blot. The Ea-RIT showed that the DE- I peak of the two emerged peaks in DEAE Fast Flow ion exchange chromatography, and the H-Ⅱ peak of the two arisen peaks in Heparin Fast Flow affinity chromatography were the EPF activity peak. The SDS-PAGE profile further revealed there was only one band with a molecular weight of 38 100 kD for the extraction of the H-Ⅱ peak, and Western blot analysis showed that the matching of antibody and antigen was good. The quantity of the protein was 0.615 mg with a recovery rate was 0.034%, which was much higher than the traditional four-step method. The result indicated that the optimized method for the isolation and purification of EPF from the early pregnancy women serum is feasible, which can greatly improve the purification efficiency.%目的 对正常早孕妇女血清中早孕因子(EPF)的分离纯化过程进行优化,以提高EPF的回收率.方法 依次采用DEAEFast Flow离子交换色谱和Heparin Fast Flow亲和色谱,从妊娠12周内的正常早孕妇女混合血清中提纯EPF,采用活性玫瑰花结抑制试验(Ea-RIT)检测各阶段产物的EPF活性,SDS-聚丙烯酰胺凝胶(SDS-PAGE)电泳鉴定纯化物并测定其相对分子质量,用Western blot鉴定其特异性.结果 经Ea-RIT检测后,DEAE Fast Flow

  17. Surface processes during purification of InP quantum dots

    OpenAIRE

    2014-01-01

    Recently, a new simple and fast method for the synthesis of InP quantum dots by using phosphine as phosphorous precursor and myristic acid as surface stabilizer was reported. Purification after synthesis is necessary to obtain samples with good optical properties. Two methods of purification were compared and the surface processes which occur during purification were studied. Traditional precipitation with acetone is accompanied by a small increase in photoluminescence. It occurs that during ...

  18. Hybrid quantum and classical methods for computing kinetic isotope effects of chemical reactions in solutions and in enzymes.

    Science.gov (United States)

    Gao, Jiali; Major, Dan T; Fan, Yao; Lin, Yen-Lin; Ma, Shuhua; Wong, Kin-Yiu

    2008-01-01

    A method for incorporating quantum mechanics into enzyme kinetics modeling is presented. Three aspects are emphasized: 1) combined quantum mechanical and molecular mechanical methods are used to represent the potential energy surface for modeling bond forming and breaking processes, 2) instantaneous normal mode analyses are used to incorporate quantum vibrational free energies to the classical potential of mean force, and 3) multidimensional tunneling methods are used to estimate quantum effects on the reaction coordinate motion. Centroid path integral simulations are described to make quantum corrections to the classical potential of mean force. In this method, the nuclear quantum vibrational and tunneling contributions are not separable. An integrated centroid path integral-free energy perturbation and umbrella sampling (PI-FEP/UM) method along with a bisection sampling procedure was summarized, which provides an accurate, easily convergent method for computing kinetic isotope effects for chemical reactions in solution and in enzymes. In the ensemble-averaged variational transition state theory with multidimensional tunneling (EA-VTST/MT), these three aspects of quantum mechanical effects can be individually treated, providing useful insights into the mechanism of enzymatic reactions. These methods are illustrated by applications to a model process in the gas phase, the decarboxylation reaction of N-methyl picolinate in water, and the proton abstraction and reprotonation process catalyzed by alanine racemase. These examples show that the incorporation of quantum mechanical effects is essential for enzyme kinetics simulations.

  19. Methods for demonstration of enzyme activity in muscle fibres at the muscle/bone interface in demineralized tissue

    DEFF Research Database (Denmark)

    Kirkeby, S; Vilmann, H

    1981-01-01

    A method for demonstration of activity for ATPase and various oxidative enzymes (succinic dehydrogenase, alpha-glycerophosphate dehydrogenase, and lactic dehydrogenase) in muscle/bone sections of fixed and demineralized tissue has been developed. It was found that it is possible to preserve...... with the aid of a mapping of presence of phosphomonoesterases on bone surfaces, the method may be used to study possible biochemical interactions between bone and muscle tissue at the muscle/bone interface....

  20. Purification of methanol dehydrogenase from mouth methylotrophic bacteria of tropical region

    OpenAIRE

    Waturangi, D.; Marko, N.; M. T. Suhartono2)

    2011-01-01

    Aims: Purification of methanol dehydrogenase (MDH) from methylotrophic bacteria was conducted to obtain pure enzyme for further research and industrial applications due to the enzyme’s unique activity that catalyzes oxidation of methanol as an important carbon source in methylotrophic bacteria.Methodology and Results: The enzyme was screened from methylotrophic bacteria isolated from human mouth. Purification of this enzyme was conducted using ammonium sulphate precipitation followed by catio...

  1. Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes

    DEFF Research Database (Denmark)

    Trujillo, L E; Pupo, E; Miranda, F;

    1996-01-01

    We evaluated the use of two radiolabeled lambda DNA/Hpa II substrates to detect 5'-->3', 3'-->5' single and double stranded DNA dependent exonuclease and phosphatase activities found as contaminants in restriction and modifying enzyme preparations. Looking for the meaning of the radioactive assay...

  2. Application of an Improved Enzyme-Linked Immunosorbent Assay Method for Serological Diagnosis of Canine Leishmaniasis

    NARCIS (Netherlands)

    N. Santarem; R. Silvestre; L. Cardoso; H. Schallig; S.G. Reed; A. Cordeiro-da-Silva

    2010-01-01

    Accurate diagnosis of canine leishmaniasis (CanL) is essential toward a more efficient control of this zoonosis, but it remains problematic due to the high incidence of asymptomatic infections. In this study, we present data on the development of enzyme-linked immunosorbent assay (ELISA)-based techn

  3. Enzyme-linked immunospot: an alternative method for the detection of interferon gamma in Johne's disease

    DEFF Research Database (Denmark)

    Begg, Douglas J.; de Silva, Kumudika; Bosward, Katrina;

    2009-01-01

    To date, the sensitivity of the interferon gamma (IFN-) enzyme-linked immunosorbent assay (ELISA) to detect Johne's disease (JD) has been poor, especially in the early stages of disease. To improve the sensitivity of IFN- detection in the early stages of infection, an alternate assay needs...

  4. Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes

    DEFF Research Database (Denmark)

    Trujillo, L E; Pupo, E; Miranda, F

    1996-01-01

    We evaluated the use of two radiolabeled lambda DNA/Hpa II substrates to detect 5'-->3', 3'-->5' single and double stranded DNA dependent exonuclease and phosphatase activities found as contaminants in restriction and modifying enzyme preparations. Looking for the meaning of the radioactive assay...

  5. Elaboration of new method of enzyme adsorption on silicalite and nano beta zeolite for amperometric biosensor creation

    Directory of Open Access Journals (Sweden)

    Soldatkin O. O.

    2014-07-01

    Full Text Available Aim. Optimization of a new method of enzyme immobilization for amperometric biosensor creation. Methods. The amperometric biosensor with glucose oxidase immobilized on zeolites as bioselective elements and platinum disk electrode as transducers of biochemical signal into the electric one was used in the work. Results. The biosensors based on glucose oxidase adsorbed on zeolites were characterized by a higher sensitivity to glucose and a better inter-reproducibility. The best analytical characteristics were obtained for the biosensors based on nano beta zeolite. It has been found that an increase in the amount of zeolite on the surface of amperometric transducer may change such biosensor parameters as sensitivity to the substrate and duration of the analysis. Conclusions. The proposed method of enzyme immobilization by adsorption on zeolites is shown to be quite promising in the development of amperometric biosensors and therefore should be further investigated.

  6. Enzymes: principles and biotechnological applications.

    Science.gov (United States)

    Robinson, Peter K

    2015-01-01

    Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyse a wide range of commercially important processes. This chapter covers the basic principles of enzymology, such as classification, structure, kinetics and inhibition, and also provides an overview of industrial applications. In addition, techniques for the purification of enzymes are discussed.

  7. Enzymes: principles and biotechnological applications

    Science.gov (United States)

    Robinson, Peter K.

    2015-01-01

    Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyse a wide range of commercially important processes. This chapter covers the basic principles of enzymology, such as classification, structure, kinetics and inhibition, and also provides an overview of industrial applications. In addition, techniques for the purification of enzymes are discussed. PMID:26504249

  8. Enzyme detection by microfluidics

    DEFF Research Database (Denmark)

    2013-01-01

    Microfluidic-implemented methods of detecting an enzyme, in particular a DNA-modifying enzyme, are provided, as well as methods for detecting a cell, or a microorganism expressing said enzyme. The enzyme is detected by providing a nucleic acid substrate, which is specifically targeted...

  9. FN-Identify: Novel Restriction Enzymes-Based Method for Bacterial Identification in Absence of Genome Sequencing.

    Science.gov (United States)

    Awad, Mohamed; Ouda, Osama; El-Refy, Ali; El-Feky, Fawzy A; Mosa, Kareem A; Helmy, Mohamed

    2015-01-01

    Sequencing and restriction analysis of genes like 16S rRNA and HSP60 are intensively used for molecular identification in the microbial communities. With aid of the rapid progress in bioinformatics, genome sequencing became the method of choice for bacterial identification. However, the genome sequencing technology is still out of reach in the developing countries. In this paper, we propose FN-Identify, a sequencing-free method for bacterial identification. FN-Identify exploits the gene sequences data available in GenBank and other databases and the two algorithms that we developed, CreateScheme and GeneIdentify, to create a restriction enzyme-based identification scheme. FN-Identify was tested using three different and diverse bacterial populations (members of Lactobacillus, Pseudomonas, and Mycobacterium groups) in an in silico analysis using restriction enzymes and sequences of 16S rRNA gene. The analysis of the restriction maps of the members of three groups using the fragment numbers information only or along with fragments sizes successfully identified all of the members of the three groups using a minimum of four and maximum of eight restriction enzymes. Our results demonstrate the utility and accuracy of FN-Identify method and its two algorithms as an alternative method that uses the standard microbiology laboratories techniques when the genome sequencing is not available.

  10. FN-Identify: Novel Restriction Enzymes-Based Method for Bacterial Identification in Absence of Genome Sequencing

    Science.gov (United States)

    Ouda, Osama; El-Refy, Ali; El-Feky, Fawzy A.; Mosa, Kareem A.

    2015-01-01

    Sequencing and restriction analysis of genes like 16S rRNA and HSP60 are intensively used for molecular identification in the microbial communities. With aid of the rapid progress in bioinformatics, genome sequencing became the method of choice for bacterial identification. However, the genome sequencing technology is still out of reach in the developing countries. In this paper, we propose FN-Identify, a sequencing-free method for bacterial identification. FN-Identify exploits the gene sequences data available in GenBank and other databases and the two algorithms that we developed, CreateScheme and GeneIdentify, to create a restriction enzyme-based identification scheme. FN-Identify was tested using three different and diverse bacterial populations (members of Lactobacillus, Pseudomonas, and Mycobacterium groups) in an in silico analysis using restriction enzymes and sequences of 16S rRNA gene. The analysis of the restriction maps of the members of three groups using the fragment numbers information only or along with fragments sizes successfully identified all of the members of the three groups using a minimum of four and maximum of eight restriction enzymes. Our results demonstrate the utility and accuracy of FN-Identify method and its two algorithms as an alternative method that uses the standard microbiology laboratories techniques when the genome sequencing is not available. PMID:26880910

  11. ISOLATION AND PURIFICATION OF LYSOZYME FROM THE HEN EGG WHITE

    Directory of Open Access Journals (Sweden)

    S. S.

    2015-12-01

    Full Text Available The aim of the research was the development of the method of lysozyme isolation from hen egg proteins. Lysozyme was isolated by differential heat denaturation of proteins with changing of the medium pH value, followed by neutralization, dialysis and additional purification by gel chromatography on Sephadex G-50. Activity was determined by bacteriolytic method (with Micrococcus lysodeikticus 4698 as a substrate. The enzyme purity and molecular mass were determined using SDS-electrophoresis and massspectrometry. The method of lysozyme isolation from hen egg proteins with the enzyme yield of 3.2 ± 0.2% and bacteriolytic activity of 22 025 ± 1 500 U/mg is modified. According to electrophoresis data, the isolated enzyme is characterized by high degree of purity (~95–98% and is comparable with lysozyme of AppliChem company by main physical and chemical characteristics. The obtaining product is stored in a crystalline form at low temperature (–24 оC for 9 months. The proposed method allows obtaining active and stable lysozyme with high purity from hen egg protein in laboratory conditions for the usage in biotechnology.

  12. A robust and efficient method for estimating enzyme complex abundance and metabolic flux from expression data

    OpenAIRE

    Barker, Brandon E.; Sadagopan, Narayanan; Wang, Yiping; Smallbone, Kieran; Myers, Christopher R.; Xi, Hongwei; Locasale, Jason W.; Gu, Zhenglong

    2014-01-01

    A major theme in constraint-based modeling is unifying experimental data, such as biochemical information about the reactions that can occur in a system or the composition and localization of enzyme complexes, with highthroughput data including expression data, metabolomics, or DNA sequencing. The desired result is to increase predictive capability resulting in improved understanding of metabolism. The approach typically employed when only gene (or protein) intensities are available is the cr...

  13. Catalytic Enzyme-Based Methods for Water Treatment and Water Distribution System Decontamination. 1. Literature Survey

    Science.gov (United States)

    2006-06-01

    polyacrylamide 238 were very widely used, although not in the food industry where its toxicity was an issue. Since the production polymers such as polyacrylamide ...technology for OPAA as well. In addition to the type of synthetic polymers commonly thought of in regards to enzyme entrapment ( polyacrylamide and...entrapment can be diffusional limitations as well as steric hindrance, especially when the substrates are large macromolecular materials such as starch and

  14. Measurement of deoxyribonuclease I activity in human tissues and body fluids by a single radial enzyme-diffusion method.

    Science.gov (United States)

    Nadano, D; Yasuda, T; Kishi, K

    1993-03-01

    In the single radial enzyme-diffusion (SRED) method for assay of deoxyribonuclease I, a precisely measured volume of the enzyme solution is dispensed into a circular well in an agarose gel layer in which DNA and ethidium bromide are uniformly distributed. A circular dark zone is formed as the enzyme diffuses from the well radially into the gel and digests substrate DNA. The diameter of the dark circle of hydrolyzed DNA increases in size with time and correlates linearly with the amount of enzyme applied to the well. Thus, the SRED can be used for quantitation of deoxyribonuclease I with a limit of detection of 2 x 10(-6) unit. This corresponds to 1 pg of purified urine deoxyribonuclease I. We measured the deoxyribonuclease I activity of 17 different human tissues and body fluids from healthy donors. Urine samples showed the greatest activity, 6.0 +/- 2.2 kilo-units/g protein (mean +/- SD). Serum deoxyribonuclease I activity was 4.4 +/- 1.8 units/L.

  15. A simple enzymic method for the synthesis of adenosine 5'-[alpha-32P]triphosphate on a preparative scale.

    Science.gov (United States)

    Martin, B R; Voorheis, H P

    1977-03-01

    A simple, rapid and inexpensive method is described for the enzymic synthesis of [alpha-32P]ATP from [32P]Pi on a preparative scale with an overall yield of 53%. The final product contained all of the detectable radioactivity (less than 99.9%) in the alpha position and has been shown to behave identically with commerically availabe [alpha-32P]ATP during the synthesis of 3':5'-cyclic AMP in the reaction catalysed by adenylate cyclase.

  16. Purification technology of molten aluminum

    Institute of Scientific and Technical Information of China (English)

    孙宝德; 丁文江; 疏达; 周尧和

    2004-01-01

    Various purification methods were explored to eliminate the dissolved hydrogen and nonmetallic inclusions from molten aluminum alloys. A novel rotating impeller head with self-oscillation nozzles or an electromagnetic valve in the gas circuit was used to produce pulse gas currents for the rotary impeller degassing method. Water simulation results show that the size of gas bubbles can be decreased by 10%-20% as compared with the constant gas current mode. By coating ceramic filters or particles with active flux or enamels, composite filters were used to filter the scrap A356 alloy and pure aluminum. Experimental results demonstrate that better filtration efficiency and operation performance can be obtained. Based on numerical calculations, the separation efficiency of inclusions by high frequency magnetic field can be significantly improved by using a hollow cylinder-like separator or utilizing the effects of secondary flow of the melt in a square separator. A multi-stage and multi-media purification platform based on these methods was designed and applied in on-line processing of molten aluminum alloys. Mechanical properties of the processed scrap A356 alloy are greatly improved by the composite purification.

  17. Enzymes for improved biomass conversion

    Science.gov (United States)

    Brunecky, Roman; Himmel, Michael E.

    2016-02-02

    Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

  18. Enzymes for improved biomass conversion

    Energy Technology Data Exchange (ETDEWEB)

    Brunecky, Roman; Himmel, Michael E.

    2016-02-02

    Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

  19. Purification and characterization of α-Amylase from Miswak Salvadora persica

    OpenAIRE

    Mohamed, Saleh A.; Almulaiky, Yaaser Q.; Youssri M. Ahmed; Al-Bar, Omar AM; Ibrahim, Ibrahim H.

    2014-01-01

    Background The miswak (Salvadora persica) is a natural toothbrush. It is well known that very little information has been reported on enzymes in miswak as medicinal plant. Recently, we study peroxidase in miswak. In the present study, the main goal of this work is to purify and characterize α-amylase from miswak. The second goal is to study the storage stability of α-amylase in toothpaste. Method The purification method included chromatographaphy of miswak α-amylase on DEAE-Sepharose column a...

  20. Phenylalanine ammonia-lyase, flavanone 3β-hydroxylase and flavonol synthase enzyme activity by a new in vitro assay method in berry fruits.

    Science.gov (United States)

    Flores, Gema; De la Peña Moreno, Fernando; Blanch, Gracia Patricia; Del Castillo, Maria Luisa Ruiz

    2014-06-15

    An HPLC method for the determination of phenylalanine ammonia-lyase, flavanone 3β-hydroxylase and flavonol synthase enzyme activity is proposed. This method is based on the determination of the compounds produced and consumed on the enzymatic reaction in just one chromatographic analysis. Optimisation of the method considered kinetic studies to establish the incubation time to perform the assay. The method here described proved to be an interesting approach to measure the activities of the three enzymes simultaneously increasing the rapidity, selectivity and sensitivity over other exiting methods. The enzyme activity method developed was applied to strawberry, raspberry, blackberry, redcurrant and blackcurrant fruits.

  1. Membrane adsorbers as purification tools for monoclonal antibody purification.

    Science.gov (United States)

    Boi, Cristiana

    2007-03-15

    Downstream purification processes for monoclonal antibody production typically involve multiple steps; some of them are conventionally performed by bead-based column chromatography. Affinity chromatography with Protein A is the most selective method for protein purification and is conventionally used for the initial capturing step to facilitate rapid volume reduction as well as separation of the antibody. However, conventional affinity chromatography has some limitations that are inherent with the method, it exhibits slow intraparticle diffusion and high pressure drop within the column. Membrane-based separation processes can be used in order to overcome these mass transfer limitations. The ligand is immobilized in the membrane pores and the convective flow brings the solute molecules very close to the ligand and hence minimizes the diffusional limitations associated with the beads. Nonetheless, the adoption of this technology has been slow because membrane chromatography has been limited by a lower binding capacity than that of conventional columns, even though the high flux advantages provided by membrane adsorbers would lead to higher productivity. This review considers the use of membrane adsorbers as an alternative technology for capture and polishing steps for the purification of monoclonal antibodies. Promising industrial applications as well as new trends in research will be addressed.

  2. 壳聚糖絮凝沉降法精制抗疲劳颗粒的研究%Study on purification of anti-fatigue granule by chitosan flocculating setting method

    Institute of Scientific and Technical Information of China (English)

    高燕; 林桂涛; 盛华刚; 张超

    2011-01-01

    目的:优选壳聚糖精制抗疲劳颗粒的最佳纯化工艺.方法:采用正交试验考察药液浓缩程度、pH值、壳聚糖用量对壳聚糖絮凝沉降法精制抗疲劳颗粒的影响,优选出壳聚糖精制抗疲劳颗粒的最佳工艺.结果:最佳纯化工艺为药液浓度为1.0 g生药·mL-1、1 mL药液加入相当于壳聚糖2 mg的壳聚糖溶液、药液pH值不调.结论:壳聚糖澄清剂可以用于抗疲劳颗粒的纯化.%Objective:To optimize the best purification process of anti-fatigue granule by the chitosan. Methods: Adopting the orthogonal design method to study the effect of three factors including the physic liquor concentrated degree,pH value and the amount of chitosan on refining the extract of anti-fatigue granule by the chitosan flocculating setting method. Optimize the optimum purification process of chitosan. Results;The optimum purification process was the mass concentration contained 1.0 g-ml-1,1 mL of liquid added is 2 mg of chitosan solution,pH remain unchanged. Conclusion: Chitosan can be used to the purification process of anti-fatigue granule.

  3. Solvent engineering and other reaction design methods for favouring enzyme-catalysed synthesis

    DEFF Research Database (Denmark)

    Zeuner, Birgitte

    for FAE-catalysed acylation reactions was reviewed. FAE type A from Aspergillus niger and an FAE from a commercial preparation from Humicola insolens, Depol 740L, could not catalyse the esterification of arabinose or xylose with hydroxycinnamates in IL-buffer systems or in surfactantless microemulsion......, the biocatalytic productivity was increased more than 9-fold as a result of the enzyme recovery. In conclusion, where the use of non-conventional media is required for catalysis, e.g. in the thermodynamically controlled FAE-catalysed esterification, careful selection of both solvent system and the FAE itself...

  4. Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes

    Science.gov (United States)

    Smith, R.E.; Dolbeare, F.A.

    1980-10-21

    Analysis of proteinases is accomplished using conventional amino acid containing aromatic amine substrates. Aromatic amines such as 4-methoxy-2-naphthylamine (4M2NA), 2-naphthylamine, aminoisophthalic acid dimethyl ester, p-nitroaniline, 4-methoxy-1-aminofluorene and coumarin derivatives resulting from enzymatic hydrolysis of the substrate couples with aromatic aldehydes such as 5-nitrosalicylaldehyde (5-NSA), benzaldehyde and p-nitrobenzaldehyde to produce Schiff-base complexes which are water insoluble. Certain Schiff-base complexes produce a shift from blue to orange-red (visible) fluorescence. Such complexes are useful in the assay of enzymes. No Drawings

  5. Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes

    Science.gov (United States)

    Smith, Robert E.; Dolbeare, Frank A.

    1979-01-01

    Analysis of proteinases is accomplished using conventional amino acid containing aromatic amine substrates. Aromatic amines such as 4-methoxy-2-naphthylamine (4M2NA), 2-naphthylamine, aminoisophthalic acid dimethyl ester, p-nitroaniline, 5-methoxy-1-aminofluorene and coumarin derivatives resulting from enzymatic hydrolysis of the substrate couples with aromatic aldehydes such as 5-nitrosalicylaldehyde (5-NSA), benzaldehyde and p-nitrobenzaldehyde to produce Schiff-base complexes which are water insoluble. Certain Schiff-base complexes produce a shift from blue to orange-red (visible) fluorescence. Such complexes are useful in the assay of enzymes.

  6. Purification and characterization of M-177, a 177 kDa allergen, from the house dust mite Dermatophagoides farinae

    Directory of Open Access Journals (Sweden)

    Akihiro Fujikawa

    1999-01-01

    Full Text Available A high molecular weight allergen, M-177, was recently discovered in the house dust mite, Dermatophagoides farinae (D. farinae. The aims of this study were to develop a conventional purification procedure for M-177 and then to analyze some of the immunochemical properties of M-177. Mite extracts obtained from purified mite bodies were a suitable material for preparing M-177, because the purified mite extract contained large amounts of M-177. The purification of this allergen from the extract was achieved by ethanol fractionation, anion exchange chromatography, and gel filtration chromatography. The purified antigen was immunochemically equivalent to that of a preparation obtained by a previous affinity method using an anti-Mag 3-immobilized column. The yield of this purification procedure was 36.8% of the initial amount of M-177 in the extract, 40-fold greater than that of the previous immunoaffinity method. Our purification method was useful for preparing this allergen. The purified M-177 reacted in skin tests in 11 of 16 miteallergic patients, compared to 10 of 16 with Der f 2. The amount of M-177 in the purified mite extract determined by enzyme-linked immunosorbent assay inhibition was as much as 0.95% of the total protein, which was higher than the amounts of Der f 1 (0.52% and Der f 2 (0.32%. The potent allergenic activity and large amount of M-177 in the mites indicate that it is an important mite allergen.

  7. Simple and rapid human papillomavirus genotyping method by restriction fragment length polymorphism analysis with two restriction enzymes.

    Science.gov (United States)

    Chen, Linghan; Watanabe, Ken; Haruyama, Takahiro; Kobayashi, Nobuyuki

    2013-07-01

    Cervical cancer, the third most common cancer that affects women worldwide, is caused by the human papillomavirus (HPV) and is treatable when detected at an early stage. To date, more than 100 different HPV types have been described, and the development of simple, low-cost, and accurate methods to distinguish HPV genotypes is highly warranted. In this study, an HPV genotyping assay based on polymerase chain reaction (PCR) was evaluated. This method involved the use of MY09/11 primers followed by restriction fragment length polymorphism (RFLP) analysis with the restriction enzymes HpyCH4V and NlaIII. Cervical specimens preserved using CytoRich Blue fluid were collected from 1,134 female volunteers for HPV detection, and 1,111 valid samples were amplified using PCR. The PCR method was sensitive enough to detect 25 copies of HPV18, and three copies of HPV16. Out of 202 PCR-positive samples, HPV genotypes were determined in 189 samples (93.6%) by this RFLP method. Results were then evaluated further by capillary sequencing method. Concordant results between the two tests were as high as 96.0%. Thirteen samples, which tested negative with RFLP, were verified as non-specific amplifications with PCR. In conclusion, this PCR-RFLP method using restriction enzymes HpyCH4V and NlaIII is simple, non-labor intensive, and is applicable for the inexpensive determination of HPV genotypes in clinical samples.

  8. The Enzyme Portal: a case study in applying user-centred design methods in bioinformatics.

    Science.gov (United States)

    de Matos, Paula; Cham, Jennifer A; Cao, Hong; Alcántara, Rafael; Rowland, Francis; Lopez, Rodrigo; Steinbeck, Christoph

    2013-03-20

    User-centred design (UCD) is a type of user interface design in which the needs and desires of users are taken into account at each stage of the design process for a service or product; often for software applications and websites. Its goal is to facilitate the design of software that is both useful and easy to use. To achieve this, you must characterise users' requirements, design suitable interactions to meet their needs, and test your designs using prototypes and real life scenarios.For bioinformatics, there is little practical information available regarding how to carry out UCD in practice. To address this we describe a complete, multi-stage UCD process used for creating a new bioinformatics resource for integrating enzyme information, called the Enzyme Portal (http://www.ebi.ac.uk/enzymeportal). This freely-available service mines and displays data about proteins with enzymatic activity from public repositories via a single search, and includes biochemical reactions, biological pathways, small molecule chemistry, disease information, 3D protein structures and relevant scientific literature.We employed several UCD techniques, including: persona development, interviews, 'canvas sort' card sorting, user workflows, usability testing and others. Our hope is that this case study will motivate the reader to apply similar UCD approaches to their own software design for bioinformatics. Indeed, we found the benefits included more effective decision-making for design ideas and technologies; enhanced team-working and communication; cost effectiveness; and ultimately a service that more closely meets the needs of our target audience.

  9. Plasmodium serine hydroxymethyltransferase as a potential anti-malarial target: inhibition studies using improved methods for enzyme production and assay

    Directory of Open Access Journals (Sweden)

    Sopitthummakhun Kittipat

    2012-06-01

    Full Text Available Abstract Background There is an urgent need for the discovery of new anti-malarial drugs. Thus, it is essential to explore different potential new targets that are unique to the parasite or that are required for its viability in order to develop new interventions for treating the disease. Plasmodium serine hydroxymethyltransferase (SHMT, an enzyme in the dTMP synthesis cycle, is a potential target for such new drugs, but convenient methods for producing and assaying the enzyme are still lacking, hampering the ability to screen inhibitors. Methods Production of recombinant Plasmodium falciparum SHMT (PfSHMT and Plasmodium vivax SHMT (PvSHMT, using auto-induction media, were compared to those using the conventional Luria Bertani medium with isopropyl thio-β-D-galactoside (LB-IPTG induction media. Plasmodium SHMT activity, kinetic parameters, and response to inhibitors were measured spectrophotometrically by coupling the reaction to that of 5,10-methylenetetrahydrofolate dehydrogenase (MTHFD. The identity of the intermediate formed upon inactivation of Plasmodium SHMTs by thiosemicarbazide was investigated by spectrophotometry, high performance liquid chromatography (HPLC, and liquid chromatography-mass spectrometry (LC-MS. The active site environment of Plasmodium SHMT was probed based on changes in the fluorescence emission spectrum upon addition of amino acids and folate. Results Auto-induction media resulted in a two to three-fold higher yield of Pf- and PvSHMT (7.38 and 29.29 mg/L compared to that produced in cells induced in LB-IPTG media. A convenient spectrophotometric activity assay coupling Plasmodium SHMT and MTHFD gave similar kinetic parameters to those previously obtained from the anaerobic assay coupling SHMT and 5,10-methylenetetrahydrofolate reductase (MTHFR; thus demonstrating the validity of the new assay procedure. The improved method was adopted to screen for Plasmodium SHMT inhibitors, of which some were originally designed

  10. Gas purification process

    Energy Technology Data Exchange (ETDEWEB)

    Hoelter, H.; Gresch, H.; Igelbuescher, H.; Dewert, H.

    1987-08-13

    To avoid the problems of reheating in a wet process as well as the problems of higher gas supply in a dry process, the invention proposes to separate the raw gas in two component currents, one of which undergoes wet purification while the other is led through a dry purification process. The two component currents are mixed before entering the stack. The dry chemisorption masses added in substoichiometric doses are treated in a milk-of-lime processing stage, after which the reacted and non-reacted chemisorption masses are treated by wet purification and then by oxidation.

  11. The Borexino purification system

    Science.gov (United States)

    Benziger, Jay

    2014-05-01

    Purification of 278 tons of liquid scintillator and 889 tons of buffer shielding for the Borexino solar neutrino detector is performed with a system of combined distillation, water extraction, gas stripping and filtration. The purification system removed K, U and Th by distillation of the pseudocumene solvent and the PPO fluor. Noble gases, Rn, Kr and Ar were removed by gas stripping. Distillation was also employed to remove optical impurities and reduce the attenuation of scintillation light. The success of the purification system has facilitated the first time real time detection of low energy solar neutrinos.

  12. Comparison of four functionalization methods of gold nanoparticles for enhancing the enzyme-linked immunosorbent assay (ELISA)

    Science.gov (United States)

    Ciaurriz, Paula; Fernández, Fátima; Tellechea, Edurne; Moran, Jose F

    2017-01-01

    The enzyme-linked immunosorbent assay (ELISA) technique is based on the specific recognition ability of the molecular structure of an antigen (epitope) by an antibody and is likely the most important diagnostic technique used today in bioscience. With this methodology, it is possible to diagnose illness, allergies, alimentary fraud, and even to detect small molecules such as toxins, pesticides, heavy metals, etc. For this reason, any procedures that improve the detection limit, sensitivity or reduce the analysis time could have an important impact in several fields. In this respect, many methods have been developed for improving the technique, ranging from fluorescence substrates to methods for increasing the number of enzyme molecules involved in the detection such as the biotin–streptavidin method. In this context, nanotechnology has offered a significant number of proposed solutions, mainly based on the functionalization of nanoparticles from gold to carbon which could be used as antibody carriers as well as reporter enzymes like peroxidase. However, few works have focused on the study of best practices for nanoparticle functionalization for ELISA enhancement. In this work, we use 20 nm gold nanoparticles (AuNPs) as a vehicle for secondary antibodies and peroxidase (HRP). The design of experiments technique (DOE) and four different methods for biomolecule loading were compared using a rabbit IgG/goat anti-rabbit IgG ELISA model (adsorption, directional, covalent and a combination thereof). As a result, AuNP probes prepared by direct adsorption were the most effective method. AuNPs probes were then used to detect gliadin, one of the main components of wheat gluten, the protein composite that causes celiac disease. With this optimized approach, our data showed a sensitivity increase of at least five times and a lower detection limit with respect to a standard ELISA of at least three times. Additionally, the assay time was remarkably decreased. PMID:28243563

  13. INFLUENCE OF SURFACE TREATMENT AND PURIFICATION METHODS OF CO-115M GLASS-CERAMICS ON OPTICAL CONTACT STRENGTH

    Directory of Open Access Journals (Sweden)

    N. V. Tikhmenev

    2016-07-01

    Full Text Available Subject of Research.We present findings of the optical contact for details made of СО-115Мglass-ceramics brand mark. The optical contact is the main method of joining parts made of CO-115M glass-ceramics brand mark in commercially available laser gyros. The existing technology has a number of unresolved issues related to the durability of the optical contact, that determine the tightness of the laser sensor internal volume. Method. Mechanical strength control of the optical contact consisted in the measurement of specific tear force of the connection. Mechanical strength tests of the optical contact were carried out with the use of RMI-250 tensile testing machine. The evenly increasing load of 50 N/s was applied to the samples in mechanical tests. The value with the occurence of the optical contact destruction was registered. Main Results. We have shown that one of the main factors determining the mechanical strength of the joint is cleanliness of the surfaces being connected. Comparison of the influence of different surface cleaning methods for optical elements on the optical contact durability has been given. The negative impact of even short-term storage of optical parts after washing on the assembly strength has been shown. The additional operation of mechanical polishing of surfaces of stored optical parts before connection enabled significantly reducing the scatter of the optical contact mechanical strength. We have also established experimentally that the heating of assembly of optical elements under vacuum at a temperature of 300°C leads to the twofold increase in the optical contact strength, while the optical contact remains separable. Practical Relevance. The carried out studies make it possible to improve the technical and operational characteristics of the laser gyroes. The use of additional mechanical cleaning of surfaces of optical parts and vacuum heating of the assembly in the process of laser sensor production may

  14. A simple method for production and purification of soluble and biologically active recombinant human leukemia inhibitory factor (hLIF) fusion protein in Escherichia coli.

    Science.gov (United States)

    Imsoonthornruksa, Sumeth; Noisa, Parinya; Parnpai, Rangsun; Ketudat-Cairns, Mariena

    2011-02-20

    Mouse embryonic stem cells (mESCs) rely on a cytokine named leukemia inhibitory factor (LIF) to maintain their undifferentiated state and pluripotency. However, the progress of mESC research is restricted and limited to highly funded laboratories due to the cost of commercial LIF. Here we presented the homemade hLIF which is biologically active. The hLIF cDNA was cloned into two different vectors in order to produce N-terminal His₆-tag and Trx-His₆-tag hLIF fusion proteins in Origami(DE3) Escherichia coli. The His₆-hLIF fusion protein was not as soluble as the Trx-His₆-hLIF fusion protein. One-step immobilized metal affinity chromatography (IMAC) was done to recover high purity (> 95% pure) His₆-hLIF and Trx-His₆-hLIF fusion proteins with the yields of 100 and 200 mg/l of cell culture, respectively. The hLIF fusion proteins were identified by Western blot and verified by mass spectrometry (LC/MS/MS). The hLIF fusion proteins specifically promote the proliferation of TF-1 cells in a dose-dependent manner. They also demonstrate the potency to retain the morphology of undifferentiated mESCs, in that they were positive for mESC markers (Oct-4, Sox-2, Nanog, SSEA-1 and alkaline phosphatase activity). These results demonstrated that the N-terminal fusion tags of the His₆-hLIF and Trx-His₆-hLIF fusion proteins do not interfere with their biological activity. This expression and purification approach to produce recombinant hLIF is a simple, reliable, cost effective and user-friendly method.

  15. Methods to determine the transcriptomes of trypanosomes in mixtures with mammalian cells: the effects of parasite purification and selective cDNA amplification.

    Directory of Open Access Journals (Sweden)

    Julius Mulindwa

    2014-04-01

    Full Text Available Patterns of gene expression in cultured Trypanosoma brucei bloodstream and procyclic forms have been extensively characterized, and some comparisons have been made with trypanosomes grown to high parasitaemias in laboratory rodents. We do not know, however, to what extent these transcriptomes resemble those in infected Tsetse flies - or in humans or cattle, where parasitaemias are substantially lower. For clinical and field samples it is difficult to characterize parasite gene expression because of the large excess of host cell RNA. We have here examined two potential solutions to this problem for bloodstream form trypanosomes, assaying transcriptomes by high throughput cDNA sequencing (RNASeq. We first purified the parasites from blood of infected rats. We found that a red blood cell lysis procedure affected the transcriptome substantially more than purification using a DEAE cellulose column, but that too introduced significant distortions and variability. As an alternative, we specifically amplified parasite sequences from a mixture containing a 1000-fold excess of human RNA. We first purified polyadenylated RNA, then made trypanosome-specific cDNA by priming with a spliced leader primer. Finally, the cDNA was amplified using nested primers. The amplification procedure was able to produce samples in which 20% of sequence reads mapped to the trypanosome genome. Synthesis of the second cDNA strand with a spliced leader primer, followed by amplification, is sufficiently reproducible to allow comparison of different samples so long as they are all treated in the same way. However, SL priming distorted the abundances of the cDNA products and definitely cannot be used, by itself, to measure absolute mRNA levels. The amplification method might be suitable for clinical samples with low parasitaemias, and could also be adapted for other Kinetoplastids and to samples from infected vectors.

  16. A simple method for the preparation and purification of C1 complement cleaved beta 2-microglobulin from human serum

    DEFF Research Database (Denmark)

    Nissen, Mogens Holst; Johansen, B; Bjerrum, Ole Jannik

    1997-01-01

    A simple method is described for the preparation of proteolytically processed forms of beta 2-microglobulin suitable for structural and biological studies. PEG 6000 was added to the serum of healthy individuals to precipitate the C1 complement complex from C1 esterase inhibitor (C1-inh). After...... cleaved beta 2-microglobulin, whereby Lys58-cleaved beta 2-microglobulin was obtained. The proteolytically processed forms were subsequently purified by G-75 Sephadex gel filtration followed by chromatofocusing. A yield of 10-40% of proteolytically processed beta 2-microglobulin was obtained. Only one...

  17. Investigation of acid red 88 oxidation in water by means of electro-Fenton method for water purification.

    Science.gov (United States)

    Özcan, Ali; Gençten, Metin

    2016-03-01

    In this study, electro-Fenton method was applied to acid red 88 (AR88) containing aqueous solutions for the removal of it from water. The mineralization of AR88 has been achieved by oxidation with hydroxyl radicals. These radicals were produced simultaneously by the electro-Fenton method using an electrochemical cell including a carbon felt cathode and a platinum anode. Applied current and concentrations of catalyst and supporting electrolyte were optimized to obtain the best effective parameters of 500 mA, 0.1 mM and 75 mM, respectively. The absolute rate constant for the oxidation reaction of AR88 with hydroxyl radical was determined as (1.57 ± 0.06) x 10(10) M(-1) s(-1). Total organic carbon (TOC) analysis was performed to determine whether the organics were converted to carbon dioxide or not. A two-hour electrolysis at 500 mA is enough to remove 87% of initial TOC values of 0.25 mM AR88 solution. Electro-Fenton treatment of AR88 led to the formation of five aromatic intermediates, five short-chain carboxylic acids and three inorganic ions. Identified intermediates and complete mineralization of AR88 allowed us to propose a mineralization pathway for first time in the literature.

  18. The Enzyme Portal: a case study in applying user-centred design methods in bioinformatics

    Science.gov (United States)

    2013-01-01

    User-centred design (UCD) is a type of user interface design in which the needs and desires of users are taken into account at each stage of the design process for a service or product; often for software applications and websites. Its goal is to facilitate the design of software that is both useful and easy to use. To achieve this, you must characterise users’ requirements, design suitable interactions to meet their needs, and test your designs using prototypes and real life scenarios. For bioinformatics, there is little practical information available regarding how to carry out UCD in practice. To address this we describe a complete, multi-stage UCD process used for creating a new bioinformatics resource for integrating enzyme information, called the Enzyme Portal (http://www.ebi.ac.uk/enzymeportal). This freely-available service mines and displays data about proteins with enzymatic activity from public repositories via a single search, and includes biochemical reactions, biological pathways, small molecule chemistry, disease information, 3D protein structures and relevant scientific literature. We employed several UCD techniques, including: persona development, interviews, ‘canvas sort’ card sorting, user workflows, usability testing and others. Our hope is that this case study will motivate the reader to apply similar UCD approaches to their own software design for bioinformatics. Indeed, we found the benefits included more effective decision-making for design ideas and technologies; enhanced team-working and communication; cost effectiveness; and ultimately a service that more closely meets the needs of our target audience. PMID:23514033

  19. It's a dirty job--A robust method for the purification and de novo genome assembly of Cryptosporidium from clinical material.

    Science.gov (United States)

    Andersson, Sofia; Sikora, Per; Karlberg, Maria L; Winiecka-Krusnell, Jadwiga; Alm, Erik; Beser, Jessica; Arrighi, Romanico B G

    2015-06-01

    We have developed a novel strategy for the purification of Cryptosporidium oocysts from clinical samples using IMS and PCR amplification of target DNA to facilitate uniform coverage genome sequencing and de novo assembly. Our procedure could also be used for other microbial pathogens from clinical specimens.

  20. Discussion on Purification Performance Evaluation Methods of Car Air Purifier%对车载空气净化器净化性能评价方法的探讨

    Institute of Scientific and Technical Information of China (English)

    杨瑾; 闻真; 孙立

    2016-01-01

    30 car air purifiers (in view of family car) are selected in 3 m3 experiment module for testing, clean air delivery rate (CADR) and purification efficiency are used to evaluate the purifying effect of car air purifier on formaldehyde and particle pollutants, and the paper discusses the method differences. Testing result shows that it is proportional to both of them, when CADR formaldehyde≥12 m3/h and CADR particles≥15 m3/h, CADR can reflect the differences in purification capacity, and it can help consumers understand the real purification capacity of car air purifier in combination with purification efficiency.%选取30个车载空气净化器(针对家庭用车)在3 m3实验舱中进行测试,通过“洁净空气量(CADR)”和“净化效率”2种方法来评价车载空气净化器对甲醛及颗粒物污染物的净化效果,并就方法的差异展开探讨。检测结果表明,两者成正比关系,当CADR 甲醛≥12 m3/h、CADR 颗粒物≥15 m3/h时,CADR更能体现净化能力差异,结合净化效率,能帮助消费者了解车载空气净化器真实的净化能力。

  1. Application of a new purification method of West-Kazakhstan chestnut soil microbiota DNA for metagenomic analysis

    Science.gov (United States)

    Sergaliev, N. Kh.; Kakishev, M. G.; Zhiengaliev, A. T.; Volodin, M. A.; Andronov, E. E.; Pinaev, A. G.

    2015-04-01

    A method for the extraction of soil microbial DNA has been tested on chestnut soils (Kastanozems) of the West Kazakhstan region. The taxonomic analysis of soil microbiome libraries has shown that the phyla Actinobacteria and Proteobacteria constitute the largest part of microbial communities in the analyzed soils. The Archaea form an appreciable part of the microbiome in the studied samples. In the underdeveloped dark chestnut soil, their portion is higher than 11%. This is of interest, as the proportion of Archaea in the soil communities of virgin lands usually does not exceed 5%. In addition to the phyla mentioned above, there are representatives of the phyla Acidobacteria, Bacteroidetes, Firmicutes, Gemmatimonadales, Planctomycetes, and Verrucomicrobia, which are all fairly common in soil communities.

  2. Enhanced recovery and purification of P(3HB-co-3HHx) from recombinant Cupriavidus necator using alkaline digestion method.

    Science.gov (United States)

    Anis, Siti Nor Syairah; Nurhezreen, M I; Sudesh, K; Amirul, A A

    2012-06-01

    A simple, efficient and economical method for the recovery of P(3HB-co-3HHx) was developed using various chemicals and parameters. The initial content of P(3HB-co-3HHx) in bacterial cells was 50-60 wt%, whereas the monomer composition of 3HHx used in this experiments was 3-5 mol%. It was found that sodium hydroxide (NaOH) was the most effective chemical for the recovery of biodegradable polymer. High polyhydroxyalkanoate purity and recovery yield both in the range of 80-90 wt% were obtained when 10-30 mg/ml of cells were incubated in NaOH at the concentration of 0.1 M for 60-180 min at 30 °C and polished using 20 % (v/v) of ethanol.

  3. Development of methods for the purification of {sup 67}Ga and {sup 68}Ga for biomolecules labeling; Desenvolvimento de metodos de purificacao do {sup 67}Ga e {sup 68}Ga para a marcacao de biomoleculas

    Energy Technology Data Exchange (ETDEWEB)

    Costa, Renata Ferreira

    2012-07-01

    For more than fifty years, the long-lived {sup 68}Ge/{sup 68}Ga generators have been in development, obtaining {sup 68}Ga without the need of having in house cyclotron, which is a considerable convenience for PET centers that have no nearby cyclotrons. {sup 68}Ga decays 89% by positron emission and low photon emission (1077 keV) and the physical half life of 67.7 minutes is compatible with the pharmacokinetics of low biomolecular weight substances like peptides and antibody fragments. Moreover, its established metallic chemistry allows it to be stably bound to the carrier peptide sequence via a suitable bifunctional chelator, such as DOTA. All these reasons together with the technology of PET/CT allowed advances in molecular imaging, in particular in the diagnosis of neuroendocrine diseases. However, the eluate from the commercial {sup 68}Ge/{sup 68}Ga generators still contains high levels of long lived {sup 68}Ge, besides other metallic impurities, which competes with {sup 68}Ga with a consequent reduction of the labeling yield of biomolecules, such as Fe{sup 3+} and Zn{sup 2+}. Thus, the lower the amount of impurities in the eluate, the competition between the radiolabeled and unlabeled peptide by the receptor will be smaller and the quality of imaging will be better, a subsequent purification step is needed after the generator elution. The aim of this work is to evaluate different purifications methods of {sup 68}Ga to label biomolecules, with emphasis on the study of the chemical impurities contained in the eluate and to develop a new purification method. Several purification methods were studied. Many cationic resin were tested simulating the commercial process. {sup 68}Ga is adsorbed in cationic resin, which is not commercial available and eluted in acid/acetone solution. The use of minor particles of cationic resin AG50W-X4 (200-400 mesh) showed the best results. An innovate method was the extraction chromatography, which is based on the absorption of

  4. 强辐射催化法提纯多晶硅%Purification of silicon by the method of strong radiation catalysis

    Institute of Scientific and Technical Information of China (English)

    陈应天; 何祚庥

    2011-01-01

    Among others, the removal of boron is the key issue to effectively purify metallurgical silicon into solar grade silicon. An innovative slagging process is proposed in this article after reviewing many previous studies. In the process, strong radiation is employed as catalysis to promote the chemical reaction. The authors have for the first time unveiled the secrets including the newly developed high temperature tools, the materials used, the method of mixing materials before purification, the process for separating silicon from residuals after purification, etc.. In the meantime, the authors also propose a postulate to explain the mechanism of the catalysis process. Except the removal of boron, the processes to remove other impurities, such as phosphorous and residual metals using electron beam and magnetic floating directional solidification are also discussed for the completion of a new industrial process to fabricate pure silicon. In the article, the authors also extend their discussion to the possibility of using the proposed non - imaging heliostat in other fields ; to the possibility to further manufacture electronic grade silicon using the same process ; to the possibility to obtain more experimental evidences for the postulate of strong radiation catalysis and to other subjects related to authors' claimed creations. It is sincerely hoped that the publication of the innovations reported in this article may provide an alternative way in the route map for the use of renewable energy of low cost and low carbon emission.%将低纯度的金属硅,提纯成可用于制造太阳能电池的高纯硅材料的主要关键,是去除材料中的硼杂质.本文提出了一种采用特殊的造渣过程以去除硼杂质的新方法.在这种新方法中,为了促进快速的化学反应,采用高密度的光子作为催化剂,以达到太阳能级硅材料的标准.本文对使用这种新的强辐射催化法炼硅的高温工具、冶炼方法、

  5. Enzymatic pretreatment of wood chips for energy reductions in TMP production. A method for ranking of enzymes; Enzymatisk foerbehandling av flis foer energibesparing vid TMP tillverkning. Metod foer rankning av enzymer

    Energy Technology Data Exchange (ETDEWEB)

    Viforr, Silvia

    2010-11-15

    The production of thermomechanical pulp (TMP) demands high levels of energy. This, together with current expensive energy prices of nowadays results in significant costs, which is the reason why there is a demand for processes that require less energy. One way of reducing energy consumption in TMP refining could be to pretreat the wood chips with enzymes before the subsequent refining step. However, enzymes molecules are relatively large, which limits the impregnation process, and so the pores in the fibre walls are not large enough to fit the size of the enzymes. By mechanically pretreating wood chips in a screw feeder and press equipment, this opens the wood structure significantly which increases enzyme penetration. If enzymes are used for reducing energy consumption in TMP processes, it is necessary to optimise the enzymatic effect during the pretreatment of wood chips. It is very expensive to evaluate completely the effect of enzymes in large scale refining. Thus there is a need for other relevant methods for rapidly and effectively evaluating the energy saving effects when it comes to refining enzymatic pretreated wood chips. The aim of this project was to find a method for ranking of enzymes for pretreatment of chips for energy savings at TMP production. This method was to be independent of the type of enzyme used and of the type of pretreated wood chips involved. In order to asses the method for ranking enzymes being used in the pretreatment of chips to reduce energy input during refining, a comparison between the method and a mill trial was carried out in the mill trial. A known chemical pretreatment was used; here it was sulphonation of the wood chips before refining with low sulphite levels. Further, a laboratory wing refiner was used as an evaluation equipment. The trial started with the running conditions for a wing refiner that the best correspond with industrial refining. An evaluation was made on the effect of enzymatic pretreatment on energy

  6. Insights into the Thiamine Diphosphate Enzyme Activation Mechanism: Computational Model for Transketolase Using a Quantum Mechanical/Molecular Mechanical Method.

    Science.gov (United States)

    Nauton, Lionel; Hélaine, Virgil; Théry, Vincent; Hecquet, Laurence

    2016-04-12

    We propose the first computational model for transketolase (TK), a thiamine diphosphate (ThDP)-dependent enzyme, using a quantum mechanical/molecular mechanical method on the basis of crystallographic TK structures from yeast and Escherichia coli, together with experimental kinetic data reported in the literature with wild-type and mutant TK. This model allowed us to define a new route for ThDP activation in the enzyme environment. We evidenced a strong interaction between ThDP and Glu418B of the TK active site, itself stabilized by Glu162A. The crucial point highlighted here is that deprotonation of ThDP C2 is not performed by ThDP N4' as reported in the literature, but by His481B, involving a HOH688A molecule bridge. Thus, ThDP N4' is converted from an amino form to an iminium form, ensuring the stabilization of the C2 carbanion or carbene. Finally, ThDP activation proceeds via an intermolecular process and not by an intramolecular one as reported in the literature. More generally, this proposed ThDP activation mechanism can be applied to some other ThDP-dependent enzymes and used to define the entire TK mechanism with donor and acceptor substrates more accurately.

  7. Salivary aldehyde dehydrogenase--reversible oxidation of the enzyme and its inhibition by caffeine, investigated using fluorimetric method.

    Science.gov (United States)

    Wierzchowski, Jacek; Pietrzak, Monika; Szelag, Malgorzata; Wroczyński, Piotr

    2008-05-01

    We have applied fluorimetric method to monitor aldehyde dehydrogenase (ALDH*) activity in human saliva samples to study inactivation, reactivation and inhibition of the enzyme. Saliva samples were collected to buffer stock solution, containing various thiols, and assayed in the presence of the fluorogenic substrate 6-dimethylamino-2-naphthaldehyde and NAD(+). Fluorescence of the produced 6-dimethylamino-2-naphthalene carboxylate was used to measure the reaction rate. Kinetic parameters for the highly fluorogenic substrate, 6-dimethylamino-2-naphthaldehyde were measured, with apparent K(m) of 7.9 microM at pH 7.3. The apparent K(m) for NAD(+) was 1.2 microM. The observed ALDH activity is unstable in the absence of thiols, but can be stabilized by 1mM glutathione, and inactivated enzyme can be re-activated within 10 min by treatment of 0.5 mM DTT. Two-assay procedure was applied to measure degree of inactivation of ALDH in saliva samples. It was found that degree of ALDH inactivation in fresh samples, stabilized by glutathione, is between 0% and 90%, with average value ca. 40%. Caffeine and theophylline were shown to be moderate inhibitors of salivary ALDH. Oxidation of the salivary ALDH in fresh saliva may be reliably measured using fluorimetric two-assay procedure. Preliminary statistics indicate that in most individuals this enzyme is partially inactive. Inhibition of the salivary ALDH by caffeine may have consequences for nutrition safety.

  8. Purification and characterization of the membrane-bound quinoprotein glucose dehydrogenase of Gluconacetobacter diazotrophicus PAL 5.

    Science.gov (United States)

    Sará-Páez, Martin; Contreras-Zentella, Martha; Gómez-Manzo, Saúl; González-Valdez, Alejandra Abigail; Gasca-Licea, Rolando; Mendoza-Hernández, Guillermo; Escamilla, José Edgardo; Reyes-Vivas, Horacio

    2015-02-01

    Acetic acid bacteria oxidize a great number of substrates, such as alcohols and sugars, using different enzymes that are anchored to the membrane. In particular, Gluconacetobacter diazotrophicus is distinguished for its N2-fixing activity under high-aeration conditions. Ga. diazotrophicus is a true endophyte that also has membrane-bound enzymes to oxidize sugars and alcohols. Here we reported the purification and characterization of the membrane-bound glucose dehydrogenase (GDHm), an oxidoreductase of Ga. diazotrophicus. GDHm was solubilized and purified by chromatographic methods. Purified GDHm was monomeric, with a molecular mass of 86 kDa. We identified the prosthetic group as pyrroloquinoline quinone, whose redox state was reduced. GDHm showed an optimum pH of 7.2, and its isoelectric point was 6.0. This enzyme preferentially oxidized D-glucose, 2-deoxy-D-glucose, D-galactose and D-xylose; its affinity towards glucose was ten times greater than that of E. coli GDHm. Finally, Ga. diazotrophicus GDHm was capable of reducing quinones such as Q 1, Q 2, and decylubiquinone; this activity was entirely abolished in the presence of micromolar concentrations of the inhibitor, myxothiazol. Hence, our purification method yielded a highly purified GDHm whose molecular and kinetic parameters were determined. The possible implications of GDHm activity in the mechanism for reducing competitor microorganisms, as well as its participation in the respiratory system of Ga. diazotrophicus, are discussed.

  9. Immunoaffinity purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from human erythrocytes

    Institute of Scientific and Technical Information of China (English)

    Driss Mountassif; Tarik Baibai; Latifa Fourrat; Adnane Moutaouakkil; Abdelghani Iddar; M'Hammed Sa(i)d El Kebbaj; Abdelaziz Soukri

    2009-01-01

    A new procedure utilizing immunoaffinity column chromatography has been used for the purification of glyceraldehyde-3-phosphate dehydrogenase(GAPDH,EC 1.2.1.12)from human erythrocytes.The comparison between this rapid method(one step)and the traditional procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography shows that the new method gives a highest specific activity with a highest yield in a short time.The characterization of the purified GAPDH reveals that the native enzyme is a homotetramer of ~150 kDa with an absolute specificity for the oxidized form of nicotinamide adenine dinucleotide(NAD+).Western blot analysis using purified monospecific poly clonal antibodies raised against the purified GAPDH showed a singie 36 kDa band corresponding to the enzyme subunit.Studies on the effect of temperature and pH on enzyme activity revealed optimal values of about 43℃ and 8.5, respectively.The kinetic par ameters were also calculated:the Vmax was 4.3 U/mg and the Km values against G3P and NAD+ were 20.7and 17.8μM,respectively.The new protocol described represents a simple,economic,and reproducible tool for the purification Of GAPDH and can be used for other proteins.

  10. Overview of the purification of recombinant proteins.

    Science.gov (United States)

    Wingfield, Paul T

    2015-04-01

    When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches, many of which were described and mentioned throughout Current Protocols in Protein Science. In the interim, there has been a shift toward an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein, and whether to engineer a self-cleavage system or simply leave them on. We will briefly address some of these issues. Also, although this overview focuses on E.coli, protein expression and purification, other commonly used expression systems are mentioned and, apart from cell-breakage methods, protein purification methods and strategies are essentially the same.

  11. A scintillator purification system for the Borexino solar neutrino detector

    Science.gov (United States)

    Benziger, J.; Cadonati, L.; Calaprice, F.; Chen, M.; Corsi, A.; Dalnoki-Veress, F.; Fernholz, R.; Ford, R.; Galbiati, C.; Goretti, A.; Harding, E.; Ianni, Aldo; Ianni, Andrea; Kidner, S.; Leung, M.; Loeser, F.; McCarty, K.; McKinsey, D.; Nelson, A.; Pocar, A.; Salvo, C.; Schimizzi, D.; Shutt, T.; Sonnenschein, A.

    2008-03-01

    Purification of the 278 tons of liquid scintillator and 889 tons of buffer shielding for the Borexino solar neutrino detector is performed with a system that combines distillation, water extraction, gas stripping, and filtration. This paper describes the principles of operation, design, and construction of that purification system, and reviews the requirements and methods to achieve system cleanliness and leak-tightness.

  12. A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.

    Science.gov (United States)

    Herget, Meike; Scheibinger, Mirko; Guo, Zhaohua; Jan, Taha A; Adams, Christopher M; Cheng, Alan G; Heller, Stefan

    2013-01-01

    Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

  13. Co-expression as a convenient method for the production and purification of core histones in bacteria.

    Science.gov (United States)

    Anderson, Megan; Huh, Joon H; Ngo, Thien; Lee, Alice; Hernandez, Genaro; Pang, Joy; Perkins, Jennifer; Dutnall, Robert N

    2010-08-01

    Co-expression offers an important strategy for producing multiprotein complexes for biochemical and biophysical studies. We have found that co-expression of histones H2A and H2B (from yeast, chicken or Drosophila) leads to production of soluble heterodimeric H2AH2B complexes. Drosophila histones H3 and H4 can also be produced as a soluble (H3H4)(2) heterotetrameric complex if they are co-expressed with the histone chaperone Asf1. The soluble H2AH2B and (H3H4)(2) can be purified by simple chromatographic techniques and have similar properties to endogenous histones. Our methods should facilitate histone production for studies of chromatin structure and regulatory proteins that interact with histones. We describe a simple strategy for constructing co-expression plasmids, based on the T7 RNA polymerase system, which is applicable to other systems. It offers several advantages for quickly creating plasmids to express two or more proteins and for testing different combinations of proteins for optimal complex production, solubility or activity. Copyright 2010 Elsevier Inc. All rights reserved.

  14. The Blood Compatibilities of Blood Purification Membranes and Other Materials Developed in Japan

    OpenAIRE

    Takaya Abe; Karen Kato; Tomoaki Fujioka; Tadao Akizawa

    2011-01-01

    The biocompatibilities in blood purification therapy are defined as “a concept to stipulate safety of blood purification therapy by an index based on interaction in the body arising from blood purification therapy itself.” The biocompatibilities are associated with not only materials to be used but also many factors such as sterilization method and eluted substance. It is often evaluated based on impacts on cellular pathways and on humoral pathways. Since the biocompatibilities of blood purif...

  15. Production and partial purification of streptokinase from Streptococcus pyogenes

    Directory of Open Access Journals (Sweden)

    Freeda Felsia X

    2011-11-01

    Full Text Available Normal 0 false false false MicrosoftInternetExplorer4 Streptokinase is as effective as recombinant tissue plasminogen activator (tPA in treating acute myocardial infarction and it is certainly more cost-effective. In view of the relatively recent availability of the competing recombinant tPA, skepticism is being expressed about the continued viability of streptokinase therapy. Despite this research on streptokinase continues, and it remains a vital affordable therapy especially in the world’s poorer healthcare systems. Our present study focused on production of streptokinase from Streptococcus pyogenes species and partial purification of streptokinase by ammonium sulfate precipitation, dialysis and column chromatography. The enzyme was quantified by Lowry’s method and its electrophoretic mobility and molecular weight were determined by SDS-PAGE.

  16. 高效分离纯化藻蓝蛋白新法%New method for efficient separation and purification of C-phycocyanin

    Institute of Scientific and Technical Information of China (English)

    廖晓霞; 张学武

    2011-01-01

    An efficient method was developed for the purification of C-phycocyanin (C-PC), which involved three steps:chitosan affinity precipitation,activated charcoal absorption and DEAE Sephadex A-25 chromatography. As compared with conventional methods,this method was cheap, time-saving and easy to operate. The purity(A620/A280) of C- phycocyanin obtained after chitosan- activated charcoal treatment was increased from 0.93 to 2.78,which could be improved to 4.3 after being subjected to DEAE Sephadex A-25 chromatography. Analyzed by SDS -PAGE electrophoresis,phycocyanin migrated as two bands corresponding to its two subunits(α and β)and the molecular weights were 16.5ku and 17.6ku, respectively. The absorption spectra and fluorescence emission specra of purified protein showed peaks at 620nm and 643nm, respectively,which was characteristic of C-phycocyanin.%采用壳聚糖亲和沉淀-活性炭吸附-DEAE Sephadex A-25柱层析三步法分离纯化藻蓝蛋白,与传统方法相比具有成本低、耗时短、操作便捷等优点.壳聚糖和活性炭结合使用,可使藻蓝蛋白纯度(A62>/A280)由0.93提高至2.78.通过DEAE Sephadex A-25葡聚糖凝胶柱层析后,可得到高纯度藻蓝蛋白,纯度达4.3.纯化后的藻蓝蛋白经聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定,产生分子量分别为16.5ku和17.6ku的α,β亚基条带.吸收光谱和荧光发射光谱扫描表明所得蛋白在620、643nm分别具有藻蓝蛋白特征吸收峰和荧光发射峰.

  17. A simple method for purification of deodorizer distillate from Indian rice (Oryza Sativa bran oil and preparation of phytosterols

    Directory of Open Access Journals (Sweden)

    Raja Rajan, R. G.

    2014-12-01

    Full Text Available Samples of rice bran oil deodorizer distillates (RBO DOD-1 and RBO DOD-2 were studied for their physicochemical characteristics. The samples were semisolid and had a dark color. The free fatty acid values were 59.2% and 86.0%, the unsaponifiable matter was 18.7% and 7.75% and the phytosterol contents were 8.71% and 4.22%, respectively for the deodorizer distillates studied. A simple method of silica gel percolation was developed to purify DOD to obtain phytosterol concentrate fractions (PCF and a brown color and bad odor fraction (BCBOF. The color values were reduced by 72.8% and 73.0% of lovibond units in the PCF for DOD-1 and DOD-2 respectively, had no bad odor and were increased in the phytosterol concentration to 12.4% and 5.9%. The PCF was further processed to prepare high purity phytosterols. An HPLC analysis of the phytosterol mixture showed it to be formed by β-sitosterol (38.2%, stigmasterol (34.9%, campesterol (9.5% and other sterols (17.4%.Se estudiaron las características físico-químicas de muestras de destilados de desodorización de aceites de salvado de arroz (RBO DOD-1 y RBO DOD-2. Las muestras eran semi-sólidas y tenían un color oscuro. Los valores de ácidos grasos libres fueron 59,2% y 86,0%, materia insaponificable 18,7% y 7,75% y contenido de fitoesteroles de 8,71% y 4,22%, respectivamente, para los destilados de desodorización estudiados. Se desarrolló un método simple de filtración mediante sílica gel para purificar DOD y obtener concentrados de fitosteroles (PCF y una fracción de color marrón y olor desagradable (BCBOF. Los valores de color se redujeron en un 72,8% y el 73,0% de unidades Lovibond en el PCF para DOD-1 y DOD-2, respectivamente, no tenían mal olor y aumentaron su concentración en fitoesteroles al 12,4% y 5,9%. El PCF se procesó adicionalmente para preparar fitosteroles de alta pureza. El análisis por HPLC mostró que la mezcla de fitosteroles estaba formada por β-sitosterol (38

  18. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes.

    Directory of Open Access Journals (Sweden)

    Carola Engler

    Full Text Available We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call 'Golden Gate shuffling', is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.

  19. Surface processes during purification of InP quantum dots

    Directory of Open Access Journals (Sweden)

    Natalia Mordvinova

    2014-08-01

    Full Text Available Recently, a new simple and fast method for the synthesis of InP quantum dots by using phosphine as phosphorous precursor and myristic acid as surface stabilizer was reported. Purification after synthesis is necessary to obtain samples with good optical properties. Two methods of purification were compared and the surface processes which occur during purification were studied. Traditional precipitation with acetone is accompanied by a small increase in photoluminescence. It occurs that during the purification the hydrolysis of the indium precursor takes place, which leads to a better surface passivation. The electrophoretic purification technique does not increase luminescence efficiency but yields very pure quantum dots in only a few minutes. Additionally, the formation of In(OH3 during the low temperature synthesis was explained. Purification of quantum dots is a very significant part of postsynthetical treatment that determines the properties of the material. But this subject is not sufficiently discussed in the literature. The paper is devoted to the processes that occur at the surface of quantum dots during purification. A new method of purification, electrophoresis, is investigated and described in particular.

  20. Necessity of Purification during Bacterial DNA Extraction with Environmental Soils.

    Science.gov (United States)

    Lim, Hyun Jeong; Choi, Jung-Hyun; Son, Ahjeong

    2017-08-08

    Complexity and heterogeneity of soil samples have often implied the inclusion of purification steps in conventional DNA extraction for PCR assays. Unfortunately the purification steps are also time and labor intensive. Therefore the necessity of DNA purification was re-visited and investigated for a variety of environmental soil samples that contained various amounts of PCR inhibitors. Bead beating and centrifugation was used as the baseline (without purification) method for DNA extraction. Its performance was compared with that of conventional DNA extraction kit (with purification). The necessity criteria for DNA purification were established with environmental soil samples. Using lysis conditions at 3000 rpm for 3 minutes with 0.1 mm glass beads, centrifugation time of 10 minutes and 1:10 dilution ratio, the baseline method outperformed conventional DNA extraction on cell seeded sand samples. Further investigation with PCR inhibitors (i.e., humic acids, clay, and magnesium) showed that sand samples containing less than 10 ug/g humic acids and 70% clay may not require purifications. Interestingly, the inhibition pattern of magnesium ion was different from other inhibitors due to the complexation interaction of magnesium ion with DNA fragments. It was concluded that DNA extraction method without purification is suitable for soil samples that have less than 10 ug/g of humic acids, less than 70% clay content and less than 0.01% magnesium ion content.

  1. Surface processes during purification of InP quantum dots.

    Science.gov (United States)

    Mordvinova, Natalia; Emelin, Pavel; Vinokurov, Alexander; Dorofeev, Sergey; Abakumov, Artem; Kuznetsova, Tatiana

    2014-01-01

    Recently, a new simple and fast method for the synthesis of InP quantum dots by using phosphine as phosphorous precursor and myristic acid as surface stabilizer was reported. Purification after synthesis is necessary to obtain samples with good optical properties. Two methods of purification were compared and the surface processes which occur during purification were studied. Traditional precipitation with acetone is accompanied by a small increase in photoluminescence. It occurs that during the purification the hydrolysis of the indium precursor takes place, which leads to a better surface passivation. The electrophoretic purification technique does not increase luminescence efficiency but yields very pure quantum dots in only a few minutes. Additionally, the formation of In(OH)3 during the low temperature synthesis was explained. Purification of quantum dots is a very significant part of postsynthetical treatment that determines the properties of the material. But this subject is not sufficiently discussed in the literature. The paper is devoted to the processes that occur at the surface of quantum dots during purification. A new method of purification, electrophoresis, is investigated and described in particular.

  2. Purification of contaminated groundwater by membrane technology

    Energy Technology Data Exchange (ETDEWEB)

    Youn, In Soo; Chung, Chin Ki; Kim, Byoung Gon [Korea Institute of Geology Mining and Materials, Taejon (Korea, Republic of)

    1996-12-01

    The objective of this study is to apply the membrane separation technology to the purification of contaminated ground water in Korea. Under this scope, the purification was aimed to the drinking water level. The scale of the membrane system was chosen to a small filtration plant for local clean water supplies and/or heavy purifiers for buildings and public uses. The actual conditions of ground water contamination in Korea was surveyed to determine the major components to remove under the drinking water requirements. To set up a hybrid process with membrane methods, conventional purification methods were also investigated for the comparison purpose. The research results are summarized as follows : 1) Contamination of the groundwater in Korea has been found to be widespread across the country. The major contaminant were nitrate, bacteria, and organic chlorides. Some solvents and heavy metals are also supposed to exist in the ground water of industrial complexes, cities, and abandoned mines. 2) The purification methods currently used in public filtration plants appear not to be enough for new contaminants from recent industrial expanding. The advanced purification technologies generally adopted for this problem have been found to be unsuitable due to their very complicated design and operation, and lack of confidence in the purification performance. 3) The reverse osmosis tested with FilmTec FT30 membrane was found to remove nitrate ions in water with over 90 % efficiency. 4) The suitable membrane process for the contaminated groundwater in Korea has been found to be the treatments composed of activated carbon, microfiltration, reverse osmosis or ultrafiltration, and disinfection. The activated carbon treatment could be omitted for the water of low organic contaminants. The microfiltration and the reverse osmosis treatments stand for the conventional methods of filtration plants and the advanced methods for hardly removable components, respectively. It is recommended

  3. Development of constructed wetland using hydroponic biofilter method for purification of hyper-eutrophic lake water; Fueiyoka kosui no joka no tameno suiko seibutsu rokaho wo mochiita jinko shicchi no kaihatsu

    Energy Technology Data Exchange (ETDEWEB)

    Aizaki, M. [Shimane Univ., Matsue (Japan)] Nakasato, H. [Top Ecology Co. Ltd., Tokyo (Japan)

    1997-09-10

    Applying the hydroponic biofilter method as a direct purification method for a hyper-eutrophic lake water, an experiment was carried out at the Tsuchiura Port on Lake Kasumigaura to obtain data for constructing a hydrophilic artificial wetland. Purification of hyper-eutrophic lake water containing a large amount of water blooms in summer was attempted applying the hydroponic biofilter method for which hydrophyte is used. As a result, it was clarified, by applying the hydroponic biofilter method, that capturing effect of suspended substances can be achieved in the rooting zone, captured suspended substances are decomposed at high rate, and the revolved nutrient salt can be absorbed and assimilated by the use of plants having high growth rates. Ipomoea aquatica had the highest removal activity, followed by nasturtium officinal, menthe spicata, and oenanthe javanica. As a result, it became clear that a constructed wetland made with the hydroponic biofilter method can be applied as a direct purifying method for hyper-eutrophic lake water by selecting appropriate plants in accordance with season. 18 refs., 1 fig., 4 tabs.

  4. Expression, purification and crystallization of class C acid phosphatases from Francisella tularensis and Pasteurella multocida

    Science.gov (United States)

    Singh, Harkewal; Felts, Richard L.; Ma, Li; Malinski, Thomas J.; Calcutt, Michael J.; Reilly, Thomas J.; Tanner, John J.

    2009-01-01

    Class C nonspecific acid phosphatases are bacterial enzymes that are secreted across the cytoplasmic membrane and hydrolyze a variety of phosphomono­esters at acidic pH. These enzymes are of interest for the development of improved vaccines and clinical diagnostic methods. In one case, the category A pathogen Francisella tularensis, the class C phosphatase plays a role in bacterial fitness. Here, the cloning, expression, purification and crystallization methods for the class C acid phosphatases from F. tularensis and Pasteurella multocida are reported. Crystals of the F. tularensis enzyme diffracted to 2.0 Å resolution and belonged to space group C2221, with one enzyme molecule in the asymmetric unit. Crystals of the P. multocida enzyme diffracted to 1.85 Å resolution and belonged to space group C2, with three molecules in the asymmetric unit. Diffraction patterns from crystals of the P. multocida enzyme exhibited multiple interpenetrating reciprocal-space lattices, indicating epitaxial twinning. Despite this aberrance, autoindexing was robust and the data could be satisfactorily processed to 1.85 Å resolution using MOSFLM and SCALA. PMID:19255471

  5. Comparison of two methods (microscopy and enzyme-linked immunosorbent assay) for the diagnosis of amebiasis.

    Science.gov (United States)

    Tanyuksel, Mehmet; Yilmaz, Hasan; Ulukanligil, Mustafa; Araz, Engin; Cicek, Mutalip; Koru, Ozgur; Tas, Zeynep; Petri, William A

    2005-07-01

    Diagnosis of amebiasis is usually performed on a clinical basis alone in most endemic countries having limited economic resources. This epidemiological study was conducted using modern diagnostic tests for amebiasis in the southeastern region of Turkey, an endemic area for amebiasis. The population of this study included patients with symptomatic diarrhea/dysentery attending both Yuzuncu Yil University, Van and Harran University, Sanliurfa, Turkey. A total of 380 stool specimens were collected and examined for Entamoeba by light microscopy (fresh, lugol, and trichrome staining) and stool antigen detection based- enzyme-linked immunosorbent assay (EIA) test (TechLab Entamoeba histolytica II). 24% (91/380) of stool specimens were positive for E. histolytica/Entamoeba dispar trophozoites/cysts microscopically using trichrome staining. 13% (51/380) of the stool specimens were found to be positive for E. histolytica by the EIA test, including 15% (14/91) of microscopy (+) stool specimens and 13% (37/289) of microscopy (-) stool specimens. Enteric parasites were common in these populations with 66% (251/380) of the study population harboring more than one parasite. In addition to the 13% (51/380) of patients determined to have E. histolytica by EIA, eighty-six patients (22.6%) had Blastocystis hominis, 54 (14.2%) Entamoeba coli, 44 (11.5%) Giardia lamblia, 16 (4.2%) Chilomastix mesnili, 15 (3.9%) Iodamoeba bütschlii, 12 (3.1%) Hymenolepis nana, 9 (2.3%) Endolimax nana, 9 (2.3%) Dientamoeba fragilis, and 8 (2.1%) had Ascaris lumbricoides. We concluded that E. histolytica infection was found in 13% of the patients presenting with diarrhea in Van and Sanliurfa Turkey.

  6. Purification and characterization of tyrosinase from walnut leaves (Juglans regia).

    Science.gov (United States)

    Zekiri, Florime; Molitor, Christian; Mauracher, Stephan G; Michael, Claudia; Mayer, Rupert L; Gerner, Christopher; Rompel, Annette

    2014-05-01

    Polyphenol oxidase (PPO) is a type-3 copper enzyme catalyzing the oxidation of phenolic compounds to their quinone derivates, which are further converted to melanin, a ubiquitous pigment in living organisms. In this study a plant originated tyrosinase was isolated from walnut leaves (Juglans regia) and biochemically characterized. It was possible to isolate and purify the enzyme by means of an aqueous two-phase extraction method followed by chromatographic purification and identification. Interestingly, the enzyme showed a rather high monophenolase activity considering that the main part of plant PPOs with some exceptions solely possess diphenolase activity. The average molecular mass of 39,047 Da (Asp(101)→Arg(445)) was determined very accurately by high resolution mass spectrometry. This proteolytically activated tyrosinase species was identified as a polyphenol oxidase corresponding to the known jrPPO1 sequence by peptide sequencing applying nanoUHPLC-ESI-MS/MS. The polypeptide backbone with sequence coverage of 96% was determined to start from Asp(101) and not to exceed Arg(445).

  7. An Efficient and Safe Method for Hemoglobin Purification%一种安全高效的血红蛋白纯化方法

    Institute of Scientific and Technical Information of China (English)

    臧家涛; 刘建仓; 徐竞; 李东红; 刘良明

    2011-01-01

    Objective: To build up a hemoglobin purification method which could meet the needs of safety, efficiency and scale amplification. Methods; Packed red blood cells were lysed by dialyzing against 10 mmol/L Tris-HCl (pH 8. 0) containing 0. 15% (M/V) ascorbic acid at 4°C for 3 h with the buffer changed once an hour, and then centrifuged at 10 000 g for 10 min (4℃). The supernatant was treated with ammonium sulfate by the two-step salting-out operation to the saturation of 20% and 46% (M/V, calibrated at OX,) subsequently, and then the precipitated protein was re-suspended using the equalizing buffer (25 mmol/L Tris, 10 mmol/L NaCl pH 8. 5) with the equal volume to that of the packed red blood cells. After dialyzing against the equalizing buffer for at 4℃ for 3 h with the same changing interval, the protein solution was diluted with the equalizing buffer by the ration of 1:2 (V/V) and centrifuged at 12 000 g for 20 min (4℃). The supernatant after centrifugation, namely the rough hemoglobin extract, was loaded to an 50 mm * 200 mm ( d * h) Q sapharose FF anion exchange chromatography column pre-equalized with the equalizing buffer and then hemoglobin was eluted using 25 mmol/L Tris, 80 mmol/ L NaCl pH 8. 5. The purity of samples from different processed was analyzed by SDS-PAGE and HPLC, and the oxygen-binding status was characterized by the UV-Vis spectrum scanning and blood-gas indexes, which were provided by the Thermo Multiscan Spectrum and ABL800 Flex Blood-gas Analyzer respectively. Iipoprotein concentration was tested by the phosphorus determination method and LPS was determined using the stachypleus amebocyte lysate with the sensitivity of 0. 3 EU/ml, 0. 1 EU/ml and 0. 03 EU/ml. Results more than 98% lipids were removed in the rough hemoglobin extracts compared with hemoglobin extracts prepared by the conventional method, and this rough hemoglobin extracts could easily go through the 0.45μm cellulose nitrate membrane. In the following anion exchange

  8. One-step purification of phosphinothricin acetyltransferase using reactive dye-affinity chromatography.

    Science.gov (United States)

    Wang, Cunxi; Lee, Thomas C; Crowley, Kathleen S; Bell, Erin

    2015-01-01

    Reactive dye purification is an affinity purification technique offering unique selectivity and high purification potential. Historically, purification of phosphinothricin acetyltransferase (PAT) has involved several steps of precipitation and column chromatography. Here, we describe a novel purification method that is simple, time-saving, inexpensive, and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein can be purified to homogeneity from E. coli or plant tissue with high recovery efficiency.

  9. Purification, crystallization and preliminary X-ray characterization of the human GTP fucose pyrophosphorylase

    Energy Technology Data Exchange (ETDEWEB)

    Quirk, Stephen; Seley-Radtke, Katherine L., E-mail: kseley@umbc.edu [Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Chemistry 405C, Baltimore, MD 21250 (United States)

    2006-04-01

    The human GTP fucose pyrophosphohydrolase protein has been crystallized via the hanging-drop technique over a reservoir of polyethylene glycol (MW 8000) and ethylene glycol. The orthorhombic crystals diffract to 2.8 Å resolution. The human nucleotide-sugar metabolizing enzyme GTP fucose pyrophosphorylase (GFPP) has been purified to homogeneity by an affinity chromatographic procedure that utilizes a novel nucleoside analog. This new purification regime results in a protein preparation that produces significantly better crystals than traditional purification methods. The purified 66.6 kDa monomeric protein has been crystallized via hanging-drop vapor diffusion at 293 K. Crystals of the native enzyme diffract to 2.8 Å and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}. There is a single GFPP monomer in the asymmetric unit, giving a Matthews coefficient of 2.38 Å{sup 3} Da{sup −1} and a solvent content of 48.2%. A complete native data set has been collected as a first step in determining the three-dimensional structure of this enzyme.

  10. Direct Purification of Pectinase from Mango (Mangifera Indica Cv. Chokanan Peel Using a PEG/Salt-Based Aqueous Two Phase System

    Directory of Open Access Journals (Sweden)

    Abdul Manap Mohd Yazid

    2011-10-01

    Full Text Available An Aqueous Two-Phase System (ATPS was employed for the first time for the separation and purification of pectinase from mango (Mangifera Indica Cv. Chokanan peel. The effects of different parameters such as molecular weight of the polymer (polyethylene glycol, 2,000–10,000, potassium phosphate composition (12–20%, w/w, system pH (6–9, and addition of different concentrations of neutral salts (0–8%, w/w on partition behavior of pectinase were investigated. The partition coefficient of the enzyme was decreased by increasing the PEG molecular weight. Additionally, the phase composition showed a significant effect on purification factor and yield of the enzyme. Optimum conditions for purification of pectinase from mango peel were achieved in a 14% PEG 4000-14% potassium phosphate system using 3% (w/w NaCl addition at pH 7.0. Based on this system, the purification factor of pectinase was increased to 13.2 with a high yield of (97.6%. Thus, this study proves that ATPS can be an inexpensive and effective method for partitioning of pectinase from mango peel.

  11. Direct purification of pectinase from mango (Mangifera Indica Cv. Chokanan) peel using a PEG/salt-based Aqueous Two Phase System.

    Science.gov (United States)

    Mehrnoush, Amid; Sarker, Md Zaidul Islam; Mustafa, Shuhaimi; Yazid, Abdul Manap Mohd

    2011-10-10

    An Aqueous Two-Phase System (ATPS) was employed for the first time for the separation and purification of pectinase from mango (Mangifera Indica Cv. Chokanan) peel. The effects of different parameters such as molecular weight of the polymer (polyethylene glycol, 2,000-10,000), potassium phosphate composition (12-20%, w/w), system pH (6-9), and addition of different concentrations of neutral salts (0-8%, w/w) on partition behavior of pectinase were investigated. The partition coefficient of the enzyme was decreased by increasing the PEG molecular weight. Additionally, the phase composition showed a significant effect on purification factor and yield of the enzyme. Optimum conditions for purification of pectinase from mango peel were achieved in a 14% PEG 4000-14% potassium phosphate system using 3% (w/w) NaCl addition at pH 7.0. Based on this system, the purification factor of pectinase was increased to 13.2 with a high yield of (97.6%). Thus, this study proves that ATPS can be an inexpensive and effective method for partitioning of pectinase from mango peel.

  12. Photocatalytic materials and technologies for air purification.

    Science.gov (United States)

    Ren, Hangjuan; Koshy, Pramod; Chen, Wen-Fan; Qi, Shaohua; Sorrell, Charles Christopher

    2017-03-05

    Since there is increasing concern for the impact of air quality on human health, the present work surveys the materials and technologies for air purification using photocatalytic materials. The coverage includes (1) current photocatalytic materials for the decomposition of chemical contaminants and disinfection of pathogens present in air and (2) photocatalytic air purification systems that are used currently and under development. The present work focuses on five main themes. First, the mechanisms of photodegradation and photodisinfection are explained. Second, system designs for photocatalytic air purification are surveyed. Third, the photocatalytic materials used for air purification and their characteristics are considered, including both conventional and more recently developed photocatalysts. Fourth, the methods used to fabricate these materials are discussed. Fifth, the most significant coverage is devoted to materials design strategies aimed at improving the performance of photocatalysts for air purification. The review concludes with a brief consideration of promising future directions for materials research in photocatalysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

    Science.gov (United States)

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.; Henriksen, Emily D.

    2010-12-28

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  14. Thermophilic and thermoacidophilic metabolism genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    Science.gov (United States)

    Thompson, Vicki S; Apel, William A; Reed, David W; Lee, Brady D; Thompson, David N; Roberto, Francisco F; Lacey, Jeffrey A

    2014-05-20

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for modulating or altering metabolism in a cell using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

  15. Thermophilic and thermoacidophilic sugar transporter genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    Science.gov (United States)

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

    2013-01-29

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for transporting sugars across cell membranes using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

  16. Thermophilic and thermoacidophilic glycosylation genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A

    2017-06-14

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for glycosylating and/or post-translationally modifying proteins using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

  17. Thermophilic and thermoacidophilic biopolymer degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A; Henriksen, Emily D.

    2016-08-02

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  18. Thermophilic and thermoacidophilic glycosylation genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

    2016-01-12

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for glycosylating and/or post-translationally modifying proteins using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

  19. Thermophilic and thermoacidophilic metabolism genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Vicki S.; Apel, William A.; Reed, David William; Lee, Brady D.; Thompson, David N.; Roberto, Francisco F.; Lacey, Jeffrey A.

    2015-12-29

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for modulating or altering metabolism in a cell using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

  20. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A; Henriksen, Emily D

    2013-07-30

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.