Sample records for plasmid purification process

  1. Anion exchange purification of plasmid DNA using expanded bed adsorption. (United States)

    Ferreira, G N; Cabral, J M; Prazeres, D M


    Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity of process streams is of major concern in the purification of plasmids, since it can cause high back pressures in column operations, thus limiting the throughput. In order to avoid these high back pressures, expanded bed anion exchange chromatography was evaluated as an alternative to fixed bed chromatography. A Streamline 25 column filled with 100 ml of Streamline QXL media, was equilibrated with 0.5 M NaCl in TE (10 mM Tris, 1 mM EDTA, pH = 8.0) buffer at an upward flow of 300 cmh-1, E. coli lysates (obtained from up to 3 liters of fermentation broth) were injected in the column. After washing out the unbound material, the media was allowed to sediment and the plasmid was eluted with 1 M NaCl in TE buffer at a downward flow of 120 cmh-1. Purification factors of 36 +/- 1 fold, 26 +/- 0.4 plasmid purity, and close to 100% yields were obtained when less than one settled column volume of plasmid feed was injected. However, both recovery yield and purity abruptly decreased when larger amounts were processed-values of 35 +/- 2 and 5 +/- 0.7 were obtained for the recovery yield and purity, respectively, when 250 ml of feedstock were processed. In these cases, gel clogging and expansion collapse were observed. The processing of larger volumes, thus larger plasmid quantities, was only possible by performing an isopropanol precipitation step prior to the chromatographic step. This step led to an enhancement of the purification step.

  2. Purification process

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    Marshall, A.


    A process for the removal of hydrogen sulphide from gases or liquid hydrocarbons, comprises contacting the gas or liquid hydrocarbon with an aqueous alkaline solution, preferably having a pH value of 8 to 10, comprising (A) an anthraquinone disulphonic acid or a water-soluble sulphonamide thereof (B) a compound of a metal which can exist in at least two valency states and (C) a sequestering agent.

  3. Gas purification process

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    Hoelter, H.; Gresch, H.; Igelbuescher, H.; Dewert, H.


    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.

  4. Large-scale purification of pharmaceutical-grade plasmid DNA using tangential flow filtration and multi-step chromatography. (United States)

    Sun, Bo; Yu, XiangHui; Yin, Yuhe; Liu, Xintao; Wu, Yongge; Chen, Yan; Zhang, Xizhen; Jiang, Chunlai; Kong, Wei


    The demand for pharmaceutical-grade plasmid DNA in vaccine applications and gene therapy has been increasing in recent years. In the present study, a process consisting of alkaline lysis, tangential flow filtration, purification by anion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography was developed. The final product met the requirements for pharmaceutical-grade plasmid DNA. The chromosomal DNA content was <1 μg/mg plasmid DNA, and RNA was not detectable by agarose gel electrophoresis. Moreover, the protein content was <2 μg/mg plasmid DNA, and the endotoxin content was <10 EU/mg plasmid DNA. The process was scaled up to yield 800 mg of pharmaceutical-grade plasmid DNA from approximately 2 kg of bacterial cell paste. The overall yield of the final plasmid DNA reached 48%. Therefore, we have established a rapid and efficient production process for pharmaceutical-grade plasmid DNA. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography. (United States)

    Hilbrig, Frank; Freitag, Ruth


    Hydroxyapatite and related stationary phases increasingly play a role in the downstream processing of high-value biological materials, such as recombinant proteins, therapeutic antibodies and pharmaceutical-grade plasmid DNA. Chromatographic hydroxyapatite is an inorganic, ceramic material identical in composition, if not in structure, to calcium phosphate found in human bones and teeth. The interaction of hydroxyapatite with biomacromolecules is complex and highly dynamic, which can make predicting performance difficult, but also allows the design of very selective isolation processes. This review discusses the currently commercially available chromatographic materials, different retention mechanisms supported by these materials and differential exploitation for the design of highly specific isolation procedures. The state of the art of antibody purification by hydroxy- and fluoroapatite is reviewed together with tested routines for method development and implementation. Finally, the isolation of plasmid DNA is discussed, since the purification of DNA therapeutics at a sufficiently large scale is an emerging need in bioprocess development and perhaps the area in bioseparation where apatite chromatography can make its most important contribution to date.

  6. Purification of transfection-grade plasmid DNA from bacterial cells with superparamagnetic nanoparticles (United States)

    Chiang, Chen-Li; Sung, Ching-Shan


    The functionalized magnetic nanobeads were used to develop a rapid protocol for extracting and purifying transfection-grade plasmid DNA from bacterial culture. Nanosized superparamagnetic nanoparticles (Fe 3O 4) were prepared by chemical coprecipitation method using Fe 2+, Fe 3+ salt, and ammonium hydroxide under a nitrogen atmosphere. The surface of Fe 3O 4 nanoparticles was modified by coating with the multivalent cationic agent, polyethylenimine (PEI). The PEI-modified magnetic nanobeads were employed to simplify the purification of plasmid DNA from bacterial cells. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of Escherichia coli. The loaded nanobeads are recovered by magnetically driven separation and regenerated by exposure to the elution buffer with optimal ionic strength (1.25 M) and pH (9.0). Up to approximately 819 μg of high-purity (A 260/A 280 ratio=1.86) plasmid DNA was isolated from 100 ml of overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and animal cell transfection applications with success. The PEI-modified magnetic nanobead delivers significant time-savings, overall higher yields and better transfection efficiencies compared to anion-exchange and other methods. The results presented in this report show that PEI-modified magnetic nanobeads are suitable for isolation and purification of transfection-grade plasmid DNA.

  7. Purification of supercoiled plasmid DNA from clarified bacterial lysate by arginine-affinity chromatography: effects of spacer arms and ligand density. (United States)

    Bai, Jin-Shan; Bai, Shu; Shi, Qing-Hong; Sun, Yan


    Efficient loading on a chromatographic column is the dilemma of the process development faced by engineers in plasmid DNA purification. In this research, novel arginine-affinity chromatographic beads were prepared to investigate the effect of spacer arm and ligand density to their chromatographic performance for the purification of plasmid. The result indicated that dynamic binding capacity for plasmid increased with an increasing ligand density and carbon number of spacer arm, and the highest binding capacity for plasmid of 6.32 mg/mL bead was observed in the column of arginine bead with a ligand density of 47 mmol/L and 10-atom carbon spacer. Furthermore, this arginine bead exhibited better selectivity to supercoiled (sc) plasmid. The evidence of a linear gradient elution suggested further that the binding of plasmid on arginine beads was driven by electrostatic interaction and hydrogen bonding. Hence, sc plasmid could successfully be purified from clarified lysate by two-stepwise elution of salt concentration. By the refinement of the elution scheme and loading volume of clarified lysate, the column of arginine bead with a ligand density of 47 mmol/L exhibited the highest recovery yield and a much higher productivity among arginine-affinity columns. Therefore, reshaped arginine beads provided more feasible and practical application in the preparation of sc plasmid from clarified lysate. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Binding and elution strategy for improved performance of arginine affinity chromatography in supercoiled plasmid DNA purification. (United States)

    Sousa, F; Prazeres, D M F; Queiroz, J A


    New interesting strategies for plasmid DNA (pDNA) purification were designed, exploiting affinity interactions between amino acids and nucleic acids. The potential application of arginine-based chromatography to purify pDNA has been recently described in our work; however, to achieve higher efficiency and selectivity in arginine affinity chromatography, it is essential to characterize the behaviour of binding/elution of supercoiled (sc) isoforms. In this study, two different strategies based on increased sodium chloride (225-250 mm) or arginine (20-70 mm) stepwise gradients are described to purify sc isoforms. Thus, it was proved that well-defined binding/elution conditions are crucial to enhance the purification performance, resulting in an improvement of the final plasmids yields and transfection efficiency, as this could represent a significant impact on therapeutic applications of the purified sc isoform. Copyright (c) 2008 John Wiley & Sons, Ltd.

  9. Sewage Purification Business Process Management

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    Esad Ahmetagić


    Full Text Available This paper presents the current level of drainage and sewage purification facilities built in the Autonomous Province of Vojvodina, a territorial unit of the Republic of Serbia. It also points out the issues related to organized business management in companies involved in this business.The management of business processes in sewage purification involves a comprehensive cycle: business organizing process, issues of standard, investments, workforce, and information system design as factors in establishing an effective organization of business processes. The definition of gap existing between the current approach to organizing business activities and the need to establish an approach based on knowledge, information technologies, and effective business process management points to the necessity for organization redesign and standard definition in business process management. Sewage purification business process management in Vojvodina, the Republic of Serbia has been elaborated through theoretical presentation and a practical example realized by electronic ISO 9001:2008 system of quality management in public water utility company JKP "Vodokanal" Sombor.

  10. Validation and scale-up of plasmid DNA purification by phenyl-boronic acid chromatography. (United States)

    Gomes, A Gabriela; Azevedo, Ana M; Aires-Barros, M Raquel; Prazeres, D Miguel F


    This study addresses the feasibility of scaling-up the removal of host cell impurities from plasmid DNA (pDNA)-containing Escherichia coli lysates by phenyl-boronic (PB) acid chromatography using columns packed with 7.6 and 15.2 cm(3) of controlled porous glass beads (CPG) derivatized with PB ligands. Equilibration was performed with water at 10 cm(3) /min and no conditioning of the lysate feed was required. At a ratio of lysate feed to adsorbent volume of 1.3, 93-96% of pDNA was recovered in the flow through while 66-71% of impurities remained bound (~2.5-fold purification). The entire sequence of loading, washing, elution, and re-equilibration was completed in 20 min. Run-to-run consistency was observed in terms of chromatogram features and performance (yield, purification factor, agarose electrophoresis) across the different amounts of adsorbent (0.75-15.2 cm(3) ) by performing successive injections of lysates prepared independently and containing 3.7 or 6.1 kbp plasmids. The column productivity at large scale was 4 dm(3) of alkaline lysate per hour per dm(3) of PB-CPG resin. The method is rapid, reproducible, simple, and straightforward to scale-up. Furthermore, it is capable of handling heavily contaminated samples, constituting a good alternative to purification techniques such as isopropanol precipitation, aqueous two-phase systems, and tangential flow filtration. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Gas purification process. Verfahren zur Gasreinigung

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    Hoelter, H.; Gresch, H.; Igelbuescher, H.; Dewert, H.


    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.

  12. 基于色谱法的超螺旋质粒DNA纯化与分析进展%Advances on Purification Process and Analysis of Supercoiled Plasmid DNA Based on Chromatography

    Institute of Scientific and Technical Information of China (English)

    李亮; 柳方方; 宛煜嵩; 金芜军


    质粒DNA含有独立复制的遗传结构,是基因工程的常用工具,广泛应用于分子生物学基础研究、农业转基因检测、医疗诊断与基因治疗等领域。质粒DNA的构型一般分为超螺旋、开口环状及线性3种。初步纯化的质粒DNA溶液中常混有3种构型,并且掺杂一定量的蛋白质、RNA、内毒素以及宿主基因组DNA,这些杂质会影响后续的应用,因此质粒DNA需要进一步精细纯化。该文对质粒DNA的新应用领域、基于色谱的精细纯化技术及产物质量分析体系进行了综述,并展望了高纯度质粒DNA精细纯化及产物分析的发展方向。%Plasmid DNA,a common tool of genetic engineering containing the independent genetic structure of the replication,is widely used in basic research of molecular biology,genetically modi-fied detection,medical diagnosis and gene therapy. Plasmid DNA conformations are generally divided into three types:supercoiled,open circle and linear. Crude plasmid DNA often mixes with three configurations,and the amount of protein,RNA,endotoxin with the host genome DNA. These im-purities need to be further refined as they affect subsequent applications. The new applications of plasmid DNA,refined technologies and products based on chromatography system,and quality anal-ysis of purified plasmid DNA is reviewed in this paper. Finally,the trends of technology of high pu-rity plasmid DNA and analysis of refined product are presented.

  13. Purification of plasmid (pVaxLacZ by hydrophobic interaction chromatography

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    Keila Aparecida Moreira


    Full Text Available This paper describes a method for the plasmid DNA purification, which includes an ammonium sulphate precipitation, followed by hydrophobic interaction chromatography (HIC using Phenyl Sepharose 6 Fast Flow (low sub. The use of HIC took advantage of the more hydrophobic character of single stranded nucleic acid impurities as compared with double-stranded plasmid DNA.O DNA plasmidial tem sua aplicação na terapia gênica, tendo que apresentar elevado grau de pureza. Um método de purificação foi descrito neste trabalho, que inclui precipitação com sulfato de amônio seguida da cromatografia de interação hidrofóbica, usando a resina phenyl sepharose 6 fast flow. Ocorreu aumento da pureza das amostras analisadas, em torno de 30% após a precipitação com sulfato de amônio. Houve significativa redução dos contaminantes após a eluição das amostras na cromatografia de interação. O maior rendimento obtido foi 53% e fator de purificação 3,1 vezes após a eluição na cromatografia de interação hidrofóbica. A vantagem deste método é a possibilidade de explorar o caráter hidrofóbico, constituído de material genético de fita simples, tais como RNA e DNA genômico e proteínas, que podem interferir na liberação deste material para possíveis aplicações em terapia gênica.

  14. Purification of plasmid DNA from clarified and non-clarified Escherichia coli lysates by berenil pseudo-affinity chromatography. (United States)

    Caramelo-Nunes, C; Gabriel, M F; Almeida, P; Marcos, J C; Tomaz, C T


    In this study, berenil was tested as a ligand, specifically to purify plasmids of different sizes pVAX1-LacZ (6.05 Kbp) and pCAMBIA-1303 (12.361 Kbp) from clarified Escherichia coli alkaline lysates. For this purpose, chromatographic experiments were performed using Sepharose derivatized with berenil. The results showed that both pDNA molecules are completely purified using lower amounts of salt in the eluent than those previously reported for other pseudo-affinity and hydrophobic interaction chromatography based processes. Total retention of all lysate components was achieved with 1.3M ammonium sulphate in the eluent buffer and pDNA elution was obtained by decreasing the salt concentration to 0.55 M. All impurities were eluted after decreasing the concentration to 0M. The recovery yield for pCAMBIA-1303 (45%) was lower than that obtained for pVAX1-LacZ (85%), however the larger pDNA showed a higher purity level. Purification of pVAX1-LacZ was also performed using non-clarified E. coli process streams, replacing the clarification step with a second chromatographic run on the berenil-Sepharose. Using the same binding and elution conditions as before, a pure plasmid sample was obtained with a 33% yield and with all host impurity levels in accordance with the requirements established by the regulatory agencies. These results suggest that this chromatographic method is a promising alternative to purify pDNA for therapeutic use. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Nanospines incorporation into the structure of the hydrophobic cryogels via novel cryogelation method: an alternative sorbent for plasmid DNA purification. (United States)

    Üzek, Recep; Uzun, Lokman; Şenel, Serap; Denizli, Adil


    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.

  16. Production and purification of plasmid DNA vaccines: is there scope for further innovation? (United States)

    Xenopoulos, Alex; Pattnaik, Priyabrata


    The demand for plasmid DNA (pDNA) has vastly increased over the past decade in response to significant advances that have been made in its application for gene therapy and vaccine development. Plasmid DNA-based vaccines are experiencing a resurgence due to success with prime-boost immunization strategies. The challenge has always been poor productivity and delivery of pDNA. Plasmid DNA-based vaccines have traditionally required milligram scale of GMP-grade product for vaccination due to the relatively low efficacy and duration of gene expression. However, efforts to increase pDNA vaccine effectiveness are evolving in genetic manipulations of bacterial host, improvements in product recovery and innovative delivery methods. This review summarizes recent advances in large-scale pDNA vaccine manufacturing, ranging from upstream processing, downstream processing and formulation, as such information is usually not available to the scientific community. The article will highlight technology gaps and offer insight on further scope of innovation.

  17. Purification process for vertically aligned carbon nanofibers (United States)

    Nguyen, Cattien V.; Delziet, Lance; Matthews, Kristopher; Chen, Bin; Meyyappan, M.


    Individual, free-standing, vertically aligned multiwall carbon nanotubes or nanofibers are ideal for sensor and electrode applications. Our plasma-enhanced chemical vapor deposition techniques for producing free-standing and vertically aligned carbon nanofibers use catalyst particles at the tip of the fiber. Here we present a simple purification process for the removal of iron catalyst particles at the tip of vertically aligned carbon nanofibers derived by plasma-enhanced chemical vapor deposition. The first step involves thermal oxidation in air, at temperatures of 200-400 degrees C, resulting in the physical swelling of the iron particles from the formation of iron oxide. Subsequently, the complete removal of the iron oxide particles is achieved with diluted acid (12% HCl). The purification process appears to be very efficient at removing all of the iron catalyst particles. Electron microscopy images and Raman spectroscopy data indicate that the purification process does not damage the graphitic structure of the nanotubes.

  18. Poly(hydroxyethyl methacrylate) based magnetic nanoparticles for plasmid DNA purification from Escherichia coli lysate

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    Percin, Is Latin-Small-Letter-Dotless-I k [Department of Biology, Hacettepe University, Ankara (Turkey); Karakoc, Veyis [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey); Akgoel, Sinan [Department of Biochemistry, Ege University, Izmir (Turkey); Aksoez, Erol [Department of Biology, Hacettepe University, Ankara (Turkey); Denizli, Adil, E-mail: [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey)


    The aim of this study is to prepare poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine) [PHEMAH] magnetic nanoparticles for plasmid DNA (pDNA) purification from Escherichia coli (E. coli) cell lysate. Magnetic nanoparticles were produced by surfactant free emulsion polymerization. mPHEMAH nanoparticles were characterized by elemental analysis, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), vibrating sample magnetometer (VSM), electron spin resonance (ESR), thermogravimetric analyses (TGA) and transmission electron microscopy (TEM). Surface area, average particle size and size distribution were also performed. Specific surface area of the mPHEMAH nanoparticles was found to be 1180 m{sup 2}/g. Elemental analysis of MAH for nitrogen was estimated as 0.18 mmol/g polymer. The amount of pDNA adsorbed onto the mPHEMAH nanoparticles first increased and then reached a saturation value at around 1.0 mg/mL of pDNA concentration. Compared with the mPHEMA nanoparticles (50 {mu}g/g polymer), the pDNA adsorption capacity of the mPHEMAH nanoparticles (154 mg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The maximum pDNA adsorption was achieved at 25 Degree-Sign C. The overall recovery of pDNA was calculated as 92%. The mPHEMAH nanoparticles could be used six times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH nanoparticles promise high selectivity for pDNA. - Highlights: Black-Right-Pointing-Pointer Magnetic nanoparticles have several advantages over conventional adsorbents. Black-Right-Pointing-Pointer MAH acted as the pseudospecific ligand, ligand immobilization step was eliminated. Black-Right-Pointing-Pointer pDNA adsorption amount was 154 mg/g. Black-Right-Pointing-Pointer Fifty-fold capacity increase was obtained when compared to conventional matrices.

  19. Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology

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    Li Li


    Full Text Available Abstract Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1 DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2 Miniprep DNA from E. coli can be eluted with as little as 5 μl water, leading to high DNA concentrations (>1 μg/μl for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3 Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4 High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.

  20. Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology. (United States)

    Li, Jian-Feng; Li, Li; Sheen, Jen


    Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2) Miniprep DNA from E. coli can be eluted with as little as 5 mul water, leading to high DNA concentrations (>1 mug/mul) for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG)-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3) Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4) High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.

  1. Research of the thorium purification at monazite refinement processes (United States)

    Shagalov, V. V.; Sobolev, V. I.; Turinskaya, M. V.; Malin, A. V.


    This paper is aimed to the research of the thorium purification processes at monazite refinement processes. We have investigated different solution containing thorium with different mix of rare-earth elements. It was found that the application of cation resin is well- recommended if we want to reach the highest yields of thorium purification process.

  2. Surface processes during purification of InP quantum dots



    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 ...

  3. Human papillomavirus DNA from warts for typing by endonuclease restriction patterns: purification by alkaline plasmid methods. (United States)

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


    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.

  4. Metal chelate affinity precipitation of RNA and purification of plasmid DNA (United States)

    Balan, Sindhu; Murphy, Jason; Galaev, Igor; Kumar, Ashok; Fox, George E.; Mattiasson, Bo; Willson, Richard C.


    The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine 'tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.

  5. Metal chelate affinity precipitation of RNA and purification of plasmid DNA (United States)

    Balan, Sindhu; Murphy, Jason; Galaev, Igor; Kumar, Ashok; Fox, George E.; Mattiasson, Bo; Willson, Richard C.


    The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine 'tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.


    Institute of Scientific and Technical Information of China (English)

    白金山; 白姝


    非病毒载体质粒DNA已被广泛应用于基因治疗和DNA疫苗,目前迫切需要开发其大规模制备和分离纯化方法.亲和色谱是一种高分辨率、高选择性的分离技术,在蛋白质、抗体、核酸等生物大分子的分离纯化方面显示了良好的应用前景.本文综述了亲和色谱技术在超螺旋质粒DNA分离纯化中的研究进展,总结了各种亲和色谱方法分离超螺旋质粒DNA的机理和优缺点,并展望了亲和纯化技术在质粒DNA生产和制备中的应用前景.%Non-viral vector,plasmid DNA has been widely used in gene therapy and DNA vaccines.It is imperative to develop large-scale preparation and purification methods of plasmid DNA at present.As a separation technology of high resolution and high selectivity,affinity chromatography shows great application potential in terms of separation and purification of biological macromolecules such as proteins,antibodies,nucleic acids and so on.The domestic and foreign research progress of High Performance Liquid Chromatography (HPLC) technology,used in separation and purification of supercoiled plasmid DNA was reviewed in this paper.The advantages and disadvantages of various affinity chromatographic methods for separating supercoiled plasmid DNA were also summarized.At last,the affinity chromatography technology for preparation and purification of plasmid DNA was prospected.


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    С. Бойченко


    Full Text Available Sorption properties of carbonic sorbents on natural raw materials for purification of waste waters frompetroleum products are investigated. Temperature influence on sumption properties of sorbents on naturalraw materials to increase the purification degree of water ecosystem is studied. Mathematical model ofpurification process of oil contaminated waters is developed



    С. Бойченко; Кучер, О.; Л. Павлюх


    Sorption properties of carbonic sorbents on natural raw materials for purification of waste waters frompetroleum products are investigated. Temperature influence on sumption properties of sorbents on naturalraw materials to increase the purification degree of water ecosystem is studied. Mathematical model ofpurification process of oil contaminated waters is developed

  9. Surface processes during purification of InP quantum dots

    Directory of Open Access Journals (Sweden)

    Natalia Mordvinova


    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.

  10. Surface processes during purification of InP quantum dots. (United States)

    Mordvinova, Natalia; Emelin, Pavel; Vinokurov, Alexander; Dorofeev, Sergey; Abakumov, Artem; Kuznetsova, Tatiana


    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.

  11. Process for purification of petroleum oil fractions

    Energy Technology Data Exchange (ETDEWEB)

    Gaile, A.A.; Proskuryakov, V.A.; Semenov, L.V.; Ul' chenkova, L.M.; Volkova, N.I.


    In the process for removal of polycyclic aromatic hydrocarbons, asphatic tarry materials, and heterocyclic compounds from petroleum oil fractions by extraction with a selective solvent, with the aim of increasing the degree of purification and increasing the oil fraction yield, cyanomethyl acetate (I) or its aqueous solution is used as the solvent. I possesses a combination of high selectivity with sufficiently high dissolving capacity in relation to the undesirable oil fraction components. The extraction properties of I were studied both on artificial mixtures modelling the hydrocarbon part of petroleum oil fractions (tridecane + ..cap alpha..-methylnaphthalene) and on industrial oil fractions. The H/sub 2/O content in I can be from 0 to 10%. By reducing the water content of the extractant the yield and quality of the purified oil can be regulated. The solvent: feedstock ratio with the use of I or mixtures of it with H/sub 2/O is advisably maintained within the limits 0.5-3:1. Example -- Results are presented for a one-stage extraction of ..cap alpha..-methylnaphthalene from a mixture with tridecane at 20/sup 0/ and with a solvent:feedstock weight ratio of 1.5 in comparison with the use of furfurol as the selective solvent. In the proposed process the degree of extraction of ..cap alpha..-methylnaphthalene is 57% as against 55.6%; the tridecane content in the raffinate is 80.9% as against 79.3%; the degree of tridecane extraction is 97.5% as against 92%, and the raffinate yield is 78.5% as against 75.3%. In a multistage extraction process the advantages of I will be still more substantial.

  12. Design strategies for integrated protein purification processes: challenges, progress and outlook

    NARCIS (Netherlands)

    Nfor, B.; Ahamed, T.; Dedem, G.; Wielen, van der L.; Sandt, van de E.; Eppink, M.H.M.; Ottens, M.


    The key to successful and efficient protein purification is the selection of the most appropriate purification techniques and their combination in a logical way to obtain the desired purification in the minimum number of steps. However, the rationalization of protein purification process development

  13. Zein purification: the process, the product, market potential (United States)

    The objectives of this article intend to give an overview of a zein purification, decolorization and deodorization process, methodologies to assess those properties and applications of the purified product. The process involves column filtration of commercial zein solutions through a combination of ...

  14. Separation process design for isolation and purification of natural products

    DEFF Research Database (Denmark)

    Malwade, Chandrakant R.

    selection of separation techniques and operating conditions. The key factor in designing separation processes with multiple unit operations is to determine the synergy between them which in turn demands molecular level understanding of process streams. Therefore, the methodology is fortified with process......, thereby providing process information crucial for determining synergistic effects between different unit operations. In this work, the formulated methodology has been used to isolate and purify artemisinin, an antimalarial drug, from dried leaves of the plant Artemisia annua. A process flow sheet...... is generated consisting of maceration, flash column chromatography and crystallization unit operations for extraction, partial purification and final purification of artemisinin, respectively. PAT framework is used extensively to characterize the process streams at molecular level and the generated process...

  15. Use of MagneSil(TM) paramagnetic particles for plasmid purification, PCR cleanup, and purification of dideoxy and big dye DNA sequencing reactions (United States)

    Bitner, Rex M.; White, Doug; Krueger, Steven K.; Bjerke, Michael; Butler, Braeden; Smith, Craig


    Traditional anion exchange purification of nucleic acids requires the elution of the DNA or RNA in a salt solution, necessitating the precipitation or desalting of the nucleic acid prior to many molecular biology applications. A pH dependent anion exchange purification method is described which allows the purification of nucleic acids at one pH, followed by the elution of the nucleic acid in a low salt buffer at a second, higher pH. The benefits of this method include the avoidance of alcohol washes and the drying steps required for alcohol removal, as well as the benefits of anion exchange purification without the need for desalting of the purified DNA or RNA.

  16. Chromatographic HPV-16 E6/E7 plasmid vaccine purification employing L-histidine and 1-benzyl-L-histidine affinity ligands. (United States)

    Amorim, Lúcia F A; Gaspar, Rita; Pereira, Patrícia; Černigoj, Urh; Sousa, Fani; Queiroz, João António; Sousa, Ângela


    Affinity chromatography based on amino acids as interacting ligands was already indicated as an alternative compared to ion exchange or hydrophobic interaction for plasmid DNA purification. Understanding the recognition mechanisms occurring between histidine-based ligands and nucleic acids enables more efficient purification of a DNA vaccine, as the binding and elution conditions can be adjusted in order to enhance the purification performance. Decreasing pH to slightly acidic conditions increases the positive charge of histidine ligand, what influences the type of interaction between chromatographic support and analytes. This was proven in this work, where hydrophobic effects established in the presence of ammonium sulfate were affected at pH 5.0 in comparison to pH 8.0, while electrostatic and cation-π interactions were intensified. Histidine ligand at pH 5.0 interacts with phosphate groups or aromatic rings of plasmid DNA. Due to different responses of RNA and pDNA on mobile phase changes, the elution order between RNA and pDNA was changed with mobile phase pH decrease from 8.0 to 5.0. The phenomenon was more evident with L-histidine ligand due to more hydrophilic character, leading to an improved selectivity of L-histidine-modified chromatographic monolith, allowing the product recovery with 99% of purity (RNA removal). With the 1-benzyl- L-histidine ligand, stronger and less selective interactions with the nucleic acids were observed due to the additional hydrophobicity associated with the phenyl aromatic ring. Optimization of sample displacement chromatography parameters (especially (NH4 )2 SO4 concentration) at slightly acidic pH enabled excellent isolation of pDNA, by the removal of RNA in a negative mode, with binding capacities above 1.5 mg pDNA per mL of chromatographic support. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Scale-Up of Protein Purification: Downstream Processing Issues. (United States)

    Milne, John Joseph


    Separation science continues to occupy the central position in the overall strategy for the downstream processing and purification of therapeutic protein products for human use. Increasing product titers from mammalian cell culture and new emerging classes of biopharmaceuticals has presented a challenge to the industry to identify ways of improving the robustness and economics of chromatography processes. In commercial manufacturing, there is always a need to increase the scale of the chromatography operations which are typically developed and optimized in small-scale laboratory experiments. This review discusses the key factors in the chromatography process that need to be considered as the scale of the purification step is increased in order to maintain the purity and integrity of the product purified at smaller scale.

  18. Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci

    Energy Technology Data Exchange (ETDEWEB)

    Pollet, Rebecca M.; Ingle, James D.; Hymes, Jeff P.; Eakes, Thomas C.; Eto, Karina Yui; Kwong, Stephen M.; Ramsay, Joshua P.; Firth, Neville; Redinbo, Matthew R.; Christie, P. J.


    mechanism of antimicrobial resistance transfer in bacteria such asStaphylococcus aureusis an important step toward potentially slowing the spread of antimicrobial-resistant infections. This work establishes protein-DNA interactions essential for the transfer of theStaphylococcus aureusmultiresistance plasmid pSK41 by its relaxase, NES. This enzyme also processed variantoriT-like sequences found on numerous plasmids previously considered nontransmissible, suggesting that in conjunction with an uncharacterized accessory protein, these plasmids may be transferred horizontally via a relaxase intransmechanism. These findings have important implications for our understanding of staphylococcal resistance plasmid evolution.

  19. Sodium citrate and potassium phosphate as alternative adsorption buffers in hydrophobic and aromatic thiophilic chromatographic purification of plasmid DNA from neutralized lysate. (United States)

    Bonturi, Nemailla; Radke, Vanessa Soraia Cortez Oliveira; Bueno, Sônia Maria Alves; Freitas, Sindélia; Azzoni, Adriano Rodrigues; Miranda, Everson Alves


    The number of studies on gene therapy using plasmid vectors (pDNA) has increased in recent years. As a result, the demand for preparations of pDNA in compliance with recommendations of regulatory agencies (EMEA, FDA) has also increased. Plasmid DNA is often obtained through fermentation of transformed Escherichia coli and purification by a series of unit operations, including chromatography. Hydrophobic interaction chromatography (HIC) and thiophilic aromatic chromatography (TAC), both using ammonium sulfate buffers, are commonly employed with success. This work was aimed at studying the feasibility of utilizing alternative salts in the purification of pDNA from neutralized lysate with phenyl-agarose (HIC) and mercaptopyrimidine-agarose (TAC) adsorbents. Their selectivity toward sc pDNA was evaluated through adsorption studies using 1.5 mol/L sodium citrate and 2.0 mol/L potassium phosphate as adsorption buffers. Chromatography with mercaptopyrimidine-agarose adsorbent and 1.5 mol/L sodium citrate was able to recover 91.1% of the pDNA with over 99.0% removal of gDNA and endotoxin. This represents a potential alternative for the primary recovery of sc pDNA. However, the most promising result was obtained using 2.0 mol/L potassium phosphate buffer and a mercaptopyrimidine-agarose column. In a single chromatographic step, this latter buffer/adsorbent system recovered 68.5% of the pDNA with 98.8% purity in accordance with the recommendations of regulatory agencies with regard to RNA and endotoxin impurity.

  20. Platform Technology for Developing Purification Processes

    NARCIS (Netherlands)

    Eppink, M.H.M.; Schreurs, R.; Gijsen, A.; Verhoeven, K.


    To shorten time to market for new therapeutic proteins, new and fast methods, such as high throughput screening, are needed to speed up downstream processing. The platform technology discussed in this article includes a structural approach that can be used as a general procedure to purify therapeuti

  1. Optimization of supercoiled HPV-16 E6/E7 plasmid DNA purification with arginine monolith using design of experiments. (United States)

    Almeida, A M; Queiroz, J A; Sousa, F; Sousa, A


    The progress of DNA vaccines is dependent on the development of suitable chromatographic procedures to successfully purify genetic vectors, such as plasmid DNA. Human Papillomavirus is associated with the development of tumours due to the oncogenic power of E6 and E7 proteins, produced by this virus. The supercoiled HPV-16 E6/E7 plasmid-based vaccine was recently purified with the arginine monolith, with 100% of purity, but only 39% of recovery was achieved. Therefore, the present study describes the application of experimental design tools, a newly explored methodology in preparative chromatography, in order to improve the supercoiled plasmid DNA recovery with the arginine monolith, maintaining the high purity degree. In addition, the importance and influence of pH in the pDNA retention to the arginine ligand was also demonstrated. The Composite Central Face design was validated and the recovery of the target molecule was successfully improved from 39% to 83.5%, with an outstanding increase of more than double, while maintaining 100% of purity.

  2. Investigation of purification process stresses on erythropoietin peptide mapping profile. (United States)

    Sepahi, Mina; Kaghazian, Hooman; Hadadian, Shahin; Norouzian, Dariush


    Full compliance of recombinant protein peptide mapping chromatogram with the standard reference material, is one of the most basic quality control tests of biopharmaceuticals. Changing a single amino acid substitution or side chain diversity for a given peptide changes protein hydrophobicity and causes peak shape or retention time alteration in a peptide mapping assay. In this work, the effect of different stresses during the recombinant erythropoietin (EPO) purification process, including pH 4, pH 5, and room temperature were checked on product peptide mapping results. Cell culture harvest was purified under stress by different chromatographic techniques consisting of gel filtration, anionic ion exchange, concentration by ultrafiltration, and high resolution size exclusion chromatography. To induce more pH stresses, the purified EPO was exposed to pH stress 4 and 5 by exchanging buffer by a 10 KDa dialysis sac overnight. The effects of temperature and partial deglycosylation (acid hydrolysis) on purified EPO were also studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and peptide mapping analysis. Removal of sialic acid by mild hydrolysis was performed by exposure to two molar acetic acid at 80°C for 3 h. No significant effect was observed between intact and stressed erythropoietin peptide mapping profiles and SDS-PAGE results. To validate the sensibility of the technique, erythropoietin was partially acid hydrolyzed and significant changes in the chromatographic peptide map of the intact form and a reduction on its molecular weight were detected, which indicates some partial deglycosylation. Purification process does not alter the peptide mapping profile and purification process stresses are not the cause of peptide mapping noncompliance.

  3. Process for the biological purification of waste water

    DEFF Research Database (Denmark)


    Process for the biological purification of waste water by the activated sludge method, the waste water being mixed with recirculated sludge and being subjected to an anaerobic treatment, before the waste water thus treated is alternately subjected to anoxic and aerobic treatments and the waste...... water thus treated is led into a clarification zone for settling sludge, which sludge is recirculated in order to be mixed with the crude waste water. As a result, a simultaneous reduction of the content both of nitrogen and phosphorus of the waste water is achieved....

  4. Model-based design of peptide chromatographic purification processes. (United States)

    Gétaz, David; Stroehlein, Guido; Butté, Alessandro; Morbidelli, Massimo


    In this work we present a general procedure for the model-based optimization of a polypeptide crude mixture purification process through its application to a case of industrial relevance. This is done to show how much modeling can be beneficial to optimize complex chromatographic processes in the industrial environment. The target peptide elution profile was modeled with a two sites adsorption equilibrium isotherm exhibiting two inflection points. The variation of the isotherm parameters with the modifier concentration was accounted for. The adsorption isotherm parameters of the target peptide were obtained by the inverse method. The elution of the impurities was approximated by lumping them into pseudo-impurities and by regressing their adsorption isotherm parameters directly as a function of the corresponding parameters of the target peptide. After model calibration and validation by comparison with suitable experimental data, Pareto optimizations of the process were carried out so as to select the optimal batch process.

  5. Process development and optimisation of lactic acid purification using electrodialysis. (United States)

    Madzingaidzo, L; Danner, H; Braun, R


    Cell free sodium lactate solutions were subjected to purification based on mono- and bi-polar electrodialysis. Lactate concentration in the product stream increased to a maximum of 15% during mono-polar electrodialysis. Stack energy consumption averaged 0.6 kW h kg(-1) lactate transported at current efficiencies in the 90% range. Under optimum feed concentration (125 g l(-1)) and process conditions (auto-current mode with conductivity setpoints of minimum 5 and maximum 40 mS cm(-1)), lactate flux reached 300 g m(-2) h(-1) and water flux were low for mono-polar electrodialysis averaging 0.3 kg H(2)O per M lactate transported. Glucose in the concentrate stream solutions was reduced to concentrate stream solutions. After mono-polar electrodialysis, the concentrated sodium lactate solutions were further purified using bi-polar electrodialysis. Water transport during bi-polar electrodialysis reached figures of 0.070 - 0.222 kg H(2)O per M lactate. Free lactic acid concentration reached 16% with lactate flux of up to 300 g m(-2) h(-1). Stack energy consumption ranged from 0.6 to 1 kW h per kg lactate. Under optimised process conditions current efficiency during bi-polar electrodialysis was consistently around 90%. Glucose was further reduced from 2 to solution. Acetic acid impurity remained at around 1 g l(-1). Significant reduction in colour and minerals in the product streams was observed during electrodialysis purification.

  6. Fed-batch microbioreactor platform for scale down and analysis of a plasmid DNA production process. (United States)

    Bower, Diana M; Lee, Kevin S; Ram, Rajeev J; Prather, Kristala L J


    The rising costs of bioprocess research and development emphasize the need for high-throughput, low-cost alternatives to bench-scale bioreactors for process development. In particular, there is a need for platforms that can go beyond simple batch growth of the organism of interest to include more advanced monitoring, control, and operation schemes such as fed-batch or continuous. We have developed a 1-mL microbioreactor capable of monitoring and control of dissolved oxygen, pH, and temperature. Optical density can also be measured online for continuous monitoring of cell growth. To test our microbioreactor platform, we used production of a plasmid DNA vaccine vector (pVAX1-GFP) in Escherichia coli via a fed-batch temperature-inducible process as a model system. We demonstrated that our platform can accurately predict growth, glycerol and acetate concentrations, as well as plasmid copy number and quality obtained in a bench-scale bioreactor. The predictive abilities of the micro-scale system were robust over a range of feed rates as long as key process parameters, such as dissolved oxygen, were kept constant across scales. We have highlighted plasmid DNA production as a potential application for our microbioreactor, but the device has broad utility for microbial process development in other industries as well. Copyright © 2012 Wiley Periodicals, Inc.

  7. Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes

    Directory of Open Access Journals (Sweden)

    Štrukelj Borut


    Full Text Available Abstract Background Recombinant protein production in Escherichia coli cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have an important impact on the success of productivity. It was recognised that a method for accurate and rapid quantification of plasmid copy number is necessary for optimization and better understanding of this process. Lately, qPCR is becoming the method of choice for this purpose. In the presented work, an improved qPCR method adopted for PCN determination in various fermentation processes was developed. Results To avoid experimental errors arising from irreproducible DNA isolation, whole cells, treated by heating at 95°C for 10 minutes prior to storage at -20°C, were used as a template source. Relative quantification, taking into account different amplification efficiencies of amplicons for chromosome and plasmid, was used in the PCN calculation. The best reproducibility was achieved when the efficiency estimated for specific amplicon, obtained within one run, was averaged. It was demonstrated that the quantification range of 2 log units (100 to 10000 bacteria per well enable quantification in each time point during fermentation. The method was applied to study PCN variation in fermentation at 25°C and the correlation between PCN and protein accumulation was established. Conclusion Using whole cells as a template source and relative quantification considering different PCR amplification efficiencies are significant improvements of the qPCR method for PCN determination. Due to the approaches used, the method is suitable for PCN determination in fermentation processes using various media and conditions.

  8. New process of silicon carbide purification intended for silicon passivation (United States)

    Barbouche, M.; Zaghouani, R. Benabderrahmane; Benammar, N. E.; Aglieri, V.; Mosca, M.; Macaluso, R.; Khirouni, K.; Ezzaouia, H.


    In this work, we report on a new, efficient and low cost process of silicon carbide (SiC) powder purification intended to be used in photovoltaic applications. This process consists on the preparation of porous silicon carbide layers followed by a photo-thermal annealing under oxygen atmosphere and chemical treatment. The effect of etching time on impurities removal efficiency was studied. Inductively coupled plasma atomic emission spectrometry (ICP-AES) results showed that the best result was achieved for an etching time of 10 min followed by gettering at 900 °C during 1 h. SiC purity is improved from 3N (99.9771%) to 4N (99.9946%). Silicon carbide thin films were deposited onto silicon substrates by pulsed laser deposition technique (PLD) using purified SiC powder as target. Significant improvement of the minority carrier lifetime was obtained encouraging the use of SiC as a passivation layer for silicon.

  9. A novel purification process for dodecanedioic acid by molecular distillation

    Institute of Scientific and Technical Information of China (English)

    Jiang Yu; Xigang Yuan; Aiwu Zeng


    A novel purification process is involved to obtain the high purity [N 99%(by mass)] dodecanedioic acid (DC12). It involves a re-crystal ization followed by molecular distil ation from the crude product. The objective of this study is to investigate general conditions, feed rate, distil ing temperature and vacuum, necessary for centrifugal distil-lation of DC12. Under the optimum conditions, distilling temperature 180 °C, pressure 30 Pa and feed flow rate 700 ml·h−1, the purity of DC12 in the residence reached 97.55%with a yield of 53.18%by the analysis of gas chromatography. Multiple-pass distillation made a considerable contribution by improving the purity to 99.22%. Additionally, the effect of pretreatment (re-crystallization) on distillation process was revealed through a series of comparative experiments.

  10. A Heparin Purification Process Removes Spiked Transmissible Spongiform Encephalopathy Agent. (United States)

    Bett, Cyrus; Grgac, Ksenija; Long, Dianna; Karfunkle, Michael; Keire, David A; Asher, David M; Gregori, Luisa


    In 2000, bovine heparin was withdrawn from the US market for fear of contamination with bovine spongiform encephalopathy (BSE) agent, the cause of variant Creutzfeldt-Jakob disease in humans. Thus, US heparin is currently sourced only from pig intestines. Availability of alternative sources of crude heparin, a life-saving drug, would benefit public health. Bovine heparin is an obvious option, but BSE clearance by the bovine heparin manufacturing process should be evaluated. To this end, using hamster 263K scrapie as a surrogate for BSE agent, we applied a four-step bench-scale heparin purification protocol resembling a typical heparin manufacturing process to investigate removal of the spiked scrapie agent. We removed aliquots from each step and analyzed them for residual abnormal prion protein (PrP(TSE)) using a sensitive in vitro method, real-time quaking-induced conversion (RT-QuIC) assay, and for infectivity using animal bioassays. The purification process reduced infectivity by 3.6 log10 and removed PrP(TSE), measured as seeding activity, by 3.4 log10. NaOH treatment was the most effective removal step tested. We also investigated NaOH at different concentrations and pH: the results showed that as much as 5.2 log10 of PrP(TSE) seeding activity was removed at pH 12.5. Thus, changes to the concentration, treatment time, and temperature of alkaline extraction might further improve removal. Our results, using a basic heparin manufacturing process, inform efforts to reintroduce safe bovine heparin in the USA.

  11. Separation process design for isolation and purification of natural products

    DEFF Research Database (Denmark)

    Malwade, Chandrakant R.

    Natural products are defined as secondary metabolites produced by plants and form a vast pool of compounds with unlimited chemical and functional diversity. Many of these secondary metabolites are high value added chemicals that are frequently used as ingredients in food, cosmetics, pharmaceutica...... information is used to further optimize the process flow sheet. Chemometric methods have been used to extract molecular level information for understanding the process streams in relation to their separation operations....... and other consumer products. Therefore, process technology towards industrial scale production of such high value chemicals from plants has significant value. Natural products can be obtained in pure form via synthetic or semi-synthetic route, but due to their complicated nature these methods have not been...... developed to the extent of industrial production for majority of natural products. Thus, isolation and purification of such natural products from plants is the most viable way to obtain natural products in pure form. This PhD project is mainly concerned with the design of separation process to isolate...

  12. Purification and detoxification of petroleum refinery wastewater by electrocoagulation process. (United States)

    Gousmi, N; Sahmi, A; Li, H Z; Poncin, S; Djebbar, R; Bensadok, K


    The treatment of synthetic oily wastewater having the characteristics of a typical petroleum refinery wastewater (PRW) by electrocoagulation (EC) using iron and aluminum electrodes was conducted in an electrolytic reactor equipped with fluid recirculation. During the treatment, the emulsion stability was followed by the measurement of Zeta potential and particle sizes. Effects of some operating conditions such as electrodes material, current density and electrolysis time on removal efficiencies of turbidity, and chemical oxygen demand (COD) were investigated in detail. The PRW purification by the EC process was found to be the most effective using aluminum as the anode and cathode, current density of 60 A/m(2) and 30 min of electrolysis time. Under these conditions, the process efficiencies were 83.52% and 99.94%, respectively, for COD and turbidity removals which correspond to final values of 96 mg O2/L and 0.5 NTU. A moderate energy consumption (0.341 kWh) was needed to treat 1 m(3) of PRW. Besides, the ecotoxicity test proved that toxic substances presented in the PRW, and those inhibiting the germination growth of whet, were eliminated by the EC technique.

  13. Influence of zone purification process on TlBr crystals for radiation detector fabrication

    Energy Technology Data Exchange (ETDEWEB)

    Hitomi, Keitaro [Department of Electronics, Tohoku Institute of Technology, 35-1 Yagiyama Kasumi-cho, Taihaku-ku, Sendai 982-8577 (Japan)], E-mail:; Onodera, Toshiyuki; Shoji, Tadayoshi [Department of Electronics, Tohoku Institute of Technology, 35-1 Yagiyama Kasumi-cho, Taihaku-ku, Sendai 982-8577 (Japan)


    Thallium bromide (TlBr) is a wide gap compound semiconductor and is a promising material for fabrication of nuclear radiation detectors. In this study, the conventional zone refining method was employed to reduce the concentration of impurities in the TlBr crystals. In order to evaluate the efficiency of the zone purification, the zone purification process was repeated up to 300 times. The resistivity, the charge transport properties, and the spectroscopic performance of TlBr detectors fabricated from the crystals zone purified 1 time, 100 times, and 300 times were compared in this study in order to clarify the effectiveness of the zone purification process.

  14. Purification process influence on green tea extracts’ polyphenol content and antioxidant activity

    National Research Council Canada - National Science Library

    Anna Gramza-Michałowska; Józef Korczak; Marzanna Hęś


    The research examined green tea ethanol extract, subjected to different purification processes with use of active carbon, bleaching earth, and mixture of acetone, acetic acid, water, with or without...

  15. Integrated separation process for isolation and purification of biosuccinic acid. (United States)

    Kurzrock, Tanja; Schallinger, Stefan; Weuster-Botz, Dirk


    Biotechnologically produced succinic acid has the potential to displace maleic acid and its uses. Therefore, it is of high interest for the chemical, pharmaceutical, and food industry.In addition to optimized production strains and fermentation processes, an efficient separation of succinic acid from the aqueous fermentation broth is indispensable to compete with the current petrochemical production of succinic acid. Isolation and purification of succinic acid from an Escherichia coli fermentation broth were studied with two amine-based reactive extraction systems: (i) trihexylamine in 1-octanol and (ii) diisooctylamine and dihexylamine in a mixture of 1-octanol and 1-hexanol. Back extraction of succinic acid from the organic phase was carried out using an aqueous trimethylamine solution. The trimethylammonium succinate generated after back extraction was split with an evaporation-based crystallization.The focus was on process integration, for example, reuse of the applied amines for extraction and back extraction. It was shown that the maximum trimethylamine concentration for back extraction should not exceed the stoichiometric amount (2 mol trimethylamine/mol the succinic acid in the organic phase) to ensure maximal extraction yields with the reused organic phase in subsequent extractions. Moreover, mixer-settler extraction and back extraction of succinic acid were scaled up from the milliliter- to the liter-scale making use of liquid–liquid centrifuges. The overall yield was 83.5% of the succinic acid from thefermentation supernatant. The final purity of the succinic acid crystals was 99.5%. Organic phase and amines can easily be recycled and reused. © 2011 American Institute of Chemical Engineers

  16. Research progress of novel adsorption processes in water purification:A review

    Institute of Scientific and Technical Information of China (English)


    As an effective, efficient, and economic approach for water purification, adsorbents and adsorption processes have been widely studied and applied in different aspects for a long time. In the recent years, a lot of novel adsorption processes have been developed for enhancing the efficiency of removing the organic and inorganic contaminants from water. This article reviews some new adsorbents and advanced adsorption methods that specialize in their compositions, structures, functions, and characteristics used in water treatment. The review emphasizes adsorption/catalytic oxidation process, adsorption/catalytic reduction process, adsorption coupled with redox process, biomimetic sorbent and its sorption behaviors of POPs, and modified adsorbents and their water purification efficiency.

  17. [Biocenotic dynamics of liquid sewage in the process of its biological purification at aeration stations]. (United States)

    Kalina, G P; Vinogradova, L A; Gipp, E K


    A study was made of biological purification of sewage at the aeration stations on the quantitative composition of the main indicator microbes--of bacteria of the coliform group and of the fecal coliform bacilli, enterococci, Proteus, and also pathogenic enterobacteria. There was found a difference in the behaviour of different species of Proteus, i.e. reduction in the process of purification in the numbers of Pr. mirabilis, and a sharp elevation of Pr. morganii content. There was noted an insignificant amount of Pr. vulgaris both before and after the biological purification. It was found that dynamics of biocenosis was influenced by air temperature at the time of collection of the samples. A possibility of reproduction of coliform bacilli serving as one of the factors of autopurification of sewage during the biological purification was confirmed.

  18. Process considerations related to the microencapsulation of plasmid DNA via ultrasonic atomization. (United States)

    Ho, Jenny; Wang, Huanting; Forde, Gareth M


    An effective means of facilitating DNA vaccine delivery to antigen presenting cells is through biodegradable microspheres. Microspheres offer distinct advantages over other delivery technologies by providing release of DNA vaccine in its bioactive form in a controlled fashion. In this study, biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres containing polyethylenimine (PEI) condensed plasmid DNA (pDNA) were prepared using a 40 kHz ultrasonic atomization system. Process synthesis parameters, which are important to the scale-up of microspheres that are suitable for nasal delivery (i.e., less than 20 microm), were studied. These parameters include polymer concentration; feed flowrate; volumetric ratio of polymer and pDNA-PEI (plasmid DNA-polyethylenimine) complexes; and nitrogen to phosphorous (N/P) ratio. PDNA encapsulation efficiencies were predominantly in the range 82-96%, and the mean sizes of the particle were between 6 and 15 microm. The ultrasonic synthesis method was shown to have excellent reproducibility. PEI affected morphology of the microspheres, as it induced the formation of porous particles that accelerate the release rate of pDNA. The PLGA microspheres displayed an in vitro release of pDNA of 95-99% within 30 days and demonstrated zero order release kinetics without an initial spike of pDNA. Agarose electrophoresis confirmed conservation of the supercoiled form of pDNA throughout the synthesis and in vitro release stages. It was concluded that ultrasonic atomization is an efficient technique to overcome the key obstacles in scaling-up the manufacture of encapsulated vaccine for clinical trials and ultimately, commercial applications.

  19. Materials measurement and accounting in an operating plutonium conversion and purification process. Phase I. Process modeling and simulation. [PUCSF code

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, C.C. Jr.; Ostenak, C.A.; Gutmacher, R.G.; Dayem, H.A.; Kern, E.A.


    A model of an operating conversion and purification process for the production of reactor-grade plutonium dioxide was developed as the first component in the design and evaluation of a nuclear materials measurement and accountability system. The model accurately simulates process operation and can be used to identify process problems and to predict the effect of process modifications.

  20. Decisional tool to assess current and future process robustness in an antibody purification facility. (United States)

    Stonier, Adam; Simaria, Ana Sofia; Smith, Martin; Farid, Suzanne S


    Increases in cell culture titers in existing facilities have prompted efforts to identify strategies that alleviate purification bottlenecks while controlling costs. This article describes the application of a database-driven dynamic simulation tool to identify optimal purification sizing strategies and visualize their robustness to future titer increases. The tool harnessed the benefits of MySQL to capture the process, business, and risk features of multiple purification options and better manage the large datasets required for uncertainty analysis and optimization. The database was linked to a discrete-event simulation engine so as to model the dynamic features of biopharmaceutical manufacture and impact of resource constraints. For a given titer, the tool performed brute force optimization so as to identify optimal purification sizing strategies that minimized the batch material cost while maintaining the schedule. The tool was applied to industrial case studies based on a platform monoclonal antibody purification process in a multisuite clinical scale manufacturing facility. The case studies assessed the robustness of optimal strategies to batch-to-batch titer variability and extended this to assess the long-term fit of the platform process as titers increase from 1 to 10 g/L, given a range of equipment sizes available to enable scale intensification efforts. Novel visualization plots consisting of multiple Pareto frontiers with tie-lines connecting the position of optimal configurations over a given titer range were constructed. These enabled rapid identification of robust purification configurations given titer fluctuations and the facility limit that the purification suites could handle in terms of the maximum titer and hence harvest load.

  1. Biochemistry in an Industrial Context: Methods of Protein Purification and Downstream Processing. (United States)

    Weathers, Pamela J.


    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)

  2. Extraction and purification methods in downstream processing of plant-based recombinant proteins. (United States)

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


    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.

  3. Single-step affinity purification of enzyme biotherapeutics: a platform methodology for accelerated process development. (United States)

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


    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).

  4. Establishment of a design space for biopharmaceutical purification processes using DoE. (United States)

    Amadeo, Ignacio; Mauro, Laura; Ortí, Eduardo; Forno, Guillermina


    Recent trends in the pharmaceutical sector are changing the way protein purification processes are designed and executed, moving from operating the process in a fixed point to allowing a permissible region in the operating space known as design space. This trend is driving product development to design quality into the manufacturing process (Quality by Design) and not to rely exclusively on testing quality in the product. A typical purification step has numerous operating parameters that can impact its performance. Therefore, optimization and robustness analysis in purification processes can be time-consuming since they are mainly grounded on experimental work. A valuable approach consists in the combination of an adequate risk analysis technique for selecting the relevant factors influencing process performance and the design of experiment methodology. The latter allows for many process variables which can be studied at the same time; thus, the number of tests will be reduced in comparison with the conventional approach based on trial and error. These multivariate studies permit a detailed exploration in the experimental range and lay the foundation of Quality by Design principles application. This article outlines a recommended sequence of activities toward the establishment of an expanded design space for a purification process.

  5. Monitoring bacterial processes by Fourier transform infrared spectroscopy : Helicobacter pylori drug inactivation and plasmid bioproduction in recombinant Escherichia coli cultures


    Scholz, Teresa; Lopes, Vitor V.; Calado, Cecília R. C.


    Fourier transform infrared (FTIR) spectroscopy is evaluated as a tool to monitor two bacterial processes: strain discrimination and drug inactivation studies with the gastric pathogen Helicobacter pylori and the plasmid production process based on high-density cultures of recombinant Escherichia coli. Results show, that after evaluation of different incubation conditions of H.pylori with the drug model, the application of principal component analysis to the FTIR spectra assembles the samples ...

  6. Current trends in separation of plasmid DNA vaccines: a review. (United States)

    Ghanem, Ashraf; Healey, Robert; Adly, Frady G


    Plasmid DNA (pDNA)-based vaccines offer more rapid avenues for development and production if compared to those of conventional virus-based vaccines. They do not rely on time- or labour-intensive cell culture processes and allow greater flexibility in shipping and storage. Stimulating antibodies and cell-mediated components of the immune system are considered as some of the major advantages associated with the use of pDNA vaccines. This review summarizes the current trends in the purification of pDNA vaccines for practical and analytical applications. Special attention is paid to chromatographic techniques aimed at reducing the steps of final purification, post primary isolation and intermediate recovery, in order to reduce the number of steps necessary to reach a purified end product from the crude plasmid.

  7. Purification of precursors of Yb3+-doped YLF crystals by solvent extraction and electrochemical processing (United States)

    Boncher, William L.; Judge, Elizabeth; Sansinena, Jose-Maria; Dirmyer, Matthew R.; Hehlen, Markus P.


    Optical refrigeration by laser irradiation of YLiF4:Yb3+ (YLF:Yb) crystals has been shown to be strongly deteriorated by impurities, which absorb energy at the laser wavelength, and relax non-radiatively, negating cooling produced from anti-Stokes fluorescence. We aim to increase the efficiency of optical refrigeration through materials purification. We start with the purest sources commercially available and process them in a cleanroom environment. Our method proceeds through electrochemical purification, separating out the transition metal impurities by their redox potentials, and can be scaled up to produce the amounts of material needed for crystal growth.

  8. Study of Mass Transfer in Gas Blowing Processes for Silicon Purification (United States)

    Altenberend, Jochen; Chichignoud, Guy; Delannoy, Yves


    Boron removal processes are crucial to make the metallurgical route for silicon refining for solar cells competitive and thus reduce the cost of solar energy. The rate-limiting step was investigated in silicon purification processes for boron removal based on gas blowing, to gain better understanding that should help to improve the design of such processes. We calculate the boron concentration in the off-gas that corresponds to chemical equilibrium between the gas and silicon. The real concentration in the off-gas ranges between 9 and 30 pct of this theoretical value calculated using Gibbs free energies reported in literature. Purification experiments with varying temperature and hydrogen concentration were done to evaluate whether limited chemical reaction rates induce deviation from chemical equilibrium. The experiments and data from literature show that the chemical reactions at the surface of the melt are close to chemical equilibrium, thus the purification rate is limited by mass transfer in the gas phase near the interface. Based on this, recommendations for the design of a gas blowing purification process are given.

  9. Downstream Processing Technologies/Capturing and Final Purification : Opportunities for Innovation, Change, and Improvement. A Review of Downstream Processing Developments in Protein Purification. (United States)

    Singh, Nripen; Herzer, Sibylle


    Increased pressure on upstream processes to maximize productivity has been crowned with great success, although at the cost of shifting the bottleneck to purification. As drivers were economical, focus is on now on debottlenecking downstream processes as the main drivers of high manufacturing cost. Devising a holistically efficient and economical process remains a key challenge. Traditional and emerging protein purification strategies with particular emphasis on methodologies implemented for the production of recombinant proteins of biopharmaceutical importance are reviewed. The breadth of innovation is addressed, as well as the challenges the industry faces today, with an eye to remaining impartial, fair, and balanced. In addition, the scope encompasses both chromatographic and non-chromatographic separations directed at the purification of proteins, with a strong emphasis on antibodies. Complete solutions such as integrated USP/DSP strategies (i.e., continuous processing) are discussed as well as gains in data quantity and quality arising from automation and high-throughput screening (HTS). Best practices and advantages through design of experiments (DOE) to access a complex design space such as multi-modal chromatography are reviewed with an outlook on potential future trends. A discussion of single-use technology, its impact and opportunities for further growth, and the exciting developments in modeling and simulation of DSP rounds out the overview. Lastly, emerging trends such as 3D printing and nanotechnology are covered. Graphical Abstract Workflow of high-throughput screening, design of experiments, and high-throughput analytics to understand design space and design space boundaries quickly. (Reproduced with permission from Gregory Barker, Process Development, Bristol-Myers Squibb).

  10. The importance of the ammonia purification process in ammonia-water absorption systems

    Energy Technology Data Exchange (ETDEWEB)

    Fernandez-Seara, Jose [Area de Maquinas y Motores Termicos, Escuela Tecnica Superior de Ingenieros Industriales, Universidad de Vigo, Campus Lagoas-Marcosende No. 9, 36200 Vigo (Spain)]. E-mail:; Sieres, Jaime [Area de Maquinas y Motores Termicos, Escuela Tecnica Superior de Ingenieros Industriales, Universidad de Vigo, Campus Lagoas-Marcosende No. 9, 36200 Vigo (Spain)


    Practical experience in working with ammonia-water absorption systems shows that the ammonia purification process is a crucial issue in order to obtain an efficient and reliable system. In this paper, the detrimental effects of the residual water content in the vapour refrigerant are described and quantified based on the system design variables that determine the effectiveness of the purification process. The study has been performed considering a single stage system with a distillation column with complete condensation. The ammonia purification effectiveness of the column is analysed in terms of the efficiencies in the stripping and rectifying sections and the reflux ratio. By varying the efficiencies from 0 to 1, systems with neither the rectifying nor stripping section, with either the rectifying or stripping section, or with both sections can be considered. The impact of the ammonia purification process on the absorption system performance is studied based on the column efficiencies and reflux ratio; and its effects on refrigerant concentration, system COP, system pressures and main system mass flow rates and concentrations are analysed. When the highest efficiency rectifying sections are used a combination of generation temperature and reflux ratio which leads to optimum COP values is found. The analysis covers different operating conditions with air and water cooled systems from refrigeration to air conditioning applications by changing the evaporation temperature. The importance of rectification in each kind of application is evaluated.

  11. Oil quality of passion fruit seeds subjected to a pulp-waste purification process


    Suelen Alvarenga Regis; Eder Dutra de Resende; Rosemar Antoniassi


    Passion fruit seeds must be clean and dry before the extraction processing to obtain high-quality oil for edible and cosmetic purposes. This research studies the viability of a cleaning process of seeds by evaluating the oil quality. The research examined 2 maturation stages of the fruit and one purification process of the seeds, compared to the control. The oil quality was evaluated by fatty acid composition, acidity, peroxide value and oxidative stability. The pulp waste suffered a thermal ...

  12. Optimization of a recombinant human growth hormone purification process using quality by design. (United States)

    Ortiz-Enriquez, Carolina; Romero-Díaz, Alexis de Jesús; Hernández-Moreno, Ana V; Cueto-Rojas, Hugo F; Miranda-Hernández, Mariana P; López-Morales, Carlos A; Pérez, Néstor O; Salazar-Ceballos, Rodolfo; Cruz-García, Norberto; Flores-Ortiz, Luis F; Medina-Rivero, Emilio


    This work describes a strategy to optimize a downstream processing of a recombinant human growth hormone (rhGH) by incorporating a quality by design approach toward meeting higher quality specifications. The optimized process minimized the presence of impurities and degradation by-products during manufacturing by the establishment of in-process controls. Capillary zone electrophoresis, reverse phase, and size-exclusion chromatographies were used as analytical techniques to establish new critical process parameters for the solubilization, capture, and intermediate purification steps aiming to maintain rhGH quality by complying with pharmacopeial specifications. The results indicated that the implemented improvements in the process allowed the optimization of the specific recovery and purification of rhGH without compromising its quality. In addition, this optimization facilitated the stringent removal of the remaining impurities in further polishing stages, as demonstrated by the analysis of the obtained active pharmaceutical ingredient.


    Directory of Open Access Journals (Sweden)

    L. Pilinevich


    Full Text Available The paper presents results of the investigations on the water purification process with the help of photocatalysis using the photocatalyst which is developed on the basis of porous titanium with the layer of nanoparticle layer of titanium dioxide and an experimental plant. The investigations results have shown high efficiency of the developed photocatalytic materials and a water purification plants

  14. Processing and characterization of porous electrochemical cells for flue gas purification

    DEFF Research Database (Denmark)

    He, Zeming; Andersen, Kjeld Bøhm; Keel, Li


    In the present work, porous electrode materials lanthanum strontium manganate (LSM)-gadolinium-doped cerium oxide (CGO) and electrochemical cells LSM-CGO + CGO were fabricated via the processes of slurry preparation, tape casting and lamination, and sintering. Graphite, wheat starch, and polyamide......, flat, and crack-free electrochemical cells were successfully achieved using the present ceramic processing route. The produced cells could potentially be used for flue gas purification....

  15. [Chromatographic separation of plasmid DNA by anion-exchange cryogel]. (United States)

    Guo, Yantao; Shen, Shaochuan; Yun, Junxian; Yao, Kejian


    Plasmid DNA (pDNA) is used as an important vector for gene therapy, and its wide application is restricted by the purity and yield. To obtain high-purity pDNA, a chromatographic method based on anion-exchange supermacroporous cryogel was explored. The anion-exchange cryogel was prepared by grafting diethylaminoethyl-dextran to the epoxide groups of polyacrylamide-based matrix and pUC19 plasmid was used as a target to test the method. The plasmid was transferred into Escherichia coli DH5alpha, cultivated, harvested and lysed. The obtained culture was centrifuged and the supernatant was used as the plasmid feedstock, which was loaded into the anion-exchange cryogel bed for chromatographic separation. By optimizing the pH of running buffer and the elution conditions, high-purity pDNA was obtained by elution with 0.5 mol/L sodium chloride solution at pH 6.6. Compared to the traditional methods for purification of pDNA, animal source enzymes and toxic reagents were not involved in the present separation process, ensuring the safety of both the purification operations and the obtained pDNA.

  16. High-throughput process development of purification alternatives for the protein avidin. (United States)

    Diederich, Patrick; Hoffmann, Marc; Hubbuch, Jürgen


    With an increased number of applications in the field of the avidin-biotin technology, the resulting demand for highly-purified protein avidin has drawn our attention to the purification process of avidin that naturally occurs in chicken egg white. The high-throughput process development (HTPD) methodology was exploited, in order to evaluate purification process alternatives to commonly used ion-exchange chromatography. In a high-throughput format, process parameters for aqueous two-phase extraction, selective precipitation with salts and polyethylene glycol, and hydrophobic interaction and mixed-mode column chromatography experiments were performed. The HTPD strategy was complemented by a high-throughput tandem high-performance liquid chromatography assay for protein quantification. Suitable conditions for the separation of avidin from the major impurities ovalbumin, ovomucoid, ovotransferrin, and lysozyme were identified in the screening experiments. By combination of polyethylene glycol precipitation with subsequent resolubilization and separation in a polyethylene glycol/sulfate/sodium chloride two-phase system an avidin purity of 77% was obtained with a yield >90% while at the same time achieving a significant reduction of the process volume. The two-phase extraction and precipitation results were largely confirmed in larger scale with scale-up factors of 230 and 133, respectively. Seamless processing of the avidin enriched bottom phase was found feasible by using mixed-mode chromatography. By gradient elution a final avidin purity of at least 97% and yield >90% was obtained in the elution pool. The presented identification of a new and beneficial alternative for the purification of the high value protein thus represents a successful implementation of HTPD for an industrially relevant purification task. © 2015 American Institute of Chemical Engineers.

  17. Plasmid Biopharmaceuticals. (United States)

    Prazeres, Duarte Miguel F; Monteiro, Gabriel A


    Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its mission to produce pharmaceutical proteins, antibodies, vaccines, industrial enzymes, and molecular diagnostics, to name a few key products. Furthermore, plasmids have gradually stepped up in the past 20 years as useful biopharmaceuticals in the context of gene therapy and DNA vaccination interventions. This review provides a concise coverage of the scientific progress that has been made since the emergence of what are called today plasmid biopharmaceuticals. The most relevant topics are discussed to provide researchers with an updated overview of the field. A brief outline of the initial breakthroughs and innovations is followed by a discussion of the motivation behind the medical uses of plasmids in the context of therapeutic and prophylactic interventions. The molecular characteristics and rationale underlying the design of plasmid vectors as gene transfer agents are described and a description of the most important methods used to deliver plasmid biopharmaceuticals in vivo (gene gun, electroporation, cationic lipids and polymers, and micro- and nanoparticles) is provided. The major safety issues (integration and autoimmunity) surrounding the use of plasmid biopharmaceuticals is discussed next. Aspects related to the large-scale manufacturing are also covered, and reference is made to the plasmid products that have received marketing authorization as of today.

  18. A new, integrated, continuous purification process template for monoclonal antibodies: Process modeling and cost of goods studies. (United States)

    Xenopoulos, Alex


    An evolving biopharmaceutical industry requires advancements in biomanufacturing that offer increased productivity and improved economics without sacrificing process robustness. Accordingly, we have developed a new monoclonal antibody purification template comprised of flocculation-based clarification, capture by continuous multi-column protein A chromatography and flow-through polishing. The new process offers a robust, single-use manufacturing solution while significantly reducing overall cost of goods. Modeling studies verify that the individual clarification, capture and polishing solutions offer significant advantages as stand-alone unit operations. These technologies were also designed to be integrated into a continuous purification template. Process modeling studies have been used to highlight both cost and operational advantages of the new process template. Depending on scale, savings of more than 20% and 60% were seen for commercial and clinical operation, respectively. Integrating the technologies into a continuous process consistently offered additional cost advantages. During template development, process modeling was instrumental in highlighting the importance of identifying technologies that provided high product yield and purification factors. Additionally, high product concentration and eliminating the need for intermediate product dilution emerged as important considerations for newly developed unit operations. Combining experimental work with insights from modeling can significantly improve the outcome of product and process development.

  19. Performance Assessment of SOFC Systems Integrated with Bio-Ethanol Production and Purification Processes

    Directory of Open Access Journals (Sweden)

    Sumittra Charojrochkul


    Full Text Available The overall electrical efficiencies of the integrated systems of solid oxide fuel cell (SOFC and bio-ethanol production with purification processes at different heat integration levels were investigated. The simulation studies were based on the condition with zero net energy. It was found that the most suitable operating voltage is between 0.7 and 0.85 V and the operating temperature is in the range from 973 to 1173 K. For the effect of percent ethanol recovery, the optimum percent ethanol recovery is at 95%. The most efficient case is the system with full heat integration between SOFC and bio-ethanol production and purification processes with biogas reformed for producing extra hydrogen feed for SOFC which has the overall electrical efficiency = 36.17%. However more equipment such as reformer and heat exchangers are required and this leads to increased investment cost.

  20. An integrated process for purification of lysozyme, ovalbumin, and ovomucoid from hen egg white. (United States)

    Roy, Ipsita; Rao, M V S; Gupta, Munishwar N


    This article describes an integrated process for simultaneous purification of lysozyme, ovalbumin, and ovomucoid from hen egg white. The crude egg white extract was passed through a cation exchanger Streamline trade mark SP and the bound lysozyme was eluted with 5% ammonium carbonate, pH 9.0, containing 1 M NaCl after elution of avidin. This partially purified lysozyme was further purified 639-fold on dye-linked cellulose beads. Ovalbumin and ovomucoid did not bind to Streamline SP. Ovalbumin could be precipitated from this unbound fraction by 5% trichloroacetic acid, and ovomucoid was removed from the supernatant by precipitation with ethanol. The yields of lysozyme, ovomucoid, and ovalbumin were 77, 94, and 98%, respectively. All the purified proteins showed single bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis. All the steps are easily scalable, and the process described here can be used for large-scale simultaneous purification of these proteins in the pure form.

  1. Supercritical nitrogen processing for the purification of reactive porous materials. (United States)

    Stadie, Nicholas P; Callini, Elsa; Mauron, Philippe; Borgschulte, Andreas; Züttel, Andreas


    Supercritical fluid extraction and drying methods are well established in numerous applications for the synthesis and processing of porous materials. Herein, nitrogen is presented as a novel supercritical drying fluid for specialized applications such as in the processing of reactive porous materials, where carbon dioxide and other fluids are not appropriate due to their higher chemical reactivity. Nitrogen exhibits similar physical properties in the near-critical region of its phase diagram as compared to carbon dioxide: a widely tunable density up to ~1 g ml(-1), modest critical pressure (3.4 MPa), and small molecular diameter of ~3.6 Å. The key to achieving a high solvation power of nitrogen is to apply a processing temperature in the range of 80-150 K, where the density of nitrogen is an order of magnitude higher than at similar pressures near ambient temperature. The detailed solvation properties of nitrogen, and especially its selectivity, across a wide range of common target species of extraction still require further investigation. Herein we describe a protocol for the supercritical nitrogen processing of porous magnesium borohydride.

  2. Characterization of a biopharmaceutical protein and evaluation of its purification process using automated capillary Western blot. (United States)

    Xu, Dong; Mane, Sarthak; Sosic, Zoran


    This paper describes the application of an automated size-based capillary Western blot system (Sally instrument) from ProteinSimple, Inc., for biopharmaceutical fusion-Fc protein characterization and evaluation of its purification process. The fusion-Fc protein column purification from an excess of single chain Fc polypeptide and removal of an enzyme coexpressed for protein maturation have been demonstrated using an automated capillary Western system. The clearance of a selected host cell protein (HCP) present in cell culture of fusion-Fc protein was also quantitatively monitored throughout the protein purification process. Additionally, the low levels of fusion-Fc product-related impurities detected by traditional slab gel Western blot were confirmed by the automated capillary Western system. Compared to the manual approach, the automated capillary Western blot provides the advantages of ease of operation, higher sample throughput, greater linearity range, and higher precision for protein quantitation. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. An improved strategy for easy process monitoring and advanced purification of recombinant proteins. (United States)

    Miladi, Baligh; Dridi, Cyrine; El Marjou, Ahmed; Boeuf, Guilhem; Bouallagui, Hassib; Dufour, Florence; Di Martino, Patrick; Elm'selmi, Abdellatif


    In this work, a multifunctional expression cassette, termed Multitags, combining different and complementary functionalities, was designed and used to monitor the expression and the purification of two model proteins (Pfu DNA polymerase and Myosin-VIIa- and Rab-Interracting protein : MyRIP). Multitags contains two affinity purification tags, a polyhistidine sequence (10× His) and the streptavidin-binding peptide (SBP) and as a marker tag the heme-binding domain of rat cytochrome b5 followed by the TEV cleavage site. Using the Multitags as fusion partner, more than 90 % of both fusion proteins were produced in soluble form when expressed in Escherichia coli KRX. In addition, high purity (99 %) of recombinant proteins was achieved after two consecutive affinity purification steps. The expression cassette also demonstrated an accurate monitoring capability comparable to that of a dual recognition-based method. The choice of the SBP tag was considered as an integral process that included a method for tag removal. Thus, an immobilized TEV protease fixed on streptavidin-agarose matrix was used for the cleavage of fusion proteins. After digestion, both unprocessed fusion proteins and Multitags were retained on the proteolytic column via their SBP sequence, allowing cleavage and recovery of target proteins on one step. This combined approach may accelerate the development of optimized production processes, while insuring high product quality and a low production cost.

  4. Construction of High Expression Plasmid of Human Augmenter of Liver Regeneration( hALR), Expression and Purification of hALR

    Institute of Scientific and Technical Information of China (English)


    Experimental evidence has been presented to suggest that the human augmenter of liver regeneration (hALR)serves as a hepatotrophic growth factor during liver regeneration and as a generalized growth factor during pancreas transplant/regeneration. A prokaryotic expression plasmid, pRSET/6his-c-myc-hALR was constructed, by cloning synthesized hALR cDNA into pRSET/6his-c-myc that was improved on the basis of pRSET B by the group. As a result, the protein was highly expressed in E. coli BL21. The recombinant hALR was over 60% of the total protein in E. coli. Its validity was confirmed by meansof Western Blotting. The protein was purified by Ni-NTA affinity chromatography and this FAD-dependent sulfhydryl oxidase activity was measured.

  5. Purification process of natural graphite as anode for Li-ion batteries: chemical versus thermal (United States)

    Zaghib, K.; Song, X.; Guerfi, A.; Rioux, R.; Kinoshita, K.

    The intercalation of Li ions in natural graphite that was purified by chemical and thermal processes was investigated. A new chemical process was developed that involved a mixed aqueous solution containing 30% H 2SO 4 and 30% NH xF y heated to 90 °C. The results of this process are compared to those obtained by heating the natural graphite from 1500 to 2400 °C in an inert environment (thermal process). The first-cycle coulombic efficiency of the purified natural graphite obtained by the chemical process is 91 and 84% after the thermal process at 2400 °C. Grinding the natural graphite before or after purification had no significant effect on electrochemical performance at low currents. However, grinding to a very small particle size before purification permitted optimization of the size distribution of the particles, which gives rise to a more homogenous electrode. The impurities in the graphite play a role as microabrasion agents during grinding which enhances its hardness and improves its mechanical properties. Grinding also modifies the particle morphology from a 2- to a 3-D structure (similar in shape to a potato). This potato-shaped natural graphite shows high reversible capacity at high current densities (about 90% at 1 C rate). Our analysis suggests that thermal processing is considerably more expensive than the chemical process to obtain purified natural graphite.

  6. Sample displacement chromatography of plasmid DNA isoforms. (United States)

    Černigoj, Urh; Martinuč, Urška; Cardoso, Sara; Sekirnik, Rok; Krajnc, Nika Lendero; Štrancar, Aleš


    Sample displacement chromatography (SDC) is a chromatographic technique that utilises different relative binding affinities of components in a sample mixture and has been widely studied in the context of peptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) or linear isoform. Since displacement is more efficient when mass transfer between stationary and mobile chromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobicities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoform separation was shown to be dependent on column selectivity for individual isoform, column efficiency and on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative mode elution often operate in parallel, therefore negative mode elution additionally influences the efficiency of the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNA homogeneity and a dynamic binding capacity of over 1mg/mL at a relatively low concentration of AS. SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes, and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used. This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, which is compatible with continuous, multicolumn chromatography systems, and could therefore be used to increase productivity of pDNA production in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Fast and scalable purification of a therapeutic full-length antibody based on process crystallization. (United States)

    Smejkal, Benjamin; Agrawal, Neeraj J; Helk, Bernhard; Schulz, Henk; Giffard, Marion; Mechelke, Matthias; Ortner, Franziska; Heckmeier, Philipp; Trout, Bernhardt L; Hekmat, Dariusch


    The potential of process crystallization for purification of a therapeutic monoclonal IgG1 antibody was studied. The purified antibody was crystallized in non-agitated micro-batch experiments for the first time. A direct crystallization from clarified CHO cell culture harvest was inhibited by high salt concentrations. The salt concentration of the harvest was reduced by a simple pretreatment step. The crystallization process from pretreated harvest was successfully transferred to stirred tanks and scaled-up from the mL-scale to the 1 L-scale for the first time. The crystallization yield after 24 h was 88-90%. A high purity of 98.5% was reached after a single recrystallization step. A 17-fold host cell protein reduction was achieved and DNA content was reduced below the detection limit. High biological activity of the therapeutic antibody was maintained during the crystallization, dissolving, and recrystallization steps. Crystallization was also performed with impure solutions from intermediate steps of a standard monoclonal antibody purification process. It was shown that process crystallization has a strong potential to replace Protein A chromatography. Fast dissolution of the crystals was possible. Furthermore, it was shown that crystallization can be used as a concentrating step and can replace several ultra-/diafiltration steps. Molecular modeling suggested that a negative electrostatic region with interspersed exposed hydrophobic residues on the Fv domain of this antibody is responsible for the high crystallization propensity. As a result, process crystallization, following the identification of highly crystallizable antibodies using molecular modeling tools, can be recognized as an efficient, scalable, fast, and inexpensive alternative to key steps of a standard purification process for therapeutic antibodies.

  8. A flow-through chromatography process for influenza A and B virus purification. (United States)

    Weigel, Thomas; Solomaier, Thomas; Peuker, Alessa; Pathapati, Trinath; Wolff, Michael W; Reichl, Udo


    Vaccination is still the most efficient measure to protect against influenza virus infections. Besides the seasonal wave of influenza, pandemic outbreaks of bird or swine flu represent a high threat to human population. With the establishment of cell culture-based processes, there is a growing demand for robust, economic and efficient downstream processes for influenza virus purification. This study focused on the development of an economic flow-through chromatographic process avoiding virus strain sensitive capture steps. Therefore, a three-step process consisting of anion exchange chromatography (AEC), Benzonase(®) treatment, and size exclusion chromatography with a ligand-activated core (LCC) was established, and tested for purification of two influenza A virus strains and one influenza B virus strain. The process resulted in high virus yields (≥68%) with protein contamination levels fulfilling requirements of the European Pharmacopeia for production of influenza vaccines for human use. DNA was depleted by ≥98.7% for all strains. The measured DNA concentrations per dose were close to the required limits of 10ng DNA per dose set by the European Pharmacopeia. In addition, the added Benzonase(®) could be successfully removed from the product fraction. Overall, the presented downstream process could potentially represent a simple, robust and economic platform technology for production of cell culture-derived influenza vaccines. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR. (United States)

    Plotka, Magdalena; Wozniak, Mateusz; Kaczorowski, Tadeusz


    Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a

  10. TiO2-Based Photocatalytic Process for Purification of Polluted Water: Bridging Fundamentals to Applications

    Directory of Open Access Journals (Sweden)

    Chuan Wang


    Full Text Available Recent years have witnessed a rapid accumulation of investigations on TiO2-based photocatalysis, which poses as a greatly promising advanced oxidation technology for water purification. As the ability of this advanced oxidation process is well demonstrated in lab and pilot scales to decompose numerous recalcitrant organic compounds and microorganism as well in water, further overpass of the hurdles that stand before the real application has become increasingly important. This review focuses on the fundamentals that govern the actual water purification process, including the fabrication of engineered TiO2-based photocatalysts, process optimization, reactor design, and economic consideration. The state of the art of photocatalyst preparation, strategies for process optimization, and reactor design determines the enhanced separation of photo-excited electron-hole (e-h pairs on the TiO2 surface. For the process optimization, the kinetic analysis including the rate-determining steps is in need. For large-scale application of the TiO2-based photocatalysis, economics is vital to balance the fundamentals and the applied factors. The fundamentals in this review are addressed from the perspective of a bridge to the real applications. This review would bring valuably alternative paradigm to the scientists and engineers for their associated research and development activities with an attempt to push the TiO2-based photocatalysis towards industrially feasible applications.

  11. In situ NIR spectroscopy monitoring of plasmid production processes effect of producing strain, medium composition and the cultivation strategy


    Lopes, Marta B.; Gonçalves, Geisa A. L.; Felício-Silva, Daniel; Prather, Kristala L. J.; Monteiro, Gabriel; Prazeres, Duarte M. F.; Calado, Cecília Ribeiro da Cruz


    BACKGROUNDWhile the pharmaceutical industry keeps an eye on plasmid DNA production for new generation gene therapies, real-time monitoring techniques for plasmid bioproduction are as yet unavailable. This work shows the possibility of in situ monitoring of plasmid production in Escherichia coli cultures using a near infrared (NIR) fiber optic probe. RESULTSPartial least squares (PLS) regression models based on the NIR spectra were developed for predicting bioprocess critical variables su...

  12. A High-Throughput Process for the Solid-Phase Purification of Synthetic DNA Sequences. (United States)

    Grajkowski, Andrzej; Cieślak, Jacek; Beaucage, Serge L


    An efficient process for the purification of synthetic phosphorothioate and native DNA sequences is presented. The process is based on the use of an aminopropylated silica gel support functionalized with aminooxyalkyl functions to enable capture of DNA sequences through an oximation reaction with the keto function of a linker conjugated to the 5'-terminus of DNA sequences. Deoxyribonucleoside phosphoramidites carrying this linker, as a 5'-hydroxyl protecting group, have been synthesized for incorporation into DNA sequences during the last coupling step of a standard solid-phase synthesis protocol executed on a controlled pore glass (CPG) support. Solid-phase capture of the nucleobase- and phosphate-deprotected DNA sequences released from the CPG support is demonstrated to proceed near quantitatively. Shorter than full-length DNA sequences are first washed away from the capture support; the solid-phase purified DNA sequences are then released from this support upon reaction with tetra-n-butylammonium fluoride in dry dimethylsulfoxide (DMSO) and precipitated in tetrahydrofuran (THF). The purity of solid-phase-purified DNA sequences exceeds 98%. The simulated high-throughput and scalability features of the solid-phase purification process are demonstrated without sacrificing purity of the DNA sequences. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.


    Directory of Open Access Journals (Sweden)

    Dechechi E.C.


    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.

  14. Research and Development of Crystal Purification for Product of Uranium Crystallization Process

    Energy Technology Data Exchange (ETDEWEB)

    Yano, K. [Japan Atomic Energy Agency - JAEA (Japan)


    Uranium crystallization has been developed as a part of advanced aqueous reprocessing for FBR spent fuel. Although the purity of uranyl nitrate hexahydrate (UNH) crystal from the crystallization process is supposed to meet a specification of FBR blanket fuel, an improvement of its purity is able to reduce the cost of fuel fabrication and storage (in case interim storage of recovered uranium is required). In this work, UNH crystal purification was developed as additional process after crystallization. Contamination of the crystal is caused by mother solution and solid state impurities. They are inseparable by washing and filtration. Mother solution on the surface of UNH crystals is removable by washing, but it is difficult to remove that in an obstructed part of crystalline aggregate by washing. Major elements of solid state impurities are cesium and barium. Cesium precipitates with tetravalent plutonium as a double nitrate, Cs{sub 2}Pu(NO{sub 3}){sub 6}. Barium crystallizes as Ba(NO{sub 3}){sub 2} because of its low solubility in nitric acid solution. It is difficult to separate their particle from UNH crystal by solid-liquid separation such as simple filtration. As a kind of crystal purification, there are some methods using sweating. Sweating is a phenomenon that a crystal melts partly below its melting point and it is caused by depression of freezing point due to impurity. It is considerably applicable for removal of mother solution. Concerning the solid state impurities, which has higher melting point than that of UNH crystal, it is supposed that they are separable by melting UNH crystal and filtration. The behaviors of impurities and applicability of sweating and melting-filtration operations to the purification for UNH crystal were investigated experimentally on a beaker and an engineering scale. With regard to behaviors of impurities, the conditions of cesium and barium precipitation were surveyed and it was clarified that there were most impurities on the


    Directory of Open Access Journals (Sweden)

    R. T. Zanardo

    Full Text Available Abstract The main virulence factor of Streptococcus pneumoniae is the capsular polysaccharide (PS, which is the antigen of all current vaccines that are prepared with PS purified from serotypes prevalent in the population. In this work, three purification strategies were evaluated and a new process was developed for purification of serotype 14 PS (PS14, responsible for 39.8% of diseases in children of 0-6 years old in Brazil. The developed method consists of cell separation by tangential microfiltration, concentration of the microfiltrate by tangential ultrafiltration (50 kDa, diafiltration in the presence of sodium dodecyl sulfate using a 30 kDa ultrafiltration membrane, precipitation with 5% trichloroacetic acid, precipitation with 20% and 60% ethanol, and anion exchange chromatography. The required purity regarding nucleic acids (≤ 2% and proteins (≤ 3% was achieved, resulting in a relative purity of 439 mg PS14/mg nucleic acids and 146 mg PS14/mg proteins. The final polysaccharide recovery was 65%, which is higher than the recovery of the majority of processes described in the literature.

  16. 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


    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.

  17. Persistence Mechanisms of Conjugative Plasmids

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Hansen, Lars H.; Sørensen, Søren Johannes


    Are plasmids selfish parasitic DNA molecules or an integrated part of the bacterial genome? This chapter reviews the current understanding of the persistence mechanisms of conjugative plasmids harbored by bacterial cells and populations. The diversity and intricacy of mechanisms affecting...... the successful propagation and long-term continued existence of these extra-chromosomal elements is extensive. Apart from the accessory genetic elements that may provide plasmid-harboring cells a selective advantage, special focus is placed on the mechanisms conjugative plasmids employ to ensure their stable...... maintenance in the host cell. These importantly include the ability to self-mobilize in a process termed conjugative transfer, which may occur across species barriers. Other plasmid stabilizing mechanisms include the multimer resolution system, active partitioning, and post-segregational-killing of plasmid...

  18. Model-based design space determination of peptide chromatographic purification processes. (United States)

    Gétaz, David; Butté, Alessandro; Morbidelli, Massimo


    Operating a chemical process at fixed operating conditions often leads to suboptimal process performances. It is important in fact to be able to vary the process operating conditions depending upon possible changes in feed composition, products requirements or economics. This flexibility in the manufacturing process was facilitated by the publication of the PAT initiative from the U.S. FDA [1]. In this work, the implementation of Quality-by-design in the development of a chromatographic purification process is discussed. A procedure to determine the design space of the process using chromatographic modeling is presented. Moreover, the risk of batch failure and the critical process parameters (CPP) are assessed by modeling. The ideal cut strategy is adopted and therefore only yield and productivity are considered as critical quality attributes (CQA). The general trends in CQA variations within the design space are discussed. The effect of process disturbances is also considered. It is shown that process disturbances significantly decrease the design space and that only simultaneous and specific changes in multiple process parameters (i.e. critical process parameters (CPP) lead to batch failure. The reliability of the obtained results is proven by comparing the model predictions to suitable experimental data. The case study presented in this work proves the reliability of process development using a model-based approach.

  19. Heterogeneous glycoform separation by process chromatography: I: Monomer purification and characterization. (United States)

    Li, Yi; Xu, Xuankuo; Shupe, Alan; Yang, Rong; Bai, Kevin; Das, Tapan; Borys, Michael C; Li, Zheng Jian


    Fc fusion proteins with high and low sialylation were purified and separated by preparative ion-exchange and hydrophobic interaction chromatography. Heterogeneity in sialylation and glycosylation led to variation in surface charge and hydrophobicity, and resulted in multiple distinct glycoform populations in response to various purification conditions. Monomer with high sialic acid content has higher surface charge and adsorbs stronger to ion-exchange resin, while the less sialylated monomer interacts more favorably with hydrophobic resin. Extensive biophysical characterization was carried out for purified monomers at different level of sialylation. In general, different monomeric glycoforms have different surface charge and hydrophobicity, different thermal stability, and different aggregation propensity. The surface charge corresponds well with sialic acid content, as evidenced by electrophoresis, N-link domain analysis, and zeta potential results. The sialylation also contributes to minor modification of protein size, molecular mass and tertiary structure. Notably, fluorescence emission spectra and thermal transition became less distinguishable when the monomers containing low and high sialic acid were prepared in high ionic strength solution. Such finding reiterates the fact that the electrostatic forces, which are largely dependent on sialic acid content of protein, plays a dominant role in many intra- and inter-molecular interactions. Overall, the characterization data agreed well with separation behaviors and provided valuable insight to control of glycoform profile in purification process. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Comparison of purification processes of natural gas obtained from three different regions in the world

    Institute of Scientific and Technical Information of China (English)

    Tansel Koyun; Mehmet Kunduz; Hakan F. Oztop; Ibrahim Ucgul


    Natural gases obtained from different regions in the world as Scholen-Germany,Saudi Arabia and Iran were purified with a package code and the obtained results were compared in this study.For purification process,both natural gases flowing in a vertical pipe and monoethanolamine (MEA) flowing as a film from the internal surface of a pipe were examined together.Both fluids were flown in a vertical and laminar regime.Binary diffusion coefficients,Schmidt numbers (Sc) and dynamical viscosities were calculated individually for three types of natural gases.It is demonstrated that the chemical absorption method by MEA process is the most appropriate method at high Damk(o)hler (Da) numbers particularly for natural gases containing high concentrations of CO2 and H2S.

  1. Purification technology of flue gas from remelting process of aluminum alloy tailings

    Institute of Scientific and Technical Information of China (English)

    李彩亭; 曾光明; 魏先勋; 袁兴中; 王丽平


    Through a practical example of treatment of the flue gas from the remelting process of aluminum alloy tailings, the design and calculation method of exhaust hood, as well as the principles and the equipments of dust removal, smoke abatement and harmful gas elimination were studied. Combination of centrifugal and wet dust removal can purify the dust high efficiently. The carbon black and harmful gases in the flue gas can be removed by adding a small quantity of activator to the absorption solution. The application results are that the dedusting efficiency is 97.43%, Cl2 control efficiency is 88.03%, the exhaust fume blackness is lower than Ringelman number I, and the purification device resistance is 1126Pa.

  2. Physico-chemical Modification of the Fibrous Filter Nozzles for Purification Processes of Water and Air (United States)

    Bordunov, S. V.; Galtseva, O. V.; Natalinova, N. M.; Rogachev, A. A.; Zhang, Ruizhi


    A set of experiments to study physical and chemical modification of the surface of fibers is conducted to expand the area of their application for purification of water, gas and air (including that in conditions of space). The possibility of modification of filter nozzles in the process of fiber formation by particles of coal of BAU type, copper sulfide and silver chloride is experimentally shown. The fraction of the copper sulfide powder less than 50 microns in size was crushed in a spherical mill; it was deposited on fiber at air temperature of 50° C and powder consumption of 0.5 g/l of air. The resulting material contained 6–18 CuS particles per 1 cm of the fiber length. An effective bactericidal fibrous material can be produced using rather cheap material – CuS and relatively cheap natural compounds of sulphides and oxides of heavy metals.

  3. Analytical characterization of complex, biotechnological feedstocks by pH gradient ion exchange chromatography for purification process development. (United States)

    Kröner, Frieder; Hanke, Alexander T; Nfor, Beckley K; Pinkse, Martijn W H; Verhaert, Peter D E M; Ottens, Marcel; Hubbuch, Jürgen


    The accelerating growth of the market for proteins and the growing interest in new, more complex molecules are bringing new challenges to the downstream process development of these proteins. This results in a demand for faster, more cost efficient, and highly understood downstream processes. Screening procedures based on high-throughput methods are widely applied nowadays to develop purification processes for proteins. However, screening highly complex biotechnological feedstocks, such as complete cell lysates containing target proteins often expressed with a low titre, is still very challenging. In this work we demonstrate a multidimensional, analytical screening approach based on pH gradient ion exchange chromatography (IEC), gel electrophoresis and protein identification via mass spectrometry to rationally characterize a biotechnological feedstock for the purpose of purification process development. With this very simple characterization strategy a two-step purification based on consecutive IEC operations was rapidly laid out for the purification of a diagnostic protein from a cell lysate reaching a purity of ∼80%. The target protein was recombinantly produced using an insect cell expression system. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Industrial compatible re-growth of vertically aligned multiwall carbon nanotubes by ultrafast pure oxygen purification process

    DEFF Research Database (Denmark)

    Bu, Ian Y.Y.; Hou, Kai; Engstrøm, Daniel Southcott


    Reproducible high-yield purification process of multiwalled carbon nanotubes (CNTs) was developed by thermal annealing in ultrapure oxygen. The optimized condition involves thermal annealing via a PID controlled heater in high purity oxygen at temperature of 450°C for 180s, which burns out...

  5. Multi-scale flowsheet simulation of an integrated continuous purification-downstream pharmaceutical manufacturing process. (United States)

    Sen, Maitraye; Chaudhury, Anwesha; Singh, Ravendra; John, Joyce; Ramachandran, Rohit


    Properties of active pharmaceutical ingredients influence the critical quality attributes (CQAs) of final solid dosage forms (e.g. tablets). In the last decade, continuous manufacturing has been shown to be a promising alternative to batch processing in the pharmaceutical industry. Therefore, a quantitative model-based analysis of the influence of upstream API properties on downstream processing quality metrics will lead to enhanced QbD in pharmaceutical drug product manufacturing (Benyahia et al., 2012). In this study, a dynamic flowsheet simulation of an integrated API purification step (crystallization), followed by filtration and drying, with a downstream process (powder mixing) is presented. Results show that the temperature profile of a cooling crystallization process influences the crystal size distribution which in turn impacts the RSD and API concentration of the powder mixing process, which in turn has a direct effect on tablet properties (Boukouvala et al., 2012). A hybrid PBM-DEM model is also presented to demonstrate the coupling of particle-scale information with process-scale information leading to enhanced elucidation of the dynamics of the overall flowsheet simulation. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Development of a novel and efficient cell culture flocculation process using a stimulus responsive polymer to streamline antibody purification processes. (United States)

    Kang, Yun Kenneth; Hamzik, James; Felo, Michael; Qi, Bo; Lee, Julia; Ng, Stanley; Liebisch, Gregory; Shanehsaz, Behnam; Singh, Nripen; Persaud, Kris; Ludwig, Dale L; Balderes, Paul


    Recent advances in mammalian cell culture processes have significantly increased product titers, but have also resulted in substantial increases in cell density and cellular debris as well as process and product related impurities. As such, with improvements in titer, corresponding improvements in downstream processing are essential. In this study we have developed an alternative antibody harvest process that incorporates flocculation using a novel stimulus responsive polymer, benzylated poly(allylamine), followed by depth filtration. As tested on multiple antibodies, this process demonstrates high process yield, improved clearance of cells and cell debris, and efficient reduction of aggregates, host cell proteins (HCP) and DNA. A wide operating window was established for this novel flocculation process through design of experiments condition screening and optimization. Residual levels of impurities in the Protein A eluate were achieved that potentially meet requirements of drug substance and thus alleviate the burden for further impurities removal in subsequent chromatography steps. In addition, efficient clearance of residual polymer was demonstrated using a fluorescence tagged polymer in the presence of a stimulus reagent. The mechanism of HCP and aggregates removal during flocculation was also explored. This novel and efficient process can be easily integrated into current mAb purification platforms, and may overcome downstream processing challenges. © 2013 Wiley Periodicals, Inc.

  7. Purification and utilization of garlic processing wastewater in lotus pond wetlands

    Institute of Scientific and Technical Information of China (English)

    Jun PANG; Xue-ling FENG; Xiu-feng WANG


    Based on the experiments of utilization of garlic processing wastewater in a lotus pond, this study demonstrates that lotus pond wetlands have a remarkable ability to remove organic pollutants and decrease chemical oxygen demand (CODCr), biochemical oxygen demand (BOD5), and suspended substances (SS) in garlic processing wastewater. Results also show evident effects of lotus roots on absorption of NH3-N. The pH value in a lotus pond with wastewater discharged was relatively stable. The water quality in the lotus pond reached the classⅡ emission standard, according to the Integrated Wastewater Discharge Standard (GB8978-1996), seven days after pretreated garlic processing wastewater had been discharged into the lotus pond. Garlic processing wastewater irrigation does not produce pollution in the pond sediment and has no negative effect on the growth of lotus roots. Due to utilization of garlic processing wastewater, the output of lotus roots increased by 3.0% to 8.3%, and the quality of lotus roots was improved. Therefore, better purification and utilization results can be achieved.

  8. Successful Integration of Membrane Technologies in a Conventional Purification Process of Tannery Wastewater Streams

    Directory of Open Access Journals (Sweden)

    Angelo Chianese


    Full Text Available The aim of this work is to design and integrate an optimized batch membrane process in a conventional purification process used for the treatment of tannery wastewater. The integration was performed by using two spiral wound membrane modules in series, that is, nanofiltration and reverse osmosis, as substitutes to the biological reactor. The membrane process was designed in terms of sensible fouling issues reduction, which may be observed on the nanofiltration membrane if no optimization is performed. The entity of the fouling phenomena was estimated by pressure cycling measurements, determining both the critical and the threshold flux on the nanofiltration membrane. The obtained results were used to estimate the need of the overdesign of the membrane plant, as well as to define optimized operating conditions in order to handle fouling issues correctly for a long period of time. Finally, the developed membrane process was compared, from a technical and economic point of view, with the conventional biological process, widely offered as an external service near tannery production sites, and, here, proposed to be substituted by membrane technologies.

  9. [Characteristics of microbial community and operation efficiency in biofilter process for drinking water purification]. (United States)

    Xiang, Hong; Lü, Xi-Wu; Yang, Fei; Yin, Li-Hong; Zhu, Guang-Can


    In order to explore characteristics of microbial community and operation efficiency in biofilter (biologically-enhanced active filter and biological activated carbon filter) process for drinking water purification, Biolog and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) techniques were applied to analyze the metabolic function and structure of microbial community developing in biofilters. Water quality parameters, such as NH; -N, NO; -N, permanganate index, UV254 and BDOC etc, were determined in inflow and outflow of biofilters for investigation of operation efficiency of the biofilters. The results show that metabolic capacity of microbial community of the raw water is reduced after the biofilters, which reflect that metabolically active microbial communities in the raw water can be intercepted by biofilters. After 6 months operation of biofilters, the metabolic profiles of microbial communities are similar between two kinds of biologically-enhanced active filters, and utilization of carbon sources of microbial communities in the two filters are 73.4% and 75.5%, respectively. The metabolic profiles of microbial communities in two biological activated carbon filters showed significant difference. The carbon source utilization rate of microbial community in granule-activated carbon filter is 79.6%, which is obviously higher than 53.8% of the rate in the columnar activated carbon filter (p water purification efficiency was not significant (p > 0.05). However, in biological activated carbon filters, granule-activated carbon is conducive to microbial growth and reproduction, and the microbial communities in the biofilter present high metabolic activities, and the removal efficiency for NH4(+)-N, permanganate index and BDOC is better than the columnar activated carbon filter(p < 0.05). The results also suggest that operation efficiency of biofilter is related to the metabolic capacity of microbial community in biofilter.

  10. A cell-free expression and purification process for rapid production of protein biologics. (United States)

    Sullivan, Challise J; Pendleton, Erik D; Sasmor, Henri H; Hicks, William L; Farnum, John B; Muto, Machiko; Amendt, Eric M; Schoborg, Jennifer A; Martin, Rey W; Clark, Lauren G; Anderson, Mark J; Choudhury, Alaksh; Fior, Raffaella; Lo, Yu-Hwa; Griffey, Richard H; Chappell, Stephen A; Jewett, Michael C; Mauro, Vincent P; Dresios, John


    Cell-free protein synthesis has emerged as a powerful technology for rapid and efficient protein production. Cell-free methods are also amenable to automation and such systems have been extensively used for high-throughput protein production and screening; however, current fluidic systems are not adequate for manufacturing protein biopharmaceuticals. In this work, we report on the initial development of a fluidic process for rapid end-to-end production of recombinant protein biologics. This process incorporates a bioreactor module that can be used with eukaryotic or prokaryotic lysates that are programmed for combined transcription/translation of an engineered DNA template encoding for specific protein targets. Purification of the cell-free expressed product occurs through a series of protein separation modules that are configurable for process-specific isolation of different proteins. Using this approach, we demonstrate production of two bioactive human protein therapeutics, erythropoietin and granulocyte-macrophage colony-stimulating factor, in yeast and bacterial extracts, respectively, each within 24 hours. This process is flexible, scalable and amenable to automation for rapid production at the point-of-need of proteins with significant pharmaceutical, medical, or biotechnological value.

  11. Oil quality of passion fruit seeds subjected to a pulp-waste purification process

    Directory of Open Access Journals (Sweden)

    Suelen Alvarenga Regis


    Full Text Available Passion fruit seeds must be clean and dry before the extraction processing to obtain high-quality oil for edible and cosmetic purposes. This research studies the viability of a cleaning process of seeds by evaluating the oil quality. The research examined 2 maturation stages of the fruit and one purification process of the seeds, compared to the control. The oil quality was evaluated by fatty acid composition, acidity, peroxide value and oxidative stability. The pulp waste suffered a thermal treatment in an alkaline water solution at 60°C for 10min and was further purified in an experimental decanter. In the control treatment, the pulp waste was processed using only water at ambient conditions. The passion fruit seeds were totally cleaned by the thermal/chemical treatment, allowing a faster drying (less than 50% of the drying time of the seeds and a bit higher yield of oil extraction (proportionally around 7.7%, without changes in quality of the oil

  12. Purification of chondroitin precursor from Escherichia coli K4 fermentation broth using membrane processing. (United States)

    Schiraldi, Chiara; Carcarino, Immacolata Loredana; Alfano, Alberto; Restaino, Odile Francesca; Panariello, Andrea; De Rosa, Mario


    Recently the possibility of producing the capsular polysaccharide K4, a fructosylated chondroitin, in fed-batch experiments was assessed. In the present study, a novel downstream process to obtain chondroitin from Escherichia coli K4 fermentation broth was developed. The process is simple, scalable and economical. In particular, downstream procedures were optimized with a particular aim of purifying a product suitable for further chemical modifications, in an attempt to develop a biotechnological platform for chondroitin sulfate production. During process development, membrane devices (ultrafiltration/diafiltration) were exploited, selecting the right cassette cut-offs for different phases of purification. The operational conditions (cross-flow rate and transmembrane pressure) used for the process were determined on an ÄKTA cross-flow instrument (GE Healthcare, USA), a lab-scale automatic tangential flow filtration system. In addition, parameters such as selectivity and throughput were calculated based on the analytical quantification of K4 and defructosylated K4, as well as the major contaminants. The complete downstream procedure yielded about 75% chondroitin with a purity higher than 90%.

  13. River Water Purification via a Coagulation-Porous Ceramic Membrane Hybrid Process

    Institute of Scientific and Technical Information of China (English)

    张荟钦; 仲兆祥; 李卫星; 邢卫红; 金万勤


    Membrane filtration technology combined with coagulation is widely used to purify river water. In this study, microfiltration (MF) and ultrafiltration (UF) ceramic membranes were combined with coagulation to treat local river water located at Xinghua, Jiangsu province, China. The operation parameters, fouling mechanism and pilot-scale tests were investigated. The results show that the pore size of membrane has small effect on the pseudo-steady flux for dead-end filtration, and the increase of flux in MF process is more than that in UF process for cross-flow filtration with the same increase of cross-flow velocity. The membrane pore size has little influence on the water quality. The analysis on membrane fouling mechanism shows that the cake filtration has significant in-fluence on the pseudo-steady flux and water quality for the membrane with pore size of 50, 200 and 500 nm. For the membrane with pore size of 200 nm and backwashing employed in our pilot study, a constant flux of 150 L·m-2·h-1 was reached during stable operation, with the removal efficiency of turbidity, total organic carbon (TOC) and UV254 higher than 99%, 45%and 48%, respectively. The study demonstrates that coagulation-porous ceramic membrane hybrid process is a reliable method for river water purification.

  14. Polonium purification

    Energy Technology Data Exchange (ETDEWEB)

    Baker, J.D.


    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.

  15. Origin and Evolution of Rickettsial Plasmids. (United States)

    El Karkouri, Khalid; Pontarotti, Pierre; Raoult, Didier; Fournier, Pierre-Edouard


    Rickettsia species are strictly intracellular bacteria that have undergone a reductive genomic evolution. Despite their allopatric lifestyle, almost half of the 26 currently validated Rickettsia species have plasmids. In order to study the origin, evolutionary history and putative roles of rickettsial plasmids, we investigated the evolutionary processes that have shaped 20 plasmids belonging to 11 species, using comparative genomics and phylogenetic analysis between rickettsial, microbial and non-microbial genomes. Plasmids were differentially present among Rickettsia species. The 11 species had 1 to 4 plasmid (s) with a size ranging from 12 kb to 83 kb. We reconstructed pRICO, the last common ancestor of the current rickettsial plasmids. pRICO was vertically inherited mainly from Rickettsia/Orientia chromosomes and diverged vertically into a single or multiple plasmid(s) in each species. These plasmids also underwent a reductive evolution by progressive gene loss, similar to that observed in rickettsial chromosomes, possibly leading to cryptic plasmids or complete plasmid loss. Moreover, rickettsial plasmids exhibited ORFans, recent gene duplications and evidence of horizontal gene transfer events with rickettsial and non-rickettsial genomes mainly from the α/γ-proteobacteria lineages. Genes related to maintenance and plasticity of plasmids, and to adaptation and resistance to stress mostly evolved under vertical and/or horizontal processes. Those involved in nucleotide/carbohydrate transport and metabolism were under the influence of vertical evolution only, whereas genes involved in cell wall/membrane/envelope biogenesis, cycle control, amino acid/lipid/coenzyme and secondary metabolites biosynthesis, transport and metabolism underwent mainly horizontal transfer events. Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via horizontal gene transfer as well as

  16. A sulfuric-lactic acid process for efficient purification of fungal chitosan with intact molecular weight. (United States)

    Naghdi, Mitra; Zamani, Akram; Karimi, Keikhosro


    The most recent method of fungal chitosan purification, i.e., two steps of dilute sulfuric acid treatment, pretreatment of cell wall at room temperature for phosphate removal and extraction of chitosan from the phosphate free cell wall at high temperature, significantly reduces the chitosan molecular weight. This study was aimed at improvement of this method. In the pretreatment step, to choose the best conditions, cell wall of Rhizopus oryzae, containing 9% phosphate, 10% glucosamine, and 21% N-acetyl glucosamine, was treated with sulfuric, lactic, acetic, nitric, or hydrochloric acid, at room temperature. Sulfuric acid showed the best performance in phosphate removal (90%) and cell wall recovery (89%). To avoid depolymerisation of chitosan, hot sulfuric acid extraction was replaced with lactic acid treatment at room temperature, and a pure fungal chitosan was obtained (0.12 g/g cell wall). Similar pretreatment and extraction processes were conducted on pure shrimp chitosan and resulted in a chitosan recovery of higher than 87% while the reduction of chitosan viscosity was less than 15%. Therefore, the sulfuric-lactic acid method purified the fungal chitosan without significant molecular weight manipulation.

  17. 异丙醚提纯工艺的研究%Research Isopropyl Ether Purification Process

    Institute of Scientific and Technical Information of China (English)

    姜丽敏; 位淑华; 张梦博


    研究了异丙醚的提纯工艺。采用酸洗的方法,用硫酸做酸化剂与异丙醇副产叠合油中的异丙醚反应,静止分离,水解,精馏得到高纯度的异丙醚。讨论了硫酸质量分数、硫酸与异丙醚的质量比、反应时间对反应的影响,所得异丙醚的质量分数大于99%。%Purification process of isopropyl ether was studied.Sulfuric acid as acidifying reagent reac-ted with isopropyl ether in composite oil which was a byproduct of isopropanol,after still separation,hydroly-sis,distillation,isopropyl ether with high purity was obtained.The content of sulfuric acid,the mass ratio of sulfuric acid to isopropyl ether,the reaction time were discussed,the obtained content of diisopropyl ether was greater than 99%.

  18. Purification of radioactive decontamination liquids from NPP Paks with reactive adsorption and ion-exchange process

    Energy Technology Data Exchange (ETDEWEB)

    Szaanya, T.; Hanaak, L.; Marton, Gy.; Salamon, T. [University of Veszprem, Veszprem (Hungary); Tilky, P. [Nuclear Power Plant, Paks (Hungary)


    In nuclear power plant Paks, Hungary, alkaline oxidative (NaOH, KMnO{sub 4}, H{sub 2}O) and acidic reductive (citric- and oxalic acid, water) liquids are using for the decontamination of primary circuit equipment (main liquid circulating pumps, steam generators, pipelines etc). The above mentioned decontamination liquids are containing {sup 110m}Ag, {sup 95}Nb, {sup 54}Mn, {sup 58} Co, {sup 60}Co, {sup 51} Cr, {sup 124} Sb radioisotopes, summarized radioactivity is between 10{sup 3}-8x10{sup 4} kBq/dm{sup 3} liquid. The decontamination liquid can be cleaned with reactive adsorption (active carbon) and ion-exchange process at elevated temperature (333-368 K) in multilayered columns. After purification the summarized radioactivity for {sup 54}Mn, {sup 60}Co, and {sup 110m}Ag are in the outlet liquid below 1 kBq/dm{sup 3}. Decontamination factor DF{approx_equal}10{sup 3}-10{sup 4}, volumetric reduction factor VRF{approx_equal}50-500.



    Zainal; Laga, Amran; Bastian, Februadi


    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...

  20. Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab. (United States)

    Spooner, Jennifer; Keen, Jenny; Nayyar, Kalpana; Birkett, Neil; Bond, Nicholas; Bannister, David; Tigue, Natalie; Higazi, Daniel; Kemp, Benjamin; Vaughan, Tristan; Kippen, Alistair; Buchanan, Andrew


    Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab.

  1. Impact of the chemicals, essential for the purification process of strict Fe-hydrogenase, on the corrosion of mild steel. (United States)

    Rouvre, Ingrid; Gauquelin, Charles; Meynial-Salles, Isabelle; Basseguy, Régine


    The influence of additional chemical molecules, necessary for the purification process of [Fe]-hydrogenase from Clostridium acetobutylicum, was studied on the anaerobic corrosion of mild steel. At the end of the purification process, the pure [Fe-Fe]-hydrogenase was recovered in a Tris-HCl medium containing three other chemicals at low concentration: DTT, dithionite and desthiobiotin. Firstly, mild steel coupons were exposed in parallel to a 0.1 M pH7 Tris-HCl medium with or without pure hydrogenase. The results showed that hydrogenase and the additional molecules were in competition, and the electrochemical response could not be attributed solely to hydrogenase. Then, solutions with additional chemicals of different compositions were studied electrochemically. DTT polluted the electrochemical signal by increasing the Eoc by 35 mV 24 h after the injection of 300 μL of control solutions with DTT, whereas it drastically decreased the corrosion rate by increasing the charge transfer resistance (Rct 10 times the initial value). Thus, DTT was shown to have a strong antagonistic effect on corrosion and was removed from the purification process. An optimal composition of the medium was selected (0.5 mM dithionite, 7.5 mM desthiobiotin) that simultaneously allowed a high activity of hydrogenase and a lower impact on the electrochemical response for corrosion tests.

  2. Generic chromatography-based purification strategies accelerate the development of downstream processes for biopharmaceutical proteins produced in plants. (United States)

    Buyel, Johannes F; Fischer, Rainer


    Plants offer a valuable alternative to cultured mammalian cells for the production of recombinant biopharmaceutical proteins. However, the target protein typically represents only a minor fraction of the total protein in the initial plant extract, which means that the development of product-specific chromatography-based purification strategies is often laborious and expensive. To address this challenge, we designed a generic downstream process that is suitable for the purification of recombinant proteins with diverse properties from plant production platforms. This was achieved by focusing on the binding behavior of tobacco host cell proteins (HCPs) to a broad set of chromatography resins under different pH and conductivity conditions. Strong cation exchanger and salt-tolerant anion exchanger resins exhibited the best resolution of tobacco HCPs among the 13 tested resins, and their selectivity was easy to manipulate through the adjustment of pH and conductivity. The advantages, such as direct capture of a target protein from leaf extract, and limitations, such as low binding capacity, of various chromatography ligands and resins are discussed. We also address the most useful applications of the chromatography ligands, namely recovery of proteins with a certain pI, in a downstream process that aims to purify diverse plant-derived biopharmaceutical proteins. Based on these results, we describe generic purification schemes that are suitable for acidic, neutral, and basic target proteins, as a first step toward the development of industrial platform processes.

  3. Air Stripping Designs and Reactive Water Purification Processes for the Lunar Surface (United States)

    Boul, Peter J.; Lange, Kevin; Conger, Bruce; Anderson, Molly


    Air stripping designs are considered to reduce the presence of volatile organic compounds in the purified water. Components of the wastewater streams are ranked by Henry's Law Constant and the suitability of air stripping in the purification of wastewater in terms of component removal is evaluated. Distillation processes are modeled in tandem with air stripping to demonstrate the potential effectiveness and utility of these methods in recycling wastewater on the Moon. Scaling factors for distillation and air stripping columns are presented to account for the difference in the lunar gravitation environment. Commercially available distillation and air stripping units which are considered suitable for Exploration Life Support are presented. The advantages to the various designs are summarized with respect to water purity levels, power consumption, and processing rates. An evaluation of reactive distillation and air stripping is presented with regards to the reduction of volatile organic compounds in the contaminated water and air. Among the methods presented, an architecture is presented for the evaluation of the simultaneous oxidation of organics in air and water. These and other designs are presented in light of potential improvements in power consumptions and air and water purities for architectures which include catalytic activity integrated into the water processor. In particular, catalytic oxidation of organics may be useful as a tool to remove contaminants that more traditional distillation and/or air stripping columns may not remove. A review of the current leading edge at the commercial level and at the research frontier in catalytically active materials is presented. Themes and directions from the engineering developments in catalyst design are presented conceptually in light of developments in the nanoscale chemistry of a variety of catalyst materials.

  4. Stress analysis of the O-element pipe during the process of flue gases purification

    Directory of Open Access Journals (Sweden)

    Nekvasil R.


    Full Text Available Equipment for flue gases purification from undesired substances is used throughout power and other types of industry. This paper deals with damaging of the O-element pipe designed to remove sulphur from the flue gases, i.e. damaging of the pipe during flue gases purification. This purification is conducted by spraying the water into the O-shaped pipe where the flue gases flow. Thus the sulphur binds itself onto the water and gets removed from the flue gas. Injection of cold water into hot flue gases, however, causes high stress on the inside of the pipe, which can gradually damage the O-element pipe. In this paper initial injection of water into hot pipe all the way to stabilization of temperature fields will be analyzed and the most dangerous places which shall be considered for fatigue will be determined.

  5. Process for purification of waste water produced by a Kraft process pulp and paper mill (United States)

    Humphrey, M. F. (Inventor)


    The water from paper and pulp wastes obtained from a mill using the Kraft process is purified by precipitating lignins and lignin derivatives from the waste stream with quaternary ammonium compounds, removing other impurities by activated carbon produced from the cellulosic components of the water, and then separating the water from the precipitate and solids. The activated carbon also acts as an aid to the separation of the water and solids. If recovery of lignins is also desired, then the precipitate containing the lignins and quaternary ammonium compounds is dissolved in methanol. Upon acidification, the lignin is precipitated from the solution. The methanol and quaternary ammonium compound are recovered for reuse from the remainder.

  6. Application of near-infrared spectroscopy combined with multivariate analysis in monitoring of crude heparin purification process. (United States)

    Zang, Hengchang; Wang, Jinfeng; Li, Lian; Zhang, Hui; Jiang, Wei; Wang, Fengshan


    Ion-exchange chromatography is a widely used purification technology in the heparin manufacturing process. To improve the efficiency and understand the process directly, a rapid and equally precise method needs to be developed to measure heparin concentration in chromatography process. Here, two robust partial least squares regression (PLS-R) models were established for quantification of heparin based on the near-infrared (NIR) spectroscopy with 80 samples of adsorption process and 76 samples of elution process. Several variables selection algorithms, including correlation coefficient method, successive projection algorithm (SPA) and interval partial least squares (iPLSs), were performed to remove non-informative variables. The results showed that the correlation coefficient of validation (Rp) and the residual predictive deviation (RPD) corresponded to 0.957 and 3.4472 for adsorption process, 0.968 and 3.9849 for elution process, respectively. The approach was found considerable potential for real-time monitoring the heparin concentration of chromatography process.

  7. Application of near-infrared spectroscopy combined with multivariate analysis in monitoring of crude heparin purification process (United States)

    Zang, Hengchang; Wang, Jinfeng; Li, Lian; Zhang, Hui; Jiang, Wei; Wang, Fengshan


    Ion-exchange chromatography is a widely used purification technology in the heparin manufacturing process. To improve the efficiency and understand the process directly, a rapid and equally precise method needs to be developed to measure heparin concentration in chromatography process. Here, two robust partial least squares regression (PLS-R) models were established for quantification of heparin based on the near-infrared (NIR) spectroscopy with 80 samples of adsorption process and 76 samples of elution process. Several variables selection algorithms, including correlation coefficient method, successive projection algorithm (SPA) and interval partial least squares (iPLSs), were performed to remove non-informative variables. The results showed that the correlation coefficient of validation (Rp) and the residual predictive deviation (RPD) corresponded to 0.957 and 3.4472 for adsorption process, 0.968 and 3.9849 for elution process, respectively. The approach was found considerable potential for real-time monitoring the heparin concentration of chromatography process.

  8. TIO2 based photocatalytic gas purification: the effects of co-catalysts and process conditions

    NARCIS (Netherlands)

    Fraters, Bindikt Daouda


    Photocatalysis is highly promising as a technology to mitigate environmental pollution. In this thesis the focus will be on air purification by photocatalytic oxidation of volatile organic compounds (VOC’s). A major challenge in photocatalysis is the low photonic efficiency, due to high electron hol

  9. TIO2 based photocatalytic gas purification: the effects of co-catalysts and process conditions

    NARCIS (Netherlands)

    Fraters, B.D.


    Photocatalysis is highly promising as a technology to mitigate environmental pollution. In this thesis the focus will be on air purification by photocatalytic oxidation of volatile organic compounds (VOC’s). A major challenge in photocatalysis is the low photonic efficiency, due to high electron

  10. Differential interactions of plasmid DNA, RNA and genomic DNA with amino acid-based affinity matrices. (United States)

    Sousa, Angela; Sousa, Fani; Queiroz, João A


    The development of a strategy to plasmid DNA (pDNA) purification has become necessary for the development of gene therapy and DNA vaccine production processes in recent years, since this nucleic acid and most of contaminants, such as RNA, genomic DNA and endotoxins, are negatively charged. An ideal separation methodology may be achieved with the use of affinity interactions between immobilized amino acids and nucleic acids. In this study, the binding behaviour of nucleic acids under the influence of different environmental conditions, such as the composition and ionic strength of elution buffer, and the temperature, is compared with various amino acids immobilized on chromatography resins. Supercoiled (sc) plasmid isoform was isolated with all matrices used, but in some cases preferential interactions with other nucleic acids were found. Particularly, lysine chromatography showed to be an ideal technology mainly on RNA purification using low salt concentration. On the other hand, arginine ligands have shown a greater ability to retain the sc isoform comparatively to the other nucleic acids retention, becoming this support more adequate to sc pDNA purification. The temperature variation, competitive elution and oligonucleotides affinity studies also allowed to recognize the dominant interactions inherent to biorecognition of pDNA molecule and the affinity matrices.

  11. A novel liquid/liquid extraction process composed of surfactant and acetonitrile for purification of polygalacturonase enzyme from Durio zibethinus. (United States)

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


    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.

  12. Chemotherapy of Bacterial Plasmids (United States)


    render them non-susceptible to K: z plasmid-encoded enzymes. (3) Development of drugs which are selective inhibitor! 1 4, of plasmid DNA replication. (4... Development of drugs which inhibit phenotypic as expression of plasmid genes, and (5) Development of drugs which are inhibitors o, drug-inactivating...Barnes [2] them non-susceptible to plasmid-encoded enzymes, tabulated data on the incidence of Gram-negative 3) development of drugs which are

  13. A Hybrid MPC-PID Control System Design for the Continuous Purification and Processing of Active Pharmaceutical Ingredients

    Directory of Open Access Journals (Sweden)

    Maitraye Sen


    Full Text Available In this work, a hybrid MPC (model predictive control-PID (proportional-integral-derivative control system has been designed for the continuous purification and processing framework of active pharmaceutical ingredients (APIs. The specific unit operations associated with the purification and processing of API have been developed from first-principles and connected in a continuous framework in the form of a flowsheet model. These integrated unit operations are highly interactive along with the presence of process delays. Therefore, a hybrid MPC-PID is a promising alternative to achieve the desired control loop performance as mandated by the regulatory authorities. The integrated flowsheet model has been simulated in gPROMSTM (Process System Enterprise, London, UK. This flowsheet model has been linearized in order to design the control scheme. The ability to track the set point and reject disturbances has been evaluated. A comparative study between the performance of the hybrid MPC-PID and a PID-only control scheme has been presented. The results show that an enhanced control loop performance can be obtained under the hybrid control scheme and demonstrate that such a scheme has high potential in improving the efficiency of pharmaceutical manufacturing operations.

  14. Plasmid segregation mechanisms

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Gerdes, Kenn; Charbon, Gitte Ebersbach


    Bacterial plasmids encode partitioning (par) loci that ensure ordered plasmid segregation prior to cell division. par loci come in two types: those that encode actin-like ATPases and those that encode deviant Walker-type ATPases. ParM, the actin-like ATPase of plasmid R1, forms dynamic filaments ...

  15. An efficient process for production and purification of hyaluronic acid from Streptococcus equi subsp. zooepidemicus. (United States)

    Rangaswamy, Vidhya; Jain, Dharmendra


    Growth of Streptococcus zooepidemicus in a 10 l bioreactor with 50 g sucrose/l and 10 g casein hydrolysate/l gave 5-6 g hyaluronic acid/l after 24-28 h. Purification of hyaluronic acid gave a recovery of 65% with the final material having an Mr of approximately 4 x 10(6) Da with less than 0.1% protein.

  16. TIO2 based photocatalytic gas purification: the effects of co-catalysts and process conditions


    Fraters, B.D.


    Photocatalysis is highly promising as a technology to mitigate environmental pollution. In this thesis the focus will be on air purification by photocatalytic oxidation of volatile organic compounds (VOC’s). A major challenge in photocatalysis is the low photonic efficiency, due to high electron hole recombination rates. One of the solutions proposed for this issue, is the use of (metal) nanoparticles as co-catalyst. However, currently knowledge on how nanoparticles affect the photocatalytic ...

  17. Community structure of microbial biofilms associated with membrane-based water purification processes as revealed using a polyphasic approach

    Energy Technology Data Exchange (ETDEWEB)

    Chen, C.L.; Chong, M.L.; Wong, M.T.; Ong, S.L.; Ng, W.J. [Dept. of Civil Engineering, National Univ. of Singapore (Singapore); Liu, W.T. [Dept. of Civil Engineering, National Univ. of Singapore (Singapore); Nanoscience and Nanotechnology Initiative, National Univ. of Singapore (Singapore); Seah, H. [Public Utilities Board (Singapore)


    The microbial communities of membrane biofilms occurring in two full-scale water purification processes employing microfiltration (MF) and reverse osmosis (RO) membranes were characterized using a polyphasic approach that employed bacterial cultivation, 16S rDNA clone library and fluorescence in situ hybridization techniques. All methods showed that the {alpha}-proteobacteria was the largest microbial fraction in the samples, followed by the {gamma}-proteobacteria. This suggested that members of these two groups could be responsible for the biofouling on the membranes studied. Furthermore, the microbial community structures between the MF and RO samples were considerably different in composition of the most predominant 16S rDNA clones and bacterial isolates from the {alpha}-proteobacteria and only shared two common groups (Bradyrhizobium, Bosea) out of more than 17 different bacterial groups observed. The MF and RO samples further contained Planctomycetes and Fibroacter/Acidobacteria as the second predominant bacterial clones, respectively, and differed in minor bacterial clones and isolates. The community structure differences were mainly attributed to differences in feed water, process configurations and operating environments, such as the pressure and hydrodynamic conditions present in the water purification systems. (orig.)

  18. Model-based process development for the purification of a modified human growth hormone using multimodal chromatography. (United States)

    Sejergaard, Lars; Karkov, Hanne Sophie; Krarup, Janus Kristian; Hagel, Anne Birgitte Bagge; Cramer, Steven M


    This study demonstrates how the multimodal Capto adhere resin can be used in concert with calcium chloride or arginine hydrochloride as mobile phase modifiers to create a highly selective purification process for a modified human growth hormone. Importantly, these processes are shown to result in significant clearance of product related aggregates and host cell proteins. Furthermore, the steric mass action model is shown to be capable of accurately describing the chromatographic process and the aggregate removal. Finally, justification of the selected operating ranges is evaluated using the model together with Latin hypercube sampling. The results in this article establish the utility of multimodal chromatography when used with appropriate mobile phase modifiers for the downstream bioprocessing of a modified human growth hormone and offer new approaches for bioprocess verification.

  19. Purification of the off-gases of the process of radioactive waste vitrification in induction melter

    Energy Technology Data Exchange (ETDEWEB)

    Gorbunov, V. A.; Katannikov, I. S.; Knyasev, O. A.; Kornev, V. I.; Lifanov, F. A.; Polkanov, M. A.; Savkin, A. E. [Moscow Scientific and Inndustrial Association RADON, Moscow (Russian Federation)


    Moscow SIA RADON has developed the method of vitrifying both radioactive ashes, arising from radioactive waste incineration, and liquid radioactive waste in induction melter. In the experimental plant the characteristics of off-gases were determined and various constructions of filters and filtering materials for dust trapping were tested. On the base of test results the plant for liquid radioactive waste vitrification has been constructed on the base of induction melter {sup c}old crucible{sup ,} equipped with modern effective dust and gas purification system, consisting of filtration unit, absorption unit and unit for nitrogen oxide catalytic reduction. (author). 3 refs., 9 tabs., 3 figs.

  20. Chemical purification of Gunungpati elephant foot yam flour to improve physical and chemical quality on processed food (United States)

    Paramita, Octavianti; Wahyuningsih, Ansori, Muhammad


    This study was aimed at improving the physicochemical quality of elephant foot yam flour in Gunungpati, Semarang by chemical purification. The utilization of elephant foot yam flour in several processed food was also discussed in this study. The flour purification discussed in this study was expected to become a reference for the manufacturers of elephant foot yam flour and its processed food in Gunungpati. This study modified the elephant foot yam flour using pre - gelatinization method. The physical and chemical quality of each elephant foot yam flour purification sample were assessed using proximate analysis. The likability test was conducted for its processed food. 20 grams of elephant foot yam flour was put into a beaker glass, then 60 ml of water was added. The suspension was then heated at a temperature of 60 ° C and 70 ° C while stirred until it was homogeneous and thickened for 10, 30 and 60 minutes. The flour which had been heated was then cooled at room temperature for 1 hour and then at a temperature of 0 ° C until it was frozen. Furthermore, flour was dried in an oven at a temperature of 60 ° C for 9 hours. The dried flour was sifted with a 80 mesh sieve. Chemical test was conducted after elephant foot yam was pre-gelatinized to determine changes in the quality flour: test levels of protein, fat, crude fiber content, moisture content, ash content and starch content. In addition, color tests and granular test on elephant foot yam flour were also conducted. The pre-gelatinization as chemical treatment on elephant foot yam flour in this study was able to change the functional properties of elephant foot yam flour towards a better processing characterized by a brighter color (L = 70, a = 6 and b = 12), the hydrolysis of polysaccharides flour into shorter chain (flour content decreased to 44%), the expansion of granules in elephant foot yam resulting in a process - ready flour, and better monolayer water content of 9%. The content of protein and fiber

  1. Purification processes of cadmium based quantum dots in aqueous medium: a comparative study (United States)

    Petris, Dimitri; Freitas, Denilson V.; dos Santos, Rayany K. V.; Dias, Jéssica M. M.; Navarro, Marcelo


    Three different purification methods for CdTe-MPA quantum dot (QD) were performed in aqueous medium: acid titration (HCl, HClO4, H2SO4 and CH3COOH), non-selective precipitation by addition of acetone and co-precipitation of the QD in the presence of inorganic salts. The QD stock solutions were prepared by an electrochemical method of synthesis, in four different heating times (1 h, 4 h, 8 h and 12 h). After purifications, the QD solids were redispersed in distilled water and analyzed by absorption and emission spectra. The λ abs and λ em of the purified QDs showed similar data observed for QD stock solutions, and the recovery rate varied from 71% to 99%. Co-precipitation method showed some advantages: quantum yield maintenance of the QD redispersed solution, longer period of storage (over 6 months) in solution and in solid state (QD embedded into the KCl crystal lattice). CdSe-MPA and CdS-MPA solutions were also purified by co-precipitation method with KCl, showing good results as observed for CdTe-MPA.

  2. Novel immobilization process of a thermophilic catalase: efficient purification by heat treatment and subsequent immobilization at high temperature. (United States)

    Xu, Juan; Luo, Hui; López, Claudia; Xiao, Jing; Chang, Yanhong


    The main goal of the present work is to investigate a novel process of purification and immobilization of a thermophilic catalase at high temperatures. The catalase, originated from Bacillus sp., was overexpressed in a recombinant Escherichia coli BL21(DE3)/pET28-CATHis and efficiently purified by heat treatment, achieving a threefold purification. The purified catalase was then immobilized onto an epoxy support at different temperatures (25, 40, and 55 °C). The immobilizate obtained at higher temperatures reached its maximum activity in a shorter time than that obtained at lower temperatures. Furthermore, immobilization at higher temperatures required a lower ionic strength than immobilization at lower temperatures. The characteristics of immobilized enzymes prepared at different temperatures were investigated. The high-temperature immobilizate (55 °C) showed the highest thermal stability, followed by the 40 °C immobilizate. And the high-temperature immobilizate (55 °C) had slightly higher operational stability than the 25 °C immobilizate. All of the immobilized catalase preparations showed higher stability than the free enzyme at alkaline pH 10.0, while the alkali resistance of the 25 °C immobilizate was slightly better than that of the 40 and 55 °C immobilizates.


    Directory of Open Access Journals (Sweden)

    Beata Fryczkowska


    Full Text Available The paper presents results of research on the use of composite membranes of polyacrylonitrile (PAN doped polyaniline (PANI to remove contaminations of industrial wastewater generated during the processing of metals. Wastewater obtained from industry was pre-treated with the flocculant Magnafloc®336, and then the supernatant solution was introduced into the ultrafiltration cell, AMICON (Millipore equipped in the previously prepared polymer membrane. Using spectrophotometer UV-Vis (HACH and atomic absorption spectrometry (AAS pollution indicators was marked before and after the integrated purification proces, to determine the degree of removal of selected ions from wastewater. As a result of flocculation from wastewater there have been removed phosphates (79%, chlorides (11-14%, sulfates (2-10% and iron (36-92%, cobalt (~ 80%, cadmium (~ 31% and nickel (~ 25%. However, the pressure membrane process almost completely removed zinc, copper and cadmium (~ 100%, iron (by a further 43-69% and phosphate anions, which was a little.

  4. Improved OMV vaccine against Neisseria meningitidis using genetically engineered strains and a detergent-free purification process. (United States)

    van de Waterbeemd, Bas; Streefland, Mathieu; van der Ley, Peter; Zomer, Bert; van Dijken, Harry; Martens, Dirk; Wijffels, René; van der Pol, Leo


    The use of detergent-extracted outer membrane vesicles (OMVs) is an established approach for development of a multivalent PorA vaccine against N. meningitidis serogroup B. Selective removal of lipopolysaccharide (LPS) decreases toxicity, but promotes aggregation and narrows the immune response. Detergent-free OMV vaccines retain all LPS, which preserves the native vesicle structure, but result in high toxicity and lower yield. The present study assessed the effects of gene mutations that attenuated LPS toxicity (lpxL1) or improved OMV yield (rmpM) in combination with the available OMV purification processes. The results substantiate that OMVs from a strain with both mutations, produced with a detergent-free process provide better vaccine characteristics than the traditional detergent-based approach. With comparable toxicity and yield, no aggregation and cross-protection against other PorA subtypes, these OMV vaccines are potentially safe and effective for parenteral use in humans. Copyright 2010 Elsevier Ltd. All rights reserved.

  5. Stress responses and replication of plasmids in bacterial cells

    Directory of Open Access Journals (Sweden)

    Wegrzyn Alicja


    Full Text Available Abstract Plasmids, DNA (or rarely RNA molecules which replicate in cells autonomously (independently of chromosomes as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage λ that are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly.

  6. Adsorption behavior of plasmid DNA onto perfusion chromatographic matrix

    Institute of Scientific and Technical Information of China (English)



    Anion exchange chromatography is the most popular chromatographic method for plasmid separa-tion.POROS RI 50 is a perfusion chromatographic support which is a reversed phase matrix and is an alterna-tive to conventional ones due to its mass transfer properties.The adsorption and elution of the pIDKE2 plasmidonto reversed phase POROS RI 50 was studied.Langmuir isotherm model was adjusted in order to get the max-imum adsorption capacity and the dissociation constant for POROS RI 50-plasmid DNA (pDNA) system.Break-through curves were obtained for volumetric flows between 0.69-3.33mL/min,given dynamic capacity up to2.3 times higher than those reported for ionic exchange matrix used during the purification process of plasmidswith similar size to that of pIDKE2.The efficiency was less than 45% for the flow conditions and initial concen-tration studied,which means that the support will not be operated under saturation circumstances.


    Directory of Open Access Journals (Sweden)

    V. A. Golybin


    Full Text Available Summary. The effect of reducing substances in the final stage of lime - carbon dioxide purification of raw juice is studied in the article. The presence of significant amounts of reducing substances in the juice of the I saturation increases chroma and calcium salts in the purified product. It is actual to apply additional techniques and methods of cleaning of production sugar-containing solutions at the final stage of lime -carbon dioxide cleaning - II saturation, that will increase the completeness of precipitation of organic and mineral non-sugars, improve the quality of the purified juice, increase the yield of white sugar and improve its quality. The effect filtroperlit as seed material for forming the structure of particles of calcium carbonate precipitate with a larger surface adsorption is studied. The effect of phosphate input for further improvement of the efficiency of adsorption in the juice purification process was also studied. The effect of flow of activated filtroperlit on II saturation filtration speed was studied. It was found out that the more non-sugars are present in the juice, the smaller electrokinetic potential has the surface sediment. Rational consumption of reagents depending on the quality of the feedstock is calculated. In the process of cleaning the juice of various technological quality, it is necessary to control the reagents flow. It was found out that for cleaning juice of satisfactory technological quality the flow of filtroperlit is 0.015 - 0.033% by weight of juice and 15% РО4 3- . When cleaning the juice obtained from sugar beet of poor quality, it is necessary to increase the filtroperlit flow up to 0.050% and phosphate up to 20 %. It is necessary to control permanently the main liming process, the maximum decomposition of reducing substances to obtain thermally stable juice.

  8. Effect of water purification process in radioactive content: analysis on small scale purification plants; Efecto del proceso de purificacion de agua en el contenido radiactivo: analisis en plantas purificadoras a pequena escala

    Energy Technology Data Exchange (ETDEWEB)

    Lopez del Rio, H.; Quiroga S, J. C.; Davila R, J. I.; Mireles G, F. [Universidad Autonoma de Zacatecas, Unidad Academica de Estudios Nucleares, Calle Cipres No. 10, Fracc. La Penuela, 98000, Zacatecas (Mexico)], e-mail:


    Water from small scale purification plants is a low cost alternative for consumers in comparison to the bottled commercial presentations. Because of its low cost per liter, the consumption of this product has increased in recent years, stimulating in turn the installation of purification systems for these small businesses. The purpose of this study was to estimate the efficiency of small scale purification systems located in the cities of Zacatecas and Guadalupe, Zacatecas, to reduce the radioactive content of water. It was measured the total alpha and beta activity in water samples of entry and exit to process, through the liquid scintillation technique. In general it was observed that the process is more efficient in removing alpha that beta activity. The fraction of total alpha activity removed varied between 27 and 100%, while between 0 and 77% of the total beta activity was removed by the analyzed plants. In all cases, the total radioactivity level was lower than the maximum permissible value settled by the official mexican standard for drinking water. (Author)


    Directory of Open Access Journals (Sweden)

    Tomasz Turek


    Full Text Available The paper presents results of research on the use of composite membranes of polyacrylonitrile (PAN doped with graphene oxide (GO to remove contaminations of galvanic wastewater. Membranes were obtained using phase inversion method from PAN and GO solution in N,N-dimethylformamide (DMF. Wastewater was pre-treated with the flocculant Magnafloc®336. Next, ultrafiltration of the treated wastewater was carried out in the ultrafiltration cell AMICON on the previously prepared PAN/GO composite membranes. Physico-chemical properties and composition of solutions before and after integrated purification process were analyzed by UV-Vis spectrophotometer and atomic absorption spectrometry (AAS. As a result of flocculation from wastewater there have been removed phosphates (97%, chlorides (5,2%, sulfates (5,9% and iron (82%. In addition, as a result of ultrafiltration was complete removal of phosphate anions (100% and iron (~91-92%, zinc (68÷84%, lead (65-98% and cadmium (~67%.

  10. High-Throughput, Automated Protein A Purification Platform with Multiattribute LC-MS Analysis for Advanced Cell Culture Process Monitoring. (United States)

    Dong, Jia; Migliore, Nicole; Mehrman, Steven J; Cunningham, John; Lewis, Michael J; Hu, Ping


    The levels of many product related variants observed during the production of monoclonal antibodies are dependent on control of the manufacturing process, especially the cell culture process. However, it is difficult to characterize samples pulled from the bioreactor due to the low levels of product during the early stages of the process and the high levels of interfering reagents. Furthermore, analytical results are often not available for several days, which slows the process development cycle and prevents "real time" adjustments to the manufacturing process. To reduce the delay and enhance our ability to achieve quality targets, we have developed a low-volume, high-throughput, and high-content analytical platform for at-line product quality analysis. This workflow includes an automated, 96-well plate protein A purification step to isolate antibody product from the cell culture fermentation broth, followed by rapid, multiattribute LC-MS analysis. We have demonstrated quantitative correlations between particular process parameters with the levels of glycosylated and glycated species in a series of small scale experiments, but the platform could be used to monitor other attributes and applied across the biopharmaceutical industry.

  11. Protocol for Initial Purification of Bacteriocin (United States)


    lysate/extract preparation , column purification, and a desalting. The peptide was tracked throughout the process using a soft agar overlay activity... PREPARATION SOLUBLE EXTRACTS COLUMN CHROMATOGRAPHY BACTERIA PURIFICATION CHARACTERIZATION...3  6.1  Preparation of Target Bacteria

  12. 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


    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

  13. Improved purification process of β- and α-trypsin isoforms by ion-exchange chromatography

    Directory of Open Access Journals (Sweden)

    Alexandre Martins Costa Santos


    Full Text Available The purpose of this work was to improve the separation and yield of pure β- and α-trypsin isoforms by ion-exchange chromatography and to characterize some physical-chemical properties of these isoforms. Purification of trypsin isoforms was performed by ion-exchange chromatography in 0.1 mol/L tris-HC buffer, pH 7.10 at 4ºC. The sample loading, salt concentration, flow rate and pH of mobile phase were varied to determine their effects on the resolution of the separation. The resolution was optimized mainly between β- and α-trypsin. Pure isoforms were obtained by chromatographying 100 mg of commercial trypsin during seven days, yielding 51 mg of high purity β-trypsin and 13 mg of α-trypsin partially pure, with small amounts of contaminating of ψ-trypsin. Thus, time and resolution of purification were optimized yielding large amounts of pure active enzymes that are useful for several research areas and biotechnology.O propósito deste trabalho foi melhorar a separação e o rendimento das isoformas puras β- e α-tripsina por meio de cromatografia de troca iônica e caracterizar algumas propriedades físico-químicas dessas isoformas. A purificação de isoformas de tripsina foi realizada em SE Sephadex, com tampão tris-HCl, pH 7,10 a 4ºC. A quantidade de amostra, a concentração salina, o fluxo e o pH da fase móvel foram variados para determinar o efeito sobre a resolução da separação. A resolução foi otimizada principalmente entre β- e α-tripsina, utilizando o pH 7,10 a 4ºC. Isoformas puras foram obtidas a partir de 100 mg de tripsina comercial bovina depois de sete dias de cromatografia, fornecendo 51,0 mg de β-tripsina totalmente pura e 13,0 mg de α-tripsina parcialmente pura, com quantidades pequenas de contaminação por ψ-Tripsina. Assim, tempo e resolução da purificação foram otimizados redendo grandes quantidades de enzimas puras e ativas que são úteis em várias áreas de pesquisa e ciências biotecnológicas.

  14. Use of capillary electrophoresis-sodium dodecyl sulfate to monitor disulfide scrambled forms of an Fc fusion protein during purification process. (United States)

    Hapuarachchi, Suminda; Fodor, Szilan; Apostol, Izydor; Huang, Gang


    Overexpression of recombinant Fc fusion proteins in Escherichia coli frequently results in the production of inclusion bodies that are subsequently used to produce fully functional protein by an in vitro refolding process. During the refolding step, misfolded proteins such as disulfide scrambled forms can be formed, and purification steps are used to remove these product-related impurities to produce highly purified therapeutic proteins. A variety of analytical methods are commonly used to monitor protein variants throughout the purification process. Capillary electrophoresis (CE)-based techniques are gaining popularity for such applications. In this work, we used a nonreduced capillary electrophoresis-sodium dodecyl sulfate (nrCE-SDS) method for the analysis of disulfide scrambled forms in a fusion protein. Under denatured nonreduced conditions, an extra post-shoulder peak was observed at all purification steps. Detailed characterization revealed that the peak was related to the disulfide scrambled forms and was isobaric with the correctly folded product. In addition, when sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used during the CE-SDS peak characterization, we observed that the migration order of scrambled forms is reversed on CE-SDS versus SDS-PAGE. This illustrates the importance of establishing proper correlation of these two techniques when they are used interchangeably to guide the purification process and to characterize proteins.

  15. Influence of the pore size of reversed phase materials on peptide purification processes. (United States)

    Gétaz, David; Dogan, Nihan; Forrer, Nicola; Morbidelli, Massimo


    The influence of the pore size of a chromatographic reversed phase material on the adsorption equilibria and diffusion of two industrially relevant peptides (i.e. a small synthetic peptide and insulin) has been studied using seven different reversed phase HPLC materials having pore sizes ranging from 90 Å to 300 Å. The stationary phase pore size distribution was obtained by inverse size exclusion measurement (iSEC). The effect of the pore size on the mass transfer properties of the materials was evaluated from Van Deemter experiments. It has been shown that the lumped mass transfer coefficient increases linearly with the average pore size. The Henry coefficient and the impurity selectivity were determined in diluted conditions. The saturation capacity of the main peptides was determined in overloaded conditions using the inverse method (i.e. peak fitting). It was shown that the adsorption equilibria of the peptides on the seven materials is well described by a surface-specific adsorption isotherm. Based on this a lumped kinetic model has been developed to model the elution profile of the two peptides in overloaded conditions and to simulate the purification of the peptide from its crude mixture. It has been found that the separation of insulin from its main impurity (i.e. desamido-insulin) was not affected by the pore size. On the other hand, in the case of the synthetic peptide, it was found that the adsorption of the most significant impurity decreases with the pore size. This decrease is probably due to an increase in silanol activity with decreasing pore size.

  16. A review: Potential and challenges of biologically activated carbon to remove natural organic matter in drinking water purification process. (United States)

    Korotta-Gamage, Shashika Madushi; Sathasivan, Arumugam


    The use of biologically activated carbon (BAC) in drinking water purification is reviewed. In the past BAC is seen mostly as a polishing treatment. However, BAC has the potential to provide solution to recent challenges faced by water utilities arising from change in natural organic matter (NOM) composition in drinking water sources - increased NOM concentration with a larger fraction of hydrophilic compounds and ever increasing trace level organic pollutants. Hydrophilic NOM is not removed by traditional coagulation process and causes bacterial regrowth and increases disinfection by-products (DBPs) formation during disinfection. BAC can offer many advantages by removing hydrophilic fraction and many toxic and endocrine compounds which are not otherwise removed. BAC can also aid the other downstream processes if used as a pre-treatment. Major drawback of BAC was longer empty bed contact time (EBCT) required for an effective NOM removal. This critical review analyses the strategies that have been adopted to enhance the biological activity of the carbon by operational means and summarises the surface modification methods. To maximize the benefit of the BAC, a rethink of current treatment plant configuration is proposed. If the process can be expedited and adopted appropriately, BAC can solve many of the current problems. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Optimizing Conditions for the Purification of Omega-3 Fatty Acids from the By-product of Tuna Canning Processing

    Directory of Open Access Journals (Sweden)

    Teti Estiasih


    Full Text Available This research studied the optimization conditions for separation and purification of omega-3 (&omega-3 fatty acids from the by-product of tuna canning processing by urea crystallization. Crystallization reaction conditions of urea inclusion (urea to fatty acid ratio (X1 and crystallization time(X2 were optimized using the Response Surface Methodology (RSM and a model was developed. Optimization results showed a quadratic polynomial regression equation of Y = 24.44X1+5.65X2-8.71XX1 2-0.19X22+1.171X1X2- 12.95. The maximum response was obtained at an urea to fatty acid ratio of 2.99:1 and a crystallization time of 23.64 h and predicted response of 90.44%. Analysis of variance showed that the urea to fatty acid ratio and crystallization time affected the response. Verification under optimal conditions showed that the purity of &omega-3 fatty acids was 89.64% and the enrichment was 2.85 fold. Verification result revealed that the predicted value from this model was reasonably close to the experimentally observed value. The urea crystallization process changed oil quality parameters including oxidation level (peroxide, anisidin, and totox values, Fe, Cu and P concentrations and moisture content and this were mostly due to the saponification process before urea crystallization.

  18. Process for purification of monoclonal antibody expressed in transgenic Lemna plant extract using dextran-coated charcoal and hexamer peptide affinity resin. (United States)

    Naik, Amith D; Menegatti, Stefano; Reese, Hannah R; Gurgel, Patrick V; Carbonell, Ruben G


    The production of therapeutic proteins using transgenic plants offers several advantages, including low production cost, absence of human pathogens, presence of glycosylation mechanisms, and the ability to fold complex therapeutic proteins into their proper conformation. However, impurities such as phenolic compounds and pigments encountered during purification are quite different from those faced during purification from mammalian cell culture supernatants. This paper deals with the development of a pretreatment and affinity separation process for the purification of a monoclonal antibody from transgenic Lemna plant extract. A pretreatment step is described using dextran-coated charcoal for the removal of pigments and phenolic compounds without reducing the antibody concentration. Then, the peptide affinity ligand HWRGWV coupled to a commercial polymethacrylate resin is used for the capture and purification of MAb from the pretreated plant extract. The final yield and purity of the MAb obtained were 90% and 96% respectively. The performance of the hexamer peptide resin after the pretreatment step was found to be similar to that obtained with a commercial Protein A resin.

  19. Chlamydial plasmids and bacteriophages. (United States)

    Pawlikowska-Warych, Małgorzata; Śliwa-Dominiak, Joanna; Deptuła, Wiesław


    Chlamydia are absolute pathogens of humans and animals; despite being rather well recognised, they are still open for discovery. One such discovery is the occurrence of extrachromosomal carriers of genetic information. In prokaryotes, such carriers include plasmids and bacteriophages, which are present only among some Chlamydia species. Plasmids were found exclusively in Chlamydia (C.) trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum and C. caviae. In prokaryotic organisms, plasmids usually code for genes that facilitate survival of the bacteria in the environment (although they are not essential). In chlamydia, their role has not been definitely recognised, apart from the fact that they participate in the synthesis of glycogen and encode proteins responsible for their virulence. Furthermore, in C. suis it was evidenced that the plasmid is integrated in a genomic island and contains the tetracycline-resistance gene. Bacteriophages specific for chlamydia (chlamydiaphages) were detected only in six species: C. psittaci, C. abortus, C. felis, C. caviae C. pecorum and C. pneumoniae. These chlamydiaphages cause inhibition of the developmental cycle, and delay transformation of reticulate bodies (RBs) into elementary bodies (EBs), thus reducing the possibility of infecting other cells in time. Plasmids and bacteriophages can be used in the diagnostics of chlamydioses; although especially in the case of plasmids, they are already used for detection of chlamydial infections. In addition, bacteriophages could be used as therapeutic agents to replace antibiotics, potentially addressing the problem of increasing antibiotic-resistance among chlamydia.

  20. 我国湿法磷酸净化技术及其工业化进展%Development in Technology and Industry of Purification of Wet-process Phosphoric Acid in China

    Institute of Scientific and Technical Information of China (English)



    分析了湿法磷酸净化的主要技术及特点,介绍了我国湿法磷酸净化技术的产业化情况,提出了湿法磷酸净化技术的发展趋势和展望。%Main technical characteristics about purification of wet-process phosphoric acid were analyzed. The industrializations of wet-process phosphoric acid purification in China were introduced. Development trends and recommendations about the industry of wet-process acid purification were given.

  1. Sucrose purification and repeated ethanol production from sugars remaining in sweet sorghum juice subjected to a membrane separation process. (United States)

    Sasaki, Kengo; Tsuge, Yota; Kawaguchi, Hideo; Yasukawa, Masahiro; Sasaki, Daisuke; Sazuka, Takashi; Kamio, Eiji; Ogino, Chiaki; Matsuyama, Hideto; Kondo, Akihiko


    The juice from sweet sorghum cultivar SIL-05 (harvested at physiological maturity) was extracted, and the component sucrose and reducing sugars (such as glucose and fructose) were subjected to a membrane separation process to purify the sucrose for subsequent sugar refining and to obtain a feedstock for repeated bioethanol production. Nanofiltration (NF) of an ultrafiltration (UF) permeate using an NTR-7450 membrane (Nitto Denko Corporation, Osaka, Japan) concentrated the juice and produced a sucrose-rich fraction (143.2 g L(-1) sucrose, 8.5 g L(-1) glucose, and 4.5 g L(-1) fructose). In addition, the above NF permeate was concentrated using an ESNA3 NF membrane to provide concentrated permeated sugars (227.9 g L(-1)) and capture various amino acids in the juice, enabling subsequent ethanol fermentation without the addition of an exogenous nitrogen source. Sequential batch fermentation using the ESNA3 membrane concentrate provided an ethanol titer and theoretical ethanol yield of 102.5-109.5 g L(-1) and 84.4-89.6%, respectively, throughout the five-cycle batch fermentation by Saccharomyces cerevisiae BY4741. Our results demonstrate that a membrane process using UF and two types of NF membranes has the potential to allow sucrose purification and repeated bioethanol production.

  2. Application of a multiwalled carbon nanotube-chitosan composite as an electrode in the electrosorption process for water purification. (United States)

    Ma, Chih-Yu; Huang, Shih-Ching; Chou, Pei-Hsin; Den, Walter; Hou, Chia-Hung


    In this study, a multiwalled carbon nanotubes-chitosan (CNTs-CS) composite electrode was fabricated to enable water purification by electrosorption. The CNTs-CS composite electrode was shown to possess excellent capacitive behaviors and good pore accessibility by electrochemical impedance spectroscopy, galvanostatic charge-discharge, and cyclic voltammetry measurements in 1 M H2SO4 electrolyte. Moreover, the CNTs-CS composite electrode showed promising performance for capacitive water desalination. At an electric potential of 1.2 V, the electrosorption capacity and electrosorption rate of NaCl ions on the CNTs-CS composite electrode were determined to be 10.7 mg g(-1) and 0.051 min(-1), respectively, which were considerably higher than those of conventional activated electrodes. The improved electrosorption performance could be ascribed to the existence of mesopores. Additionally, the feasibility of electrosorptive removal of aniline from an aqueous solution has been demonstrated. Upon polarization at 0.6 V, the CNTs-CS composite electrode had a larger electrosorption capacity of 26.4 mg g(-1) and a higher electrosorption rate of 0.006 min(-1) for aniline compared with the open circuit condition. The enhanced adsorption resulted from the improved affinity between aniline and the electrode under electrochemical assistance involving a nonfaradic process. Consequently, the CNT-CS composite electrode, exhibiting typical double-layer capacitor behavior and a sufficient potential range, can be a potential electrode material for application in the electrosorption process.

  3. Preparation of core-shell Fe{sub 3}O{sub 4}/SiO{sub 2} microspheres as adsorbents for purification of DNA

    Energy Technology Data Exchange (ETDEWEB)

    Gai Ligang; Li Zhili; Jiang Haihui; Han Xiaoyun; Ma Wanyong [Shandong Provincial Key Laboratory of Fine Chemicals, School of Chemical Engineering, Shandong Institute of Light Industry, Jinan 250353 (China); Hou Yunhua, E-mail: [Shandong Provincial Key Laboratory of Microbial Engineering, School of Food Science and Bioengineering, Shandong Institute of Light Industry, Jinan 250353 (China)


    Nearly monodisperse core-shell Fe{sub 3}O{sub 4}/SiO{sub 2} microspheres have been prepared via a glycol reduction method followed by a modified Stoeber process. The thickness of the silica shells can be tuned in the range 33-53 nm by varying the amount of tetraethyl silicate (TEOS) during syntheses. The magnetic composite microspheres were characterized with XRD, XPS, FTIR, TEM, ICP-OES and VSM, and further tested as adsorbents for purification of plasmid DNA from Escherichia coli DH5{alpha} cells. The magnetic purification of plasmid DNA leads to satisfying integrity, yield and purity in comparison with those isolated by the traditional phenol-chloroform extraction.

  4. Permissiveness of soil microbial communities towards broad host range plasmids

    DEFF Research Database (Denmark)

    Klümper, Uli

    at high frequencies from diverse donors, I showed plasmid or donor dependence of plasmid transfer to other species. Additionally, environmental factors like stress also impact the permissiveness of phylogenetic groups towards plasmids. The developed method and results increase our ability to predict......Horizontal transfer of mobile genetic elements facilitates adaptive and evolutionary processes in bacteria. Among the known mobile genetic elements, plasmids can confer their hosts with accessory adaptive traits, such as antibiotic or heavy metal resistances, or additional metabolic pathways...... and the extent of bacterial phyla permissive towards plasmid receipt are largely unknown. Historically, methods exploring the underlying genetic and environmental factors of plasmid transfer have been heavily reliant on cultivation and expression of plasmid encoded phenotypes. This has provided an incomplete...

  5. Purification of heavy metal loaded wastewater from electroplating industry under synthesis of delafossite (ABO2) by "Lt-delafossite process". (United States)

    John, Melanie; Heuss-Aßbichler, Soraya; Ullrich, Aladin; Rettenwander, Daniel


    In this study we present a new, environmental friendly and economic method, called Lt-delafossite process to treat industrial wastewater (initial Cu(2+)-concentrations of 1-15.6 g/l) by subsequent synthesis of nano-crystalline (doped) delafossite (CuFeO2) solely by precipitation and ageing at temperatures between 50 °C and 90 °C. The reached water purification rates are exclusively ≥99.99% for both wastewater models and wastewaters from electroplating industry. We succeeded to synthesize a mixture of 3R and 2H delafossite at 50 °C after 90 h and ≥70 °C after 16 h of ageing directly from industrial wastewater without any additional phases. In all cases green rust (GR), a Fe(II-III) layered double hydroxysulphate, Cu2O (cuprite) and Fe10O14(OH)2 (ferrihydrite) precipitates first. During ageing of the residues the metastable phases transform to delafossite. The residues are characterized by XRD, FTIR, SEM, TEM, VFTB and Mößbauer measurements.

  6. Grafting of cellulose acetate with ionic liquids for biofuel purification by a membrane process: Influence of the cation. (United States)

    Hassan Hassan Abdellatif, Faten; Babin, Jérôme; Arnal-Herault, Carole; David, Laurent; Jonquieres, Anne


    A new strategy was developed for grafting ionic liquids (ILs) onto cellulose acetate in order to avoid IL extraction and improve its performance for ethyl tert-butyl ether (ETBE) biofuel purification by the pervaporation membrane process. This work extended the scope of IL-containing membranes to the challenging separation of organic liquid mixtures, in which these ILs were soluble. The ILs contained the same bromide anion and different cations with increasing polar feature. The membrane properties were strongly improved by IL grafting. Their analysis in terms of structure-property relationships revealed the influence of the IL content, chemical structure and chemical physical parameters α, β, π* in the Kamlet-Taft polarity scale. The ammonium IL led to the best normalized flux of 0.182kg/m(2)h for a reference thickness of 5μm, a permeate ethanol content of 100% and an outstanding infinite separation factor for the azeotropic mixture EtOH/ETBE at 50°C.

  7. Purification of a modified cyclosporine A by co-current centrifugal partition chromatography: process development and intensification. (United States)

    Amarouche, Nassima; Boudesocque, Leslie; Sayagh, Charlotte; Giraud, Matthieu; McGarrity, John; Butte, Alessandro; Marchal, Luc; Foucault, Alain; Renault, Jean-Hugues


    Synthetic hydrophobic non-ionizable peptides are not soluble in most common solvents and are thus difficult to purify by preparative reversed-phase HPLC, normally used for industrial production. The challenge exists to develop alternative purification chromatographic processes using suitable solvents and providing good yields, high purity and sufficient productivity. A 11mer hydrophobic synthetic modified cyclosporine, showing an anti-HIV activity, was successfully purified by centrifugal partition chromatography using the biphasic solvent system heptane/ethyl acetate/acetone/methanol/water (1:2:2:1:2, v/v). A 5% co-current elution - made possible by the liquid nature of the two phases - has been used in order to avoid hydrodynamic instabilities mainly due to the physico-chemical properties of the target peptide. This original solution was developed after the study of the effect of the peptide on the hydrodynamic behavior of the two phases during the separation, and the visualization of the flow patterns using the Visual-CPC device. Critical impurities were efficiently eliminated and the peptide was recovered in high yield and high productivity achieving the specifications requirements.


    The report gives results of a study evaluating the Polyad fluidized-bed (FB) process for controlling styrene emissions at a representative fiberglass shower stall and bath tub manufacturing plan*t. he process was evaluated using a transport able unit supplied by Weatherly, Inc., ...

  9. Model-based Rational and Systematic Protein Purification Process Development: A Knowledge-based Approach

    NARCIS (Netherlands)

    Kungah Nfor, B.


    The increasing market and regulatory (quality and safety) demands on therapeutic proteins calls for radical improvement in their manufacturing processes. Addressing these challenges requires the adoption of strategies and tools that enable faster and more efficient process development. This thesis i

  10. Caprylic acid-induced impurity precipitation from protein A capture column elution pool to enable a two-chromatography-step process for monoclonal antibody purification. (United States)

    Zheng, Ji; Wang, Lu; Twarowska, Barbara; Laino, Sarah; Sparks, Colleen; Smith, Timothy; Russell, Reb; Wang, Michelle


    This article presents the use of caprylic acid (CA) to precipitate impurities from the protein A capture column elution pool for the purification of monoclonal antibodies (mAbs) with the objective of developing a two chromatography step antibody purification process. A CA-induced impurity precipitation in the protein A column elution pool was evaluated as an alternative method to polishing chromatography techniques for use in the purification of mAbs. Parameters including pH, CA concentrations, mixing time, mAb concentrations, buffer systems, and incubation temperatures were evaluated on their impacts on the impurity removal, high-molecular weight (HMW) formation and precipitation step yield. Both pH and CA concentration, but not mAb concentrations and buffer systems, are key parameters that can affect host-cell proteins (HCPs) clearance, HMW species, and yield. CA precipitation removes HCPs and some HMW species to the acceptable levels under the optimal conditions. The CA precipitation process is robust at 15-25°C. For all five mAbs tested in this study, the optimal CA concentration range is 0.5-1.0%, while the pH range is from 5.0 to 6.0. A purification process using two chromatography steps (protein A capture column and ion exchange polishing column) in combination with CA-based impurity precipitation step can be used as a robust downstream process for mAb molecules with a broad range of isoelectric points. Residual CA can be effectively removed by the subsequent polishing cation exchange chromatography.

  11. Rapid isolation of plasmid DNA by LiCl-ethidium bromide treatment and gel filtration. (United States)

    Kondo, T; Mukai, M; Kondo, Y


    We established a simple and rapid plasmid DNA purification method. Crude plasmid DNA preparations are treated with 4 M LiCl in the presence of 0.6 mg/ml ethidium bromide to precipitate RNA and proteins contained in the DNA preparations. After removal of RNA and protein precipitates, the supernatant is filtered through a Sepharose CL6B column to remove low-molecular-weight contaminants. This procedure takes only 30 min and provides pure plasmid DNA preparations that consist mainly of covalently closed circular plasmid DNA but have no detectable RNA and protein. The purified DNA preparations are susceptible to various six- and four-base-recognition restriction endonucleases, T4 DNA ligase, the Klenow fragment of DNA polymerase I, and T7 and Taq DNA polymerase. Since no special equipment is needed for this purification method, 20 or more samples of microgram to milligram levels can be treated in parallel.

  12. Purification process for .sup.153Gd produced in natural europium targets

    Energy Technology Data Exchange (ETDEWEB)

    Johnsen, Amanda M; Soderquist, Chuck Z; McNamara, Bruce K; Risher, Darrell R


    An alteration of the traditional zinc/zinc-amalgam reduction procedure which eliminates both the hazardous mercury and dangerous hydrogen gas generation. In order to avoid the presence of water and hydrated protons in the working solution, which can oxidize Eu.sup.2+ and cause hydrogen gas production, a process utilizing methanol as the process solvent is described. While methanol presents some flammability hazard in a radiological hot cell, it can be better managed and is less of a flammability hazard than hydrogen gas generation.

  13. Processing and characterization of porous electrochemical cells for flue gas purification

    DEFF Research Database (Denmark)

    He, Zeming; Andersen, Kjeld Bøhm; Keel, Li;


    In the present work, porous electrode materials lanthanum strontium manganate (LSM)-gadolinium-doped cerium oxide (CGO) and electrochemical cells LSM-CGO + CGO were fabricated via the processes of slurry preparation, tape casting and lamination, and sintering. Graphite, wheat starch, and polyamide...

  14. The study of the process of alkaline precipitation purification of solutions from silicon macroscales in the ammonium-fluoride processing of beryllium-containing materials

    Directory of Open Access Journals (Sweden)

    Dyachenko Alexander


    Full Text Available The ammonium-fluoride method of beryllium materials processing is examined. An analysis of the existing sulfuric-acid and fluoride processing scheme of beryllium-containing concentrates processing is described; advantages of the proposed ammonium-fluoride scheme and possible problems that may occur when testing a new technique are discussed. Studies on determining the effect of silicon macroscales on the behavior of beryllium in fluoride solutions at an increased pH, as well as on establishing the distribution of beryllium and silicon by phases in laboratory testing of the technology on model mixtures, are described. As a result of the studies, it is found that precipitation purification of solutions from silicon with a solution of ammonia is possible at a temperature of 20-25 °C. The paper presents data on the effect of an excessive concentration of ion-fluoride in a solution on the pH level of silicon precipitation; and on the hydrolysis degree of beryllium and silicon during the precipitation.

  15. Evaluation of Effectiveness Technological Process of Water Purification Exemplified on Modernized Water Treatment Plant at Otoczna

    Directory of Open Access Journals (Sweden)

    Jordanowska Joanna


    Full Text Available The article presents the work of the Water Treatment Plant in the town of Otoczna, located in the Wielkopolska province, before and after the modernization of the technological line. It includes the quality characteristics of the raw water and treated water with particular emphasis on changes in the quality indicators in the period 2002 -2012 in relation to the physicochemical parameters: the content of total iron and total manganese, the ammonium ion as well as organoleptic parameters(colour and turbidity. The efficiency of technological processes was analysed, including the processes of bed start up with chalcedonic sand to remove total iron and manganese and ammonium ion. Based on the survey, it was found that the applied modernization helped solve the problem of water quality, especially the removal of excessive concentrations of iron, manganese and ammonium nitrogen from groundwater.

  16. Purification of wet process phosphoric acid by decreasing iron and uranium using white silica sand

    Energy Technology Data Exchange (ETDEWEB)

    El-Bayaa, A.A., E-mail: [Chemistry Department, Faculty of Science, Al-Azhar University (Girls), Nasr City, Cairo (Egypt); Badawy, N.A.; Gamal, A.M.; Zidan, I.H.; Mowafy, A.R. [Chemistry Department, Faculty of Science, Al-Azhar University (Girls), Nasr City, Cairo (Egypt)


    Natural white silica sand as an adsorbent has been developed to reduce the concentration of iron and uranium ions as inorganic impurities in crude Egyptian phosphoric acid. Several parameters such as adsorbate concentration, adsorbent dose, volume to weight ratio and temperature, were investigated. Equilibrium isotherm studies were used to evaluate the maximum sorption capacity of adsorbent. Thermodynamic parameters showed the exothermic nature of the process and the negative entropy reflects the affinity of the adsorbent material towards each metal ion.

  17. 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)


    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.

  18. Electron beam silicon purification

    Energy Technology Data Exchange (ETDEWEB)

    Kravtsov, Anatoly [SIA ' ' KEPP EU' ' , Riga (Latvia); Kravtsov, Alexey [' ' KEPP-service' ' Ltd., Moscow (Russian Federation)


    Purification of heavily doped electronic grade silicon by evaporation of N-type impurities with electron beam heating was investigated in process with a batch weight up to 50 kilos. Effective temperature of the melt, an indicative parameter suitable for purification process characterization was calculated and appeared to be stable for different load weight processes. Purified material was successfully approbated in standard CZ processes of three different companies. Each company used its standard process and obtained CZ monocrystals applicable for photovoltaic application. These facts enable process to be successfully scaled up to commercial volumes (150-300 kg) and yield solar grade silicon. (copyright 2014 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  19. Purification process for MUFA- and PUFA-based monoacylglycerols from edible oils. (United States)

    González-Fernández, M J; Ramos-Bueno, R P; Rodríguez-García, I; Guil-Guerrero, J L


    Important health benefits have been attributed to monoacylglycerols (MAGs) due to their various physiological functions, owing to which they become candidates for use as functional foods in order to prevent the onset of certain diseases such as colon cancer. In this work, six edible oils, namely: olive, linseed, sunflower, evening primrose, DHASCO(®) and ARASCO(®) have been processed to obtain different MUFA- and PUFA- based MAGs. First, the oils were hydrolyzed by means of an enzymatic process using porcine pancreatic lipase and then the reaction products were fractionated by using a liquid chromatography column containing silica gel as stationary phase in order to purify the MAGs-enriched fraction. A second chromatography process was performed using silver nitrate coated silica gel as stationary phase, in order to obtain the different MUFA- and PUFA-based MAGs from the corresponding oils. Overall, MAGs based on oleic, linoleic, α-linolenic, γ-linolenic, arachidonic and docosahexaenoic acids have been isolated in high yields and purities (92.6, 97.4, 95.3, 90.9, 100 and 95.3% purity, respectively). Positional distribution was determined by means of (1)H NMR, which revealed a mix of 1(3) and 2-MAGs in variable proportions in the different MAGs. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  20. Survey of processes for high temperature-high pressure gas purification. [52 references

    Energy Technology Data Exchange (ETDEWEB)

    Meyer, J.P.; Edwards, M.S.


    In order to ensure the optimum operating efficiency of a combined-cycle electric power generating system, it is necessary to provide gas treatment processes capable of operating at high temperatures (> 1000/sup 0/F) and high pressures (> 10 atm (absolute)). These systems will be required to condition the inlet stream to the gas turbine to suitable levels of gas purity (removal of particulate matter, sulfur, nitrogen, and alkali metal compounds) to be compatible with both environmental and machine constraints. A survey of the available and developmental processes for the removal of these various contaminant materials has been conducted. Based on the data obtained from a variety of sources, an analysis has been performed to evaluate the performance of a number of potential cleanup processes in view of the overall system needs. The results indicate that commercially available, reliable, and economically competitive hot-gas cleanup systems (for the removal of H/sub 2/S, particulate matter, alkali, and nitrogen compounds) capable of conditioning raw product gas to the levels required for turbine use will not be available for some time.

  1. Chemical hot gas purification for biomass gasification processes; Chemische Heissgasreinigung bei Biomassevergasungsprozessen

    Energy Technology Data Exchange (ETDEWEB)

    Stemmler, Michael


    The German government decided to increase the percentage of renewable energy up to 20 % of all energy consumed in 2020. The development of biomass gasification technology is advanced compared to most of the other technologies for producing renewable energy. So the overall efficiency of biomass gasification processes (IGCC) already increased to values above 50 %. Therefore, the production of renewable energy attaches great importance to the thermochemical biomass conversion. The feedstock for biomass gasification covers biomasses such as wood, straw and further energy plants. The detrimental trace elements released during gasification of these biomasses, e.g. KCl, H{sub 2}S and HCl, cause corrosion and harm downstream devices. Therefore, gas cleaning poses an especial challenge. In order to improve the overall efficiency this thesis aims at the development of gas cleaning concepts for the allothermic, water blown gasification at 800 C and 1 bar (Guessing-Process) as well as for the autothermic, water and oxygen blown gasification at 950 C and 18 bar (Vaernamo-Process). Although several mechanisms for KCl- and H{sub 2}S-sorption are already well known, the achievable reduction of the contamination concentration is still unknown. Therefore, calculations on the produced syngas and the chemical hot gas cleaning were done with a thermodynamic process model using SimuSage. The syngas production was included in the calculations because the knowledge of the biomass syngas composition is very limited. The results of these calculations prove the dependence of syngas composition on H{sub 2}/C-ratio and ROC (Relative Oxygen Content). Following the achievable sorption limits were detected via experiments. The KCl containing syngases were analysed by molecular beam mass spectrometry (MBMS). Furthermore, an optimised H{sub 2}S-sorbent was developed because the examined sorbents exceeded the sorption limit of 1 ppmv. The calculated sorption limits were compared to the limits

  2. Separation and Purification of Mineral Salts from Spacecraft Wastewater Processing via Electrostatic Beneficiation (United States)

    Miles, John D., II; Lunn, Griffin


    Electrostatic separation is a class of material processing technologies commonly used for the sorting of coarse mixtures by means of electrical forces acting on charged or polarized particles. Most if not all of the existing tribo-electrostatic separators had been initially developed for mineral ores beneficiation. It is a well-known process that has been successfully used to separate coal from minerals. Potash (potassium) enrichment where underground salt mines containing large amounts of sodium is another use of this techno logy. Through modification this technology can be used for spacecraft wastewater brine beneficiation. This will add in closing the gap beeen traveling around Earth's Gravity well and long-term space explorations. Food has been brought on all man missions, which is why plant growth for food crops continues to be of interest to NASA. For long-term mission considerations food productions is one of the top priorities. Nutrient recovery is essential for surviving in or past low earth orbit. In our advance bio-regenerative process instead of nitrogen gas produced; soluble nitrate salts that can be recovered for plant fertilizer would be produced instead. The only part missing is the beneficiation of brine to separate the potassium from the sodium. The use of electrostatic beneficiation in this experiment utilizes the electrical charge differences between aluminum and dried brine by surface contact. The helixes within the aluminum tribocharger allows for more surface contact when being agitated. When two materials are in contact, the material with the highest affinity for electrons becomes negatively charged, while the other becomes positively charged. This contact exchange of charge may cause the particles to agglomerate depending on their residence time within the tribocharger, compromising the efficiency of separation. The aim of this experiment is to further the development in electrostatic beneficiation by optimizing the separation of ersatz and

  3. Purification Process of Lake Water%湖水净化处理工艺

    Institute of Scientific and Technical Information of China (English)

    黄亮; 赵子玲


    某人工湖水由于富营养成分较多,致使水体透明度下降,甚至有臭味散发。利用“过滤+消毒”的湖水净化组合工艺,使湖水经处理后可以湖内循环回用。%A artificial lake due to the more nutritious ingredients, resulting in water transparency decreased, and even smell distributing. By the means of combination decontaminate process of filter and disinfection, the lake water will be 1oo13 back to the lake.

  4. Mechanisms of plasmid segregation: have multicopy plasmids been overlooked? (United States)

    Million-Weaver, Samuel; Camps, Manel


    Plasmids are self-replicating pieces of DNA typically bearing non-essential genes. Given that plasmids represent a metabolic burden to the host, mechanisms ensuring plasmid transmission to daughter cells are critical for their stable maintenance in the population. Here we review these mechanisms, focusing on two active partition strategies common to low-copy plasmids: par systems type I and type II. Both involve three components: an adaptor protein, a motor protein, and a centromere, which is a sequence area in the plasmid that is recognized by the adaptor protein. The centromere-bound adaptor nucleates polymerization of the motor, leading to filament formation, which can pull plasmids apart (par I) or push them towards opposite poles of the cell (par II). No such active partition mechanisms are known to occur in high copy number plasmids. In this case, vertical transmission is generally considered stochastic, due to the random distribution of plasmids in the cytoplasm. We discuss conceptual and experimental lines of evidence questioning the random distribution model and posit the existence of a mechanism for segregation in high copy number plasmids that moves plasmids to cell poles to facilitate transmission to daughter cells. This mechanism would involve chromosomally-encoded proteins and the plasmid origin of replication. Modulation of this proposed mechanism of segregation could provide new ways to enhance plasmid stability in the context of recombinant gene expression, which is limiting for large-scale protein production and for bioremediation.

  5. Plasmid-to-plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.


    No recombination between plasmids was observed after conjugal transfer of a plasmid into a cell carrying another plasmid. Two types of such recombination took place after transformation, one type being Rec/sup +/ dependent and suggesting a preferred site of recombination. The other much rarer type was at least partially Rec/sup +/ independent.

  6. Plasmid interference for curing antibiotic resistance plasmids in vivo. (United States)

    Kamruzzaman, Muhammad; Shoma, Shereen; Thomas, Christopher M; Partridge, Sally R; Iredell, Jonathan R


    Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing ('addiction') systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative 'interference plasmids' were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored.

  7. The use of ultrafiltration and nanofiltration membranes for the purification of cork processing wastewater. (United States)

    Benítez, F Javier; Acero, Juan L; Leal, Ana I; González, Manuel


    Filtration experiments in batch concentration mode (with recycling of the retentate stream) of the cork processing wastewater were performed in laboratory filtration membrane equipment, by using four commercial membranes: two UF membranes with MWCO of 20,000 and 5000 Da, and two NF membranes with an approximate MWCO of 150-300 Da. The filtration experiments of the selected wastewater were performed by modifying the most important operating variables: transmembrane pressure, tangential velocity, temperature, and the nature and MWCO of the membranes. The evolution of the cumulative permeate volumes and permeate fluxes with processing time were analysed, and it was established that the steady-state permeate flux was reached for a volume retention factor of 2. The effect of the mentioned operating conditions on this steady-state permeate flux was discussed. The effectiveness of the filtration treatments was determined by the evaluation of the rejection coefficients for several parameters, which measure the global pollutant content of the effluent: COD, absorbance at 254 nm, tannic content, color, and ellagic acid. Finally, the resistances in series model was used for the evaluation of the resistances to the permeate flux, and it was concluded that the contribution to the total resistance of the fouling resistance (combined external plus internal) was higher than the inherent resistance of the clean membrane.

  8. [Purification of the wastewater of quartz processing by mineral-based porous granulation material]. (United States)

    Wang, En-Wen; Lei, Shao-Min; Zhang, Shi-Chun; Huang, Teng


    A mineral-based porous granulation material (MPGM) was prepared for absorbing the heavy metal ions from quartz processing wastewater. Analytic results of the MPGM were illustrated by the techniques of BET, SEM and FT-IR, which revealed the excellent properties of multi-aperture distribution, large specific surface area, low loss ratio and so on; the N2 adsorption-desorption isotherm was type-III with H4 hysteresis loop; the functional groups were dominated by groups of layer silicate mineral. A batch adsorption study was carried out with varied adsorbent dosage, initial pH and reaction time. The results showed that the residual ion concentrations of Fe, Zn, Mn and As were decreased from 77.760, 3.700, 2.789 and 0.963 mg x L(-1) to 3.421, 0.574, 0.126 and 0.034 mg x L(-1), respectively. MPGM might be re-utilized after desorption by 1.0 mol x L(-1) NaCl for 12 hours, and the ideal adsorption performance was maintained after 5 recyclings. The adsorption equilibrium and kinetics followed Langmuir and typical pseudo-first-order/pseudo-second-order adsorption models. Thermodynamic parameters of ΔG(θ) 0, ΔSθ > 0 showed that the adsorptions were spontaneous and endothermic processes in the temperature range of 15-45 degrees C.

  9. Ionic behavior of treated water at a water purification plant


    Yanagida, Kazumi; Kawahigashi, Tatsuo


    [Abstract] Water at each processing stage in a water purification plant was extracted and analyzed to investigate changes of water quality. Investigations of water at each processing stage at the water purification plant are discussed herein.

  10. Ionic behavior of treated water at a water purification plant


    Yanagida, Kazumi; Kawahigashi, Tatsuo


    [Abstract] Water at each processing stage in a water purification plant was extracted and analyzed to investigate changes of water quality. Investigations of water at each processing stage at the water purification plant are discussed herein.

  11. Optimization of purification processes to remove polycyclic aromatic hydrocarbons (PAHs) in polluted raw fish oils. (United States)

    Yebra-Pimentel, Iria; Fernández-González, Ricardo; Martínez-Carballo, Elena; Simal-Gándara, Jesús


    Fish oils are one of the main sources of health promoting nutrients such as n-3 fatty acids in animal and human diet. Nevertheless, they could be an important source of persistent organic pollutants (POPs). Different strategies of decontamination processes to reduce polycyclic aromatic hydrocarbon (PAH) levels in fish oils, such as solvent extraction (ethanol) and adsorbent extraction using commercially available (activated carbon) and sustainable adsorbents (mussel shell and wood ashes), were compared. Adsorption conditions were evaluated and optimized by an experimental design and the experimental results were adjusted to response surfaces. In this way, PAH removals increased with increasing of individual PAH molecular weight and they range from 80% to 100% using activated carbon and from 10% to 100% using wood ashes. Pine wood ashes showed similar removal rates to activated carbon (87%-100%) excluding F (51%) and P (42%). No PAH removal was observed using mussel shell ashes. Ethanol extraction was also optimized and showed a good performance in the extraction of PAHs. However, it does affect their ω-3 fatty acid contents. Finally, real oil samples from different fishing areas: Spain, South America, and North Europe were selected for the decontamination experiments under experimental conditions previously optimized.

  12. Optimization of electrocoagulation (EC) process for the purification of a real industrial wastewater from toxic metals. (United States)

    Gatsios, Evangelos; Hahladakis, John N; Gidarakos, Evangelos


    In the present work, the efficiency evaluation of electrocoagulation (EC) in removing toxic metals from a real industrial wastewater, collected from Aspropyrgos, Athens, Greece was investigated. Manganese (Mn), copper (Cu) and zinc (Zn) at respective concentrations of 5 mg/L, 5 mg/L and 10 mg/L were present in the wastewater (pH=6), originated from the wastes produced by EBO-PYRKAL munitions industry and Hellenic Petroleum Elefsis Refineries. The effect of operational parameters such as electrode combination and distance, applied current, initial pH and initial metal concentration, was studied. The results indicated that Cu and Zn were totally removed in all experiments, while Mn exhibited equally high removal percentages (approximately 90%). Decreasing the initial pH and increasing the distance between electrodes, resulted in a negative effect on the efficiency and energy consumption of the process. On the other hand, increasing the applied current, favored metal removal but resulted in a power consumption increase. Different initial concentrations did not affect metal removal efficiency. The optimal results, regarding both cost and EC efficiency, were obtained with a combination of iron electrodes, at 2 cm distance, at initial current of 0.1 A and pH=6. After 90 min of treatment, maximum removal percentages obtained were 89% for Mn, 100% for Cu and 100% for Zn, at an energy consumption of 2.55 kWh/m(3).

  13. Unified Model of Purification Units in Hydrogen Networks

    Institute of Scientific and Technical Information of China (English)

    吴思东; 王彧斐; 冯霄


    Purification processes are widely used in hydrogen networks of refineries to increase hydrogen reuse. In refineries, hydrogen purification techniques include hydrocarbon, hydrogen sulfide and CO removal units. In addi-tion, light hydrocarbon recovery from the hydrogen source streams can also result in hydrogen purification. In order to simplify the superstructure and mathematical model of hydrogen network integration, the models of different pu-rification processes are unified in this paper, including mass balance and the expressions for hydrogen recovery and impurity removal ratios, which are given for all the purification units in refineries. Based on the proposed unified model, a superstructure of hydrogen networks with purification processes is constructed.

  14. Plasmid interference for curing antibiotic resistance plasmids in vivo (United States)

    Kamruzzaman, Muhammad; Shoma, Shereen; Thomas, Christopher M.; Partridge, Sally R.


    Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing (‘addiction’) systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative ‘interference plasmids’ were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored. PMID:28245276

  15. Continuous processing of recombinant proteins: integration of refolding and purification using simulated moving bed size-exclusion chromatography with buffer recycling. (United States)

    Wellhoefer, Martin; Sprinzl, Wolfgang; Hahn, Rainer; Jungbauer, Alois


    Continuous processing of recombinant proteins was accomplished by combining continuous matrix-assisted refolding and purification by tandem simulated moving bed (SMB) size-exclusion chromatography (SEC). Recombinant proteins, N(pro) fusion proteins from inclusion bodies were dissolved with NaOH and refolded in the SMB system with a closed-loop set-up with refolding buffer as the desorbent buffer and buffer recycling of the refolding buffer of the raffinate by tangential flow filtration. For further purification of the refolded proteins, a second SMB operation also based on SEC was added. The whole system could be operated isocratically with refolding buffer as the desorbent buffer, and buffer recycling could also be applied in the purification step. Thus, a significant reduction in buffer consumption was achieved. The system was evaluated with two proteins, the N(pro) fusion pep6His and N(pro) fusion MCP-1. Refolding solution, which contained residual N(pro) fusion peptide, the cleaved autoprotease N(pro), and the cleaved target peptide was used as feed solution. Full separation of the cleaved target peptide from residual proteins was achieved at a purity and recovery in the raffinate and extract, respectively, of approximately 100%. In addition, more than 99% of the refolding buffer of the raffinate was recycled. A comparison of throughput, productivity, and buffer consumption of the integrated continuous process with two batch processes demonstrated that up to 60-fold higher throughput, up to 180-fold higher productivity, and at least 28-fold lower buffer consumption can be obtained by the integrated continuous process, which compensates for the higher complexity. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Effect of plasmid R391 and other IncJ plasmids on the survival of Escherichia coli after UV irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Pembroke, J.T.; Stevens, E. (University Coll., Galway (Ireland))


    The presence of the IncJ plasmids R391, R997, R705, R706, R748, and R749 was shown to sensitize Escherichia coli AB1157 and both its uvr A and lexA derivatives to UV irradiation. No alteration in post-irradiation survival was observed in a recA mutant containing these plasmids, compared with the non-plasmid-containing recA strain. Analysis of recombination frequency in Hfr crosses to recA/sup +/ cells containing plasmid R391 indicated a reduction in recombination frequency compared with that obtained in similar crosses to a non-plasmid-containing strain. This effect was not due to plasmid-encoded restriction or entry exclusion systems and therefore must be considered as a real block in recombination. When cells containing plasmid R391 were irradiated and allowed to photoreactivate, an increase in survival was observed which was comparable to that observed in the non-plasmid-containing derivative. This indicated that post-irradiation processing of UV-induced damage, or lack of such processing, by mechanisms other than photoreactivation was responsible for the UV sensitivity associated with plasmid R391.

  17. Genomic comparison of archaeal conjugative plasmids from Sulfolobus

    DEFF Research Database (Denmark)

    Greve, Bo Bjørn


    All of the known self-transmissable plasmids of the Archaea have been found in the genus Sulfolobus. To gain more insight into archaeal conjugative processes, four newly isolated self-transmissable plasmids, pKEF9, pHVE14, pARN3 and pARN4, were sequenced and subjected to a comparative sequence...

  18. Production and pharmaceutical formulation of plasmid DNA vaccines

    NARCIS (Netherlands)

    van der Heijden, I.


    Research leading to the thesis ‘Production and pharmaceutical formulation of plasmid DNA vaccines‘ can be divided into two parts. The first part describes the development of a Good Manufacturing Practice (GMP) compliant plasmid DNA production process of pDNA vaccines for the treatment of Human papil

  19. Production and pharmaceutical formulation of plasmid DNA vaccines

    NARCIS (Netherlands)

    van der Heijden, I.


    Research leading to the thesis ‘Production and pharmaceutical formulation of plasmid DNA vaccines‘ can be divided into two parts. The first part describes the development of a Good Manufacturing Practice (GMP) compliant plasmid DNA production process of pDNA vaccines for the treatment of Human papil

  20. The SELEX Air Purification System (United States)


    REPORT Final Report for the SELEX Air Purification System 14. ABSTRACT 16. SECURITY CLASSIFICATION OF: A new air purification technology ( SELEX ) was...developed and demonstrated. The SELEX system utilizes an array of electrospray wick aerosol sources for particle ionization and an electrostatic...precipitator for particle collection. The particle ionization process does not produce ozone and the SELEX technology provides a unique combination of

  1. Extraction and purification of high added value compounds from by-products of the winemaking chain using alternative/nonconventional processes/technologies. (United States)

    Yammine, Sami; Brianceau, Sylène; Manteau, Sébastien; Turk, Mohammad; Ghidossi, Rémy; Vorobiev, Eugène; Mietton-Peuchot, Martine


    Grape byproducts are today considered as a cheap source of valuable compounds since existent technologies allow the recovery of target compounds and their recycling. The goal of the current article is to explore the different recovery stages used by both conventional and alternative techniques and processes. Alternative pre-treatments techniques reviewed are: ultrasounds, pulsed electric fields and high voltage discharges. In addition, nonconventional solvent extraction under high pressure, specifically, supercritical fluid extraction and subcritical water extraction are discussed. Finally alternative purification technologies, for example membrane processing were also examined. The intent is to describe the mechanisms involved by these alternative technologies and to summarize the work done on the improvement of the extraction process of phenolic compounds from winery by-products. With a focus on the developmental stage of each technology, highlighting the research need and challenges to be overcome for an industrial implementation of these unitary operations in the overall extraction process. A critical comparison of conventional and alternative techniques will be reviewed for ethe pre-treatment of raw material, the diffusion of polyphenols and the purification of these high added value compounds. This review intends to give the reader some key answers (costs, advantages, drawbacks) to help in the choice of alternative technologies for extraction purposes.

  2. Abundance and diversity of ammonia-oxidizing archaea and bacteria on granular activated carbon and their fates during drinking water purification process. (United States)

    Niu, Jia; Kasuga, Ikuro; Kurisu, Futoshi; Furumai, Hiroaki; Shigeeda, Takaaki; Takahashi, Kazuhiko


    Ammonia is a precursor to trichloramine, which causes an undesirable chlorinous odor. Granular activated carbon (GAC) filtration is used to biologically oxidize ammonia during drinking water purification; however, little information is available regarding the abundance and diversity of ammonia-oxidizing archaea (AOA) and bacteria (AOB) associated with GAC. In addition, their sources and fates in water purification process remain unknown. In this study, six GAC samples were collected from five full-scale drinking water purification plants in Tokyo during summer and winter, and the abundance and community structure of AOA and AOB associated with GAC were studied in these two seasons. In summer, archaeal and bacterial amoA genes on GACs were present at 3.7 × 10(5)-3.9 × 10(8) gene copies/g-dry and 4.5 × 10(6)-4.2 × 10(8) gene copies/g-dry, respectively. In winter, archaeal amoA genes remained at the same level, while bacterial amoA genes decreased significantly for all GACs. No differences were observed in the community diversity of AOA and AOB from summer to winter. Phylogenetic analysis revealed high AOA diversity in group I.1a and group I.1b in raw water. Terminal-restriction fragment length polymorphism analysis of processed water samples revealed that AOA diversity decreased dramatically to only two OTUs in group I.1a after ozonation, which were identical to those detected on GAC. It suggests that ozonation plays an important role in determining AOA diversity on GAC. Further study on the cell-specific activity of AOA and AOB is necessary to understand their contributions to in situ nitrification performance.

  3. Development of production and purification processes of recombinant fragment of pneumococcal surface protein A in Escherichia coli using different carbon sources and chromatography sequences. (United States)

    Carvalho, Rimenys Junior; Cabrera-Crespo, Joaquin; Tanizaki, Martha Massako; Gonçalves, Viviane Maimoni


    Pneumococcal surface protein A (PspA) is essential for Streptococcus pneumoniae virulence and its use either as a novel pneumococcal vaccine or as carrier in a conjugate vaccine would improve the protection and the coverage of the vaccine. Within this context, the development of scalable production and purification processes of His-tagged recombinant fragment of PspA from clade 3 (rfPspA3) in Escherichia coli BL21(DE3) was proposed. Fed-batch production was performed using chemically defined medium with glucose or glycerol as carbon source. Although the use of glycerol led to lower acetate production, the concentration of cells were similar at the end of both fed-batches, reaching high cell density of E. coli (62 g dry cell weight/L), and the rfPspA3 production was higher with glucose (3.48 g/L) than with glycerol (2.97 g/L). A study of downstream process was also carried out, including cell disruption and clarification steps. Normally, the first chromatography step for purification of His-tagged proteins is metal affinity. However, the purification design using anion exchange followed by metal affinity gave better results for rfPspA3 than the opposite sequence. Performing this new design of chromatography steps, rfPspA3 was obtained with 95.5% and 75.9% purity, respectively, from glucose and glycerol culture. Finally, after cation exchange chromatography, rfPspA3 purity reached 96.5% and 90.6%, respectively, from glucose and glycerol culture, and the protein was shown to have the expected alpha-helix secondary structure.

  4. Single-step purification of recombinant proteins using elastin-like peptide-mediated inverse transition cycling and self-processing module from Neisseria meningitides FrpC. (United States)

    Liu, Wen-Jun; Wu, Qian; Xu, Bi; Zhang, Xin-Yu; Xia, Xiao-Li; Sun, Huai-Chang


    Purification of recombinant proteins is a major task and challenge in biotechnology and medicine. In this paper we report a novel single-step recombinant protein purification system which was based on elastin-like peptide (ELP)-mediated reversible phase transition and FrpC self-processing module (SPM)-mediated cleavage. After construction of a SPM-ELP fusion expression vector, we cloned the coding sequence for green fluorescent protein (GFP), the Fc portion of porcine IgG (pFc) or human β defensin 3 (HBD3) into the vector, transformed the construct into Escherichia coli, and induced the fusion protein expression with IPTG. The target-SPM-ELP fusion proteins GFP-SPM-ELP, Fc-SPM-ELP and HBD3-SPM-ELP were expressed in a soluble form and efficiently purified from the clarified cell extracts by two rounds of inverse transition cycling (ITC). Under the optimized conditions, the SPM-mediated cleavage efficiencies for the three fusion proteins ranged from 92% to 93%. After an additional round of ITC, the target proteins GFP, pFc and HBD3 were recovered with purities ranging from 90% to 100% and yields ranging from 1.1 to 36mg/L in shake flasks. The endotoxin levels in all of the three target proteins were proteins were functionally active with the expected molecular weights. These experimental results confirmed the high specificity and efficiency of SPM-mediated cleavage, and suggested the applicability of SPM-ELP fusion system for purification of recombinant proteins.

  5. Toxin Plasmids of Clostridium perfringens (United States)

    Li, Jihong; Adams, Vicki; Bannam, Trudi L.; Miyamoto, Kazuaki; Garcia, Jorge P.; Uzal, Francisco A.; Rood, Julian I.


    SUMMARY In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  6. Design and optimization of a chromatographic purification process for Streptococcus pneumoniae serotype 23F capsular polysaccharide by a Design of Experiments approach. (United States)

    Ji, Yu; Tian, Yang; Ahnfelt, Mattias; Sui, Lili


    Multivalent pneumococcal vaccines were used worldwide to protect human beings from pneumococcal diseases. In order to eliminate the toxic organic solutions used in the traditional vaccine purification process, an alternative chromatographic process for Streptococcus pneumoniae serotype 23F capsular polysaccharide (CPS) was proposed in this study. The strategy of Design of Experiments (DoE) was introduced into the process development to solve the complicated design procedure. An initial process analysis was given to review the whole flowchart, identify the critical factors of chromatography through FMEA and chose the flowthrough mode due to the property of the feed. A resin screening study was then followed to select candidate resins. DoE was utilized to generate a resolution IV fractional factorial design to further compare candidates and narrow down the design space. After Capto Adhere was selected, the Box-Behnken DoE was executed to model the process and characterize all effects of factors on the responses. Finally, Monte Carlo simulation was used to optimize the process, test the chosen optimal conditions and define the control limit. The results of three scale-up runs at set points verified the DoE and simulation predictions. The final results were well in accordance with the EU pharmacopeia requirements: Protein/CPS (w/w) 1.08%; DNA/CPS (w/w) 0.61%; the phosphorus content 3.1%; the nitrogen 0.315% and the Methyl-pentose percentage 47.9%. Other tests of final pure CPS also met the pharmacopeia specifications. This alternative chromatographic purification process for pneumococcal vaccine without toxic organic solvents was successfully developed by the DoE approach and proved scalability, robustness and suitability for large scale manufacturing.

  7. Optimization of Purification Process of Polysaccharide in Compound Qianyu Water Decoction%复方前愈水煎液中多糖的纯化∗

    Institute of Scientific and Technical Information of China (English)

    李梓盟; 张柯达; 王晖; 吴金虎


    目的:优选复方前愈水煎液中纯化多糖最佳工艺条件。方法采用水提醇沉、复溶、Sevag法除蛋白、透析法进行纯化,以苯酚-硫酸分光光度法测定多糖纯度,在单因素实验基础上,通过正交实验考察复溶固液比、除蛋白次数、透析时间等条件对复方前愈多糖纯度的影响。结果复方前愈多糖最佳纯化工艺条件为:固液比为1∶40( g/mL,W/V)、脱蛋白10次、透析18 h,在此条件下纯化多糖纯度可达69.04%,转移率51.84%。结论优选的纯化工艺流程简便易行,可用于复方前愈水煎剂中多糖的纯化。%Objective To optimize the technological conditions of polysaccharide purification from compound Qianyu water decoction. Methods Water extraction and alcohol precipitation, resolution, Sevag method and dialysis method were used to purify polysaccharide.The purity of polysaccharide was measured with the phenol-sulfuric acid spectrophotometry.On the basis of single factor test, effects of redissolved solid-liquid ratio, number of protein removal, and dialytic time on polysaccharide purity of Qianyu were investigated by orthogonal test. Results The best conditions for purification of polysaccharide in Qianyu were as follows: liquid-solid ratio was 1:40(g/mL, W/V), remove protein for 10 times, and dialysis for 18 h.The content of polysaccharide could reach 69.04%, and the transfer rate was 51.84%. Conclusion The optimized purification process was simple and accurate.It can be used for polysaccharide purification in compound Qianyu water decoction.

  8. Conjugative plasmids of Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Emilia Pachulec

    Full Text Available Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones and with and without different tetM determinants (Dutch and American type tetM determinants have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233 or containing Dutch (pEP5289 or American (pEP5050 type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1alpha, beta, gamma, delta and epsilon subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids

  9. Hydrogen purification by periodic adsorption

    Energy Technology Data Exchange (ETDEWEB)

    Barg, Christian; Secchi, Argimiro R.; Trierweiler, Jorge O. [Rio Grande do Sul Univ., Porto Alegre, RS (Brazil). Dept. de Engenharia Quimica]. E-mail:;;


    The periodic adsorption processes have been widely used for industrial applications, mainly because it spends less energy than the usual gas separation processes, like the cryogenic distillation. The largest commercial application of periodic adsorption processes is the pressure swing adsorption (PSA) applied to hydrogen purification. Although its wide use in the chemical and petrochemical industry, there are no reports in the open literature about complete modeling studies of a complex commercial unit, with multiple adsorbents and multiple beds and several feed components. This study has as objective the modeling, optimization and dynamical analysis of an industrial PSA unit for hydrogen purification. (author)

  10. Optimisation of the extraction and purification of chondroitin sulphate from head by-products of Prionace glauca by environmental friendly processes. (United States)

    Vázquez, José Antonio; Blanco, María; Fraguas, Javier; Pastrana, Lorenzo; Pérez-Martín, Ricardo


    The goal of the present work was to optimise the different environmental friendly processes involved in the extraction and purification of chondroitin sulphate (CS) from Prionace glauca head wastes. The experimental development was based on second order rotatable designs and evaluated by response surface methodology combined with a previous kinetic approach. The sequential stages optimised were: (1) the enzymatic hydrolysis of head cartilage catalysed by alcalase (55.7 °C/pH 8.2); (2) the chemical treatment of enzyme hydrolysates by means of alkaline-hydroalcoholic saline solutions (NaOH: 0.54 M, EtOH: 1.17 v, NaCl: 2.5%) to end the protein hydrolysis and to precipitate and selectively redissolve CS versus the peptidic material and (3) the selective purification and concentration of CS and the concomitant protein permeation of extracts which were obtained from previous treatment using ultrafiltration and diafiltration (UF-DF) technologies at two different cut-offs.

  11. Optimization of the purification process of Haemophilus influenzae type b polysaccharide%b型流感嗜血杆菌多糖纯化工艺的优化

    Institute of Scientific and Technical Information of China (English)

    王伟; 马雷钧; 沈坚; 陈晓怿; 马相虎


    目的 优化b型流感嗜血杆菌(Haemophilus influenzae type b,Hib)多糖纯化工艺,替代传统的苯酚结合乙醇方法.方法 采用十六烷基三甲基溴化铵提取粗糖,以脱氧胆酸钠结合乙醇多级纯化进一步纯化粗糖,通过超滤浓缩和除菌过滤获得纯化的Hib多糖.采用优化的纯化工艺以中试和放大规模各纯化3批Hib多糖,按照《中华人民共和国药典》2010年版三部的要求检测纯化的Hib多糖.结果 中试和放大规模纯化的各3批Hib多糖的相对分子质量分别为621 800、634 400、659 900和597 200、612 100、583 400,均符合规定的标准.中试和放大规模纯化的各3批Hib多糖的细菌内毒素含量分别为2.0、0.5、0.7 EU/μg和4.0、2.0、1.0 EU/μg,均明显低于规定的标准.鉴别试验显示,中试和放大规模纯化的各批Hib多糖均可与标准Hib抗血清形成明显的沉淀线.结论 优化的Hib多糖纯化工艺具有较好的稳定性,且易于工艺放大,可替代传统工艺用于Hib多糖的纯化.%Objective To optimize the purification process of Haemophilus influenzae type b (Hib) polysaccharide for replacing the traditional combination of phenol and ethanol.Methods Crude Hib polysaccharides were extracted with Cetavlon and further purified with the combination of sodium deoxycholate and ethanol multistage purification,and purified polysaccharides were obtained by ultrafiltration and sterilizing filtration.Three batches of purified Hib polysaccharides were prepared with the optimized purification precess in pilot and other 3 batches in amplifying scales.Prepared Hib polysaccharides were detected in accordance with the Chinese Pharmacopoeia 2010 edition (Volume Ⅲ).Results The relative molecular masses of each 3 batches of Hib polysaccharides purified in pilot and amplifying scales were 621 800,634 400,659 900 and 597 200,612 100,583 400,respectively,and all met standard requirement.The contents of bacterial endotoxin of each 3 batches

  12. Evaluation of microbial triglyceride oil purification requirements for the CelTherm process: an efficient biochemical pathway to renewable fuels and chemicals. (United States)

    Linnen, Michael; Seames, Wayne; Kubatova, Alena; Menon, Suresh; Alisala, Kashinatham; Hash, Sara


    CelTherm is a biochemical process to produce renewable fuels and chemicals from lignocellulosic biomass. The present study's objective was to determine the level of treatment/purity of the microbial triacylglyceride oil (TAG) necessary to facilitate fuel production. After a unique microbe aerobically synthesizes TAG from biomass-derived sugars, the microbes were harvested and dried then crude TAG was chemically extracted from the residual biomass. Some TAGs were further purified to hydrotreating process requirements. Both grades were then noncatalytically cracked into a petroleum-like intermediate characterized by gas chromatography. Experiments were repeated using refined soybean oil for comparison to previous studies. The products from crude microbial TAG cracking were then further refined into a jet fuel product. Fuel tests indicate that this jet fuel corresponds to specifications for JP-8 military turbine fuel. It was thus concluded that the crude microbial TAG is a suitable feedstock with no further purification required, demonstrating CelTherm's commercial potential.

  13. Phenotypic plasticity in bacterial plasmids. (United States)

    Turner, Paul E


    Plasmid pB15 was previously shown to evolve increased horizontal (infectious) transfer at the expense of reduced vertical (intergenerational) transfer and vice versa, a key trade-off assumed in theories of parasite virulence. Whereas the models predict that susceptible host abundance should determine which mode of transfer is selectively favored, host density failed to mediate the trade-off in pB15. One possibility is that the plasmid's transfer deviates from the assumption that horizontal spread (conjugation) occurs in direct proportion to cell density. I tested this hypothesis using Escherichia coli/pB15 associations in laboratory serial culture. Contrary to most models of plasmid transfer kinetics, my data show that pB15 invades static (nonshaking) bacterial cultures only at intermediate densities. The results can be explained by phenotypic plasticity in traits governing plasmid transfer. As cells become more numerous, the plasmid's conjugative transfer unexpectedly declines, while the trade-off between transmission routes causes vertical transfer to increase. Thus, at intermediate densities the plasmid's horizontal transfer can offset selection against plasmid-bearing cells, but at high densities pB15 conjugates so poorly that it cannot invade. I discuss adaptive vs. nonadaptive causes for the phenotypic plasticity, as well as potential mechanisms that may lead to complex transfer dynamics of plasmids in liquid environments. PMID:15166133

  14. Plasmid Rolling-Circle Replication. (United States)

    Ruiz-Masó, J A; MachóN, C; Bordanaba-Ruiseco, L; Espinosa, M; Coll, M; Del Solar, G


    Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

  15. 莪葛胶囊提取纯化工艺研究%Study on Process of Extraction and Purification of E'ge Capsule

    Institute of Scientific and Technical Information of China (English)

    陈武; 周洪亮; 陈最鹏; 刘志辉


    Objective To optimize process of extraction and purification orE'ge Capsule. Methods Orthogonal design was used to optimize extracting process. The extracting rate of volatile oil was index in extraction of Blue Turmeric Rhizoma, and the extracting rate of puerarin and pueraria flavonid were index in extraction of Lobed Kudzuvine Root; AB-8 macroporous adsorptive resins were used to purify pueraria flavonid. Results The optimum process were as follows: Blue Turmeric Rhizoma was comminuted to 20 mu powder, 8 times amount of water, refluxing and extracting for 5 h; Lobed Kudzuvine Root was extracted by 8 times amount of water for 50minutes, 3 times altogether; the purity of puerarin and pueraria flavonid can respectively reach to 27% and 65% after Purification. Conclusion The optimal process is feasible, which can be used in industrial production.%目的 优选莪葛胶囊提取纯化工艺.方法 以挥发油提取率为指标,正交试验法优选莪术挥发油提取工艺;以葛根素和葛根总黄酮得率为指标,正交试验筛选葛根总黄酮提取工艺,采用AB-8大孔吸附树脂纯化葛根总黄酮.结果 优选的工艺条件为:莪术粉碎成20目粗粉,加8倍量水提取5h;葛根以8倍量水煎煮3次,每次50 min;经大孔吸附树脂纯化,葛根素和葛根总黄酮纯度分别可达27%和65%.结论 该工艺可行,适合工业化生产.

  16. Purification and characterization of a thermostable glucoamylase ...

    African Journals Online (AJOL)



    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.

  17. Conjugation efficiency depends on intra and intercellular interactions between distinct plasmids: Plasmids promote the immigration of other plasmids but repress co-colonizing plasmids. (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco


    Conjugative plasmids encode the genes responsible for the synthesis of conjugative pili and plasmid transfer. Expression of the conjugative machinery (including conjugative pili) may be costly to bacteria, not only due to the energetic/metabolic cost associated with their expression but also because they serve as receptors for certain viruses. Consequently, the presence of two plasmids in the same cell may be disadvantageous to each plasmid, because they may impose a higher fitness cost on the host. Therefore, plasmids may encode mechanisms to cope with co-resident plasmids. Moreover, it is possible that the transfer rate of a plasmid is affected by the presence of a distinct plasmid in the recipient cell. In this work, we measured transfer rates of twelve natural plasmids belonging to seven incompatibility groups in three situations, namely when: (i) donor cells contain a plasmid and recipient cells are plasmid-free; (ii) donor cells contain two unrelated plasmids and recipient cells are plasmid-free; and (iii) half of the cells contain a given plasmid and the other half contain another, unrelated, plasmid. In the third situation, recipient cells of a plasmid are the donor cells of the other plasmid. We show that there are more negative interactions (reduction of a plasmid's conjugative efficiency) between plasmids if they reside in the same cell than if they reside in different cells. However, if plasmids interacted intercellularly, the transfer rate of one of the plasmids was often higher (when the unrelated conjugative plasmid was present in the recipient cell) than if the recipient cell was plasmid-free - a positive effect. Experimental data retrieved from the study of mutant plasmids not expressing conjugative pili on the cell surface suggest that positive effects result from a higher efficiency of mating pair formation. Overall, our results suggest that negative interactions are significantly more frequent when plasmids occupy the same cell. Such

  18. 重组人促红细胞生成素纯化工艺的优化%Optimized Purification Process of Human Recombinant Erythropoietin

    Institute of Scientific and Technical Information of China (English)

    李干祥; 聂东宋; 杨葆生; 朱宁


    采用堆积床生物反应器,用无血清培养基培养分泌重组人促红细胞生成素(rhEPO)的工程细胞株ZK9703.所收集的上清,采用阴离子交换层析-反相层析-分子筛层析三步纯化工艺路线,分别用Q-Sepharose XL-C4-S-200(方法Ⅰ)和DEAE Sepharose FF-Source-S -200( 方法Ⅱ)纯化3批产品,所得EPO纯度达98%以上,体外比活性大于1.3×105 IU/mg.方法Ⅰ、方法Ⅱ纯化过程的EPO体外活性回收率分别为23.56%和28.57%.本纯化方法Ⅱ工艺纯化日程短,分离效果好,EPO体内、体外活性回收率较高,更适合于大规模生产重组人促红细胞生成素.%Gene-engineered CHO cell line ZK9703 with the capacity to express huma n erythropoietin were cultured in packed bed bioreactor in a perfusion culture m ode. Harvest medium was collected and purified with a three-step purificati on pr ocession-exchanger, reverse phase and molecular sieve chromatogra phy. Three lot s of harvest medium was purified with a purification processQ-Sep harose XL, C4 reverse phase and S-200( the first method), three lots of harvest medium wa s pu rified with a purification processDEAE FF, source reverse phase an d S-200(the second method),the final products were analyzed for its purity and specific in vitro biological activity. Its purity is over 98%, and its specific in vit ro bio logical activity is over 1.3×105 IU/mg.The total recovery of EPO activity wa s 23.56% (by the fist method) and 28.57%.( by the second method), respectively. Th e results show that the second method of purification process is rapid and effic ient with high recovery, and could be more useful in large-scale production of rhEPO.

  19. Plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.


    DNA recombination in exponential phase and competent Haemophilus influenzae was measured by an electron microscopic assay that relies on the conversion of plasmid RSF0885 monomers into multimeric forms. Dimer circles were present at a frequency of 2% in plasmid preparations from competent Rd (wild-type) cells; multimers were present at a frequency of 0.2% in preparations from exponential phase cells. Thus, plasmid recombination was stimulated in competent cells. Multimer formation occurred efficiently in cells of the transformation defective mutant rec2, implying that the rec2 gene product is not required for plasmid recombination. However, the absence of multimer plasmids in preparations from competent cells of the transformation defective mutant rec1 suggests that the rec1 gene product is required. Digestion of purified plasmids with restriction endonuclease PvuII, which makes a single cut in the monomer, revealed the presence of recombination intermediates composed of two linear plasmids joined to form two pairs of arms resembling the Greek letter chi. Length measurements of these arms taken from a population of recombination intermediates gave evidence that the plasmids were joined at sites of homology. The distributions of individual DNA strands, at the intersections of the four arms, could be resolved in some recombination intermediates and were of two types. The first type of junction appeared as a single-stranded arm appended to each corner. The second type of junction consisted of a single strand of DNA linking the two linear plasmids at a site of homology. The single-stranded linker was frequently situated at the edge of a short gap on one of the plasmids in the pair. The fine structures of the recombinational joints have been interpreted in terms of previously proposed models of recombination.

  20. Accelerated purification of colloidal silica sols (United States)

    Bahnsen, E. B.; Garofalini, S.; Pechman, A.


    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.

  1. Impact of purification and fractionation process on the chemical structure and physical properties of locust bean gum. (United States)

    Sébastien, Gillet; Christophe, Blecker; Mario, Aguedo; Pascal, Laurent; Michel, Paquot; Aurore, Richel


    Crude locust bean gum (CLBG) was purified and fractionated into two parts: the first was obtained by solubilization in water at 25°C (GM25) and the second consisted in a further extraction at 80°C on the residual impoverished fraction (GM80). The complete structural characterization has shown that GM80 possessed relatively longer chain lengths than GM25, a slightly lower degree of galactose substitution and a somewhat sharper galactosyl distribution in substituted and unsubstituted regions. A physical behavior analysis was carried out on solubilization kinetics, viscosity, viscoelasticity and formation of associated gels with xanthan or carrageenan. The average structure of GM80 generated larger intra-chain, inter-chain and inter-molecular interactions, resulting in the appearance of a stronger network. Small structural differences therefore generated very different physical behaviors. This study thus allowed to establish, in a precise and complete manner, fractionation-purification-structure-function relationships of galactomannans extracted from carob.

  2. Production and purification of xylooligosaccharides from oil palm empty fruit bunch fibre by a non-isothermal process. (United States)

    Ho, Ai Ling; Carvalheiro, Florbela; Duarte, Luís C; Roseiro, Luísa B; Charalampopoulos, Dimitris; Rastall, Robert A


    Oil palm empty fruit bunches (OPEFB) fibre, a by-product generated from non-woody, tropical perennial oil palm crop was evaluated for xylooligosaccharides (XOS) production. Samples of OPEFB fibre were subjected to non-isothermal autohydrolysis treatment using a temperature range from 150 to 220 °C. The highest XOS concentration, 17.6g/L which relayed from solubilisation of 63 g/100 g xylan was achieved at 210 °C and there was a minimum amount of xylose and furfural being produced. The chromatographic purification which was undertaken to purify the oligosaccharide-rich liquor resulted in a product with 74-78% purity, of which 83-85% was XOS with degree of polymerisation (DP) between 5 and 40.

  3. Complex nature of enterococcal pheromone-responsive plasmids. (United States)

    Wardal, Ewa; Sadowy, Ewa; Hryniewicz, Waleria


    Pheromone-responsive plasmids constitute a unique group of approximately 20 plasmids identified, as yet, only among enterococcal species. Several of their representatives, e.g. pAD1, pCF10, pPD1 and pAM373 have been extensively studied. These plasmids possess a sophisticated conjugation mechanism based on response to sex pheromones--small peptides produced by plasmid-free recipient cells. Detailed analysis of regulation and function of the pheromone response process revealed its great complexity and dual role--in plasmid conjugation and modulation of enterococcal virulence. Among other functional modules identified in pheromone plasmids, the stabilization/partition systems play a crucial role in stable maintenance of the plasmid molecule in host bacteria. Among them, the par locus of pAD1 is one of the exceptional RNA addiction systems. Pheromone-responsive plasmids contribute also to enterococcal phenotype being an important vehicle of antibiotic resistance in this genus. Both types of acquired vancomycin resistance determinants, vanA and vanB, as well many other resistant phenotypes, were found to be located on these plasmids. They also encode two basic agents of enterococcal virulence, i.e. aggregation substance (AS) and cytolysin. AS participates in mating-pair formation during conjugation but can also facilitate the adherence ofenterococci to human tissues during infection. The second protein, cytolysin, displays hemolytic activity and helps to invade eukaryotic cells. There are still many aspects of the nature of pheromone plasmids that remain unclear and more detailed studies are needed to understand their uniqueness and complexity.

  4. Plasmid DNA Manufacturing for Indirect and Direct Clinical Applications. (United States)

    Schmeer, Marco; Buchholz, Tatjana; Schleef, Martin


    Plasmid DNA is currently gaining increasing importance for clinical research applications in gene therapy and genetic vaccination. For direct gene transfer into humans, good manufacturing practice (GMP)-grade plasmid DNA is mandatory. The same holds true if the drug substance contains a genetically modified cell, for example chimeric antigen receptor (CAR) T cells, where these cells as well as the contained plasmids are used. According to the responsible regulatory agencies, they have to be produced under full GMP. On the other hand, for GMP production of, for example, mRNA or viral vectors (lentiviral vectors, adeno-associated virus vectors, etc.), in many cases, High Quality Grade plasmid DNA is accepted as a starting material. The manufacturing process passes through different production steps. To ensure the right conditions are used for the plasmid, a pilot run must be conducted at the beginning. In this step, a followed upscaling with respect to reproducibility and influences on product quality is performed. Subsequently, a cell bank of the transformed productions strain is established and characterized. This cell bank is used for the cultivation process. After cell harvesting and lysis, several chromatography steps are conducted to receive a pure plasmid product. Depending on the respective required quality grade, the plasmid product is subject to several quality controls. The last step consists of formulation and filling of the product.

  5. In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing

    DEFF Research Database (Denmark)

    Carattoli, Alessandra; Zankari, Ea; García-Fernández, Aurora


    In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft...... genomes of multidrug-resistant Enterobacteriaceae species by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the family Enterobacteriaceae in the NCBI nucleotide database were collected to build a consensus database for integration...... sequences identified in the 559 fully sequenced plasmids. For plasmid multilocus sequence typing (pMLST) analysis, a database that is updated weekly was generated from and integrated into a Web tool called pMLST. Both databases were evaluated using draft genomes from a collection...

  6. The conjugative plasmid of a bean-nodulating Sinorhizobium fredii strain is assembled from sequences of two Rhizobium plasmids and the chromosome of a Sinorhizobium strain

    Directory of Open Access Journals (Sweden)

    Brom Susana


    cointegrate from R. etli to a S. fredii strain, and at least two recombination events among the R. etli plasmids and the S. fredii genome. As in R. etli CFN42, the S. fredii GR64 transmissible plasmid is required for the conjugative transfer of the symbiotic plasmid. In spite of the similarity in the conjugation related genes, the transfer process of these plasmids shows a host-specific behaviour.

  7. Controlling Points of Air Purification System in Design and Construction Process%净化空调系统在设计施工过程中的控制要点

    Institute of Scientific and Technical Information of China (English)



    随着高新技术的发展,对生产环境的洁净要求也越来越严格,净化空调的应用越来越广泛。该文在阐述洁净等级的划分之后,根据实际经验提出净化空调系统设计存在的问题,并分析了系统施工过程应注意的问题。%With the development of high and new technology, requirements on production environment are getting increasingly stricter, and application of air purification system is also getting increasingly wider. The classification of purification level is elaborated, the existing problems in air purification sys-tem design are presented in accordance with actual experience and some attention worth issues in construction process are also analyzed.

  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. 低聚木糖的超滤纯化生产工艺优化%The optimization of xylooligosaccharides ultrafiltration purification processes

    Institute of Scientific and Technical Information of China (English)

    章茹; 曹济; 刘辉; 冯斐


    以纯化分离秸秆酶解液中低聚木糖为目的,以蛋白、木质素去除率和低聚木糖的透过率为评价指标,采用工业化超滤膜分离装置,考察料液浓度、操作压力、温度、pH值对超滤除杂的影响.单因素实验确定初步的工艺条件后,Box-Behnken模型优化得最佳超滤纯化工艺条件为:料液浓度2.24°Bx,操作压力0.18 MPa,温度25℃,pH 7.0.在此条件下,低聚木糖的透过率为92.1%,蛋白去除率67.9%,木质素去除率67.8%,透过液澄清透亮.响应面法分析各因素对超滤提纯效果的影响,其强弱性依次为:料液浓度、pH值、操作压力、操作温度.%Ultrafiltration membrane separation was used to purify xylooligosaccharides (XOS) from straw enzymatic hydrolysis.Protein,lignin removal rates and XOS transmittance were used as evaluation indices,the influence of feed concentration,operating pressure,temperature,and pH on the purification efficiencies were studied.Preliminary extraction conditions were studied by single factor experiment,and then optimized by Box-Behnken model.The results were:feed concentration 2.24 ° Bx,operating pressure 0.18MPa,temperature 25℃,pH 7.0.Under these conditions,XOS transmittance was 92.1%,protein removal rate was 67.9%,and lignin removal rate was 67.8% The factors of ultrafiltration purification were analyzed by response surface method,the order of importance was:feed concentration,pH value,operating pressure,and operating temperature.This research provides reference for XOS purification process by ultrafiltration separation method.

  10. Bacterial mitosis: ParM of plasmid R1 moves plasmid DNA by an actin-like insertional polymerization mechanism. (United States)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette; Jensen, Rasmus B; Roepstorff, Peter; Gerdes, Kenn


    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system act together to generate the force required for plasmid movement during segregation. ParR protein binds cooperatively to the centromeric parC DNA region, thereby forming a complex that interacts with the filament-forming actin-like ParM protein in an ATP-dependent manner, suggesting that plasmid movement is powered by insertional polymerization of ParM. Consistently, we find that segregating plasmids are positioned at the ends of extending ParM filaments. Thus, the process of R1 plasmid segregation in E. coli appears to be mechanistically analogous to the actin-based motility operating in eukaryotic cells. In addition, we find evidence suggesting that plasmid pairing is required for ParM polymerization.

  11. Performance Modeling and Cost Analysis of a Pilot-Scale Reverse Osmosis Process for the Final Purification of Olive Mill Wastewater (United States)

    Ochando-Pulido, Javier Miguel; Hodaifa, Gassan; Victor-Ortega, Maria Dolores; Martinez-Ferez, Antonio


    A secondary treatment for olive mill wastewater coming from factories working with the two-phase olive oil production process (OMW-2) has been set-up on an industrial scale in an olive oil mill in the premises of Jaén (Spain). The secondary treatment comprises Fenton-like oxidation followed by flocculation-sedimentation and filtration through olive stones. In this work, performance modelization and preliminary cost analysis of a final reverse osmosis (RO) process was examined on pilot scale for ulterior purification of OMW-2 with the goal of closing the loop of the industrial production process. Reduction of concentration polarization on the RO membrane equal to 26.3% was provided upon increment of the turbulence over the membrane to values of Reynolds number equal to 2.6 × 104. Medium operating pressure (25 bar) should be chosen to achieve significant steady state permeate flux (21.1 L h−1 m−2) and minimize membrane fouling, ensuring less than 14.7% flux drop and up to 90% feed recovery. Under these conditions, irreversible fouling below 0.08 L h−2 m−2 bar−1 helped increase the longevity of the membrane and reduce the costs of the treatment. For 10 m3 day−1 OMW-2 on average, 47.4 m2 required membrane area and 0.87 € m−3 total costs for the RO process were estimated. PMID:24957058

  12. Performance modeling and cost analysis of a pilot-scale reverse osmosis process for the final purification of olive mill wastewater. (United States)

    Ochando-Pulido, Javier Miguel; Hodaifa, Gassan; Victor-Ortega, Maria Dolores; Martinez-Ferez, Antonio


    A secondary treatment for olive mill wastewater coming from factories working with the two-phase olive oil production process (OMW-2) has been set-up on an industrial scale in an olive oil mill in the premises of Jaén (Spain). The secondary treatment comprises Fenton-like oxidation followed by flocculation-sedimentation and filtration through olive stones. In this work, performance modelization and preliminary cost analysis of a final reverse osmosis (RO) process was examined on pilot scale for ulterior purification of OMW-2 with the goal of closing the loop of the industrial production process. Reduction of concentration polarization on the RO membrane equal to 26.3% was provided upon increment of the turbulence over the membrane to values of Reynolds number equal to 2.6 × 104. Medium operating pressure (25 bar) should be chosen to achieve significant steady state permeate flux (21.1 L h-1 m-2) and minimize membrane fouling, ensuring less than 14.7% flux drop and up to 90% feed recovery. Under these conditions, irreversible fouling below 0.08 L h-2 m-2 bar-1 helped increase the longevity of the membrane and reduce the costs of the treatment. For 10 m3 day-1 OMW-2 on average, 47.4 m2 required membrane area and 0.87 € m-3 total costs for the RO process were estimated.

  13. Plasmid and chromosome partitioning: surprises from phylogeny

    DEFF Research Database (Denmark)

    Gerdes, Kenn; Møller-Jensen, Jakob; Bugge Jensen, Rasmus


    Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids and chr...

  14. The extraction and purification process of Qing Feng Teng in the Compound Qufeng sustained - release tablets%复方祛风缓释片中青风藤的提取纯化工艺研究

    Institute of Scientific and Technical Information of China (English)

    方达任; 张志


    目的 优选复方祛风缓释片中青风藤的提取纯化工艺.方法 以青藤碱作为指标,采用正交试验和单因素相结合的方法优选青风藤提取纯化工艺.结果 青藤碱最佳提取工艺为:70%的乙醇6倍量提取2次,每次2h;纯化工艺为:加1% HCl调pH值为2,氯仿萃取5次.结论 该提取纯化工艺科学、合理.%Objective Optimization of the extraction and purification process of Qing Feng Teng in the Compound Qufeng sustained -release tablets. Method With sinomenine as an indicator,orthogonal test and single factors combined to optimize of extraction and purification process of Qing Feng Teng. Result The best extraction of Sinomenine is; 70% ethanol 6 times capacity of Caulis Sinomenii, extracted 2 times, 2 hours per time;the purification technology are; using dilute HC1 to adjust PH to 2, then adding chloroform to extract 5 times. Conclusion The extraction and purification is scientific and rational.

  15. Scaling-up recombinant plasmid DNA for clinical trial: current concern, solution and status. (United States)

    Ismail, Ruzila; Allaudin, Zeenathul Nazariah; Lila, Mohd-Azmi Mohd


    Gene therapy and vaccines are rapidly developing field in which recombinant nucleic acids are introduced in mammalian cells for enhancement, restoration, initiation or silencing biochemical function. Beside simplicity in manipulation and rapid manufacture process, plasmid DNA-based vaccines have inherent features that make them promising vaccine candidates in a variety of diseases. This present review focuses on the safety concern of the genetic elements of plasmid such as propagation and expression units as well as their host genome for the production of recombinant plasmid DNA. The highlighted issues will be beneficial in characterizing and manufacturing plasmid DNA for save clinical use. Manipulation of regulatory units of plasmid will have impact towards addressing the safety concerns raised in human vaccine applications. The gene revolution with plasmid DNA by alteration of their plasmid and production host genetics will be promising for safe delivery and obtaining efficient outcomes.

  16. Improved purification process for cholera toxin and its application to the quantification of residual toxin in cholera vaccines. (United States)

    Jang, Hyun; Kim, Hyo Seung; Kim, Jeong Ah; Seo, Jin Ho; Carbis, Rodney


    A simplified method for the purification of cholera toxin was developed. The 569B strain of Vibrio cholerae, a recognized hyper-producer of cholera toxin, was propagated in a bioreactor under conditions that promote the production of the toxin. The toxin was separated from the bacterial cells using 0.2-microm crossflow microfiltration, the clarified toxin was passed through the membrane into the permeate, and the bacterial cells were retained in the retentate. The 0.2-microm permeate was then concentrated 3-fold and diafiltered against 10 mM phosphate buffer, pH 7.6, using 30-kDa crossflow ultrafiltration. The concentrated toxin was loaded onto a cation exchange column, the toxin was bound to the column, and most of the impurities were passed unimpeded through the column. The toxin was eluted with a salt gradient of phosphate buffer, pH7.0, containing 1.0M NaCl. The peak containing the toxin was assayed for cholera toxin and protein and the purity was determined to be 92%. The toxin peak had a low endotoxin level of 3.1 EU/microg of toxin. The purified toxin was used to prepare antiserum against whole toxin, which was used in a G(M1) ganglioside-binding ELISA to determine residual levels of toxin in an oral inactivated whole-cell cholera vaccine. The G(M1) ganglioside-binding ELISA was shown to be very sensitive and capable of detecting as little as 1 ng/ml of cholera toxin.

  17. Purification of rhamnolipid using colloidal magnetic nanoparticles

    African Journals Online (AJOL)



    Jul 6, 2009 ... separation and purification of bio-molecules, particularly, .... of model Rhamnolipid by ion exchange processes. The ..... dynamic diameter of the particles is 35.6 nm. ... solubility of rhaminolipid was decreased and amount of.

  18. Affinity purification of recombinant human plasminogen activator ...

    African Journals Online (AJOL)

    Purpose: To develop processes for effective isolation and purification of recombinant human plasminogen ... three hybridoma strains were superior for producing PR-mAbs (C1, C4, C8). ..... characterization of a polyol- responsive monoclonal.

  19. Rapid Purification of Glycerol by-product from Biodiesel Production through Combined Process of Microwave Assisted Acidification and Adsorption via Chitosan Immobilized with Yeast

    Directory of Open Access Journals (Sweden)

    N. Saifuddin


    Full Text Available Biodiesel is a proven alternative to the petroleum diesel fuel. During biodiesel production, glycerol is produced as a by-product. This by-product consist of impureties such as soap, salts, sodium catalyst and so on. Traditionally, two of the most conventional techniques that is applied to glycerol purification are distillation and ion-exchange. These techniques are, however, still expensive to generate pure glycerol. Recently, several alternative “combination” treatment procedures have been used. These treatment has several advantages over others methods such as producing large amounts of glycerol-rich layer that requires simple treatments and not causing any high operational cost. In this study, the combination treatment process have been used in order to reach high glycerol content. Basically, these stages starts with using microwave assisted acidification process and the next process utilizing a bioadsorbent synthesized from dead yeast cells immobilized on chitosan. The final yield of glycerol was about 93.1-94.2% (w/w.

  20. Purification and characterisation of processive-type endoglucanase and β-glucosidase from Aspergillus ochraceus MTCC 1810 through saccharification of delignified coir pith to glucose. (United States)

    Asha, P; Divya, Jose; Bright Singh, I S


    The study describes purification and characterisation of processive-type endoglucanase and β-glucosidase from Aspergillus ochraceus MTCC 1810 through bioconversion of delignified coir pith to fermentable glucose. The purified processive endoglucanase (AS-HT-Celuz A) and β-glucosidase (AS-HT-Celuz B) were found to have molecular mass of ≈78-kDa and 43-kDa respectively with optimum endoglucanase (35.63U/ml), total cellulase (28.15FPU/ml) and β-glucosidase (15.19U/ml) activities at 40°C/pH 6. The unique feature of AS-HT-Celuz A is the multiple substrate specificity and processivity towards both amorphous and crystalline cellulose. Zymogram indicated both endo and exoglucanase activities residing in different binding sites of a single protein exhibiting sequential synergy with its own β-glucosidase. Accordingly, the identified enzymes could be implemented as synergistic cellulases for complete cellulose saccharification which still considered an unresolved issue in bio-refineries.

  1. Mechanisms of Evolution in High-Consequence Drug Resistance Plasmids

    Directory of Open Access Journals (Sweden)

    Susu He


    Full Text Available The dissemination of resistance among bacteria has been facilitated by the fact that resistance genes are usually located on a diverse and evolving set of transmissible plasmids. However, the mechanisms generating diversity and enabling adaptation within highly successful resistance plasmids have remained obscure, despite their profound clinical significance. To understand these mechanisms, we have performed a detailed analysis of the mobilome (the entire mobile genetic element content of a set of previously sequenced carbapenemase-producing Enterobacteriaceae (CPE from the National Institutes of Health Clinical Center. This analysis revealed that plasmid reorganizations occurring in the natural context of colonization of human hosts were overwhelmingly driven by genetic rearrangements carried out by replicative transposons working in concert with the process of homologous recombination. A more complete understanding of the molecular mechanisms and evolutionary forces driving rearrangements in resistance plasmids may lead to fundamentally new strategies to address the problem of antibiotic resistance.

  2. Biobased monoliths for adenovirus purification. (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


    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.

  3. Optimization of Purification Process for Total Flavonoids from Baeckea frutescens L.%岗松总黄酮的纯化工艺优选

    Institute of Scientific and Technical Information of China (English)

    邱宏聪; 刘布鸣; 孙翠


    Objective:To optimize purification process of total flavonoids from Baeckea frutescens macroporous adsorption resin.Method:Different types of resin were selected with absorption and desorption ratio of total flavonoids as indexes,the content of total flavonoids was determined by UV,single factor test was adopted to investigate purification technology of total flavonoids by macroporous adsorption resin.Result:AB-8 type resin was adopted,its optimum process conditions were as followings:the concentration of sample solution 0.3 g ·mL-1,absorption velocity 1 BV · h-1,sample volume 3 BV,eluted by 5 BV 50% ethanol at flow rate of 2 BV ·h-1 Under these conditions,purity of total flavonoids was up to 52.33%.Conclusion:Optimized process was rational,feasible and suitable for industrial production.%目的:优选岗松总黄酮的大孔吸附树脂纯化工艺.方法:以总黄酮吸附率和洗脱率为指标,采用UV测定总黄酮含量,筛选最佳大孔吸附树脂,并通过单因素试验考察岗松总黄酮的大孔树脂纯化工艺.结果:采用AB-8型大孔吸附树脂对岗松总黄酮进行纯化,其最佳工艺条件为上样液生药质量浓度0.3 g·mL-1,上样流速1 BV·h-1,上样体积3 BV,加50%乙醇5BV以2 BV·h-1的流速洗脱,总黄酮纯度达52.33%.结论:优选的工艺合理可行,适用于工业化生产.

  4. Increase of the processing capacity through modification and enlargement of the assets areas preparation and waste water purification in the fermentation plant Kirchstockach; Durchsatzsteigerung der Vergaerungsanlage Kirchstockach durch Umbau und Erweiterung der Anlagenbereiche Aufbereitung und Prozesswasserreinigung

    Energy Technology Data Exchange (ETDEWEB)

    Kirschenhofer, M. [LRA Muenchen (Germany). Tiefbau, Verkehrsplanung, Abfallwirtschaft; Kroner, T. [ia GmbH - Wissensmanagement und Ingenieurleistungen, Muenchen (Germany). Bereich Kommunale Abfallwirtschaft und Energie; Niefnecker, U. [M. Ganser GmbH und Co. Entsorgungsbetriebe KG, Brunnthal/Kirchstockach (Germany)


    At the fermentation plant Kirchstockach the operations for the rectification of deficiencies and process optimisations were completed in 2004. Now process results of 2005 show the success of the performed actions. In the asset area of preparations the existing rake discharge system was removed and the use of the new discharge reservoir with a drainage coil conveyor system minimises deadlock times and rises preparations throughput. With the new set-up of the light material presses the process procedure was optimised, too. The installation of the new process water reservoir was conditional on the non-uniform hydraulic load of the waste-water purification, which results from the operation of the facility. With the higher buffer capacity, realised by the new process water reservoir, a uniform hydraulic load of the purification system and an optimised process control was implemented. With the optimised performance of the wastewater purification wastewater thresholds are guaranteed now and it is possible to realise the increased throughput of the preparation in the complete system of the fermentation plant Kirchstockach. (orig.)

  5. 浅谈石灰烟道气法卤水净化工艺的应用%Brief Discussion on the Application of Lime Flue Gas Method for Brine Purification Process

    Institute of Scientific and Technical Information of China (English)



    石灰烟道气卤水净化生产工艺因其环保、低耗已被各大制盐生产企业广泛应用,但在应用过程中均或多或少出现一些问题。本文主要介绍我厂石灰烟道气卤水净化生产的操作和控制,并对生产过程中存在的一些问题加以探讨。%Lime Flue Gas brine purification process is widely used by each salt manufacturing enterprises for the advantage of environmental friendly and low cost but in the application process were more or less some problems. This paper mainly introduces the operation and control of the brine purification with lime flue gas, and discusses some problems in the process of production.

  6. Development and qualification of a high sensitivity, high throughput Q-PCR assay for quantitation of residual host cell DNA in purification process intermediate and drug substance samples. (United States)

    Zhang, Wei; Wu, Meng; Menesale, Emily; Lu, Tongjun; Magliola, Aeona; Bergelson, Svetlana


    Methods of high sensitivity, accuracy and throughput are needed for quantitation of low level residual host cell DNA in purification process intermediates and drug substances of therapeutic proteins. In this study, we designed primer/probe sets targeting repetitive Alu repeats or Alu-equivalent sequences in the human, Chinese hamster and murine genomes. When used in quantitative polymerase chain reactions (Q-PCRs), these primer/probe sets showed high species specificity and gave significantly higher sensitivity compared to those targeting the low copy number GAPDH gene. This allowed for detection of residual host cell DNA of much lower concentrations and, for some samples, eliminated the need for DNA extraction. By combining the high sensitivity Alu Q-PCR with high throughput automated DNA extraction using an automated MagMAX magnetic particle processor, we successfully developed and qualified a highly accurate, specific, sensitive and efficient method for the quantitation of residual host cell DNA in process intermediates and drug substances of multiple therapeutic proteins purified from cells of multiple species. Compared to the previous method using manual DNA extraction and primer/probe sets targeting the GAPDH gene, this new method increased our DNA extraction throughput by over sevenfold, and lowered the lower limit of quantitation by up to eightfold.

  7. A one-step miniprep for the isolation of plasmid DNA and lambda phage particles.

    Directory of Open Access Journals (Sweden)

    George Lezin

    Full Text Available Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E. coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.

  8. Plasmid copy number noise in monoclonal populations of bacteria (United States)

    Wong Ng, Jérôme; Chatenay, Didier; Robert, Jérôme; Poirier, Michael Guy


    Plasmids are extra chromosomal DNA that can confer to their hosts’ supplementary characteristics such as antibiotic resistance. Plasmids code for their copy number through their own replication frequency. Even though the biochemical networks underlying the plasmid copy number (PCN) regulation processes have been studied and modeled, no measurement of the heterogeneity in PCN within a whole population has been done. We have developed a fluorescent-based measurement system, which enables determination of the mean and noise in PCN within a monoclonal population of bacteria. Two different fluorescent protein reporters were inserted: one on the chromosome and the other on the plasmid. The fluorescence of these bacteria was measured with a microfluidic flow cytometry device. We show that our measurements are consistent with known plasmid characteristics. We find that the partitioning system lowers the PCN mean and standard deviation. Finally, bacterial populations were allowed to grow without selective pressure. In this case, we were able to determine the plasmid loss rate and growth inhibition effect.

  9. Study on Purification Process of Water Extract of Kunshen Tablet with ZTC1+1-ⅡClarificant%ZTC1+1-Ⅱ澄清剂用于昆参片水提液的纯化研究

    Institute of Scientific and Technical Information of China (English)

    朱立俏; 盛华刚


    目的:研究纯化昆参片水提液的工艺.方法:以纯化前后昆参片水提液中总多糖含量和浸膏率为考察指标,比较乙醇沉淀法、壳聚糖澄清剂和ZTC1+1-Ⅱ澄清剂对提取液的纯化效果,并用单因素考察法确定ZTC1+1-Ⅱ澄清剂的最佳纯化工艺.结果:ZTC1+1-Ⅱ澄清剂优于乙醇沉淀法和壳聚糖澄清剂,最佳纯化工艺为澄清剂用量5%B+2.5%A,药液浓度0.2g/mL,搅拌速度100r/min.结论:ZTC1+1-Ⅱ澄清剂可用于纯化昆参片水提液.%Objective To study the purification process of the water extract of Kunshen Tablet with ZTC1+1-Ⅱ clarificant. Methods The purification efficacy of ethanol precipitation, chitosan clarificant and ZTC 1+1-Ⅱ clarificant was compared, using the content of total polysaccharides and dry extract rate as performance indicators. The optimum purification process was determined by single factor test. Results The purification effect of ZTC1 +1- ii clarificant was better than that of ethanol precipitation and chitosan clarificant. The optimum purification process was as follows: the concentration was 0.2g·mL-1, the amount of ZTC1+1- Ⅱ clarificant was 5%B+2.5%A, the stirring speed was 100 r· min-1. Conclusion ZTC 1 + 1- Ⅱ clarificant can be used in purification process of the water extract of Kunshen Tablet.

  10. 响应曲面法优化茶皂素提纯工艺研究%Purification Process Optimization of Camellia Saponin by Response Surface Methodology

    Institute of Scientific and Technical Information of China (English)

    赖徐倩; 仲山民; 王超; 郑剑; 常银子


    本文主要以有机溶剂法浸提油茶籽外壳粉得到的粗提液作为原料,应用絮凝-沉淀法提纯皂素.在单因素试验的基础上,利用Mintab15.0软件得到回归方程的预测模型并进行响应面分析,确定利用絮凝-沉淀法提纯皂素的最优工艺条件.絮凝试验: 加入16%的壳聚糖溶液和皂素损失率为28.46%,杂质损失量为1.39%; 沉淀试验: 氧化钙加入量1.78%、沉淀温度32.75℃和沉淀时间3.01h,沉淀率为98.99%; 释放试验: 释放温度52.2℃、释放时间3.17h和碳酸氢铵加入量4.09%,释放率为3.61%.最后,在最优提纯工艺条件下测得纯度为60.4%.%In this paper, saponin was purified by flocculation-precipitation, and crude extraction from camellia oleifera seed shell was as raw material. On the basis of single factor experiments, purification process was optimized using response surface methodology, and a mathematical regression model was established by Mintab15.0 software. Optimal purification conditions obtained from this model were as follows: 16% Chitosan solution added in crude extraction; 1.78% CaO was added in supernatant after flocculation to subside for 3.01h at 32.75℃; 4.09% NH4HCO3 was added to the sediment, and then released at 52.2℃ for 3.17h. Under conditions, content of camellia saponin was 60.4% in the final.

  11. 蛹虫草多糖纯化工艺研究%Research on Technology of Purification Process of Cordyceps militaris Polysaccharide

    Institute of Scientific and Technical Information of China (English)

    韩建华; 杨淑芳


    To research the purification process of polysaccharide from Cordyceps militaris ,the effects of deproteinization with different methods(such as sevage ,improved sevage and present clarifier methods)were evaluated in terms of the ratio of protein removing and polysaccharide holding .Cordyceps militaris polysaccharide was separated and purified by DEAE‐52 and Sephdex G‐100 chromatography .The sample purity was checked by Sephdex G‐200 chromatography and HPLC . The results show that after purification by present clarifier methods ,the removal rate of protein was 93 .4% and the reten‐tion rate of polysaccharide was 90 .7% .The elution peak CP was obtained by DEAE-52 column chromatography and then the two elution peaks of CP -1 and CP -2 were obtained by Sephdex G -100 column chromatography .It was showed that the two elution peaks purified by column chromatography were both homogeneous polysaccharides through purity in‐spection .%为优化蛹虫草多糖的纯化工艺,以蛋白去除率和多糖保留率为指标,比较Sevage法、改良的Sevage法、澄清剂法三种方法对粗多糖中蛋白杂质的去除效果。本试验依次采用DEAE-52、Sephdex G -100葡聚糖凝胶柱层析进一步分离纯化蛹虫草多糖,再经Sephdex G-200柱色谱及高效液相凝胶色谱进行纯度检验。结果显示:澄清剂法蛋白去除率为93.4%,多糖保存率为90.7%;DEAE -52柱层析得到一个洗脱峰CP ,再经Sephdex G -100柱层析后得到两个洗脱峰CP-1和CP-2。对CP-1和CP-2进行纯度检验,发现两者皆为均一性多糖。

  12. Purification of crude biodiesel using dry washing and membrane technologies

    Directory of Open Access Journals (Sweden)

    I.M. Atadashi


    Full Text Available Purification of crude biodiesel is mandatory for the fuel to meet the strict international standard specifications for biodiesel. Therefore, this paper carefully analyzed recently published literatures which deal with the purification of biodiesel. As such, dry washing technologies and the most recent membrane biodiesel purification process have been thoroughly examined. Although purification of biodiesel using dry washing process involving magnesol and ion exchange resins provides high-quality biodiesel fuel, considerable amount of spent absorbents is recorded, besides the skeletal knowledge on its operating process. Further, recent findings have shown that biodiesel purification using membrane technique could offer high-quality biodiesel fuel with less wastewater discharges. Thus, both researchers and industries are expected to benefit from the development of membrane technique in purifying crude biodiesel. As well biodiesel purification via membranes has been shown to be environmentally friendly. For these reasons, it is important to explore and exploit membrane technology to purify crude biodiesel.

  13. Kinetic Properties of Four Plasmid-Mediated AmpC β-Lactamases (United States)

    Bauvois, Cédric; Ibuka, Akiko Shimizu; Celso, Almeida; Alba, Jimena; Ishii, Yoshikazu; Frère, Jean-Marie; Galleni, Moreno


    The heterologous production in Escherichia coli, the purification, and the kinetic characterization of four plasmid-encoded class C β-lactamases (ACT-1, MIR-1, CMY-2, and CMY-1) were performed. Except for their instability, these enzymes are very similar to the known chromosomally encoded AmpC β-lactamases. Their kinetic parameters did not show major differences from those obtained for the corresponding chromosomal enzymes. However, the Km values of CMY-2 for cefuroxime, cefotaxime, and oxacillin were significantly decreased compared to those of the chromosomal AmpC enzymes. Finally, the susceptibility patterns of different E. coli hosts producing a plasmid- or a chromosome-encoded class C enzyme toward β-lactam antibiotics are mainly due to the overproduction of the β-lactamase in the periplasmic space of the bacteria rather than to a specific catalytic profile of the plasmid-encoded β-lactamases. PMID:16189104

  14. 溶气法在糖汁(浆)气浮清净工艺中的应用%Application of the Dissolved-air Flotation Technology in the Cane Juice Purification Process

    Institute of Scientific and Technical Information of China (English)



    This paper introduces the dissolved-air flotation technology and the design of the key equipment, and analyses the application of the dissolved-air flotation technology in the flotating purification process of cane juice. The results showed that the best choice is to exploit the pressed dissolved-air flotation technology for the flotating purification process of diluted cane juice.%介绍了溶气上浮法的工艺流程及主要设备的设计并分析了其应用效果。生产实践表明,利用溶气法进行充气打泡是稀汁气浮清净工艺的最好选择。

  15. Bioinspired Materials for Water Purification

    Directory of Open Access Journals (Sweden)

    Alfredo Gonzalez-Perez


    Full Text Available Water scarcity issues associated with inadequate access to clean water and sanitation is a ubiquitous problem occurring globally. Addressing future challenges will require a combination of new technological development in water purification and environmental remediation technology with suitable conservation policies. In this scenario, new bioinspired materials will play a pivotal role in the development of more efficient and environmentally friendly solutions. The role of amphiphilic self-assembly on the fabrication of new biomimetic membranes for membrane separation like reverse osmosis is emphasized. Mesoporous support materials for semiconductor growth in the photocatalytic degradation of pollutants and new carriers for immobilization of bacteria in bioreactors are used in the removal and processing of different kind of water pollutants like heavy metals. Obstacles to improve and optimize the fabrication as well as a better understanding of their performance in small-scale and pilot purification systems need to be addressed. However, it is expected that these new biomimetic materials will find their way into the current water purification technologies to improve their purification/removal performance in a cost-effective and environmentally friendly way.

  16. Co-resident plasmids travel together. (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco


    Conjugative plasmids encode genes that enable them to transfer, by conjugation, from a given host cell to another cell. Conjugative transfer, despite being an important feature of conjugative plasmids, is not constitutive for most plasmids, the reason being that genes involved in horizontal transfer are mostly repressed. Only upon their transient de-repression are plasmids able to transfer horizontally. If host cells harbour multiple plasmids, their simultaneous transfer depends on simultaneous transient de-repression of all plasmids. If de-repression of different plasmids was random and independent events, simultaneous de-repression should be a rare event because the probability of simultaneous de-repression would be the product of the probabilities of de-repression of each plasmid. Some previous observations support this hypothesis, while others show that co-transfer of plasmids is more frequent than this reasoning indicates. Here, we show that co-transfer of multiple plasmids mainly results from non-independent events: the probability that all plasmids within a cell become de-repressed is much higher than if de-repression of plasmids genes were independent. We found a simple model for the probability of co-transfer: the plasmid having the lowest conjugation rates is the one who limits co-transfer. In this sense, cells receiving the plasmid with the lower transfer rate also receive the other plasmid. If de-repression happens simultaneously on co-resident plasmids, common cues may stimulate de-repression of distinct plasmids. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. The Chlamydophila felis plasmid is highly conserved. (United States)

    Harley, Ross; Day, Sarinder; Di Rocco, Camillo; Helps, Chris


    The presence of a plasmid in the Chlamydiaceae is both species and strain specific. Knowledge of the prevalence of the plasmid in different Chlamydia species is important for future studies aiming to investigate the role of the plasmid in chlamydial biology and disease. Although strains of Chlamydophila felis with or without the plasmid have been identified, only a small number of laboratory-adapted strains have been analysed and the prevalence of the plasmid in field isolates has not been determined. This study aimed to determine the prevalence of the plasmid in C. felis-positive conjunctival and oropharyngeal clinical samples submitted for routine diagnosis of C. felis by real-time (Q)PCR. DNA extracts from four laboratory-adapted strains were also analysed. QPCR assays targeting regions of C. felis plasmid genes pCF01, pCF02 and pCF03 were developed for the detection of plasmid DNA. QPCR analysis of DNA extracts from C. felis-positive clinical samples found evidence of plasmid DNA in 591 of 595 samples representing 561 of 564 (99.5%) clinical cases. Plasmid DNA was also detected by QPCR in laboratory-adapted strains 1497V, K2487 and K2490, but not strain 905. We conclude that the plasmid is highly conserved in C. felis, and plasmid-deficient strains represent a rare but important population for future studies of chlamydial plasmid function.

  18. 预洗脱技术在组氨酸提纯工艺中的应用研究%Application Study on the Elution Fechnique in the Purification Processes of Histidine

    Institute of Scientific and Technical Information of China (English)

    焦红彦; 田立; 刘娟


    To introduce the preparing elution in the purification processes of histidine with ion exchange method, realize high purity of histidine extraction by choosing appropriate lotion concentration and the elution speed.%在离子交换法提纯组氨酸的工艺中,引入预洗脱技术,通过选择适宜的预洗液浓度和预洗脱速度实现高纯度组氨酸的提取。


    DEFF Research Database (Denmark)


    The present invention concerns the isolation of plasmids from extremely thermophilic anaerobic microorganisms and their use in genetic transformation of thermophilic and mesophilic microorganisms. More particular the invention concerns the use of thermostable plasmid vectors as tools for creating...

  20. Plasmid required for virulence of Agrobacterium tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Watson, B.; Currier, T.C.; Gordon, M.P.; Chilton, M.D.; Nester, E.W.


    The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37/sup 0/C is shown to be due to loss of a large plasmid (1.2 x 10/sup 8/ daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6, is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 x A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.

  1. Multiple Pathways of Plasmid DNA Transfer in Helicobacter pylori (United States)

    Rohrer, Stefanie; Holsten, Lea; Weiss, Evelyn; Benghezal, Mohammed; Fischer, Wolfgang; Haas, Rainer


    Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species. PMID:23029142

  2. Organic-inorganic hybrid materials in separation chemistry: a molecular approach towards design of purification processes; De la molecule au procede. Apports des materiaux hybrides organiques-inorganiques en chimie separative

    Energy Technology Data Exchange (ETDEWEB)

    Brandes, St.; Denat, F. [Centre National de la Recherche Scientifique (CNRS), 21 - Dijon (France); Meyer, M.; Guilard, R. [Universite de Bourgogne, Lab. d' Ingenierie Moleculaire pour la Separation et les Applications des Gaz (LIMSAG) UMR 5633 du CNRS, 21 - Dijon (France)


    Located on the campus of the Universite de Bourgogne and supported by the CNRS, the main particularity of the LIMSAG was its association with an industrial partner, the Air Liquide company. The main objectives of this unusual research unit in the French academic system was to conceive and develop new molecules and materials that exhibit suitable properties for the ultra-purification or the detection of gases. Beside these activities, a second research topic is dedicated to the decontamination of industrial waste streams containing either toxic (lead, cadmium) and/or radioactive metal ions (uranium, plutonium, americium). Specific sequestering agents have also been designed for the lead removal from municipal tap water. Grafted and sol-gel immobilized tetra-aza-macrocyclic complexes are used as specific adsorbents for the purification and detection of gases, while related functionalized silica-gels have been implemented in the solid/liquid extraction processes of metals. (authors)

  3. Control of higher alkynes in purification process of acetylene in natural gas to acetylene plant%浅谈天然气制乙炔净化装置中高级炔含量的控制

    Institute of Scientific and Technical Information of China (English)



    介绍了乙炔净化工艺和乙炔中甲基乙炔、1,3-丁二烯、丙二烯等高级炔脱除原理,分析了影响它们脱除的因素,在此基础上提出了控制乙炔中高级炔含量的方法并实施,实现了乙炔净化装置高负荷、长周期、稳定运行。%The purification process of acetylene and the principle of removing methyl acetylene, 1,3-butadiene, allene and other higher alkynes from acetylene were introduced, and the factors influencing the purification process were analyzed. Based on the analysis, the measures for controlling the level of higher alkynes in acetylene were proposed and implemented, which ensured that the acetylene purification unit could run in high load, long period and stable operation.

  4. The Induced Self-Purification of Creeks and Rivers

    CERN Document Server

    Mikhailovskii, V


    The clean-up of several Creeks and Rivers by induction of a self-purification process was provided. The process took place at all the sites studied with the up to 100% resulted removal of polluting agents depending on the site and nature of the contaminant. The self-purification mechanism could be used for drinking and technical water preparation.

  5. Simulation of heat pump distillation in trichlorosilane purification process%热泵精馏在三氯氢硅提纯过程中的模拟

    Institute of Scientific and Technical Information of China (English)

    黄国强; 赵虎勇; 孙帅帅


      运用化工模拟软件Aspen Plus,选用NRTL-RK物性模型和RADFRAC精馏模型,对三氯氢硅精馏塔的两种热泵流程进行了模拟计算,分别是塔顶气体直接压缩式和塔釜液体闪蒸再沸式热泵精馏。对比热泵精馏流程和常规精馏流程,结果表明:对三氯氢硅提纯而言,塔釜液体闪蒸再沸式热泵流程更有利。本研究采用双塔串行流程提纯三氯氢硅,运用塔釜液体闪蒸再沸式热泵精馏技术,优化后的主要操作参数为:T1塔回流比20,节流阀压力180 kPa,压缩机出口压力309 kPa;T2塔回流比5,节流阀压力227 kPa,压缩机出口压力310 kPa。优化后三氯氢硅的一次收率为88.75%,纯度超过99.9999%;在处理量相同情况下,与常规精馏相比,能耗费用节约82%。%Two types of heat pump distillation flow for trichlorosilane purification were simulated by chemical simulation software Aspen Plus with NRTL-RK property and RADFRAC distillation models. One model used the vapor from the top as heat pump medium and the other model used the liquid from the bottom as heat pump medium. The comparison of heat pump distillation and conventional distillation processes showed that the latter had more advantages in trichlorosilane purification. Two-column trichlorosilane refining serial process using the liquid from the bottom as heat pump medium was conducted. The main optimum operation parameters were showed below. T1 column:outlet pressure of compressor was 309 kPa,mole reflux ratio was 20 and throttle valve pressure was 180 kPa;T2 column:outlet pressure of compressor was 310 kPa,mole reflux ratio was 5 and throttle valve pressure was 227 kPa. The yield coefficient of trichlorosilane was 88.75% and its purity was more than 99.9999%. Compared with conventional distillation,energy cost saving was about 82%at the same handling capacity.

  6. Water Purification (United States)


    The Vision Catalyst Purifier employs the basic technology developed by NASA to purify water aboard the Apollo spacecraft. However, it also uses an "erosion" technique. The purifier kills bacteria, viruses, and algae by "catalytic corrosion." A cartridge contains a silver-impregnated alumina bed with a large surface area. The catalyst bed converts oxygen in a pool of water to its most oxidative state, killing over 99 percent of the bacteria within five seconds. The cartridge also releases into the pool low levels of ionic silver and copper through a controlled process of erosion. Because the water becomes electrochemically active, no electricity is required.

  7. Chlamydophila felis: plasmid detection in Italian isolates. (United States)

    Di Francesco, Antonietta; Donati, Manuela; Salvatore, Daniela; Cevenini, Roberto; Di Paolo, Maria; Baldelli, Raffaella


    Plasmids have been detected in the majority of strains in the genus Chlamydia and in many Chlamydophila species. Previous studies showed that FP Pring and FP Cello Chlamydophila felis strains have an extrachromosomial plasmid, whereas the FP Baker strain does not. Azuma et al. recently sequenced the entire genomic DNA sequence of the Japanese Cp. felis strain Fe/C-56 and described a 7,552 base pair circular plasmid. In the present study a highly conserved plasmid gene was detected in 11 Italian Cp. felis isolates, showing 100% nucleotide identity with the plasmid gene of Fe/C-56 Cp. felis strain.

  8. A three-minute synthesis and purification of ibuprofen: pushing the limits of continuous-flow processing. (United States)

    Snead, David R; Jamison, Timothy F


    In a total residence time of three minutes, ibuprofen was assembled from its elementary building blocks with an average yield of above 90% for each step. A scale-up of this five-stage process (3 bond-forming steps, one work-up, and one in-line liquid-liquid separation) provided ibuprofen at a rate of 8.09 g h(-1) (equivalent to 70.8 kg y(-1)) using a system with an overall footprint of half the size of a standard laboratory fume hood. Aside from the high throughput, several other aspects of this synthesis expand the capabilities of continuous-flow processing, including a Friedel-Crafts acylation run under neat conditions and promoted by AlCl3, an exothermic in-line quench of high concentrations of precipitation-prone AlCl3, liquid-liquid separations run at or above 200 psi to provide solvent-free product, and the use of highly aggressive oxidants, such as iodine monochloride. The use of simple, inexpensive, and readily available reagents thus affords a practical synthesis of this important generic pharmaceutical.

  9. Membrane adsorbers as purification tools for monoclonal antibody purification. (United States)

    Boi, Cristiana


    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.

  10. Cationic spin probe reporting on thermal denaturation and complexation-decomplexation of BSA with SDS. Potential applications in protein purification processes. (United States)

    Matei, Iulia; Ariciu, Ana Maria; Neacsu, Maria Victoria; Collauto, Alberto; Salifoglou, Athanasios; Ionita, Gabriela


    In this work, we present evidence on the suitability of spin probes to report on the thermal treatment of bovine serum albumin (BSA), in the temperature range 293-343 K, and indirectly monitor the release of sodium dodecyl sulfate (SDS) from its complex with BSA using a covalent gel with β-cyclodextrin (β-CD) in the network. The spin probes used, 5- and 7-doxyl-stearic acids (5-DSA, 7-DSA) or 4-(N,N'-dimethyl-N-hexadecyl)ammonium-2,2',6,6'-tetramethylpiperidine-1-oxyl iodide (CAT16), present similar, fatty acid-like structural features. Their continuous wave electron paramagnetic resonance (CW-EPR) spectra, however, reflect different dynamics when complexed with BSA: a restricted motion for 5-DSA, almost nonsensitive to the heating/cooling cycle, and a faster temperature-dependent dynamic motion for CAT16. Molecular docking allows us to rationalize these results by revealing the different binding modes of 5-DSA and CAT16. The EPR data on the temperature effect on BSA are supported by circular dichroism results projecting recovery, upon cooling, of the initial binding ability of BSA for samples heated to 323 K. The interactions occurring in BSA/SDS/β-CD systems are investigated by CW-EPR and FT-ESEEM spectroscopies. It is found that the covalent gel containing β-CD can efficiently remove SDS from the BSA/SDS complex. The gel is not permeable to BSA but it can encapsulate SDS, thus yielding the free protein in solution and allowing recovery of the native protein conformation. Collectively, the accrued knowledge supports potential applications in protein purification biotechnological processes.

  11. Purification and substrate specificities of a fructanase from Kluyveromyces marxianus isolated from the fermentation process of Mezcal. (United States)

    Arrizon, Javier; Morel, Sandrine; Gschaedler, Anne; Monsan, Pierre


    A fructanase, produced by a Kluyveromyces marxianus strain isolated during the fermentation step of the elaboration process of "Mezcal de Guerrero" was purified and biochemically characterized. The active protein was a glycosylated dimer with a molecular weight of approximately 250 kDa. The specific enzymatic activity of the protein was determined for different substrates: sucrose, inulin, Agave tequilana fructan, levan and Actilight® and compared with the activity of Fructozyme®. The hydrolysis profile of the different substrates analyzed by HPAEC-PAD showed that the enzyme has different affinities over the substrates tested with a sucrose/inulin enzymatic activity ratio (S/I) of 125. For the hydrolysis of Agave tequilana fructans, the enzyme also showed a higher enzymatic activity and specificity than Fructozyme®, which is important for its potential application in the tequila industry. Copyright © 2010 Elsevier Ltd. All rights reserved.

  12. Analysis of plasmid diversity in 96 Rhodococcus equi strains isolated in Normandy (France) and sequencing of the 87-kb type I virulence plasmid. (United States)

    Duquesne, Fabien; Hébert, Laurent; Sévin, Corinne; Breuil, Marie-France; Tapprest, Jackie; Laugier, Claire; Petry, Sandrine


    To characterize the potential epidemiological relationship between the origin of Rhodococcus equi strains and the type of their virulence plasmids, we performed a comparative analysis of virulence plasmid types encountered in 96 R. equi strains isolated from (1) autopsied horses, (2) organic samples (horse faeces, manure and straw) and (3) environmental samples. Our results revealed no clear epidemiological link between virulence plasmid type and the origin of R. equi strains isolated from horse-related environments. To understand this result, we determined the nucleotide sequence of the second most frequently isolated virulence plasmid type: a 87-kb type I (pVAPA116) plasmid and compared it with the previously sequenced (and most commonly encountered) 85-kb type I (pVAPA1037) plasmid. Our results show that the divergence between these two plasmids is mainly due to the presence of three allelic exchange loci, resulting in the deletion of two genes and the insertion of three genes in pVAPA116 compared with pVAPA1037. In conclusion, it appears that the divergence between the two sequenced rhodococcal virulence plasmids is not associated with the vap pathogenicity island and may result from an evolutionary process driven by a mobility-related invertase/resolvase invA-like gene. © 2010 ANSES. Journal compilation © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.

  13. Efficient entanglement purification for doubly entangled photon state

    Institute of Scientific and Technical Information of China (English)

    WANG Chuan; SHENG YuBo; LI XiHan; DENG FuGuo; ZHANG Wei; LONG GuiLu


    In this paper,we present an efficient purification scheme that improves the efficiency of entanglement purification of the recently proposed entanglement purification scheme for doubly entangled photon states(Phys.Rev.A,2008,77:042315).This modified scheme contains the bit-flip error correction where all the photon pairs can be kept while all the bit-flip errors are corrected and the entanglement purification of phase-flip errors where a wavelength conversion process is used.This scheme has the advantage of high efficiency and a much lower minimum fidelity of the original state.It works under existing technology.

  14. Efficient entanglement purification for doubly entangled photon state

    Institute of Scientific and Technical Information of China (English)


    In this paper,we present an efficient purification scheme that improves the efficiency of entanglement purification of the recently proposed entanglement purification scheme for doubly entangled photon states (Phys.Rev.A,2008,77:042315).This modified scheme contains the bit-flip error correction where all the photon pairs can be kept while all the bit-flip errors are corrected and the entanglement purification of phase-flip errors where a wavelength conversion process is used.This scheme has the advantage of high efficiency and a much lower minimum fidelity of the original state.It works under existing technology.

  15. Manganese functionalized silicate nanoparticles as a fenton-type catalyst for water purification by advanced oxidation processes (AOP)

    Energy Technology Data Exchange (ETDEWEB)

    Tusar, Natasa Novak [National Institute of Chemistry, Ljubljana (Slovenia); University of Nova Gorica, Nova Gorica (Slovenia); Maucec, Darja; Rangus, Mojca; Mazaj, Matjaz; Cotman, Magda; Pintar, Albin; Kaucic, Venceslav [National Institute of Chemistry, Ljubljana (Slovenia); Arcon, Iztok [University of Nova Gorica, Nova Gorica (Slovenia)


    Wet hydrogen peroxide catalytic oxidation (WHPCO) is one of the most important industrially applicable advanced oxidation processes (AOPs) for the decomposition of organic pollutants in water. It is demonstrated that manganese functionalized silicate nanoparticles with interparticle porosity act as a superior Fenton-type nanocatalyst in WHPCO as they can decompose 80% of a test organic compound in 30 minutes at neutral pH and room temperature. By using X-ray absorption spectroscopic techniques it is also shown that the superior activity of the nanocatalyst can be attributed uniquely to framework manganese, which decomposes H{sub 2}O{sub 2} to reactive hydroxyls and, unlike manganese in Mn{sub 3}O{sub 4} or Mn{sub 2}O{sub 3} nanoparticles, does not promote the simultaneous decomposition of hydrogen peroxide. The presented material thus introduces a new family of Fenton nanocatalysts, which are environmentally friendly, cost-effective, and possess superior efficiency for the decomposition of H{sub 2}O{sub 2} to reactive hydroxyls (AOP), which in turn readily decompose organic pollutants dissolved in water. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  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


    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. Technological assumptions for biogas purification. (United States)

    Makareviciene, Violeta; Sendzikiene, Egle


    Biogas can be used in the engines of transport vehicles and blended into natural gas networks, but it also requires the removal of carbon dioxide, hydrogen sulphide, and moisture. Biogas purification process flow diagrams have been developed for a process enabling the use of a dolomite suspension, as well as for solutions obtained by the filtration of the suspension, to obtain biogas free of hydrogen sulphide and with a carbon dioxide content that does not exceed 2%. The cost of biogas purification was evaluated on the basis of data on biogas production capacity and biogas production cost obtained from local water treatment facilities. It has been found that, with the use of dolomite suspension, the cost of biogas purification is approximately six times lower than that in the case of using a chemical sorbent such as monoethanolamine. The results showed travelling costs using biogas purified by dolomite suspension are nearly 1.5 time lower than travelling costs using gasoline and slightly lower than travelling costs using mineral diesel fuel.

  18. Chemical composition and molecular structure of polysaccharide-protein biopolymer from Durio zibethinus seed: extraction and purification process

    Directory of Open Access Journals (Sweden)

    Amid Bahareh


    Full Text Available Abstract Background The biological functions of natural biopolymers from plant sources depend on their chemical composition and molecular structure. In addition, the extraction and further processing conditions significantly influence the chemical and molecular structure of the plant biopolymer. The main objective of the present study was to characterize the chemical and molecular structure of a natural biopolymer from Durio zibethinus seed. A size-exclusion chromatography coupled to multi angle laser light-scattering (SEC-MALS was applied to analyze the molecular weight (Mw, number average molecular weight (Mn, and polydispersity index (Mw/Mn. Results The most abundant monosaccharide in the carbohydrate composition of durian seed gum were galactose (48.6-59.9%, glucose (37.1-45.1%, arabinose (0.58-3.41%, and xylose (0.3-3.21%. The predominant fatty acid of the lipid fraction from the durian seed gum were palmitic acid (C16:0, palmitoleic acid (C16:1, stearic acid (C18:0, oleic acid (C18:1, linoleic acid (C18:2, and linolenic acid (C18:2. The most abundant amino acids of durian seed gum were: leucine (30.9-37.3%, lysine (6.04-8.36%, aspartic acid (6.10-7.19%, glycine (6.07-7.42%, alanine (5.24-6.14%, glutamic acid (5.57-7.09%, valine (4.5-5.50%, proline (3.87-4.81%, serine (4.39-5.18%, threonine (3.44-6.50%, isoleucine (3.30-4.07%, and phenylalanine (3.11-9.04%. Conclusion The presence of essential amino acids in the chemical structure of durian seed gum reinforces its nutritional value.

  19. Postsymbiotic plasmid acquisition and evolution of the repA1-replicon in Buchnera aphidicola (United States)

    Van Ham, Roeland C. H. J.; González-Candelas, Fernando; Silva, Francisco J.; Sabater, Beatriz; Moya, Andrés; Latorre, Amparo


    Buchnera aphidicola is an obligate, strictly vertically transmitted, bacterial symbiont of aphids. It supplies its host with essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. Several lineages of Buchnera show adaptation to their nutritional role in the form of plasmid-mediated amplification of key-genes involved in the biosynthesis of tryptophan (trpEG) and leucine (leuABCD). Phylogenetic analyses of these plasmid-encoded functions have thus far suggested the absence of horizontal plasmid exchange among lineages of Buchnera. Here, we describe three new Buchnera plasmids, obtained from species of the aphid host families Lachnidae and Pemphigidae. All three plasmids belong to the repA1 family of Buchnera plasmids, which is characterized by the presence of a repA1-replicon responsible for replication initiation. A comprehensive analysis of this family of plasmids unexpectedly revealed significantly incongruent phylogenies for different plasmid and chromosomally encoded loci. We infer from these incongruencies a case of horizontal plasmid transfer in Buchnera. This process may have been mediated by secondary endosymbionts, which occasionally undergo horizontal transmission in aphids. PMID:10984505

  20. Purification of Carbon Nanotubes: Alternative Methods (United States)

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


    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.

  1. The Borexino purification system (United States)

    Benziger, Jay


    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.

  2. 萃取净化盐酸法制备磷酸工艺研究%Study of Preparation Phosphoric Acid Process by Extraction and Purification of Hydrochloric Acid Process

    Institute of Scientific and Technical Information of China (English)

    杨站平; 闫小玲; 张树帜; 王长锐


    Based on preparation crude phosphoric acid by acid hydrolysis of ground phosphate rocks with hydrochloric acid , using process of combination of tributyl phosphate and kerosene mixed solvent extraction with chemical precipitation process to carry out purification treatment for crude phosphoric acid , after refine , concentration , removal of impurities and decolorization , food grade phosphoric product is obtained . Research results show that mass fraction of prepared product phosphoric acid is 85.2%, all indicators achieved not only meet standard requirements of industrial grade phosphoric acid , but also reach qualifications of food grade phosphoric acid .%以盐酸酸解磷矿粉制得粗磷酸为基础,采用磷酸三丁酯和煤油混合溶剂萃取与化学沉淀法相结合的工艺对粗磷酸进行净化处理,通过精制、浓缩、除杂、脱色后可制得食品级磷酸产品。研究结果表明:试验制得的产品磷酸质量分数为85.2%,各项指标不仅达到了工业级磷酸标准要求,而且达到了食品级磷酸标准要求。

  3. Temperature on the Dragon's Blood Phenol Extraction and Purification Process%温度对龙血竭酚类提取物纯化工艺的影响

    Institute of Scientific and Technical Information of China (English)

    徐殿红; 孙永成; 萧伟; 陶永华; 秦建平; 陈利平


    目的:评价温度对龙血竭酚类提取物纯化过程中成分及性状的影响。方法采用高效液相色谱法、粉末堆积密度检测方法,以有效成分转移率及提取物堆密度为指标,考察纯化过程中碱溶解温度以及酸沉温度对龙血竭酚类提取物的指标成分及性状的影响。结果龙血竭酚类提取物纯化过程中碱溶解、酸沉温度均以选择65℃为最佳。结论选择优化的纯化温度,制备得到龙血竭酚类提取物各指标成分转移率较高,堆密度及性状符合要求。%Objective Temperature on the dragon's blood phenol extraction and purification process of composition and the influence of the shape.Methods Using HPLC method,powder bulk density detection method,in order to transfer rate and extract effective components bulk density as an index,purification process of alkali dissolving temperature and temperature of dragon's blood phenolic acid extract the influence of the purity and properties.Results Dragon's blood phenol extraction and purification process,acid alkali dissolve al choose to sink temperature 65℃.Conclusion Temperature purifying selection optimization,prepared from phenolic extracts including exhaust al indexes higher transfer rate,bulk density and capsule filing volume stability and meet the requirements.

  4. Persistence of Antibiotic Resistance Plasmids in Biofilms (United States)


    plasmids* in*populations*of* Gram > negative *bacteria*grown*in*biofilms*and*well>mixed*liquid*cultures.** * Task2:*Characterize*the*evolution*of*plasmid...R.! Edwards.! 2005.! Overview! of! nosocomial! infections! caused! by! gramP negative ! bacilli .!Clin.!Infect.!Dis.!41:848P854.! LoftiePEaton,!W.,!A... negative ! interaction!between!one!of! its!chromosomal!segments!and!the!plasmid! by!simply!deleting!the!appropriate!chromosomal!segment.!! 7. None

  5. DNASU plasmid and PSI:Biology-Materials repositories: resources to accelerate biological research. (United States)

    Seiler, Catherine Y; Park, Jin G; Sharma, Amit; Hunter, Preston; Surapaneni, Padmini; Sedillo, Casey; Field, James; Algar, Rhys; Price, Andrea; Steel, Jason; Throop, Andrea; Fiacco, Michael; LaBaer, Joshua


    The mission of the DNASU Plasmid Repository is to accelerate research by providing high-quality, annotated plasmid samples and online plasmid resources to the research community through the curated DNASU database, website and repository ( or The collection includes plasmids from grant-funded, high-throughput cloning projects performed in our laboratory, plasmids from external researchers, and large collections from consortia such as the ORFeome Collaboration and the NIGMS-funded Protein Structure Initiative: Biology (PSI:Biology). Through DNASU, researchers can search for and access detailed information about each plasmid such as the full length gene insert sequence, vector information, associated publications, and links to external resources that provide additional protein annotations and experimental protocols. Plasmids can be requested directly through the DNASU website. DNASU and the PSI:Biology-Materials Repositories were previously described in the 2010 NAR Database Issue (Cormier, C.Y., Mohr, S.E., Zuo, D., Hu, Y., Rolfs, A., Kramer, J., Taycher, E., Kelley, F., Fiacco, M., Turnbull, G. et al. (2010) Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community. Nucleic Acids Res., 38, D743-D749.). In this update we will describe the plasmid collection and highlight the new features in the website redesign, including new browse/search options, plasmid annotations and a dynamic vector mapping feature that was developed in collaboration with LabGenius. Overall, these plasmid resources continue to enable research with the goal of elucidating the role of proteins in both normal biological processes and disease.

  6. Plasmid profiles of Moraxella bovis isolates. (United States)

    McDonald, T J; Pugh, G W


    Two-hundred isolates of Moraxella bovis were selected at random and examined for the presence of plasmid DNA by a rapid alkaline-detergent lysis method. All isolates contained from 1 to 6 plasmids, with varying agarose-gel electrophoretic migration patterns. Most (80%) isolates carried 2 to 4 plasmids, which ranged in molecular weight from 2.6 to 80 megadaltons. Seemingly, plasmid profiles can be used as a simple, reliable epizootiologic tool to establish a strain identification scheme for M bovis.

  7. Plasmid transfer systems in the rhizobia. (United States)

    Ding, Hao; Hynes, Michael F


    Rhizobia are agriculturally important bacteria that can form nitrogen-fixing nodules on the roots of leguminous plants. Agricultural application of rhizobial inoculants can play an important role in increasing leguminous crop yields. In temperate rhizobia, genes involved in nodulation and nitrogen fixation are usually located on one or more large plasmids (pSyms) or on symbiotic islands. In addition, other large plasmids of rhizobia carry genes that are beneficial for survival and competition of rhizobia in the rhizosphere. Conjugative transfer of these large plasmids thus plays an important role in the evolution of rhizobia. Therefore, understanding the mechanism of conjugative transfer of large rhizobial plasmids provides foundations for maintaining, monitoring, and predicting the behaviour of these plasmids during field release events. In this minireview, we summarize two types of known rhizobial conjugative plasmids, including quorum sensing regulated plasmids and RctA-repressed plasmids. We provide evidence for the existence of a third type of conjugative plasmid, including pRleVF39c in Rhizobium leguminosarum bv. viciae strain VF39SM, and we provide a comparison of the different types of conjugation genes found in members of the rhizobia that have had their genomes sequenced so far.

  8. Effect of chromosome homology an plasmid transformation and plasmid conjugal transfer in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.


    The pairing between plasmid and the homologous part of the chromosome associated with plasmid establishment may differ from the pairing which results from integration of a homologous region of the plasmid into the chromosome. Thus the rate of novobiocin transformation decreases with duplication of the chromosomal portion in pMB2, but the rate of establishment of the plasmid increases with this duplication. A model to explain these data is given. 17 references, 5 figures, 4 tables.

  9. Expression and Purification of Functional Human Vascular Endothelial Growth Factor-A121; the Most Important Angiogenesis Factor

    Directory of Open Access Journals (Sweden)

    Fatemeh Kazemi-Lomedasht


    Full Text Available Purpose: Angiogenesis or formation of new blood vessels is an essential process for tumor growth, invasion and metastasis. Vascular Endothelial Growth Factor (VEGF and its receptors play an important role in angiogenesis-dependent tumors. VEGF-A is the most important factor in angiogenesis process. Human VEGF-A gene consists of eight exons that undergoes alternative exon splicing and produce five different proteins consisting of 121, 145, 165, 189 and 206 amino acids (named VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206. Methods: In this study, VEGF121 gene synthesized and cloned into the pET-26b plasmid. The recombinant plasmid was transferred into appropriate expression strain of BL-21. Expression of VEGF121 induced by IPTG (Isopropyl β-D-1-thiogalactopyranoside and confirmed by SDS-PAGE and Western-Blotting. Recombinant VEGF121 was purified by nickel affinity chromatography. HUVECs (Human Umbilical Vein Endothelia Cells cells were isolated from umbilical vein and the effect of VEGF121 on tube formation of endothelial cells was investigated. Results: SDS-PAGE and Western-Blotting results verified the purification of VEGF121. The final yield of recombinant protein was about 5mg per liter. Endothelial cell tube formation assay results showed that VEGF121 leads to tube formation of endothelial cell on matrix and induces angiogenesis in vitro. Conclusion: Recombinant VEGF121 is important factor in tube formation of endothelial cell, so it could be used in different cancer researches and angiogenesis assay.

  10. Expression, purification, and molecular analysis of the Necator americanus glutathione S-transferase 1 (Na-GST-1): a production process developed for a lead candidate recombinant hookworm vaccine antigen. (United States)

    Goud, Gaddam Narsa; Deumic, Vehid; Gupta, Richi; Brelsford, Jill; Zhan, Bin; Gillespie, Portia; Plieskatt, Jordan L; Tsao, Eric I; Hotez, Peter J; Bottazzi, Maria Elena


    The enzyme Necator americanus glutathione S-transferase 1 (Na-GST-1) belongs to a unique Nu class of GSTs and is a lead candidate antigen in a bivalent human hookworm vaccine. Here we describe the expression of Na-GST-1 in the yeast Pichia pastoris at the 20 L manufacturing scale and its purification process performed by three chromatographic steps, comprised of a Q Sepharose XL anion exchange column, followed by a Butyl Sepharose HP hydrophobic affinity column and a Superdex 75 size-exclusion column. Approximately 1.5 g of recombinant protein was recovered at an overall process yield of 51%, with a purity grade of 98% and the absence of detectable host cell protein. By mass spectrometry the recombinant protein exhibits a mass of 23,676Da, which closely matches the predicted molecular mass of the protein. The expression and purification methods described here are suitable for further scale-up product development and for its use to design formulation processes suitable to generate a vaccine for clinical testing. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. The expression of a plasmid-specified exported protein causes structural plasmid instability in Bacillus subtilis

    NARCIS (Netherlands)

    Cordes, C.; Meima, R; Twiest, B; Kazemier, B; Venema, G; vanDijl, JM; Bron, S

    The rolling-circle plasmid pGP1 was used to study the effects of the expression of a plasmid-specified exported protein on structural plasmid stability in Bacillus subtilis. pGP1 contains a fusion between the Bacillus licheniformis penP gene, encoding a C-terminally truncated penicillinase, and the

  12. Development and monitoring of a novel monoclonal antibody purification strategy


    Capito, Florian


    The studies presented in the cumulative part of this thesis illustrate the different steps to develop a polymer-driven antibody purification process. These peer-reviewed reports show in detail fundamental research, additional method development useful in the development of such a purification process as well as implementation of the final process. A strategy for analyzing copolymers, synthesized by a lab in house, was implemented with particular emphasis on copolymer composition analysis. Thi...

  13. Surveillance of Kanamycin Resistance to Escherichia coli from Swine by Digoxigenin-labled Plasmid Probe

    Institute of Scientific and Technical Information of China (English)

    YANG Han-chun; ZHAO Jing; LIU Jin-hua; ZHA Zhen-lin; CHEN Yan-hong


    A 4.34kb EcoR I fragment of kanamycin resistance plasmid from pET - 9a was purified by a DNA purification kit. The fragment was labeled with digoxigenin-dUTP with a commercial kit. A dot-blot hybridization and a colony hybridization test with the probe were successfully developed for the surveillance of Kanamycin resistance to E. coli from swine. It was shown that the methods obtained 100% concordance in a positive tate. It was indicated that the method was available for the surveillance of kanamycin resistance to E.coli from swine.

  14. Genomic and Functional Characterization of qnr-Encoding Plasmids from Municipal Wastewater Biosolid Klebsiella pneumoniae Isolates. (United States)

    Kaplan, Ella; Sela, Noa; Doron-Faigenboim, Adi; Navon-Venezia, Shiri; Jurkevitch, Edouard; Cytryn, Eddie


    plasmids concomitant to phylogenetic analysis of housekeeping genes from host Klebsiella strains, revealed that these plasmids are limited to a predominantly human-associated sub-clade of Klebsiella, suggesting that their host range is very narrow. Conversely, the pGNB2-like plasmids had a much broader host range and appeared to be associated with Klebsiella residing in natural environments. This study suggests that: (A) qnrB-harboring multidrug-resistant pKPN3-like plasmids can endure the rigorous wastewater treatment process and may therefore be disseminated to downstream environments; and (B) that small qnrS-harboring pGNB2-like plasmids are ubiquitous in wastewater treatment facilities and are most likely environmental in origin.

  15. 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)


    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

  16. Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community (United States)

    Cormier, Catherine Y.; Mohr, Stephanie E.; Zuo, Dongmei; Hu, Yanhui; Rolfs, Andreas; Kramer, Jason; Taycher, Elena; Kelley, Fontina; Fiacco, Michael; Turnbull, Greggory; LaBaer, Joshua


    The Protein Structure Initiative Material Repository (PSI-MR; provides centralized storage and distribution for the protein expression plasmids created by PSI researchers. These plasmids are a resource that allows the research community to dissect the biological function of proteins whose structures have been identified by the PSI. The plasmid annotation, which includes the full length sequence, vector information and associated publications, is stored in a freely available, searchable database called DNASU ( Each PSI plasmid is also linked to a variety of additional resources, which facilitates cross-referencing of a particular plasmid to protein annotations and experimental data. Plasmid samples can be requested directly through the website. We have also developed a novel strategy to avoid the most common concern encountered when distributing plasmids namely, the complexity of material transfer agreement (MTA) processing and the resulting delays this causes. The Expedited Process MTA, in which we created a network of institutions that agree to the terms of transfer in advance of a material request, eliminates these delays. Our hope is that by creating a repository of expression-ready plasmids and expediting the process for receiving these plasmids, we will help accelerate the accessibility and pace of scientific discovery. PMID:19906724

  17. Expression, purification, and functional characterization of recombinant PTD-SARA

    Institute of Scientific and Technical Information of China (English)

    Chen Huang; Rui Du; Peng Zhang; Hua Meng; Huiwei Jia; Yang Song; Man Li; Yingqi Zhang; Shiren Sun


    The Smad anchor for receptor activation (SARA) protein is a binding partner for Smad2/3 that plays an important role in the fibrotic promoting signaling pathway initiated by transforming growth factor-β1 (TGF-β1). The C-terminal 665-750 aa of SARA comprises the Smad-binding domain (SBD). By direct interaction through the SBD, SARA inhibits Smad2/3 phosphorylation and blocks the interaction between Smad2/3 and Smad4, thereby restrains the process of fibrosis.In this study, we constructed a SARA peptide aptamer based on the SBD sequence. The recombinant SARA aptamer,fused with a protein transduction domain (PTD-SARA), was cloned, purified from E. coli, and characterized for the first time. The full-length PTD-SARA coding sequence, created with E. coli favored codons, was cloned into a pQE-30 vector,and the recombinant plasmid was transformed into an M15 strain. After Isopropyl β-D-1-Thiogalactopyranoside (IPTG) induction and Ni2+ affinity purification, recombinant PTD-SARA was further identified by immunobiotting and protein N-terminal sequencing. Epifluorescence microscopy revealed that the recombinant PTD-SARA was transferred into the cytoplasm and nucleus more efficiently than SARA.Moreover, the recombinant PTD-SARA was found to up-regulate the level of E-cadherin and down-regulate the levels of α-SMA and phospho-Smad3 more efficiently than SARA (P< 0.05). Our work explored a method to obtain recombinant PTD-SARA protein. The recombinant PTDSARA fusion protein could enter HK2 cells (an immortalized proximal tubule epithelial cell line) more efficiently than the SARA protein and reverse the renal epithelial-to-mesenchymal transdifferentiation process that was induced by TGF-β1 more effectively than the SARA protein. Recombinant PDT-SARA is likely to be a potential candidate for clinical prevention and treatment of renal fibrosis.

  18. New and Redesigned pRS Plasmid Shuttle Vectors for Genetic Manipulation of Saccharomycescerevisiae. (United States)

    Chee, Mark K; Haase, Steven B


    We have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in the budding yeast Saccharomyces cerevisiae and the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes HIS2 and ADE1, respectively. Our pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed to increase the availability of unique restriction sites within their polylinker regions. Hence, our pRSII plasmids are a complete set of integrating, centromere and 2μ episomal plasmids with the biosynthetic marker genes ADE2, HIS3, TRP1, LEU2, URA3, HIS2, and ADE1 and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, we have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin, and bialaphos. Finally, in the process of generating the new plasmids, we have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using our updated sequences, we constructed pRS plasmid backbones with a unique restriction site for inserting new markers to facilitate future expansion of the pRS series.

  19. Differential behavior of plasmids containing chromosomal DNA insertions of various sizes during transformation and conjugation in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.


    Plasmids with chromosomal insertions were constructed by removal of a 1.1-kilobase-pair piece from the 9.8-kilobase-pair vector plasmid pDM2 by EcoRI digestion and inserting in its place various lengths of chromosomal DNA (1.7, 3.4, and 9.0 kilobase pairs) coding for resistance to novobiocin. A fourth plasmid was constructed by insertion of the largest piece of chromosomal DNA into the SmaI site of pDM2. The plasmids without inserts were taken up poorly by competent cells and thus were considered not to contain specific DNA uptake sites. The presence of even the smallest insert of chromosomal DNA caused a large increase in transformation of Rec/sup +/ and Rec/sup -/ strains. The frequency of plasmid establishment in Rec/sup +/ cells by transformation increased exponentially with increasing insert size, but in Rec/sup -/ cells there was less transformation by the larger plasmids. Conjugal transfer of these plasmids was carried out with the 35-kilobase-pair mobilizing plasmid pHD147. The frequency of establishment of plasmids by this method not only was not markedly affected by the presence of the insertions, but also decreased somewhat with increase in insert size and was independent of rec-1 and rec-2 genes. Recombination between plasmid and chromosome was readily detected after transformation, but could not be detected after transconjugation even when the recipient cells were Rec/sup +/ and made competent. These data suggested that there is a special processing of plasmid DNA that enters the competent cells in transformation that makes possible recombination of homologous regions of the plasmid with the chromosome and pairing with the chromosome that aids plasmid establishment.

  20. Microbial degradation of pyridine using Pseudomonas sp. and isolation of plasmid responsible for degradation. (United States)

    Mohan, S Venkata; Sistla, Srinivas; Guru, R Kumar; Prasad, K Krishna; Kumar, C Suresh; Ramakrishna, S V; Sarma, P N


    Pseudomonas (PI2) capable of degrading pyridine was isolated from the mixed population of the activated sludge unit which was being used for treating complex effluents, the strain was characterized. Aerobic degradation of pyridine was studied with the isolated strain and the growth parameters were evaluated. Pyridine degradation was further conformed by chromatography (HPLC) analysis. The process parameters like biomass growth and dissolved oxygen consumption were monitored during pyridine degradation. In order to conform with the plasmid capability to degrade pyridine, the requisite plasmid was isolated and transferred to DH 5alpha Escherichia coli. The subsequent biodegradation studies revealed the ability of the transformed plasmid capability to degrade the pyridine.

  1. Screening large numbers of recombinant plasmids: modifications and additions to alkaline lysis for greater efficiency

    Institute of Scientific and Technical Information of China (English)



    Selecting bacteria transformed with recombinant plasmid is a laborious step in gene cloning experiments. This selection process is even more tedious when large numbers of clones need to be screened. We describe here modifications to the ultra fast plasmid preparation method described previously by Law and Crickmore. The modified method is coupled to an efficient PCR step to rapidly determine orientation of the inserts. Compared to traditional methods of analysis requiring growth of overnight cultures, plasmid isolation and restriction enzyme digestion to determine orientation this procedure allows for the analysis and storage of a large number of recombinants within a few hours.

  2. Structural analysis of the ParR/parC plasmid partition complex

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Ringgaard, Simon; Mercogliano, Christopher P


    Accurate DNA partition at cell division is vital to all living organisms. In bacteria, this process can involve partition loci, which are found on both chromosomes and plasmids. The initial step in Escherichia coli plasmid R1 partition involves the formation of a partition complex between the DNA...... and biochemical experiments support a structural arrangement in which the centromere-like parC DNA is wrapped around a ParR protein scaffold. This structure holds implications for how ParM polymerization drives active DNA transport during plasmid partition....

  3. Factors affecting plasmid production in Escherichia coli from a resource allocation standpoint

    Directory of Open Access Journals (Sweden)

    Cunningham Drew S


    Full Text Available Abstract Background Plasmids are being reconsidered as viable vector alternatives to viruses for gene therapies and vaccines because they are safer, non-toxic, and simpler to produce. Accordingly, there has been renewed interest in the production of plasmid DNA itself as the therapeutic end-product of a bioprocess. Improvement to the best current yields and productivities of such emerging processes would help ensure economic feasibility on the industrial scale. Our goal, therefore, was to develop a stoichiometric model of Escherichia coli metabolism in order to (1 determine its maximum theoretical plasmid-producing capacity, and to (2 identify factors that significantly impact plasmid production. Results Such a model was developed for the production of a high copy plasmid under conditions of batch aerobic growth on glucose minimal medium. The objective of the model was to maximize plasmid production. By employing certain constraints and examining the resulting flux distributions, several factors were determined that significantly impact plasmid yield. Acetate production and constitutive expression of the plasmid's antibiotic resistance marker exert negative effects, while low pyruvate kinase (Pyk flux and the generation of NADPH by transhydrogenase activity offer positive effects. The highest theoretical yield (592 mg/g resulted under conditions of no marker or acetate production, nil Pyk flux, and the maximum allowable transhydrogenase activity. For comparison, when these four fluxes were constrained to wild-type values, yields on the order of tens of mg/g resulted, which are on par with the best experimental yields reported to date. Conclusion These results suggest that specific plasmid yields can theoretically reach 12 times their current experimental maximum (51 mg/g. Moreover, they imply that abolishing Pyk activity and/or transhydrogenase up-regulation would be useful strategies to implement when designing host strains for plasmid

  4. Genomic and functional characterization of qnr-encoding plasmids from municipal wastewater biosolid Klebsiella pneumoniae isolates

    Directory of Open Access Journals (Sweden)

    Ella eKaplan


    KPN3-like plasmids concomitant to phylogenetic analysis of housekeeping genes from host Klebsiella strains, revealed that these plasmids are limited to a predominantly human-associated sub-clade of Klebsiella, suggesting that their host range is very narrow. Conversely, the pGNB2-like plasmids had a much broader host range and appeared to be associated with Klebsiella residing in natural environments. This study suggests that: (A qnrB-harboring multidrug-resistant pKPN3-like plasmids can endure the rigorous wastewater treatment process and may therefore be disseminated to downstream environments; and (B that small qnrS-harboring.

  5. Cultivation-independent screening revealed hot spots of IncP-1, IncP-7 and IncP-9 plasmid occurrence in different environmental habitats.

    Directory of Open Access Journals (Sweden)

    Simone Dealtry

    Full Text Available IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC- DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC-DNA from biopurification systems (BPS which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are "hot spots" of plasmids potentially carrying catabolic genes.

  6. Renaissance of protein crystallization and precipitation in biopharmaceuticals purification. (United States)

    Dos Santos, Raquel; Carvalho, Ana Luísa; Roque, A Cecília A

    The current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand. With the upstream improvements, the bottleneck shifted towards the downstream process. New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective. Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules, overcoming the current chromatographic bottlenecks. Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules. This review focuses in the role of these two methodologies in current industrial purification processes, and highlights their potential implementation in the purification pipeline of high value therapeutic molecules, overcoming chromatographic holdups. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Accurate determination of plasmid copy number of flow-sorted cells using droplet digital PCR. (United States)

    Jahn, Michael; Vorpahl, Carsten; Türkowsky, Dominique; Lindmeyer, Martin; Bühler, Bruno; Harms, Hauke; Müller, Susann


    Many biotechnological processes rely on the expression of a plasmid-based target gene. A constant and sufficient number of plasmids per cell is desired for efficient protein production. To date, only a few methods for the determination of plasmid copy number (PCN) are available, and most of them average the PCN of total populations disregarding heterogeneous distributions. Here, we utilize the highly precise quantification of DNA molecules by droplet digital PCR (ddPCR) and combine it with cell sorting using flow cytometry. A duplex PCR assay was set up requiring only 1000 sorted cells for precise determination of PCN. The robustness of this method was proven by thorough optimization of cell sorting, cell disruption, and PCR conditions. When non plasmid-harboring cells of Pseudomonas putida KT2440 were spiked with different dilutions of the expression plasmid pA-EGFP_B, a PCN from 1 to 64 could be accurately detected. As a proof of principle, induced cultures of P. putida KT2440 producing an EGFP-fused model protein by means of the plasmid pA-EGFP_B were investigated by flow cytometry and showed two distinct subpopulations, fluorescent and nonfluorescent cells. These two subpopulations were sorted for PCN determination with ddPCR. A remarkably diverging plasmid distribution was found within the population, with nonfluorescent cells showing a much lower PCN (≤1) than fluorescent cells (PCN of up to 5) under standard conditions.

  8. Plasmid typing of Shigella sonnei epidemic strains and molecular relationship of their R-plasmids. (United States)

    Mendoza, M C; Gonzalez, A J; Mendez, F J; Hardisson, C


    We conducted a surveillance program on epidemic and/or endemic Shigella strains in Asturias (Spain), their frequency and dispersion in our community, and their R-plasmids. We analyzed initial isolates of Shigella sonnei from two epidemic outbreaks using antibiotic resistance patterns and plasmid profile analysis as epidemiological markers. We found that the 2 outbreaks were caused by different S. sonnei strains, which respectively carried one and two R-plasmids together with other plasmids. The molecular relationship among these and three other R-plasmids from two S. sonnei strains isolated during a previous outbreak, were studied by restriction enzyme analysis and DNA-DNA hybridizations. We were able to establish different levels of relationship among the six R-plasmids.

  9. Purification in a functional form of the terminal protein of Bacillus subtilis phage phi 29.



    Phage phi 29 terminal protein, p3, essentially pure, was isolated in a denatured form from viral particles, and anti-p3 antiserum was obtained. A radioimmunoassay to detect and quantitate protein p3 was developed. By using this assay, native protein p3 was highly purified from Escherichia coli cells harboring a gene 3-containing recombinant plasmid. After three purification steps, the protein was more than 96% pure; its amino acid composition was very similar to that deduced from the nucleoti...

  10. Purification in a functional form of the terminal protein of Bacillus subtilis phage ø29



    Phage phi 29 terminal protein, p3, essentially pure, was isolated in a denatured form from viral particles, and anti-p3 antiserum was obtained. A radioimmunoassay to detect and quantitate protein p3 was developed. By using this assay, native protein p3 was highly purified from Escherichia coli cells harboring a gene 3-containing recombinant plasmid. After three purification steps, the protein was more than 96% pure; its amino acid composition was very similar to that deduced from the nucleoti...

  11. Time-course determination of plasmid content in eukaryotic and prokaryotic cells using real-time PCR. (United States)

    Carapuça, Elisabete; Azzoni, Adriano R; Prazeres, Duarte M F; Monteiro, Gabriel A; Mergulhão, Filipe J M


    A Real-Time PCR method was developed to monitor the plasmid copy number (PCN) in Escherichia coli and Chinese hamster ovary (CHO) cells. E. coli was transformed with plasmids containing a ColE1 or p15A origin of replication and CHO cells were transfected with a ColE1 derived plasmid used in DNA vaccination and carrying the green fluorescent protein (GFP) reporter gene. The procedure requires neither specific cell lysis nor DNA purification and can be performed in cells, respectively. Analysis of PCN in E. coli batch cultures revealed that the maximum copy number per cell is attained in mid-exponential phase and that this number decreases on average 80% towards the end of cultivation for both types of plasmids. The plasmid content of CHO cells determined 24 h post-transfection was around 3 x 104 copies per cell although only 37% of the cells expressed GFP one day after transfection. The half-life of pDNA was 20 h and around 100 copies/cell were still detected 6 days after transfection.

  12. Purification and characterization of exonuclease-free Artemis: Implications for DNA-PK – dependent processing of DNA termini in NHEJ catalyzed DSB repair (United States)

    Pawelczak, Katherine S.; Turchi, John J.


    Artemis is a member of the β–CASP family of nucleases in the metallo-β-lactamase superfamily of hydrolases. Artemis has been demonstrated to be involved in V(D)J-recombination and in the NHEJ-catalyzed repair of DNA DSBs. In vitro, both DNA-PK independent 5’ to 3’ exonuclease activity and DNA-PK dependent endonuclease activity have been attributed to Artemis, though mutational analysis of the Artemis active site only disrupts endonuclease activity. This suggests that either the enzyme contains two different active sites, or the exonuclease activity is not intrinsic to the Artemis polypeptide. To distinguish between these possibilities, we sought to determine if it was possible to biochemically separate Artemis endonuclease activity from exonuclease activity. Recombinant [His]6–Artemis was expressed in a Baculovirus insect-cell expression system and isolated using a three-column purification methodology. Exonuclease and endonuclease activity, the ability to be phosphorylated by DNA-PK, and Artemis antibody reactivity was monitored throughout the purification and to characterize final pools of protein preparation. Results demonstrated the co-elution of exonuclease and endonuclease activity on a Ni-Agarose affinity column but separation of the two enzymatic activities upon fractionation on a hydroxyapatite column. An exonuclease free fraction of Artemis was obtained that retained DNA-PK dependent endonuclease activity, was phosphorylated by DNA-PK and reacted with an Artemis specific antibody. These data demonstrate that the exonuclease activity thought to be intrinsic to Artemis can be biochemically separated from the Artemis endonuclease. PMID:20347402

  13. Purification and characterization of exonuclease-free Artemis: Implications for DNA-PK-dependent processing of DNA termini in NHEJ-catalyzed DSB repair. (United States)

    Pawelczak, Katherine S; Turchi, John J


    Artemis is a member of the beta-CASP family of nucleases in the metallo-beta-lactamase superfamily of hydrolases. Artemis has been demonstrated to be involved in V(D)J-recombination and in the NHEJ-catalyzed repair of DNA DSBs. In vitro, both DNA-PK independent 5'-3' exonuclease activities and DNA-PK dependent endonuclease activity have been attributed to Artemis, though mutational analysis of the Artemis active site only disrupts endonuclease activity. This suggests that either the enzyme contains two different active sites, or the exonuclease activity is not intrinsic to the Artemis polypeptide. To distinguish between these possibilities, we sought to determine if it was possible to biochemically separate Artemis endonuclease activity from exonuclease activity. Recombinant [His](6)-Artemis was expressed in a Baculovirus insect-cell expression system and isolated using a three-column purification methodology. Exonuclease and endonuclease activities, the ability to be phosphorylated by DNA-PK, and Artemis antibody reactivity was monitored throughout the purification and to characterize final pools of protein preparation. Results demonstrated the co-elution of exonuclease and endonuclease activities on a Ni-agarose affinity column but separation of the two enzymatic activities upon fractionation on a hydroxyapatite column. An exonuclease-free fraction of Artemis was obtained that retained DNA-PK dependent endonuclease activity, was phosphorylated by DNA-PK and reacted with an Artemis specific antibody. These data demonstrate that the exonuclease activity thought to be intrinsic to Artemis can be biochemically separated from the Artemis endonuclease. Copyright 2010 Elsevier B.V. All rights reserved.

  14. Removal of radioactive iodine and cesium in water purification processes after an explosion at a nuclear power plant due to the Great East Japan Earthquake. (United States)

    Kosaka, Koji; Asami, Mari; Kobashigawa, Naoya; Ohkubo, Keiko; Terada, Hiroshi; Kishida, Naohiro; Akiba, Michihiro


    The presence of radionuclides at five water purification plants was investigated after an explosion at a nuclear power plant hit by the Great East Japan Earthquake on 11 March 2011. Radioactive iodine (¹³¹I) and cesium (¹³⁴Cs and ¹³⁷Cs) were detected in raw water in Fukushima and neighboring prefectures. ¹³¹I was not removed by coagulation-flocculation-sedimentation. ¹³¹I was removed by granular activated carbon (GAC) and powdered activated carbon (PAC) at a level of about 30%-40%, although ¹³¹I was not removed in some cases. This was also confirmed by laboratory-scale experiments using PAC. The removal percentages of ¹³¹I in river and pond waters by 25 mg dry/L of PAC increased from 36% to 59% and from 41% to 48%, respectively, with chlorine dosing before PAC. ¹³⁴Cs and ¹³⁷Cs were effectively removed by coagulation at both a water purification plant and in laboratory-scale experiments when turbidity was relatively high. In contrast, ¹³⁴Cs and ¹³⁷Cs in pond water with low turbidity were not removed by coagulation. This was because ¹³⁴Cs and ¹³⁷Cs in river water were present mainly in particulate form, while in pond water they were present mainly as cesium ions (¹³⁴Cs+ and ¹³⁷Cs+). However, the removal of ¹³⁴Cs and ¹³⁷Cs in pond water by coagulation increased markedly when ¹³⁴Cs and ¹³⁷Cs were mixed with sediment 24 h before coagulation.

  15. Prevalence and molecular characterization of plasmid- mediated ...

    African Journals Online (AJOL)

    lactamase genes among nosocomial Staphylococcus aureus drug resistance isolates in Taiwan. .... Table 2: Plasmid profiles of the clinical antibiotic-resistant pathogens. Strain. Profile .... Madec J. Characterization of clinical canine methicillin-.

  16. antimicrobial susceptibility and plasmids from escherichia coli ...

    African Journals Online (AJOL)


    Oct 10, 2001 ... transmission to humans of E. coli containing antibiotic resistance plasmids ... resistant micro-organisms, which may in turn transfer resistance to .... cells were washed with sterile normal saline to remove leached. Я-lactamase ...

  17. Protein diversity confers specificity in plasmid segregation. (United States)

    Fothergill, Timothy J G; Barillà, Daniela; Hayes, Finbarr


    The ParG segregation protein (8.6 kDa) of multidrug resistance plasmid TP228 is a homodimeric DNA-binding factor. The ParG dimer consists of intertwined C-terminal domains that adopt a ribbon-helix-helix architecture and a pair of flexible, unstructured N-terminal tails. A variety of plasmids possess partition loci with similar organizations to that of TP228, but instead of ParG homologs, these plasmids specify a diversity of unrelated, but similarly sized, partition proteins. These include the proteobacterial pTAR, pVT745, and pB171 plasmids. The ParG analogs of these plasmids were characterized in parallel with the ParG homolog encoded by the pseudomonal plasmid pVS1. Like ParG, the four proteins are dimeric. No heterodimerization was detectable in vivo among the proteins nor with the prototypical ParG protein, suggesting that monomer-monomer interactions are specific among the five proteins. Nevertheless, as with ParG, the ParG analogs all possess significant amounts of unordered amino acid residues, potentially highlighting a common structural link among the proteins. Furthermore, the ParG analogs bind specifically to the DNA regions located upstream of their homologous parF-like genes. These nucleoprotein interactions are largely restricted to cognate protein-DNA pairs. The results reveal that the partition complexes of these and related plasmids have recruited disparate DNA-binding factors that provide a layer of specificity to the macromolecular interactions that mediate plasmid segregation.

  18. Plasmid and chromosome segregation in prokaryotes

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Bugge Jensen, Rasmus; Gerdes, Kenn


    Recent major advances in the understanding of prokaryotic DNA segregation have been achieved by using fluorescence microscopy to visualize the localization of cellular components. Plasmids and bacterial chromosomes are partitioned in a highly dynamic fashion, suggesting the presence of a mitotic......-like apparatus in prokaryotes. The identification of chromosomal homologues of the well-characterized plasmid partitioning genes indicates that there could be a general mechanism of bacterial DNA partitioning. Udgivelsesdato: July 1...

  19. Curing of plasmid pXO1 from Bacillus anthracis using plasmid incompatibility.

    Directory of Open Access Journals (Sweden)

    Xiankai Liu

    Full Text Available The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.

  20. Simulation of Glycerol Purification Process Based on MVR Heat-pump Distillation%基于MVR热泵精馏的粗甘油脱水提纯工艺模拟研究

    Institute of Scientific and Technical Information of China (English)

    阮宗琳; 杨秀娜; 姜阳; 齐慧敏


    Based on Aspen Plus with the NRTL-RK equation, Radfrac. module and Compr. module, simulation of glycerol purification process was carried out. The differences between the conventional distillation process and two kinds of heat pump distillation processes were compared. The two kinds of heat pump distillation processes were the top steam direct compression and the tower bottom liquid phase flash compression. The results show that,for the glycerol purification process, under the same condition of processing capacity, product quality, operating pressure, reflux ratio and product purity (no less than 99%), the two MVR heat-pump distillation processes can save respectively energy by 56.5% and 54.5% than the conventional distillation process. The total energy consumption (standard oil / ton of product) of the two kinds of heat pump distillation processes can be saved as much as 58.75% and 56.67% respectively, reflecting significant energy saving effect.%利用Aspen Plus流程模拟软件,选用NRTL-RK物性模型和精馏模型格及压缩机模块对粗甘油脱水过程进行了模拟计算,分别计算了塔顶汽相出料直接压缩热泵精馏、塔底产物闪蒸压缩热泵精馏以及常规精馏,结果表明:对于粗甘油脱水提出过程来说,在相同的原料处理量、产品质量、操作压力及回流比、产品纯度(≥99%)时,两种热泵精馏工艺均比常规精馏工艺的能耗有所降低,分别节能56.5%和54.5%,总能耗(标油/吨产品)比常规精馏工艺分别节能58.75%和56.67%,具有十分显著的节能效果。

  1. Multiple plasmid interference - Pledging allegiance to my enemy's enemy. (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco


    As shown in the previous article, two distinct conjugative plasmids sometimes interact within bacterial cells, implicating changes of transfer rates. In most cases of interactions within bacteria, the transfer of one of the plasmids decreases. Less frequently, the transfer rate of one of the plasmids increases. Here we analyse what happens if three distinct conjugative plasmids colonize the same bacterial cell. Our aim is to understand how interactions between two plasmids affect the transfer rate of the third plasmid. After showing that plasmids interact in 59 out of 84 possible interactions we show that, with some exceptions, if the transfer rate of a plasmid decreases in the presence of a second plasmid, a decrease is also observed in the presence of a third plasmid. Moreover, if the conjugation rate of a plasmid increases in the presence of another, an increase is also observed if there is a third plasmid in the cell. Both types of interactions are mostly independent of the third plasmid's identity, even if sometimes the third plasmid quantitatively distorts the interaction of the other two plasmids. There is a bias towards negative intensifying interactions, which provide good news concerning the spread conjugative plasmids encoding antibiotic-resistance genes and virulence factors. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Clostridium perfringens type A–E toxin plasmids (United States)

    Freedman, John C.; Theoret, James R.; Wisniewski, Jessica A.; Uzal, Francisco A.; Rood, Julian I.; McClane, Bruce A.


    Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell. PMID:25283728

  3. Historical Events That Spawned the Field of Plasmid Biology. (United States)

    Kado, Clarence I


    This chapter revisits the historical development and outcome of studies focused on the transmissible, extrachromosomal genetic elements called plasmids. Early work on plasmids involved structural and genetic mapping of these molecules, followed by the development of an understanding of how plasmids replicate and segregate during cell division. The intriguing property of plasmid transmission between bacteria and between bacteria and higher cells has received considerable attention. The utilitarian aspects of plasmids are described, including examples of various plasmid vector systems. This chapter also discusses the functional attributes of plasmids needed for their persistence and survival in nature and in man-made environments. The term plasmid biology was first conceived at the Fallen Leaf Lake Conference on Promiscuous Plasmids, 1990, Lake Tahoe, California. The International Society for Plasmid Biology was established in 2004 (

  4. Drinking water safety and the development of purification technology

    Institute of Scientific and Technical Information of China (English)

    Li Guibai


    This paper briefly introduced the evolution of purification technology for drinking water over time. After description of the 1st generation processes in the beginning of the 20th century -- conventional processes and the 2nd generation processes in 1970s -- advanced treatment processes, a tertiary processes -- UF (ultrafiltration) based on integrated processes was proposed. Moreover, reaction measures (dosing variety of regents for different contaminants) for urban source water emergencies.were illustrated in brief. A new technology of KMnO4 and potassium permanganate composite (PPC) for drinking water purification which was developed by Harbin Institute of Technology (HIT) was concisely introduced.

  5. Preparative Purification of Liriodendrin from Sargentodoxa cuneata by Macroporous Resin


    Di-Hua Li; Yan Wang; Yuan-Shan Lv; Jun-Hong Liu; Lei Yang; Shu-Kun Zhang; Yu-Zhen Zhuo


    The preparative purification of liriodendrin from Sargentodoxa cuneata using macroporous resin combined with crystallization process was evaluated. The properties of adsorption/desorption of liriodendrin on eight macroporous resins were investigated systematically. X-5 resin was selected as the most suitable medium for liriodendrin purification. The adsorption of liriodendrin on X-5 resin fitted well with the pseudo-second-order kinetic model and Langmuir isotherm model. Dynamic adsorption/de...

  6. Biodiesel Production Using Wet and Dry Purification Methods


    DEMIR, Veli Gokhan


    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...

  7. Sodium Purification Device for Production of Tantalum Powder

    Institute of Scientific and Technical Information of China (English)


    In the process of tantalum powder production it requires pure sodium to reduce potassium fluotantalate, thus the design of a sodium purification device is improved, later it is built and commissioned. The device includes sodium transportation tank, storage tank, filter, cold trap, final storage tank, metering tank, regulating valve, argon purification system, electric control panel and instrument. Industrial purity sodium is purified, the impurities in the sodium were reduced to very

  8. Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent. (United States)

    Wooddell, Christine I; Subbotin, Vladimir M; Sebestyén, Magdolna G; Griffin, Jacob B; Zhang, Guofeng; Schleef, Martin; Braun, Serge; Huss, Thierry; Wolff, Jon A


    Various plasmids were delivered into rodent limb muscles by hydrodynamic limb vein (HLV) injection of naked plasmid DNA (pDNA). Some of the pDNA preparations caused significant muscle necrosis and associated muscle regeneration 3 to 4 days after the injection whereas others caused no muscle damage. Occurrence of muscle damage was independent of plasmid sequence, size, and encoded genes. It was batch dependent and correlated with the quantity of bacterial genomic DNA (gDNA) that copurified with the pDNA. To determine whether such an effect was due to bacterial DNA or simply to fragmented DNA, mice were treated by HLV injection with sheared bacterial or murine gDNA. As little as 20 μg of the large fragments of bacterial gDNA caused muscle damage that morphologically resembled damage caused by the toxic pDNA preparations, whereas murine gDNA caused no damage even at a 10-fold higher dose. Toxicity from the bacterial gDNA was not due to endotoxin and was eliminated by DNase digestion. We conclude that pDNA itself does not cause muscle damage and that purification methods for the preparation of therapeutic pDNA should be optimized for removal of bacterial gDNA.

  9. Fermentative production of poly (γ-glutamic acid) from renewable carbon source and downstream purification through a continuous membrane-integrated hybrid process. (United States)

    Kumar, Ramesh; Pal, Parimal


    Experimental investigations were carried out on continuous and direct production of poly-(γ-glutamic acid) in a hybrid reactor system that integrated conventional fermentative production step with membrane-based downstream separation and purification. Novelty of the integrated system lies in high degree of purity, conversion, yield and productivity of poly-(γ-glutamic acid) through elimination of substrate-product inhibitions of traditional batch production system. This new system is compact, flexible, eco-friendly and largely fouling-free ensuring steady and continuous production of poly-(γ-glutamic acid) directly from a renewable carbon source at the rate of 0.91 g/L/h. Cross-flow microfiltration membrane modules ensured almost complete separation and recycle of cells without much fouling problem. Well-screened ultrafiltration membrane module helped to concentrate poly-(γ-glutamic acid) while ensuring recovery and recycle of 96% unconverted carbon source resulting in yield of 0.6g/g along with high product purity.

  10. A food-grade process for isolation and partial purification of bacteriocins of lactic acid bacteria that uses diatomite calcium silicate. (United States)

    Coventry, M J; Gordon, J B; Alexander, M; Hickey, M W; Wan, J


    Bacteriocins, including nisin, pediocin PO2, brevicin 286, and piscicolin 126, were extracted from fermentation media by adsorption onto Micro-Cel (a food-grade diatomite calcium silicate anticaking agent) and subsequent desorption. The optimal conditions for desorption of piscicolin 126 were determined and applied to other bacteriocins, and the relative purities of the desorbed preparations were compared. Piscicolin was not successfully desorbed from Micro-Cel at pH 1.0 to 12.0, with organic solvents, or by increase of ionic strength up to 1 M NaCl. However, 25 and 75% of the bacteriocin activity was desorbed by using 1% sodium deoxycholate and 1% sodium dodecyl sulfate (SDS), respectively. Higher levels (up to 100%) of desorption were achieved by repeated elution or by an increase in surfactant concentration. Desorption of piscicolin with 1/10 volume of SDS solution resulted in a preparation with 10 times concentration in activity, equivalent to that of ammonium sulfate preparations (409,600 to 819,200 activity units/ml). Determination of organic nitrogen (N) content revealed that the desorbed piscicolin preparations were substantially free of proteinaceous substances (approximately 92 to 99%) compared with original culture supernatants and ammonium sulfate preparations. Nisin, pediocin, and brevicin were also desorbed with 1% SDS with a similar level of purification. PMID:8633875

  11. Comparison of large scale purification processes of naproxen enantiomers by chromatography using methanol-water and methanol-supercritical carbon dioxide mobile phases. (United States)

    Kamarei, Fahimeh; Vajda, Péter; Guiochon, Georges


    This paper compares two methods used for the preparative purification of a mixture of (S)-, and (R)-naproxen on a Whelk-O1 column, using either high performance liquid chromatography or supercritical fluid chromatography. The adsorption properties of both enantiomers were measured by frontal analysis, using methanol-water and methanol-supercritical carbon dioxide mixtures as the mobile phases. The measured adsorption data were modeled, providing the adsorption isotherms and their parameters, which were derived from the nonlinear fit of the isotherm models to the experimental data points. The model used was a Bi-Langmuir isotherm, similar to the model used in many enantiomeric separations. These isotherms were used to calculate the elution profiles of overloaded elution bands, assuming competitive Bi-Langmuir behavior of the two enantiomers. The analysis of these profiles provides the basis for a comparison between supercritical fluid chromatographic and high performance liquid chromatographic preparative scale separations. It permits an illustration of the advantages and disadvantages of these methods and a discussion of their potential performance.

  12. Heterogeneous profiles of a factor that renders neutrophils cytotoxic obtained from a concanavalin A-stimulated spleen cell culture in partial purification process

    Energy Technology Data Exchange (ETDEWEB)

    Fujii, Y.; Inoue, T.; Ito, M.; Kimura, S.; Fuyama, S.; Arai, S.; Naiki, M.; Sendo, F.


    Concanavalin A (Con A)-stimulated rat spleen cells were cultured in a serum-free conditioned medium. This culture supernatant contained a certain factor(s) that renders neutrophil cytotoxic for various tumor cells. The factor was tentatively termed neutrophil-activating factor (NAF). NAF activity was eluted in broad fractions by the ion exchange chromatography and the gel filtration. Moreover, on the Con A column, some NAF activities were bound to the column, but other activities passed through the column. These results showed the heterogeneity or polydispersity of NAF activity in both molecular size and charge-based separation properties. Monoclonal antibodies were produced by fusing BALB/c myeloma cells (P3-X63 Ag8.653) with spleen cells from syngeneic mice immunized with partially purified NAF (pNAF) obtained from the gel filtration. Absorbent beads which were linked with one monoclonal antibody (ANAF-10) partially absorbed NAF activity from supernatants of a Con A-stimulated spleen cell culture. By further purification of pNAF the NAF activity was concentrated about 10,000-fold. Heterogeneity of NAF activity, however, did not disappear in even this affinity chromatography. On the other hand, /sup 125/I-labeled material of the final product migrated to one major band corresponding with an m.w. of about 20,000 as determined by SDS-PAGE analysis, and NAF activity was detected in the same band.

  13. Separation and Purification of Oligosaccharide from Soybean Processing Liquid Waste by Macroreticular Resin%大孔吸附树脂分离大豆乳清废液中低聚糖技术的研究

    Institute of Scientific and Technical Information of China (English)

    李书海; 陈洪生; 唐秋菊; 刁静静; 李娟; 朱红梅


    试验研究了大孔吸附树脂对大豆乳清废液中低聚糖的分离纯化技术。以大豆低聚糖的分离度和总糖回收率为评价指标,对不同类型吸附树脂、不同洗脱流速、进料量、进料浓度和分离温度等因素进行分析,得出DSK530型树脂对大豆低聚糖的分离效果较好,且在流速2 mL·min-1、进料浓度15%、进料量15 mL条件下,大豆低聚糖的分离度达到0.55,总糖回收率达到90%以上。%The objective of the present study was to investigate separation and purification technology of oligosaccharide from soybean processing liquid waste by macroreticular resin. the separation degree of soybean oligosaccharide and total sugar recovery rate as evaluation index,the factors were analyzed that oligosaccharide purification degree of different resins,desorption velocity,feeding amount,feeding concentration and separation temperature. The results indicated DSK530 type resin was the best effective separation material and the best separation condition was desorption velocity 2 mL·min-1,feeding concentration fifteen percent,feeding amount 15 mL and the resolution of soybean oligosaccharide could obtain 0.55 and total sugar recovery rate was over ninety percent.

  14. Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids


    Jalasvuori, Matti; Friman, Ville-Petri; Nieminen, Anne; Jaana K.H. Bamford; Buckling, Angus


    Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple ant...

  15. Reverse osmosis water purification system (United States)

    Ahlstrom, H. G.; Hames, P. S.; Menninger, F. J.


    A reverse osmosis water purification system, which uses a programmable controller (PC) as the control system, was designed and built to maintain the cleanliness and level of water for various systems of a 64-m antenna. The installation operates with other equipment of the antenna at the Goldstone Deep Space Communication Complex. The reverse osmosis system was designed to be fully automatic; with the PC, many complex sequential and timed logic networks were easily implemented and are modified. The PC monitors water levels, pressures, flows, control panel requests, and set points on analog meters; with this information various processes are initiated, monitored, modified, halted, or eliminated as required by the equipment being supplied pure water.

  16. RepA and RepB exert plasmid incompatibility repressing the transcription of the repABC operon. (United States)

    Pérez-Oseguera, Angeles; Cevallos, Miguel A


    Rhizobium etli CFN42 has a multipartite genome composed of one chromosome and six large plasmids with low copy numbers, all belonging to the repABC plasmid family. All elements essential for replication and segregation of these plasmids are encoded within the repABC operon. RepA and RepB direct plasmid segregation and are involved in the transcriptional regulation of the operon, and RepC is the initiator protein of the plasmid. Here we show that in addition to RepA (repressor) and RepB (corepressor), full transcriptional repression of the operon located in the symbiotic plasmid (pRetCFN42d) of this strain requires parS, the centromere-like sequence, and the operator sequence. However, the co-expression of RepA and RepB is sufficient to induce the displacement of the parental plasmid. RepA is a Walker-type ATPase that self associates in vivo and in vitro and binds specifically to the operator region in its RepA-ADP form. In contrast, RepA-ATP is capable of binding to non-specific DNA. RepA and RepB form high molecular weight DNA-protein complexes in the presence of ATP and ADP. RepA carrying ATP-pocket motif mutations induce full repression of the repABC operon without the participation of RepB and parS. These mutants specifically bind the operator sequence in their ATP or ADP bound forms. In addition, their expression in trans exerts plasmid incompatibility against the parental plasmid. RepA and RepB expressed in trans induce plasmid incompatibility because of their ability to repress the repABC operon and not only by their capacity to distort the plasmid segregation process.

  17. Purification of nanoparticles by hollow fiber diafiltration (United States)

    Veeken, J.


    Hollow Fiber Diafiltration (Hollow Fiber Tangential Flow Filtration) is an efficient and rapid alternative to traditional methods of nanoparticle purification such as ultracentrifugation, stirred cell filtration, dialysis or chromatography. Hollow Fiber Diafiltration can be used to purify a wide range of nanoparticles including liposomes, colloids, magnetic particles and nanotubes. Hollow Fiber Diafiltration is a membrane based method where pore size determines the retention or transmission of solution components. It is a flow process where the sample is gently circulated through a tubular membrane. With controlled replacement of the permeate or (dialysate), pure nanoparticles can be attained. Hollow Fiber Diafiltration can be directly scaled up from R&D volumes to production. By adding more membrane fibers and maintaining the operating parameters, large volumes can be processed in the same time with the same pressure, and flow dynamics as bench-scale volumes. Keywords: hollow fiber, Diafiltration, filtration, purification, tangential flow filtration.

  18. Purification of Carbon Nanotubes by Proton Irradiation (United States)

    Kim, Euikwoun; Lee, Jeonggil; Lee, Younman; Jeon, Jaekyun; Kim, Jae-Yong; Kim, Jeongha; Shin, Kwanwoo; Youn, Sang-Pil; Kim, Kyeryung


    Carbon nanotubes (CNTs) exhibit variety of superior physical properties including well-defined nanodimensional structure, high electrical and thermal conductivity, and good mechanical stability against external irradiations. Further, a large specific surface area per unit weight suggests that carbon nanotubes could be excellent candidates for gas storage, purification, and separation. However, the practical application of CNTs is limited mainly due to the metallic impurities that were used as a catalyst during the fabrication process. Here, we irradiated CNTs by using high energy proton beams (35.7 MeV at the Bragg Peak). Interestingly, metallic impurities such as Fe, Ni, Co and chunk of amorphous carbon that were attached on the surface of CNTs were completely removed after the irradiation. The mechanism of such the purification process is not understood. The possible speculation will be demonstrated combined with the changes of physical properties including the appearance of the magnetism after the irradiation.

  19. Purification of tubulin from porcine brain. (United States)

    Gell, Christopher; Friel, Claire T; Borgonovo, Barbara; Drechsel, David N; Hyman, Anthony A; Howard, Jonathon


    Microtubules, polymers of the heterodimeric protein αβ-tubulin, give shape to cells and are the tracks for vesicle transport and chromosome segregation. In vitro assays to study microtubule functions and their regulation by microtubule-associated proteins require the availability of purified αβ-tubulin. In this chapter, we describe the process of purification of heterodimeric αβ-tubulin from porcine brain.

  20. Broad-Host-Range IncP-1 plasmids and their resistance potential

    Directory of Open Access Journals (Sweden)

    Magdalena ePopowska


    Full Text Available The plasmids of the incompatibility group IncP-1, also called IncP, as extrachromosomal genetic elements can transfer and replicate virtually in all Gram-negative bacteria. They are composed of backbone genes that encode a variety of essential functions and accessory genes that have implications for human health and environmental bioremediation. Broad-host-range IncP plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to a broad spectrum of antibiotics, heavy metals and quaternary ammonium compounds used as disinfectants. The backbone of these plasmids carries modules that enable them to effectively replicate, move to a new host via conjugative transfer and to be stably maintained in bacterial cells. The adaptive, resistance and virulence genes are mainly located on mobile genetic elements integrated between the functional plasmid backbone modules. Environmental studies have demonstrated the wide distribution of IncP-like replicons in manure, soils and wastewater treatment plants. They also are present in strains of pathogenic or opportunistic bacteria, which can be a cause for concern, because they may encode multiresistance. Their broad distribution suggests that IncP plasmids play a crucial role in bacterial adaptation by utilizing horizontal gene transfer. This review summarizes the variety of genetic information and physiological functions carried by IncP plasmids, which can contribute to the spread of antibiotic and heavy metal resistance while also mediating the process of bioremediation of pollutants. Due to the location of the resistance genes on plasmids with a broad host range and the presence of transposons carrying these genes it seems that the spread of these genes would be possible and quite hazardous in infection control. Future studies are required to determine the level of risk of the spread of resistance genes located on these plasmids.

  1. Distribution of small native plasmids in Streptococcus pyogenes in India. (United States)

    Bergmann, René; Nerlich, Andreas; Chhatwal, Gursharan S; Nitsche-Schmitz, D Patric


    Complete characterization of a Streptococcus pyogenes population from a defined geographic region comprises information on the plasmids that circulate in these bacteria. Therefore, we determined the distribution of small plasmids (pyogenes isolates from India, where diversity of strains and incidence rates of S. pyogenes infections are high. The collection comprised 77 emm-types. For plasmid detection and discrimination, we developed PCRs for different plasmid replication initiation protein genes, the putative repressor gene copG and bacteriocin genes dysA and scnM57. Plasmid distribution was limited to 13 emm-types. Co-detection analysis using aforementioned PCRs revealed four distinct plasmid sub-types, two of which were previously unknown. Representative plasmids pA852 and pA996 of the two uncharacterized plasmid sub-types were sequenced. These two plasmids could be assigned to the pMV158 and the pC194/pUB110 family of rolling-circle plasmids, respectively. The majority of small plasmids found in India belonged to the two newly characterized sub-types, with pA852- and pA996-like plasmids amounting to 42% and 22% of all detected plasmids, respectively. None of the detected plasmids coded for a known antibiotic resistance gene. Instead, all of the four plasmid sub-types carried known or potential bacteriocin genes. These genes may have influence on the evolutionary success of certain S. pyogenes genotypes. Notably, pA852-like plasmids were found in all isolates of the most prevalent emm-type 11.0. Together, a priori fitness of this genotype and increased fitness due to the acquired plasmids may have rendered type emm11.0 successful and caused the prevalence of pA852-like plasmids in India.

  2. Role of TLR3 in the immunogenicity of replicon plasmid-based vaccines. (United States)

    Diebold, S S; Schulz, O; Alexopoulou, L; Leitner, W W; Flavell, R A; Reis e Sousa, C


    Replicon plasmids encoding an alphavirus RNA replicase constitute an alternative to conventional DNA plasmids with promise for DNA vaccination in humans. Replicase activity amplifies the levels of transgene mRNA through a copying process involving double-stranded (ds) RNA intermediates, which contribute to vaccine immunogenicity by activating innate antiviral responses. Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs). Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines. We show that mouse CD8 alpha(+) DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells. However, we find that cytotoxic T-cell responses to a replicon plasmid intramuscular vaccine are not diminished in the absence of TLR3 in vivo. Our results underscore the potential role of TLR3 in mediating immune activation by dsRNA-bearing replicon plasmid-transfected cells and indicate that other innate sensing pathways can compensate for TLR3 absence in vivo.

  3. Molecular characterization of Syrian date palm cultivars using plasmid-like DNA markers. (United States)

    Haider, N; Nabulsi, I


    Date palm (Phoenix dactylifera L.) is one of the most important domesticated fruit trees in the Near East and North African countries. This tree has been, for several decades, in serious threat of being completely destroyed by the "Bayoud" disease caused by Fusarium oxysporum f. sp. albedinis. In this study, 18 Syrian date palm cultivars and four male trees were analyzed according to the identity of mitochondrial plasmid-like DNAs. A PCR strategy that employs plasmid-like DNAs-specific primer pair was used. These primers amplify a product of either 373-bp or 265-bp that corresponds to the S-(Bayoud-susceptible) or the R-plasmid (Bayoud-resistant), respectively. Generated data revealed that only six cultivars ('Medjool', 'Ashrasi', 'Gish Rabi', 'Khineze', and yellow- and red-'Kabkab') have the S-plasmid, suggesting their susceptibility to the fusariosis, while the remaining 12 cultivars and the four male trees contain the R-plasmid, suggesting their resistance to the fusariosis. The PCR process applied here has been proved efficient for the rapid screening for the presence of the S and R DNAs in Syrian date palm. PCR markers developed in this study could be useful for the screening of date palm lines growing in the field. The availability of such diagnostic tool for plasmid characterization in date palm would also be of great importance in establishing propagation and breeding programs of date palm in Syria.

  4. Rotating Reverse-Osmosis for Water Purification (United States)

    Lueptow, RIchard M.


    A new design for a water-filtering device combines rotating filtration with reverse osmosis to create a rotating reverse- osmosis system. Rotating filtration has been used for separating plasma from whole blood, while reverse osmosis has been used in purification of water and in some chemical processes. Reverse- osmosis membranes are vulnerable to concentration polarization a type of fouling in which the chemicals meant not to pass through the reverse-osmosis membranes accumulate very near the surfaces of the membranes. The combination of rotating filtration and reverse osmosis is intended to prevent concentration polarization and thereby increase the desired flux of filtered water while decreasing the likelihood of passage of undesired chemical species through the filter. Devices based on this concept could be useful in a variety of commercial applications, including purification and desalination of drinking water, purification of pharmaceutical process water, treatment of household and industrial wastewater, and treatment of industrial process water. A rotating filter consists of a cylindrical porous microfilter rotating within a stationary concentric cylindrical outer shell (see figure). The aqueous suspension enters one end of the annulus between the inner and outer cylinders. Filtrate passes through the rotating cylindrical microfilter and is removed via a hollow shaft. The concentrated suspension is removed at the end of the annulus opposite the end where the suspension entered.

  5. Air/Water Purification (United States)


    After 18 years of research into air/water pollution at Stennis Space Center, Dr. B. C. Wolverton formed his own company, Wolverton Environmental Services, Inc., to provide technology and consultation in air and water treatment. Common houseplants are used to absorb potentially harmful materials from bathrooms and kitchens. The plants are fertilized, air is purified, and wastewater is converted to clean water. More than 100 U.S. communities have adopted Wolverton's earlier water hyacinth and artificial marsh applications. Catfish farmers are currently evaluating the artificial marsh technology as a purification system.

  6. Water Purification Product (United States)


    Ecomaster, an affiliate of BioServe Space Technologies, this PentaPure technology has been used to purify water for our nation's Space Shuttle missions since 1981. WTC-Ecomaster of Mirneapolis, Minnesota manufactures water purification systems under the brand name PentaPure (TM). BioServe researcher Dr. George Marchin, of Kansas State University, first demonstrated the superiority of this technology and licensed it to WTC. Marchin continues to perform microgravity research in the development of new technologies for the benefit of life on Earth.

  7. [Extraction and purification method of rice DNA from rice powder containing Konjak flour]. (United States)

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


    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.

  8. Interview with Dr Robin Rothrock: the RNA purification market. 26 July 2005. (United States)

    Alger, Lynsey


    Dr Robin Rothrock, Director of Market Research at Bioinformatics LLC, talked to Lynsey Alger about the RNA purification market. The process of RNA purification is one of growing importance, not only among basic researchers, but increasingly among those involved in clinical research, particularly as purified RNA represents the base material for a vast number of innovative and widely used techniques, such as real-time and reverse transcriptase polymerase chain reaction and DNA microarrays. This interview examines the current and future status of the RNA purification market, with a focus on the different types of purification approaches available to researchers as well as the key companies involved in their production.

  9. Effect of charcoal on water purification


    Suzuki, Hirotaka; Kawahigashi, Tatsuo


    [Abstract] A natural basin system purifies water through self-purification, but the water pollution load of a river might exceed its self-purification capacity. Charcoal, which is used for other uses aside from heating, such as air purification, was evaluated experimentally for water quality purification. The experiment described herein is based on simple water quality measurements. Some experimentally obtained results are discussed.

  10. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.; LS Klinisch Onderzoek Wagenaar


    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and

  11. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.; LS Klinisch Onderzoek Wagenaar


    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and veter

  12. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.; LS Klinisch Onderzoek Wagenaar


    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and veter

  13. Purification Process of Lianhua Qingwen Capsule Water Extraction with Ceramic Membrane Filtration%陶瓷膜过滤对连花清瘟胶囊水提液纯化工艺的影响

    Institute of Scientific and Technical Information of China (English)

    刘敏彦; 张永锋; 史东霞; 许红辉; 李炳超; 李正杰; 范文成


    目的 研究不同孔径无机陶瓷膜对中药连花清瘟胶囊水提液除杂效果及主要有效成分转移率的影响.方法 以连花清瘟胶囊水提液为研究对象,采用8种不同孔径的无机陶瓷膜过滤,观察膜通量和主要有效成分的转移率.结果 经过8种不同孔径陶瓷膜过滤后,连花清瘟胶囊水提液均较过滤前明显透明澄清,出膏率也有一定程度的下降,综合考虑实际生产因素,孔径为0.2 μm的陶瓷膜管能保证在有效成分转移率和固形物去除率较高的条件下有较大的膜通量,适用于连花清瘟胶囊水提液除杂工艺.结论 陶瓷膜过滤技术可有效改善连花清瘟胶囊水提液除杂的效果,可用于中药提取液精制生产领域.%Objective To study the purification process and the retention rates of main active ingredients of Lianhua Qingwen Capsule water extraction with different pore size inorganic ceramic membrane. Methods Water extraction of Lianhua Qingwen Capsule was filtered by eight kinds of pore size inorganic ceramic membranes, the membrane flux and the retention rates of main active ingredients were observed. Results Water extraction liquid become clear and the cream rates decreased after filtered with ceramic membrane. Considering the actual production factors, the pore size of 0.2 μm ceramic membrane was selected to apply to purification process of Lianhua Qingwen Capsule water extraction, because of the higher retention rates of the active ingredients, the higher removal rates of impurities and the higher membrane flux. Conclusion Ceramic membrane filtration technology is effective to remove impurities of Lianhua Qingwen Capsule water extraction, and can be applied to the field of refining production of traditional Chinese medicine extraction.

  14. Study on Negative Pressure Microwave Spouted Puffing Processing Technology of Salted Egg White Recombinant Plasmids%咸蛋清重组粒负压微波喷动膨化工艺

    Institute of Scientific and Technical Information of China (English)

    王涛; 张慜; 王玉川; 刘亚萍


    The salted egg white and starch as the main raw material in the experiment ,this paper investigated the effect of the added amount of glutinous rice flour and corn starch,the surimi added amount,the maltodextrin added amount,water content,microwave power and jet frequency on expansion of recombinant plasmids,the expansion ratio and breaking force as index.The experimental results show that the ratio of glutinous rice flour and corn starch is 1:1,the surimi added 50%,the maltodextrin added amount 5%,water content 46%,microwave power 48W/g and spouted frequency 4/min ,with the optimum composition,the quality of the puffed productd and the expansion ration and crispness are better.%以咸蛋清和淀粉为主要原料,研究了糯米粉与玉米淀粉添加量、鱼糜添加量、麦芽糊精添加量、水分含量、微波功率及喷动频率等单因素对重组粒膨化效果的影响。实验结果表明,糯米粉与玉米淀粉比例为1∶1,鱼糜添加量50%,麦芽糊精添加量5%,水分含量为46%,微波功率48 W/g及喷动频率4次/min时得到的重组粒产品最好,产品具有较高的膨化率和脆度。

  15. Plasmids spread very fast in heterogeneous bacterial communities. (United States)

    Dionisio, Francisco; Matic, Ivan; Radman, Miroslav; Rodrigues, Olivia R; Taddei, François


    Conjugative plasmids can mediate gene transfer between bacterial taxa in diverse environments. The ability to donate the F-type conjugative plasmid R1 greatly varies among enteric bacteria due to the interaction of the system that represses sex-pili formations (products of finOP) of plasmids already harbored by a bacterial strain with those of the R1 plasmid. The presence of efficient donors in heterogeneous bacterial populations can accelerate plasmid transfer and can spread by several orders of magnitude. Such donors allow millions of other bacteria to acquire the plasmid in a matter of days whereas, in the absence of such strains, plasmid dissemination would take years. This "amplification effect" could have an impact on the evolution of bacterial pathogens that exist in heterogeneous bacterial communities because conjugative plasmids can carry virulence or antibiotic-resistance genes. PMID:12524329

  16. Increasing plasmid transformation efficiency of natural spizizen method in Bacillus Subtilis by a cell permeable peptide

    Directory of Open Access Journals (Sweden)

    Mehrdad Moosazadeh Moghaddam


    Full Text Available Introduction: Some of bacterial species are able to uptake DNA molecule from environment, the yield of this process depends on some conditions such as plasmid size and host type. In the case of Bacillus subtilis, DNA uptake has low efficacy. Using Spizizen minimal medium is common method in plasmid transformation into B. subtilis, but rate of this process is not suitable and noteworthy. The aim of this study was investigation of novel method for improvement of DNA transformation into B. subtilis based on CM11 cationic peptide as a membrane permeable agent.Materials and methods: In this study, for optimization of pWB980 plasmid transformation into B. subtilis, the CM11 cationic peptide was used. For this purpose, B. subtilis competent cell preparation in the present of different concentration of peptide was implemented by two methods. In the first method, after treatment of bacteria with different amount of peptide for 14h, plasmid was added. In the second method, several concentration of peptide with plasmid was exposed to bacteria simultaneously. Bacteria that uptake DNA were screened on LB agar medium containing kanamycin. The total transformed bacteria per microgram of DNA was calculated and compared with the control.Results: Plasmid transformation in best conditions was 6.5 folds higher than the control. This result was statistically significant (P value <0.001.Discussion and conclusion: This study showed that CM11 cationic peptide as a membrane permeable agent was able to increase plasmid transformation rate into B. subtilis. This property was useful for resolution of low transformation efficacy.

  17. Endogenous mutagenesis in recombinant sulfolobus plasmids. (United States)

    Sakofsky, Cynthia J; Grogan, Dennis W


    Low rates of replication errors in chromosomal genes of Sulfolobus spp. demonstrate that these extreme thermoacidophiles can maintain genome integrity in environments with high temperature and low pH. In contrast to this genetic stability, we observed unusually frequent mutation of the β-D-glycosidase gene (lacS) of a shuttle plasmid (pJlacS) propagated in Sulfolobus acidocaldarius. The resulting Lac(-) mutants also grew faster than the Lac(+) parent, thereby amplifying the impact of the frequent lacS mutations on the population. We developed a mutant accumulation assay and corrections for the effects of copy number and differential growth for this system; the resulting measurements and calculations yielded a corrected rate of 5.1 × 10(-4) mutational events at the lacS gene per plasmid replication. Analysis of independent lacS mutants revealed three types of mutations: (i) G · C-to-A · T transitions, (ii) slipped-strand events, and (iii) deletions. These mutations were frequent in plasmid-borne lacS expressed at a high level but not in single-copy lacS in the chromosome or at lower levels of expression in a plasmid. Substitution mutations arose at only two of 12 potential priming sites of the DNA primase of the pRN1 replicon, but nearly all these mutations created nonsense (chain termination) codons. The spontaneous mutation rate of plasmid-borne lacS was 175-fold higher under high-expression than under low-expression conditions. The results suggest that important DNA repair or replication fidelity functions are impaired or overwhelmed in pJlacS, with results analogous to those of the "transcription-associated mutagenesis" seen in bacteria and eukaryotes.

  18. Plasmid-mediated tetracycline resistance in Haemophilus ducreyi.


    Albritton, W L; Maclean, I W; Slaney, L A; Ronald, A. R.; Deneer, H G


    Clinical isolates of Haemophilus ducreyi were shown to be resistant to tetracycline. Resistance was associated in some strains with a 30-megadalton plasmid capable of transferring resistance in conjugative matings with other strains of H. ducreyi and other species of Haemophilus. Restriction endonuclease digestion patterns suggest a relationship between H. ducreyi plasmids and other tetracycline resistance plasmids in Haemophilus. The presence of plasmid-mediated resistance to the tetracyclin...

  19. Plasmid DNA entry into postmitotic nuclei of primary rat myotubes.


    Dowty, M E; Williams, P.; G. Zhang; Hagstrom, J E; Wolff, J A


    These studies were initiated to elucidate the mechanism of DNA nuclear transport in mammalian cells. Biotin- or gold-labeled plasmid and plasmid DNA expression vectors for Escherichia coli beta-galactosidase or firefly luciferase were microinjected into the cytoplasm of primary rat myotubes in culture. Plasmid DNA was expressed in up to 70% of the injected myotubes, which indicates that it entered intact, postmitotic nuclei. The nuclear transport of plasmid DNA occurred through the nuclear po...

  20. Replication of plasmids in gram-negative bacteria.



    Replication of plasmid deoxyribonucleic acid (DNA) is dependent on three stages: initiation, elongation, and termination. The first stage, initiation, depends on plasmid-encoded properties such as the replication origin and, in most cases, the replication initiation protein (Rep protein). In recent years the understanding of initiation and regulation of plasmid replication in Escherichia coli has increased considerably, but it is only for the ColE1-type plasmids that significant biochemical d...

  1. Defining process design space for a hydrophobic interaction chromatography (HIC) purification step: application of quality by design (QbD) principles. (United States)

    Jiang, Canping; Flansburg, Lisa; Ghose, Sanchayita; Jorjorian, Paul; Shukla, Abhinav A


    The concept of design space has been taking root under the quality by design paradigm as a foundation of in-process control strategies for biopharmaceutical manufacturing processes. This paper outlines the development of a design space for a hydrophobic interaction chromatography (HIC) process step. The design space included the impact of raw material lot-to-lot variability and variations in the feed stream from cell culture. A failure modes and effects analysis was employed as the basis for the process characterization exercise. During mapping of the process design space, the multi-dimensional combination of operational variables were studied to quantify the impact on process performance in terms of yield and product quality. Variability in resin hydrophobicity was found to have a significant influence on step yield and high-molecular weight aggregate clearance through the HIC step. A robust operating window was identified for this process step that enabled a higher step yield while ensuring acceptable product quality.

  2. 拳参总黄酮的纯化研究%Studies on Purificating Process of the Total Flavonoids of Polygonum Bistorta L.

    Institute of Scientific and Technical Information of China (English)

    王桂玲; 房建强; 赵雪梅; 王松


    Objective To provide a reference to further development and ulitization of the total flavanoids of Polygonum bistorta L. . Methods By the static and dynamic adsorption experiments, six types of macroporous adsorption resins( D101, HPD100,HPD400,HPD500, HPD600, AB-8) were evaluated and compared for the absorption and disaborption to find the optimum condition for the total flavanoids purification, and Rutin was used for the reference substance, 10% A1C13 for the color reagent to determine the content of the total flavanoids of Polygonum bistorta L by UV-spectrophotometric method. Results The HPD400 macroporous adsorption resin possessed optimum adsorption and elution parameters. The best technological conditions were adding 9.28 mg · mL-1 of sample with pH 3.0, being eluted with 2.9 multiple column volume of distilled water, 4.3 multiple column volume of 55% ethanol. The purified flavoniods was about 45% of total unpurified flavoniods. Conclusion The HPD400 macroporous adsorption resin fits for purifying flavoniods from Polygonum bistorta L. . This method was simple, practicable and effective.%目的 考察拳参总黄酮纯化提取条件.方法 结合静态和动态吸附实验,分别比较6种不同型号大孔树脂(D101、HPD100、HPD400、HPD500、HPD600、AB-8型)对拳参总黄酮的吸附和解吸性能,以饱和吸附量、洗脱量、总黄酮浸膏量为考察指标,对大孔树脂纯化拳参总黄酮的条件进行筛选.并以芦丁为标准品,10%三氯化铝(AlCl3)为显色剂,采用紫外分光光度法测定拳参总黄酮的含量.结果 HPD400型树脂具有最佳的吸附及洗脱参数,其最佳纯化条件浓度为9.28 mg·mL-1,pH为3.0的上样液,2.9倍柱体积纯化水,4.3倍柱体积55%乙醇洗脱.纯化后总黄酮量约为纯化前总黄酮量的45%.结论 HPD400型树脂适合拳参总黄酮的纯化.该方法 简单、易行,效果好,具有良好的应用前景.

  3. Plasmid Segregation: Spatial Awareness at the Molecular Level

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Gerdes, Kenn


    In bacteria, low-copy number plasmids ensure their stable inheritance by partition loci (par), which actively distribute plasmid replicates to each side of the cell division plane. Using time-lapse fluorescence microscopic tracking of segregating plasmid molecules, a new study provides novel insi...

  4. Cloning of Two Bacteriocin Genes from a Lactococcal Bacteriocin Plasmid

    NARCIS (Netherlands)

    Belkum, Marco J. van; Hayema, Bert Jan; Geis, Arnold; Kok, Jan; Venema, Gerard


    Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6 (60 kilobases [kb]), which specifies bacteriocin production and immunity, was analyzed with restriction endonucleases, and fragments of this plasmid were cloned into shuttle vectors based on the broad-host-range plasmid pWVO1. Two regions on p9B4

  5. Multilocus sequence typing of IncN plasmids

    DEFF Research Database (Denmark)

    García-Fernández, Aurora; Villa, Laura; Moodley, Arshnee


    categorization of IncN plasmids. METHODS: Twelve fully sequenced IncN plasmids available at GenBank were analysed in silico for selecting the loci for the IncN-specific pMLST. A total of 58 plasmids originating from different reservoirs (human, pig, poultry, cattle and horses) and geographic regions (Italy...

  6. Item Purification Does Not Always Improve DIF Detection: A Counterexample with Angoff's Delta Plot (United States)

    Magis, David; Facon, Bruno


    Item purification is an iterative process that is often advocated as improving the identification of items affected by differential item functioning (DIF). With test-score-based DIF detection methods, item purification iteratively removes the items currently flagged as DIF from the test scores to get purified sets of items, unaffected by DIF. The…

  7. I. Textural/Structural tuning and nanoparticle stabilization of copper-containing nanocomposite materials. II. Generation of reducing agents for automotive exhaust gas purification via the processing of hydrocarbons in a PACT (plasma and catalysis integrated technologies) reactor (United States)

    Xing, Yu

    This research consists of two parts. The first part deals with the preparation and properties of copper-containing nanocomposite materials. For studies of textural tuning, structural tuning, or material sintering, copper/aluminum and copper/zinc nanocomposites were prepared via various inorganic synthesis methods including conventional coprecipitation methods and a novel urea-gelation/thermal-modification method that produces narrow distributions of pore sizes, high surface areas, and significantly higher specific metal loadings. Solid-solid reaction analysis and differential scanning calorimetry (DSC) analysis were developed for the determination of the mixing homogeneities of the copper/aluminum nanocomposites. A sintering experiment at 250-600°C for 350 h under methanol-steam reforming conditions was carried out to compare the stability of supported Cu0 nanoparticles. The mixing homogeneities of CuO/Al2O3 nanocomposites significantly affected the thermal stability of their reduced Cu0 crystallites. Creation of relatively narrow distributions of pore sizes with relatively small major pore diameters (e.g., 3.5 nm) can also be used for the stabilization of supported Cu0 nanoparticles. The supported nanoparticles with a relatively small initial size cannot ensure good thermal stability. A "hereditary" character on the homogeneity of copper/aluminum nanocomposites was revealed. Stepwise reduction and reoxidation were studied for the structural tuning and purification of Cu-Al-O spinels with isotropic and gradual unit-cell contractions. The second part of the research deals with the processing of hydrocarbons. Conversion of a model hydrocarbon (n-hexane or n-octane) in an AC discharge PACT (plasma and catalysis integrated technologies) reactor was verified to be an effective method to instantly produce reducing agents (e.g., hydrogen or/and light alkanes and alkenes), at room temperature and atmospheric pressure for automotive exhaust gas purification. Effects of

  8. Molecular mechanism of immune response induced by foreign plasmid DNA after oral administration in mice

    Institute of Scientific and Technical Information of China (English)


    AIM: To study immune response induced by foreign plasmid DNA after oral administration in mice.METHODS: Mice were orally administered with 200 μg of plasmid pcDNA3 once and spleen was isolated 4 h and 18 h after administration. Total RNA was extracted from spleen and gene expression profile of BALB/c mice spleen was analyzed by using Affymetrix oligonucleotide GeneChip. Functional cluster analysis was conducted by GenMAPP software.RESULTS: At 4 h and 18 h after oral plasmid pcDNA3 administration, a number of immune-related genes,including cytokine and cytokine receptors, chemokines and chemokine receptor, complement molecule,proteasome, histocompatibility molecule, lymphocyte antigen complex and apoptotic genes, were up-regulated. Moreover, MAPPFinder results also showed that numerous immune response processes were up-regulated. In contrast, the immunoglobulin genes were down-regulated.CONCLUSION: Foreign plasmid DNA can modulate the genes expression related to immune system via the gastrointestinal tract, and further analysis of the related immune process may help understand the molecular mechanisms of immune response induced by foreign plasmid via the gastrointestinal tract.

  9. Purification Process and Oxidation Resistance of Total Flavonoids of Elaeagnus angustifolia L. Fruit%沙枣果总黄酮的纯化工艺及抗氧化性研究

    Institute of Scientific and Technical Information of China (English)

    陈亚; 梁琪; 张炎; 杨敏; 罗丽


    Based on ultrasonic extraction technology of total flavonoids of Elaeagnus angustifolia L. fruit in pre-vious studies, to study the purification process of total flavonoids Elaeagnus angustifolia L. fruit, macroporous resin as adsorbent was choosed to purify Elaeagnus angustifolia L. fruit flavonoids. Firstly, the selected test and adsorption dynamics research of the macroporous resin were conducted, the results showed that both adsorption and desorption rate of AB-8 resin were higher, and AB-8 resin was an ideal resin types for being suitable for separating total flavonoids from Elaeagnus angustifolia L.;the best condition of total flavonoids of Elaeagnus an-gustifolia L. determined by adsorption and elution dynamics experiment of AB-8 resin was:the sample quantity was 70 mL, concentration of the sample solution was 0.5 mg/mL, pH was 4.0, sample flowing rate was 1.0 mL/min. 90%ethanol of 4 BV was as the elution solvent and the flow rate was 1.5 mL/min.Under above condition, the purity of flavonoids was higher , which could reach to 65.56%, and was rough 2.84 times of crude flavonoids purity. At last, ingredient and antioxidation of total flavonoids of Elaeagnus angustifolia L. fruit were determined after being purifed. The results indicated that the anti-lipid peroxidation effect of flavonoids purification was sig-nificantly stronger than VC and PG, three kinds of free radical oxidation resistance were stronger than the PG, but weaker than VC.%在前期研究超声波辅助提取沙枣果总黄酮的工艺基础上,为探讨沙枣果总黄酮的纯化工艺,选择大孔树脂为吸附剂来分离纯化沙枣果总黄酮。先进行了大孔树脂的选择试验研究和大孔树脂静态吸附动力学研究,结果表明AB-8树脂的吸附量和解吸率都较高,是适于吸附分离沙枣果总黄酮的理想树脂类型。在此基础上,通过AB-8大孔树脂对沙枣果总黄酮动态吸附试验、动态洗脱试验确定出沙枣果总黄酮的

  10. Experimental and Simulative Studies on Vacuum Pressure Swing Adsorption Processes for CO Purification from Syngas%真空变压吸附提纯合成气中CO的实验与模拟

    Institute of Scientific and Technical Information of China (English)

    周言; 阎海宇; 李冬冬; 张东辉


    采用自制的载铜活性炭为吸附剂,实验验证了CO高吸附选择性和吸附固定床数学模型的准确性。通过数值模拟研究了多塔真空变压吸附(VPSA)提纯合成气(28.2%CO/46.9%H2/2.3%CH4/1.2%CO2/21.4%N2)(V)中CO的工艺流程。15塔数值计算结果可获得99.5%(V) CO 纯度和95.0%CO 回收率,同时吸附剂产率高达3.36 mol CO.kgads-1.h-1,而单位能耗低至0.107 kWh.Nm-3 feed。最后,系统考察了操作参数对CO吸附分离性能的影响规律。参数分析研究结果表明,解吸真空度和产品气流量都不宜过大以保证较高产品纯度和较低单位能耗,而产品纯度和回收率受进料流量和置换流量的变化波动较大,应在满足分离要求下选择最佳的进料和置换条件以获得较高的CO回收价值,从而为指导工业合成气尾气提纯CO工艺以及后续氢气纯化工艺装置的设计和调优提供了基本的操作参数。%A home-made copper-loaded activated carbon adsorbent was employed for CO adsorption. Simulation results showed high CO selectivity of the adsorbent and a mathematical model was used to predict its performance in adsorption fixed-beds, which were further confirmed by experiments. The CO purification process from syngas (28.2%CO/46.9%H2/2.3%CH4/1.2%CO2/21.4%N2)(V) by vacuum pressure swing adsorption (VPSA) was further studied based on numerical simulation. Numerical calculation results of 15 beds indicate that a CO rich product with 99.5%(V) purity and 95.0% recovery is achieved, and higher adsorbent productivity of 3.36 mol ads-1.h-1 with lower specific power consumption of 0.107 kWh.Nm-3 feed can be achieved. The relationship between operation parameters and adsorption separation performance were studied systematically. Results of parameters analysis show that it is inappropriate to choose larger desorption vacuum degree and product gas flow for higher product purity and lower specific power consumption, and the

  11. Purification of carbon nanotubes grown by thermal CVD (United States)

    Porro, S.; Musso, S.; Vinante, M.; Vanzetti, L.; Anderle, M.; Trotta, F.; Tagliaferro, A.


    We show the results of a set of purifications on carbon nanotubes (CNT) by acid and basic treatments. CNTs were obtained by thermal decomposition of camphor at 850 °C in a CVD growth system, by means of a growth process catalyzed by iron clusters originating from the addition of ferrocene in the precursors mixture. The purification procedures involved HNO 3, H 2SO 4, HSO 3Cl and NaOH for different process temperatures. As-grown CNTs showed a consistent presence of metal catalyst (about 6 wt%), evidenced by TGA. The purification treatments led to a certain amount of opening of the CNT tips, with a consequent loss of metal catalyst encapsulated in tips. This is also confirmed by BET analysis, which showed an increase of the surface area density of CNT after the purification. FT-IR and XPS revealed the presence of carboxylic groups on the CNT surface chemically modified by the harsh environment of the purification process. Among the various treatments that have been tested, the 1:3 solution of nitric and sulphuric acid was the most effective in modifying the CNT surface and inducing the formation of functional groups.

  12. Purification of GST-Tagged Proteins. (United States)

    Schäfer, Frank; Seip, Nicole; Maertens, Barbara; Block, Helena; Kubicek, Jan


    This protocol describes the purification of recombinant proteins fused to glutathione S-transferase (GST, GST-tagged proteins) by Glutathione Affinity purification. The GST tag frequently increases the solubility of the fused protein of interest and thus enables its purification and subsequent functional characterization. The GST-tagged protein specifically binds to glutathione immobilized to a matrix (e.g., agarose) and can be easily separated from a cell lysate by a bind-wash-elute procedure. GST-tagged proteins are often used to study protein-protein interactions, again making use of glutathione affinity in a procedure called a GST pull-down assay. The protocol is designed to process 200 ml of E. coli culture expressing intermediate to high amounts of a GST-tagged protein (~25 mg l(-1)). Depending on the expression rate or the available culture volume, the scale can be increased or decreased linearly. The protocol can also be used to purify GST-tagged proteins from other expression systems, such as insect or mammalian cells. Tips are provided to aid in modifying certain steps if proteins shall be recovered from alternative expression systems.

  13. Cu-TDPAT, an rht -type dual-functional metal-organic framework offering significant potential for use in H 2 and natural gas purification processes operating at high pressures

    KAUST Repository

    Wu, Haohan


    The separations of CO 2/CO/CH 4/H 2, CO 2/H 2, CH 4/H 2, and CO 2/CH 4 mixtures at pressures ranging to 7 MPa are important in a variety of contexts, including H 2 production, natural gas purification, and fuel-gas processing. The primary objective of this study is to demonstrate the selective adsorption potential of an rht-type metal-organic framework [Cu 3(TDPAT)(H 2O) 3]·10H 2O·5DMA (Cu-TDPAT), possessing a high density of both open metal sites and Lewis basic sites. Experimental high pressure pure component isotherm data for CO 2, CO, CH 4, and H 2 are combined with the Ideal Adsorbed Solution Theory (IAST) for estimation of mixture adsorption equilibrium. The separation performance of Cu-TDPAT is compared with four other microporous materials, specifically chosen in order to span a wide range of physicochemical characteristics: MgMOF-74, MIL-101, LTA-5A, and NaX. For all mixtures investigated, the capacity of Cu-TDPAT to produce the desired product, H 2 or CH 4, satisfying stringent purity requirements, in a fixed bed operating at pressures exceeding about 4 MPa, is either comparable to, or exceeds, that of other materials. © 2012 American Chemical Society.

  14. Purification Processes Development of Flavone and Terpene from Ginkgo Leaves%银杏叶中黄酮和萜类内酯分离工艺条件优化

    Institute of Scientific and Technical Information of China (English)



    Purification processes of flavone and terpene in Ginkgo leaves were developed using flash silica gel column chromatography.Four different polar solvent,as ethylether,acetic ether,acetone and ethanol,elution was made with the gel chromatographic column.The eluent was collected and the contents of flavonol glycosides and terpene lactone were measured using high performance liquid chromatography method.%用闪式硅胶柱层析分离纯化银杏叶中有效成分黄酮醇苷和萜类内酯工艺条件的优选。用不同极性溶剂(乙醚、乙酸乙酯、丙酮、乙醇)对加样品后的闪式硅胶层析柱进行洗脱,收集洗脱液,采用高效液相色谱法分别测定每份洗脱液中黄酮醇苷和萜类内酯的含量,从而筛选出应用闪式硅胶柱层析时,最佳的洗脱液种类和用量为:萜类内酯用3倍床体积的乙酸乙酯洗脱,黄酮醇苷用4倍床体积的丙酮洗脱即得。

  15. Cyclodextrin purification with hollow fibers

    Energy Technology Data Exchange (ETDEWEB)

    Berthod, A. (Univ. de Lyon 1, Villeubranne Cedex (France)); Jin, Heng Liang,; Armstrong, D.W. (Univ. of Missouri, Rolla (USA))


    Cyclodextrins are cyclic 1-4 linked oligomers of {alpha}-D-glucopyranose prepared from starch hydrolysis through enzymatic reactions. Mixtures of the three main cyclodextrins (CD), {alpha}-, {beta}-, and {gamma}-CDs, are always produced. A possible facile purification process is proposed. Permeation through hollow fibers made of a perfluorinated ionomer membrane. Nafion type, is shown to be an effective way to separate {alpha}-CD from {beta}- and {gamma}-CD. {Alpha}-CD with 95% purity was obtained after permeation through a Nafion hollow fiber of an equimolar 0.02 M solution of the three CDs. The fiber had a 56 cm{sup 2}/cm{sup 3} surface area per volume ratio. Kinetic studies and continuous extraction experiments with a 2-m coiled fiber showed that it is possible to obtain a 11.5 g {alpha}-CD solution with 92.4% purity or a 0.6 g {alpha}-CD solution with 97.2% purity, depending on the flow rate. The transport of CDs through the membrane could be due to moving water pools inside the ionomer. The small {alpha}-CD fits easily in such pools when the large {beta}- and {gamma}-CDs are excluded by steric hindrance. Temperature raises increased the permeation rates while decreasing the selectivity. The process could be scaled-up associating hollow fibers in bundle.

  16. Bacteriophages limit the existence conditions for conjugative plasmids. (United States)

    Harrison, Ellie; Wood, A Jamie; Dytham, Calvin; Pitchford, Jonathan W; Truman, Julie; Spiers, Andrew; Paterson, Steve; Brockhurst, Michael A


    Bacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome. Conjugative plasmids are infectious loops of DNA capable of transmitting DNA between bacterial cells and between species. Because plasmids often carry extra genes that allow bacteria to live in otherwise-inhospitable environments, their dynamics are central to understanding bacterial adaptive evolution. The plasmid-bacterium interaction has typically been studied in isolation, but in natural bacterial communities, bacteriophages, viruses that infect bacteria, are ubiquitous. Using experiments, mathematical models, and computer simulations we show that bacteriophages drive plasmid dynamics through their ecological and evolutionary effects on bacteria and ultimately

  17. Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids. (United States)

    Jalasvuori, Matti; Friman, Ville-Petri; Nieminen, Anne; Bamford, Jaana K H; Buckling, Angus


    Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple antibiotic-resistance genes containing plasmids are stably maintained in both Escherichia coli and Salmonella enterica in the absence of phages, while plasmid-dependent phage PRD1 causes a dramatic reduction in the frequency of antibiotic-resistant bacteria. The loss of antibiotic resistance in cells initially harbouring RP4 plasmid was shown to result from evolution of phage resistance where bacterial cells expelled their plasmid (and hence the suitable receptor for phages). Phages also selected for a low frequency of plasmid-containing, phage-resistant bacteria, presumably as a result of modification of the plasmid-encoded receptor. However, these double-resistant mutants had a growth cost compared with phage-resistant but antibiotic-susceptible mutants and were unable to conjugate. These results suggest that bacteriophages could play a significant role in restricting the spread of plasmid-encoded antibiotic resistance.

  18. Optimization of membrane protein overexpression and purification using GFP fusions

    NARCIS (Netherlands)

    Drew, David; Lerch, Mirjam; Kunji, Edmund; Slotboom, Dirk-Jan; de Gier, Jan-Willem


    Optimizing conditions for the overexpression and purification of membrane proteins for functional and structural studies is usually a Laborious and time-consuming process. This process can be accelerated using membrane protein-GFP fusions(1-3), which allows direct monitoring and visualization of mem

  19. Screening of L-histidine-based ligands to modify monolithic supports and selectively purify the supercoiled plasmid DNA isoform. (United States)

    Amorim, Lúcia F A; Sousa, Fani; Queiroz, João A; Cruz, Carla; Sousa, Ângela


    The growing demand of pharmaceutical-grade plasmid DNA (pDNA) suitable for biotherapeutic applications fostered the development of new purification strategies. The surface plasmon resonance technique was employed for a fast binding screening of l-histidine and its derivatives, 1-benzyl-L-histidine and 1-methyl-L-histidine, as potential ligands for the biorecognition of three plasmids with different sizes (6.05, 8.70, and 14 kbp). The binding analysis was performed with different isoforms of each plasmid (supercoiled, open circular, and linear) separately. The results revealed that the overall affinity of plasmids to l-histidine and its derivatives was high (KD  > 10(-8)  M), and the highest affinity was found for human papillomavirus 16 E6/E7 (K(D)  = 1.1 × 10(-10)  M and KD  = 3.34 × 10(-10)  M for open circular and linear plasmid isoforms, respectively). L-Histidine and 1-benzyl-L-histidine were immobilized on monolithic matrices. Chromatographic studies of L-histidine and 1-benzyl-L-histidine monoliths were also performed with the aforementioned samples. In general, the supercoiled isoform had strong interactions with both supports. The separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration in the eluent, in both supports, but a lower salt concentration was required in the 1-benzyl-L-histidine monolith because of stronger interactions promoted with pDNA. The efficiency of plasmid isoforms separation remained unchanged with flow rate variations. The binding capacity for pDNA achieved with the l-histidine monolith was 29-fold higher than that obtained with conventional L-histidine agarose. Overall, the combination of either L-histidine or its derivatives with monolithic supports can be a promising strategy to purify the supercoiled isoform from different plasmids with suitable purity degree for pharmaceutical applications.

  20. Electrochemical Processes

    DEFF Research Database (Denmark)

    Bech-Nielsen, Gregers


    The notes describe in detail primary and secondary galvanic cells, fuel cells, electrochemical synthesis and electroplating processes, corrosion: measurments, inhibitors, cathodic and anodic protection, details of metal dissolution reactions, Pourbaix diagrams and purification of waste water from...

  1. Efficient expression and purification of biologically active human cystatin proteins. (United States)

    Chauhan, Sakshi; Tomar, Raghuvir S


    Cystatins are reversible cysteine protease inhibitor proteins. They are known to play important roles in controlling cathepsins, neurodegenerative disease, and in immune system regulation. Production of recombinant cystatin proteins is important for biochemical and function characterization. In this study, we cloned and expressed human stefin A, stefin B and cystatin C in Escherichia coli. Human stefin A, stefin B and cystatin C were purified from soluble fraction. For cystatin C, we used various chaperone plasmids to make cystatin C soluble, as it is reported to localize in inclusion bodies. Trigger factor, GroES-GroEL, DnaK-DnaJ-GrpE chaperones lead to the presence of cystatin C in the soluble fraction. Immobilized metal affinity chromatography, glutathione sepharose and anion exchange chromatography techniques were employed for efficient purification of these proteins. Their biological activities were tested by inhibition assays against cathepsin L and H3 protease.

  2. Blood purification and hemo- perfusion

    Institute of Scientific and Technical Information of China (English)


    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.

  3. Nanomechanical Water Purification Device Project (United States)

    National Aeronautics and Space Administration — Seldon Laboratories, LLC, proposes a lightweight, low-pressure water purification device that harnesses the unique properties of carbon nanotubes and will operate...

  4. Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET, a new method for plasmid reconstruction from whole genome sequences.

    Directory of Open Access Journals (Sweden)

    Val F Lanza


    Full Text Available Bacterial whole genome sequence (WGS methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage, comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC, comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

  5. Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET), a new method for plasmid reconstruction from whole genome sequences. (United States)

    Lanza, Val F; de Toro, María; Garcillán-Barcia, M Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M; de la Cruz, Fernando


    Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

  6. Unraveling the regulatory network of IncA/C plasmid mobilization: When genomic islands hijack conjugative elements. (United States)

    Carraro, Nicolas; Matteau, Dominick; Burrus, Vincent; Rodrigue, Sébastien


    Conjugative plasmids of the A/C incompatibility group (IncA/C) have become substantial players in the dissemination of multidrug resistance. These large conjugative plasmids are characterized by their broad host-range, extended spectrum of antimicrobials resistance, and prevalence in enteric bacteria recovered from both environmental and clinical settings. Until recently, relatively little was known about the basic biology of IncA/C plasmids, mostly because of the hindrance of multidrug resistance for molecular biology experiments. To circumvent this issue, we previously developed pVCR94ΔX, a convenient prototype that codes for a reduced set of antibiotic resistances. Using pVCR94ΔX, we then characterized the regulatory pathway governing IncA/C plasmid dissemination. We found that the expression of roughly 2 thirds of the genes encoded by this plasmid, including large operons involved in the conjugation process, depends on an FlhCD-like master activator called AcaCD. Beyond the mobility of IncA/C plasmids, AcaCD was also shown to play a key role in the mobilization of different classes of genomic islands (GIs) identified in various pathogenic bacteria. By doing so, IncA/C plasmids can have a considerable impact on bacterial genomes plasticity and evolution.

  7. [Effect of plasmid pKM101 on the expression of bacterial genes not related to DNa metabolism]. (United States)

    Skavronskaya, A G; Tiganova, I G; Andreeva, I V; Rusina, O Iu


    An experimental system ensuring fusion of bacterial genes to the lac operon of the Mu dl(Aplac) phage was used. Fusion operons in which the lac operon was under the control of promoters of the elt gene, responsible for synthesis of the LT toxin, of the tetracyclin-resistance tet gene, and sfiA gene encoding filament production, was studied. Using this experimental system, plasmid pKM101 was shown to be capable of activating the expression of the above Escherichia coli and Salmonella typhimurium genes, which is manifested as the activation of beta-galactosidase synthesis. The activation of the elt gene expression by the pKM101 plasmid was also confirmed in experiments on detecting the LT toxin synthesized by bacteria carrying this plasmid. Effect of the plasmid on the activation of elt operon expression, unlike the effect of this plasmid on mutability, does not depend on the functioning of the lexA and recA genes, i.e., this is not a SOS-regulated process. The mutant plasmid pGW12, a derivative of pKM101, deficient in the mucAB genes responsible for mutagenesis, causes a more pronounced activation of the elt gene than plasmid pKM101.

  8. Preparative Purification of Liriodendrin from Sargentodoxa cuneata by Macroporous Resin. (United States)

    Li, Di-Hua; Wang, Yan; Lv, Yuan-Shan; Liu, Jun-Hong; Yang, Lei; Zhang, Shu-Kun; Zhuo, Yu-Zhen


    The preparative purification of liriodendrin from Sargentodoxa cuneata using macroporous resin combined with crystallization process was evaluated. The properties of adsorption/desorption of liriodendrin on eight macroporous resins were investigated systematically. X-5 resin was selected as the most suitable medium for liriodendrin purification. The adsorption of liriodendrin on X-5 resin fitted well with the pseudo-second-order kinetic model and Langmuir isotherm model. Dynamic adsorption/desorption tests were performed using a glass column packed with X-5 resin to optimize the separation process of liriodendrin. After one treatment with X-5 resin, the content of liriodendrin in the product was increased 48.73-fold, from 0.85% to 41.42%, with a recovery yield of 88.9%. 97.48% liriodendrin was obtained by further crystallization and determined by HPLC. The purified product possessed strong antioxidant activity. In conclusion, purification of liriodendrin might expend its further pharmacological researches and further applications in pharmacy.

  9. Role of Plasmid in Production of Acetobacter Xylinum Biofilms

    Directory of Open Access Journals (Sweden)

    Abbas Rezaee


    Full Text Available Acetobacter xylinum has the ability to produce cellulotic biofilms. Bacterial cellulose is expected to be used in many industrial or biomedical materials for its unique characteristics. A. xylinum contains a complex system of plasmid DNA molecules. A 44 kilobases (kb plasmid was isolated in wild type of A. xylinum. To improve the cellulose producing ability of A. xylinum, role of the plasmid in production of cellulose was studied. The comparisons between wild type and cured cells of A. xylinum showed that there is considerably difference in cellulose production. In order to study the relationship between plasmid and the rate of cellulose production, bacteria were screened for plasmid profile by a modified method for preparation of plasmid. This method yields high levels of pure plasmid DNA that can be used for common molecular techniques, such as digestion and transformation, with high efficiency.

  10. OPTIMASI PROSES DEASIDIFIKASI DALAM PEMURNIAN MINYAK SAWIT MERAH SKALA PILOT PLANT [Optimization of Deacidification Process in Red Palm Oil Purification on Pilot Plant Scale

    Directory of Open Access Journals (Sweden)

    I Wayan Rai Widarta1*


    Full Text Available Deacidification is one of the steps in palm oil refining process which aims to separate free fatty acids formed during post-harvest handling. It is carried out using alkali solution such as NaOH (sodium hydroxide. Carotenoids in palm oil are affected by this step. Therefore, deacidification has to be controlled to minimize the destruction of carotenoids during processing. The objective of this research was to improve deacidification process in pilot plant scale so that the process can produce lower level of free fatty acids (FFA and higher recovery of carotene in high yield neutralized red palm oil (NRPO. Characterization of physical and chemical properties of crude palm oil (CPO such as moisture content, FFA and carotene contents, saponification number, iodine value, peroxide value, and color were determined before processing. Degumming was performed before deacidification process. The 17.5% excess of NaOH was obtained from the pilot plant scale deacidification trial. The optimization of deacidification time and temperature was carried out by using central composite design (CCD. Response surface method (RSM was used to observe the influence of treatments on the FFA level reduction, carotene recovery, and NRPO yield. The result showed that the optimum deacidification condition was at 61 ± 2°C in 26 minutes, and at the 16°Be NaOH strength with 17.5% excess of NaOH. In this optimum condition, the process achieved 96.35% of FFA reduction, 87.30% of carotene recovery, and 90.16% of NRPO yield.

  11. Modeling sRNA-Regulated Plasmid Maintenance (United States)

    Klumpp, Stefan


    We study a theoretical model for the toxin-antitoxin (hok/sok) mechanism for plasmid maintenance in bacteria. Toxin-antitoxin systems enforce the maintenance of a plasmid through post-segregational killing of cells that have lost the plasmid. Key to their function is the tight regulation of expression of a protein toxin by an sRNA antitoxin. Here, we focus on the nonlinear nature of the regulatory circuit dynamics of the toxin-antitoxin mechanism. The mechanism relies on a transient increase in protein concentration rather than on the steady state of the genetic circuit. Through a systematic analysis of the parameter dependence of this transient increase, we confirm some known design features of this system and identify new ones: for an efficient toxin-antitoxin mechanism, the synthesis rate of the toxin’s mRNA template should be lower that of the sRNA antitoxin, the mRNA template should be more stable than the sRNA antitoxin, and the mRNA-sRNA complex should be more stable than the sRNA antitoxin. Moreover, a short half-life of the protein toxin is also beneficial to the function of the toxin-antitoxin system. In addition, we study a therapeutic scenario in which a competitor mRNA is introduced to sequester the sRNA antitoxin, causing the toxic protein to be expressed. PMID:28085919

  12. Modeling sRNA-regulated Plasmid Maintenance

    CERN Document Server

    Gong, Chen Chris


    We study a theoretical model for the toxin-antitoxin (hok/sok) mechanism for plasmid maintenance in bacteria. Toxin-antitoxin systems enforce the maintenance of a plasmid through post-segregational killing of cells that have lost the plasmid. Key to their function is the tight regulation of expression of a protein toxin by an sRNA antitoxin. Here, we focus on the nonlinear nature of the regulatory circuit dynamics of the toxin-antitoxin mechanism. The mechanism relies on a transient increase in protein concentration rather than on the steady state of the genetic circuit. Through a systematic analysis of the parameter dependence of this transient increase, we confirm some known design features of this system and identify new ones: for an efficient toxin-antitoxin mechanism, the synthesis rate of the toxin's mRNA template should be lower that of the sRNA antitoxin, the mRNA template should be more stable than the sRNA antitoxin, and the mRNA-sRNA complex should be more stable than the sRNA antitoxin. Moreover, ...

  13. Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes. (United States)

    McDaniel, Jonathan R; Mackay, J Andrew; Quiroz, Felipe García; Chilkoti, Ashutosh


    This paper reports a new strategy, recursive directional ligation by plasmid reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats. PRe-RDL has several unique features that stem from the use of type IIs restriction endonucleases: first, PRe-RDL is a seamless cloning method that leaves no extraneous nucleotides at the ligation junction. Because it uses type IIs endonucleases to ligate the two halves of the plasmid, PRe-RDL also addresses the major limitation of RDL in that it abolishes any restriction on the gene sequence that can be oligomerized. The reconstitution of a functional plasmid only upon successful ligation in PRe-RDL also addresses two other limitations of RDL: the significant background from self-ligation of the vector observed in RDL, and the decreased efficiency of ligation due to nonproductive circularization of the insert. PRe-RDL can also be used to assemble genes that encode different sequences in a predetermined order to encode block copolymers or append leader and trailer peptide sequences to the oligomerized gene.

  14. Nanofabrication and characterization of PVA-organofiller/Ag nanocoatings on pMAD plasmids (United States)

    Erdonmez, D.; Mosayyebi, S.; Erkan, K.; Salimi, K.; Nagizade, N.; Saglam, N.; Rzayev, Z. M. O.


    Nowadays, the most important problem in microbial researches is bacterial resistance which is carried out by DNA plasmids against antibacterial agents. The effect of antibacterial nanoparticles on bacteria is remarkable, but studies on the interactions of these particles with plasmids do not search or there are no adequate studies. We proposed that the nanoparticles, which are disrupted the self-assembled structure of plasmids, may decrease the resistance of bacteria, and therefore, increase the activity of utilized antibacterial agents. In this work, we synthesized polymer nanofiber webs samples by electrospinning technique from pure water solution of nanocomposites with different contents of silver nanoparticles, and surface morphology of nanofibers composites were characterized by SEM microscopy. Their interactions with pMAD DNA plasmids were investigated. It was demonstrated that the synthesized Ag-carrying nanohybrid composites with higher surface contacted areas were significantly inhibited the activity of plasmid DNA against bacterial resistance. Agreeing with obtained results, synthesized nanofiber coatings can be recommended for the widely applications in nanobiotechnology, nanomedicine, and bioengineering processing.

  15. Purification of Organic Wastewater Containing Cu2+ and Cr3+ by a Combined Process of Micro Electrolysis and Biofilm

    Institute of Scientific and Technical Information of China (English)

    李天成; 姜斌; 冯霞; 王大为; 袁绍军; 李鑫钢


    A complex process of micro electrolysis and biofilm was developed to continuously treat organic wastew-aters containing heavy metal ions such as Cu2+ and Cr3+,and the relevant purifying mechanism was also addressed. In detail,organic materials in wastewater could be consumed as nutritious source by biofilm composed of aerobes and anaerobes,However,for heavy metal ions (Cu2+,Cr3+),part was removed by electrodeposition,and some was adorbed on biofilm.In order to compare with the combined process of micro electrolysis and biofilm,the experimental data of micro electrolysis process (intermittent )or biofilm process (continuous)were provided,and the kinetic data of C6H12O6(glucose)biodegradation by cultured microbes or acclimated microbes were also obtained,These experimental results indicated that for wastewater initially consisted of C6H12O6(500 mg.L-1),Cu2+ and Cr3+(10mg.L6-1),after treatment,its concentrations of C6H12O6,Cu2+ and Cr3+ were lowered to the leve of 55-65 mg.L-1),after treatment,its concentrations of C6H12O6,Cu2+ and Cr3+ were lowered to the level of 55-65 mg.L-1,and less than 1mg.L-1,respectively,And the industrial reused water standards could be met by treated wastewater.

  16. Turnkey Helium Purification and Liquefaction Plant for DARWIN, Australia (United States)

    Lindemann, U.; Boeck, S.; Blum, L.; Kurtcuoglu, K.


    The Linde Group, through its Australian subsidiary BOC Limited, has signed an agreement with Darwin LNG Pty Ltd for the supply of feed-gas to Linde's new helium refining and liquefaction facility in Darwin, Australia. Linde Kryotechnik AG, located in Switzerland, has carried out the engineering and fabrication of the equipment for the turn key helium plant. The raw feed gas flow of 20'730 Nm3/h contains up to of 3 mol% helium. The purification process of the feed gas consists of partial condensation of nitrogen in two stages, cryogenic adsorption and finally catalytic oxidation of hydrogen followed by a dryer system. Downstream of the purification the refined helium is liquefied using a modified Bryton process and stored in a 30'000 gal LHe tank. For further distribution and export of the liquid helium there are two stations available for filling of truck trailers and containers. The liquid nitrogen, required for refrigeration capacity to the nitrogen removal stages in the purification process as well as for the pre-cooling of the pure helium in the liquefaction process, is generated on site during the feed gas purification process. The optimized process provides low power consumption, maximum helium recovery and a minimum helium loss.

  17. Comparative genomic hybridization analysis shows different epidemiology of chromosomal and plasmid-borne cpe-carrying Clostridium perfringens type A.

    Directory of Open Access Journals (Sweden)

    Päivi Lahti

    Full Text Available Clostridium perfringens, one of the most common causes of food poisonings, can carry the enterotoxin gene, cpe, in its chromosome or on a plasmid. C. perfringens food poisonings are more frequently caused by the chromosomal cpe-carrying strains, while the plasmid-borne cpe-positive genotypes are more commonly found in the human feces and environmental samples. Different tolerance to food processing conditions by the plasmid-borne and chromosomal cpe-carrying strains has been reported, but the reservoirs and contamination routes of enterotoxin-producing C. perfringens remain unknown. A comparative genomic hybridization (CGH analysis with a DNA microarray based on three C. perfringens type A genomes was conducted to shed light on the epidemiology of C. perfringens food poisonings caused by plasmid-borne and chromosomal cpe-carrying strains by comparing chromosomal and plasmid-borne cpe-positive and cpe-negative C. perfringens isolates from human, animal, environmental, and food samples. The chromosomal and plasmid-borne cpe-positive C. perfringens genotypes formed two distinct clusters. Variable genes were involved with myo-inositol, ethanolamine and cellobiose metabolism, suggesting a new epidemiological model for C. perfringens food poisonings. The CGH results were complemented with growth studies, which demonstrated different myo-inositol, ethanolamine, and cellobiose metabolism between the chromosomal and plasmid-borne cpe-carrying strains. These findings support a ubiquitous occurrence of the plasmid-borne cpe-positive strains and their adaptation to the mammalian intestine, whereas the chromosomal cpe-positive strains appear to have a narrow niche in environments containing degrading plant material. Thus the epidemiology of the food poisonings caused by two populations appears different, the plasmid-borne cpe-positive strains probably contaminating foods via humans and the chromosomal strains being connected to plant material.

  18. 乳酸提纯反应精馏新工艺非平衡级稳态模拟%Non-equilibrium stage static simulation of lactic acid purification reactive distillation process

    Institute of Scientific and Technical Information of China (English)

    张猛; 马利; 杨基础; 徐用懋


    Purification of lactic acid by reactive distillation (RD) is a novel technology which has many excellent characteristics compared with traditional technologies. This paper presented a non-equilibrium model for lactic acid purification RD pilot column. An Improved Separation Efficiency Function (ISEF) was proposed based on de-coupling and pseudo-homogenous assumptions, which greatly improved the solving efficiency and made this model suitable for practical application. Simulation results were consistent with the experiments in different conditions, and the simulation results outperformed the simulator ASPEN PLUS in which the equilibrium stage assumptions were adopted. The static characteristics of the pilot setup were also investigated. The analysis result could help to accelerate the commercialization of the lactic acid purification RD technology.

  19. Simultaneous SO/sub 2/ and NO/sub x/ separation in the high-temperature range using a wet purification process

    Energy Technology Data Exchange (ETDEWEB)

    Hoelter, H.; Igelbuescher, H.; Gresch, H.; Dewert, H.


    The Exxon NO/sub x/ separation process takes place in a narrow temperature window. Power demand variations and the resulting changes in the operating regime make it difficult to maintain this temperature range. In order to overcome this problem it is proposed to replace NH/sub 3/ with ammonium salts with organic and/or inorganic bonding, fine-grained and moist, which are blown into the boiler above the coal bed.


    Directory of Open Access Journals (Sweden)



    Ce plasmide appelé pSU100 a été cloné dans le vecteur de transformation pUC18 au site EcoRI chez E. coli JM103. Les profils électrophorétiques de restriction obtenus par des digestions simples, doubles et triples sous l’action de 33 endonucléases, ont contribué à l’élaboration d’une carte de restriction de ce plasmide. Cinq sites uniques ont été identifiés, ainsi que d’autres sites doubles et multiples. Une étude préliminaire du rôle physiologique de ce plasmide a permis de déceler une résistance à la kanamycine.

  1. Treatment efficiency and economic feasibility of biological oxidation, membrane filtration and separation processes, and advanced oxidation for the purification and valorization of olive mill wastewater. (United States)

    Ioannou-Ttofa, L; Michael-Kordatou, I; Fattas, S C; Eusebio, A; Ribeiro, B; Rusan, M; Amer, A R B; Zuraiqi, S; Waismand, M; Linder, C; Wiesman, Z; Gilron, J; Fatta-Kassinos, D


    Olive mill wastewater (OMW) is a major waste stream resulting from numerous operations that occur during the production stages of olive oil. The resulting effluent contains various organic and inorganic contaminants and its environmental impact can be notable. The present work aims at investigating the efficiency of (i) jet-loop reactor with ultrafiltration (UF) membrane system (Jacto.MBR), (ii) solar photo-Fenton oxidation after coagulation/flocculation pre-treatment and (iii) integrated membrane filtration processes (i.e. UF/nanofiltration (NF)) used for the treatment of OMW. According to the results, the efficiency of the biological treatment was high, equal to 90% COD and 80% total phenolic compounds (TPh) removal. A COD removal higher than 94% was achieved by applying the solar photo-Fenton oxidation process as post-treatment of coagulation/flocculation of OMW, while the phenolic fraction was completely eliminated. The combined UF/NF process resulted in very high conductivity and COD removal, up to 90% and 95%, respectively, while TPh were concentrated in the NF concentrate stream (i.e. 93% concentration). Quite important is the fact that the NF concentrate, a valuable and polyphenol rich stream, can be further valorized in various industries (e.g. food, pharmaceutical, etc.). The above treatment processes were found also to be able to reduce the initial OMW phytotoxicity at greenhouse experiments; with the effluent stream of solar photo-Fenton process to be the least phytotoxic compared to the other treated effluents. A SWOT (Strength, Weakness, Opportunities, Threats) analysis was performed, in order to determine both the strengths of each technology, as well as the possible obstacles that need to overcome for achieving the desired levels of treatment. Finally, an economic evaluation of the tested technologies was performed in an effort to measure the applicability and viability of these systems at real scale; highlighting that the cost cannot be regarded as

  2. Development of translating ribosome affinity purification for zebrafish. (United States)

    Tryon, Robert C; Pisat, Nilambari; Johnson, Stephen L; Dougherty, Joseph D


    The regulation of transcription and translation by specific cell types is essential to generate the cellular diversity that typifies complex multicellular organisms. Tagging and purification of ribosomal proteins has been shown to be an innovative and effective means of characterizing the ribosome bound transcriptome of highly specific cell populations in vivo. To test the feasibility of using translating ribosome affinity purification (TRAP) in zebrafish, we have generated both a ubiquitous TRAP line and a melanocyte-specific TRAP line using the native zebrafish rpl10a ribosomal protein. We have demonstrated the capacity to capture mRNA transcripts bound to ribosomes, and confirmed the expected enrichment of melanocyte specific genes and depletion of non-melanocyte genes when expressing the TRAP construct with a cell specific promoter. We have also generated a generic EGFP-rpl10a Tol2 plasmid construct (Tol2-zTRAP) that can be readily modified to target any additional cell populations with characterized promoters in zebrafish. Copyright © 2012 Wiley Periodicals, Inc.

  3. Plasmid transfer between bacteria in soil microcosms and the field

    Directory of Open Access Journals (Sweden)

    Eric Smit


    Full Text Available In ibis review factors influencing conjugal plasmid transfer between bacteria and the possible role of naturally occurring selftransmissible plasmide for the dissemination of recombinant DNA in soil will be discussed. In microcosm studies, plasmid transfer between various species of introduced bacteria has been detected. Moreover, plamid transfer to indigenous soil micoorganisms was observed. Soil is an oligotrophic environment and plasmid transfer occurred mainly under conditions which were nutritionally favourable for bacteria, such as in the plant rhizosphere and in the presence of clay minerais or added nutrients. Mobilizable plasmids, lacking the ability to transfer themselves, have been reported to be transferred in the presence of selftransmissible plasmids. A study comparing conjugal transfer in microcosme with those in the field revealed that the transfer rates found in microcosme and in the field were similar. Transfer of chromosomal DNA by plasmid RP4 could only be shown on filters and was not observed in soil. Transfer of plasmids carrying biodegradative genes appeared to be favoured in the presence of the compound that can be degraded. Evidence was found for the presence of naturally-occurring selftransmissible plasmids in bacteria in the rhizosphere which could mobilize recombinant plasmids.

  4. Efficient and inexpensive method for purification of heparin binding proteins. (United States)

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


    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.

  5. Biopharmaceuticals from microorganisms: from production to purification

    Directory of Open Access Journals (Sweden)

    Angela Faustino Jozala

    Full Text Available ABSTRACT The use of biopharmaceuticals dates from the 19th century and within 5-10 years, up to 50% of all drugs in development will be biopharmaceuticals. In the 1980s, the biopharmaceutical industry experienced a significant growth in the production and approval of recombinant proteins such as interferons (IFN α, β, and γ and growth hormones. The production of biopharmaceuticals, known as bioprocess, involves a wide range of techniques. In this review, we discuss the technology involved in the bioprocess and describe the available strategies and main advances in microbial fermentation and purification process to obtain biopharmaceuticals.

  6. Optimization of the purification process of wine lees through anaerobic filter reactors. Optimizacion del proceso de depuracion de vinazas de vino mediante reactores tipo filtro anaerobio

    Energy Technology Data Exchange (ETDEWEB)

    Nebot Sanz, E.; Romero Garcia, L.I.; Quiroga Alonso, J.M.; Sales Marquez, D. (Departamento de Ingenieria Quimica, Universidad de Cadiz, Cadiz (Spain))


    In this work, the optimization of thermophilic anaerobic process, using Anaerobic Filter technology was studied. Feed of the Anaerobic Filter was wine-distillery wastewaters. The experiments developed were carried out at lab-scale downflow anaerobic filter reactors. Reactors were filled with a high porous plastic media (Flocor-R). The media support entities have a high surface/volume ratio. Test were run to determine the maximum organic load attainable in the system for wich both, the depurative efficiency and the methane production were optimum. Likewise, the effect of organic load on the anaerobic filter performance were studied. (Author) 15 refs. (Author)

  7. Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10.


    Hill, K E; A. J. Weightman; Fry, J C


    This study examined the potential of bacteria from river epilithon to mobilize a recombinant catabolic plasmid, pD10, encoding 3-chlorobenzoate degradation and kanamycin resistance. Fifty-four mobilizing plasmids were exogenously isolated by triparental matings between strains of Pseudomonas putida and epilithic bacteria from the River Taff (South Wales, United Kingdom). Frequencies for mobilization ranged from 1.7 x 10(-8) to 4.5 x 10(-3) per recipient at 20 degrees C. The sizes of the mobil...

  8. 从镍电镀污泥回收的硫酸镍溶液的深度净化%Deep purification of nickel sulfate solution produced by Ni-electroplating sludge recycling process

    Institute of Scientific and Technical Information of China (English)

    吴青谚; 张贵清


    对于镍电镀污泥的硫酸浸出液所回收得到的粗硫酸镍溶液,采用镍皂化的P507萃取剂净化.考察了料液pH、相比、萃取时间、温度对萃取过程的影响,绘制萃取等温线;考察负载有机相中主要杂质离子用不同浓度H2SO4反萃的效果及规律,提出用H2SO4反萃再生有机相的方案.结果表明,以体积含量为10%的P507+磺化煤油的有机相,通过直接皂化的方法制备镍皂有机相,在皂化率80%、料液pH=2.2、相比VO/VA=1/8、温度30℃、时间5 min条件下,经过6级逆流萃取可以使料液的铁降低到40 mg/L左右;对于除铁后料液,在皂化率80%、料液pH=3.3、相比VO/VA=1/8、温度30℃、时间5 min的条件下,经过4级逆流萃取剩余杂质降至深度净化达标范围;负载有机相用4 mol/L H2SO4按VO/VA=5/1、30℃、5 min,经过3级逆流反萃可达到再生有机相的目的.反萃水相可返回至电镀污泥浸出工序回用.%The crude nickel sulfate solution recycled from nickel electroplating sludge leaching solution of sulfuric acid was purified by Nickel saponified P507 extracting agent. The extraction isotherm was drawn by analyzing the effects of feed solution pH, phase ratio, extraction time and temperature on the extraction process. The back-extraction rule of the main impurity ions by different concentrations H 2SO4 was investigated. The results show that under the following optimum conditions, that is, using 10 % P507 (volume fraction) plus sulphonated kerosene as the extractant, Nickel saponification rate is 80 %, pH= 2.2, VO/VA=1/8, in 30 ℃ and reaction time is 5 min, the concentrate of Fe3+ion can be decreased close to 40 mg/L after a six stage counter current extraction. After iron removal process the Nickel sulfate solution can be deep purified under the following optimum conditions, that is, 80 % saponification rate, pH = 3.3, VO/VA=1/8, in 30 ℃ and reaction time is 5 min, after a four stage counter

  9. Design of the water purification process of Sanya Yacheng Water Work%三亚市崖城水厂净水工艺流程设计

    Institute of Scientific and Technical Information of China (English)



    新建三亚市崖城水厂是三亚市多水源水厂之一,供水量10万m3/d,引大隆水库水为源水(Ⅰ类源水水质,属贫营养水库,仅有时长藻),经比较采用成熟,常规水处理工艺流程——机械搅拌混合,竖向折板絮凝,平流沉淀,气水反冲V型过滤,出厂水可达到或优于国标《生活饮用水卫生标准》GB5749-2006要求。%The new Yacheng Water Work is one of the source water works in Sanya City, with 100000 t/d supplying capacity. Take the Dalong Reservoir water as the source water (Class I source water quality, oligotrophic, sometimes with algae), the mature, conventional water treatment process including mechanical mixing, vertical folding flocculation, advection precipitation, gas and water hackwashing V-shaped filter was selected as the main process of Sanya Yacheng Water Work. It was expected that the product water quality could meet the requirement of "drinking water health standards" (GB5749-2006).

  10. Water Purification Systems (United States)


    Clearwater Pool Technologies employs NASA-developed silver/copper ionization to purify turtle and dolphin tanks, cooling towers, spas, water recycling systems, etc. The pool purifier consists of a microcomputer to monitor water conditions, a pair of metallic electrodes, and a rheostat controller. Ions are generated by passing a low voltage current through the electrodes; the silver ions kill the bacteria, and the copper ions kill algae. This technology has found broad application because it offers an alternative to chemical disinfectants. It was originally developed to purify water on Apollo spacecraft. Caribbean Clear has been using NASA's silver ionization technology for water purification for more than a decade. Two new products incorporate advancements of the basic technology. One is the AquaKing, a system designed for areas with no source of acceptable drinking water. Another is the Caribbean Clear Controller, designed for commercial pool and water park applications where sanitizing is combined with feedback control of pH and an oxidizer, chlorine or bromine. The technology was originally developed to purify water on Apollo spacecraft.

  11. Expression and purification of splicing proteins from mammalian cells. (United States)

    Allemand, Eric; Hastings, Michelle L


    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.

  12. Experimental Study on Purification of Low Grade Diatomite (United States)

    Xiao, Liguang; Pang, Bo


    This paper presented an innovation for purification of low grade diatomite(DE) by grinding, ultrasonic pretreatment, acid leaching of closed stirring and calcination. The optimum process parameters of DE purification were obtained, the characterizations of original and purified DE were determined by SEM and BET. The results showed that the specific surface area of DE increased from 12.65m2/g to 23.23m2/g, which increased by 45.54%. SEM analysis revealed that the pore structure of purified DE was dredged highly.

  13. Solid support resins and affinity purification mass spectrometry. (United States)

    Havis, Spencer; Moree, Wilna J; Mali, Sujina; Bark, Steven J


    Co-affinity purification-mass spectrometry (CoAP-MS) is a primary technology for elucidating the protein-protein interactions that form the basis of all biological processes. A critical component of CoAP-MS is the affinity purification (AP) of the bait protein, usually by immobilization of an antibody to a solid-phase resin. This Minireview discusses common resins, reagents, tagging methods, and their consideration for successful AP of tagged proteins. We discuss our experiences with different solid supports, their impact in AP experiments, and propose areas where chemistry can advance this important technology.

  14. Purification of histidine-tagged T4 RNA ligase from E. coli. (United States)

    Wang, Qing S; Unrau, Peter J


    Here we report the construction of a histidine-tagged T4 RNA ligase expression plasmid (pRHT4). The construct, when overexpressed in BL21 (DE3) cells, allows the preparation of large quantities of T4 RNA ligase in high purity using only a single purification column. The histidine affinity tag does not inhibit enzyme function, and we were able to purify 1-3 mg pure protein/g cell pellet. A simple purification procedure ensures that the enzyme is de-adenylated to levels comparable to those found for many commercial preparations. The purified protein has very low levels of RNase contamination and functioned normally in a variety of activity assays.

  15. An efficient procedure for the expression and purification of HIV-1 protease from inclusion bodies. (United States)

    Nguyen, Hong-Loan Thi; Nguyen, Thuy Thi; Vu, Quy Thi; Le, Hang Thi; Pham, Yen; Trinh, Phuong Le; Bui, Thuan Phuong; Phan, Tuan-Nghia


    Several studies have focused on HIV-1 protease for developing drugs for treating AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. However, large-scale expression and purification of this enzyme is difficult mainly because of its low expression and solubility. In this study, we constructed 9 recombinant plasmids containing a sequence encoding HIV-1 protease along with different fusion tags and examined the expression of the enzyme from these plasmids. Of the 9 plasmids, pET32a(+) plasmid containing the HIV-1 protease-encoding sequence along with sequences encoding an autocleavage site GTVSFNF at the N-terminus and TEV plus 6× His tag at the C-terminus showed the highest expression of the enzyme and was selected for further analysis. The recombinant protein was isolated from inclusion bodies by using 2 tandem Q- and Ni-Sepharose columns. SDS-PAGE of the obtained HIV-1 protease produced a single band of approximately 13 kDa. The enzyme was recovered efficiently (4 mg protein/L of cell culture) and had high specific activity of 1190 nmol min(-1) mg(-1) at an optimal pH of 4.7 and optimal temperature of 37 °C. This procedure for expressing and purifying HIV-1 protease is now being scaled up to produce the enzyme on a large scale for its application.

  16. Purification and separation of durene by static melt crystallization☆

    Institute of Scientific and Technical Information of China (English)

    Shan Cong; Ying Liu; Hong Li; Xingang Li; Lvhong Zhang; Lei Wang


    The purification and separation of durene from the mixture containing durene isomers were studied. Since the boiling points of tetramethyl benzene isomers are very close but their melting points are of great differences, stat-ic melt crystallization was applied to separate and purify durene from its isomers. Crystallization experiments were carried out under various operating conditions. The effects of cooling rate, crystallization temperature, sweating temperature and sweating time on the yield and purity of crystal were investigated. Orthogonal exper-imental design method was adopted to analyze the factors that may affect the yield of durene. Under the optimal crystal ization conditions, the purity of durene could reach as high as 99.06%with the yield of 75.3%through one crystal ization process. By fitting purification data based on sweating time in isothermal operations, the purifica-tion rate coefficient was obtained.

  17. Purification of artemisinin excerpt from Artemisia annua L

    Directory of Open Access Journals (Sweden)

    Chemat-Djenni Z.


    Full Text Available The active pharmaceutical substance artemisinin found in Artemisia annua L. remains the most effective remedy against malaria. The objective of this work is the purification of the active ingredient through a natural matrix composed of clay sandwiched by aluminum. The purification process is carried by adsorption of the extracts on sodium type and bridged clay type. The XRD characterization of this material shows an increase in the basal distance of 10 Å to 17 Å. This shows the success of the intercalation of aluminum polycation. We find a better purification of the extract when using aluminum bridged clay compared to sodium clay. The best performance of 80.64% adsorption is obtained at 40 ° C for the clay matrix bridged with aluminum complex.

  18. Bromelain: an overview of industrial application and purification strategies. (United States)

    Arshad, Zatul Iffah Mohd; Amid, Azura; Yusof, Faridah; Jaswir, Irwandi; Ahmad, Kausar; Loke, Show Pau


    This review highlights the use of bromelain in various applications with up-to-date literature on the purification of bromelain from pineapple fruit and waste such as peel, core, crown, and leaves. Bromelain, a cysteine protease, has been exploited commercially in many applications in the food, beverage, tenderization, cosmetic, pharmaceutical, and textile industries. Researchers worldwide have been directing their interest to purification strategies by applying conventional and modern approaches, such as manipulating the pH, affinity, hydrophobicity, and temperature conditions in accord with the unique properties of bromelain. The amount of downstream processing will depend on its intended application in industries. The breakthrough of recombinant DNA technology has facilitated the large-scale production and purification of recombinant bromelain for novel applications in the future.

  19. Magnetic purification of curcumin from Curcuma longa rhizome by novel naked maghemite nanoparticles. (United States)

    Magro, Massimiliano; Campos, Rene; Baratella, Davide; Ferreira, Maria Izabela; Bonaiuto, Emanuela; Corraducci, Vittorino; Uliana, Maíra Rodrigues; Lima, Giuseppina Pace Pereira; Santagata, Silvia; Sambo, Paolo; Vianello, Fabio


    Naked maghemite nanoparticles, namely, surface active maghemite nanoparticles (SAMNs), characterized by a diameter of about 10 nm, possessing peculiar colloidal stability, surface chemistry, and superparamagnetism, present fundamental requisites for the development of effective magnetic purification processes for biomolecules in complex matrices. Polyphenolic molecules presenting functionalities with different proclivities toward iron chelation were studied as probes for testing SAMN suitability for magnetic purification. Thus, the binding efficiency and reversibility on SAMNs of phenolic compounds of interest in the pharmaceutical and food industries, namely, catechin, tyrosine, hydroxytyrosine, ferulic acid, coumaric acid, rosmarinic acid, naringenin, curcumin, and cyanidin-3-glucoside, were evaluated. Curcumin emerged as an elective compound, suitable for magnetic purification by SAMNs from complex matrices. A combination of curcumin, demethoxycurcumin, and bis-demethoxycurcumin was recovered by a single magnetic purification step from extracts of Curcuma longa rhizomes, with a purity >98% and a purification yield of 45%, curcumin being >80% of the total purified curcuminoids.

  20. Experimental optimal single qubit purification in an NMR quantum information processor. (United States)

    Hou, Shi-Yao; Sheng, Yu-Bo; Feng, Guan-Ru; Long, Gui-Lu


    High quality single qubits are the building blocks in quantum information processing. But they are vulnerable to environmental noise. To overcome noise, purification techniques, which generate qubits with higher purities from qubits with lower purities, have been proposed. Purifications have attracted much interest and been widely studied. However, the full experimental demonstration of an optimal single qubit purification protocol proposed by Cirac, Ekert and Macchiavello [Phys. Rev. Lett. 82, 4344 (1999), the CEM protocol] more than one and half decades ago, still remains an experimental challenge, as it requires more complicated networks and a higher level of precision controls. In this work, we design an experiment scheme that realizes the CEM protocol with explicit symmetrization of the wave functions. The purification scheme was successfully implemented in a nuclear magnetic resonance quantum information processor. The experiment fully demonstrated the purification protocol, and showed that it is an effective way of protecting qubits against errors and decoherence.

  1. 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.

  2. Purification the surface of detail from biological contaminations (United States)

    Gabdrakhmanov, Az T.; Israphilov, I. H.; Galiakbarov, A. T.; Gabdrakhmanov, Al T.


    More than 70% of biodegradation occur due to the corrosion processes. A biological corrosion causes the greatest damage to the oil and gas-production industry, the Navy and pipelines, constructions of water supply, means of communication. This paper proposes an effective method of purification various surfaces from biological contaminations by using of cold plasma.

  3. Energy Efficient Bioethanol Purification by Heat Pump Assisted Extractive Distillation

    NARCIS (Netherlands)

    Kiss, Anton A.; Luo, Hao; Bildea, Costin Sorin


    The purification of bioethanol fuel requires an energy demanding separation process to concentrate the diluted streams obtained in the fermentation stage and to overcome the azeotropic behaviour of ethanol-water mixture. The classic separation sequence consists of three distillation columns that

  4. Isolation of clinical strains of Pseudomonas aeruginosa harboring different plasmids. (United States)

    Ranjbar, R; Owlia, P; Saderi, H; Bameri, Z; Izadi, M; Jonaidi, N; Morovvati, S


    Aim of this study was to investigate the presence of plasmids among the strains of P. aeruginosa isolated from clinically diagnosed cases in Tehran in 2006. A total of 38 strains of P. aeruginosa were isolated. With the exception of one isolate, all P. aeruginosa strains harbored at least one plasmid band. The electrophoretic analysis of plasmid DNAs showed different number of plasmid bands among the strains tested. The DNA band of 1.4 kbp was evident in 84.2% of the strains. Approximately 71 and 21% of the isolates harbored concomitantly two and three plasmids, respectively. Isolation of strains with diverse types of plasmids suggests the different cluster of P. aeruginosa might be disseminated during the current study period.

  5. Transformation of Haemophilus influenzae by plasmid RSF0885

    Energy Technology Data Exchange (ETDEWEB)

    Notani, N.K.; Setlow, J.K.; McCarthy, D.; Clayton, N.L.


    Plasmid RSF0885, which conferred ampicillin resistance, transformed competent Haemophilus influenzae cells with low efficiency (maximun, less than 0.01%). As judged by competition experiments and uptake of radioactivity, plasmid RSF0885 deoxyribonucleic acid was taken up into competent H. influenzae cells several orders of magnitude less efficiently than H. influenzae chromosomal deoxyribonucleic acid. Plasmid RSF0885 transformed cells with even lower efficiency than could be accounted for by the low uptake. Transformation was not affected by rec-1 and rec-2 mutations in the recipient, and strains cured of the plasmid did not show increased transformation. Plasmid molecules cut once with a restriction enzyme that made blunt ends did not transform. Transformation was favored by the closed circular form of the plasmid.

  6. A new and improved host-independent plasmid system for RK2-based conjugal transfer.

    Directory of Open Access Journals (Sweden)

    Trine Aakvik Strand

    Full Text Available Bacterial conjugation is a process that is mediated either by a direct cell-to-cell junction or by formation of a bridge between the cells. It is often used to transfer DNA constructs designed in Escherichia coli to recipient bacteria, yeast, plants and mammalian cells. Plasmids bearing the RK2/RP4 origin of transfer (oriT are mostly mobilized using the E. coli S17-1/SM10 donor strains, in which transfer helper functions are provided from a chromosomally integrated RP4::Mu. We have observed that large plasmids were occasionally modified after conjugal transfer when using E. coli S17-1 as a donor. All modified plasmids had increased in size, which most probably was a result of co-transfer of DNA from the chromosomally located oriT. It has earlier also been demonstrated that the bacteriophage Mu is silently transferred to recipient cells by these donor strains, and both occurrences are very likely to lead to mutations within the recipient DNA. Here we report the construction of a new biological system addressing both the above mentioned problems in which the transfer helper functions are provided by a plasmid lacking a functional oriT. This system is compatible with all other replicons commonly used in conjugation experiments and further enables the use of diverse bacterial strains as donors. Plasmids containing large inserts were successfully conjugated and the plasmid modifications observed when E. coli S17-1 was used as donor were eliminated by the use of the new host-independent vector system.

  7. Purification of Water by Aquatic Plants


    Morimitsu, Katsuhito; Kawahigashi, Tatsuo


    [Abstract] Water quality purification of many water systems including those occurring in rivers depends to a great degree on water quality purification activities of aquatic plants and microbes. This paper presents a discussion of results, based on laboratory experiments, of purification by aquatic plants.

  8. 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


    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。

  9. 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


    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.

  10. Conjugative botulinum neurotoxin-encoding plasmids in Clostridium botulinum.

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    Kristin M Marshall

    Full Text Available BACKGROUND: Clostridium botulinum produces seven distinct serotypes of botulinum neurotoxins (BoNTs. The genes encoding different subtype neurotoxins of serotypes A, B, F and several dual neurotoxin-producing strains have been shown to reside on plasmids, suggesting that intra- and interspecies transfer of BoNT-encoding plasmids may occur. The objective of the present study was to determine whether these C. botulinum BoNT-encoding plasmids are conjugative. METHODOLOGY/PRINCIPAL FINDINGS: C. botulinum BoNT-encoding plasmids pBotCDC-A3 (strain CDC-A3, pCLJ (strain 657Ba and pCLL (strain Eklund 17B were tagged with the erythromycin resistance marker (Erm using the ClosTron mutagenesis system by inserting a group II intron into the neurotoxin genes carried on these plasmids. Transfer of the tagged plasmids from the donor strains CDC-A3, 657Ba and Eklund 17B to tetracycline-resistant recipient C. botulinum strains was evaluated in mating experiments. Erythromycin and tetracycline resistant transconjugants were isolated from donor:recipient mating pairs tested. Transfer of the plasmids to the transconjugants was confirmed by pulsed-field gel electrophoresis (PFGE and Southern hybridizations. Transfer required cell-to-cell contact and was DNase resistant. This indicates that transfer of these plasmids occurs via a conjugation mechanism. CONCLUSIONS/SIGNIFICANCE: This is the first evidence supporting conjugal transfer of native botulinum neurotoxin-encoding plasmids in C. botulinum, and provides a probable mechanism for the lateral distribution of BoNT-encoding plasmids to other C. botulinum strains. The potential transfer of C. botulinum BoNT-encoding plasmids to other bacterial hosts in the environment or within the human intestine is of great concern for human pathogenicity and necessitates further characterization of these plasmids.

  11. Photonic plasmid stability of transformed Salmonella Typhimurium: A comparison of three unique plasmids

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    Lay Donald


    Full Text Available Abstract Background Acquiring a highly stable photonic plasmid in transformed Salmonella Typhimurium for use in biophotonic studies of bacterial tracking in vivo is critical to experimental paradigm development. The objective of this study was to determine stability of transformed Salmonella Typhimurium (S. typh-lux using three different plasmids and characterize their respective photonic properties. Results In presence of ampicillin (AMP, S. typh-lux with pCGLS-1, pAK1-lux and pXEN-1 plasmids exhibited 100% photon-emitting colonies over a 10-d study period. Photon emitters of S. typh-lux with pCGLS-1, pAK1-lux and pXEN-1 without AMP selection decreased over time (P 7 to 1 × 109 CFU, P 0.05; although photonic emissions across a range of bacterial concentrations were not different (1 × 104 to 1 × 106 CFU, P > 0.05. For very low density bacterial concentrations imaged in 96 well plates photonic emissions were positively correlated with bacterial concentration (P 3 to 1 × 105 CFU low to high were different in the 96-well plate format (P Conclusion These data characterize photon stability properties for S. typh-lux transformed with three different photon generating plasmids that may facilitate real-time Salmonella tracking using in vivo or in situ biophotonic paradigms.


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    Azam Khodashenas Pelko


    Full Text Available The Jainism emphasizes three major teachings about the purification of the soul (jiva, Ahimsa, Aparigrapha and anekantwad. Jainism, The focus of this religion has been purification of the soul by means of right conduct, right faith and right knowledge. The ultimate goal of Hinduism is Moksha or liberation (total freedom. In Hinduism, purification of the soul is a goal that one must work to attain. The Buddhism is the science of pursuing the aim of making the human mind perfect, and of purifying the human soul. The knowledge of purifying of the soul and softening of the hearts is as essential for human. They having the correct motivations means purifying our souls from hypocrisy, caprice, and heedlessness. The primary goal of Taoism may be described as the mystical intuition of the Tao, which is the way, the undivided unity, and the ultimate Reality. According to the Christianity access to truth cannot be conceived without purity of the soul

  13. Bacteriophages Limit the Existence Conditions for Conjugative Plasmids (United States)

    Wood, A. Jamie; Dytham, Calvin; Pitchford, Jonathan W.; Truman, Julie; Spiers, Andrew; Paterson, Steve; Brockhurst, Michael A.


    ABSTRACT Bacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome. PMID:26037122

  14. Plasmid genes required for microcin B17 production. (United States)

    San Millán, J L; Kolter, R; Moreno, F


    The production of the antibiotic substance microcin B17 (Mcc) is determined by a 3.5-kilobase DNA fragment from plasmid pMccB17. Several Mcc- mutations on plasmid pMccB17 were obtained by both transposon insertion and nitrosoguanidine mutagenesis. Plasmids carrying these mutations were tested for their ability to complement Mcc- insertion or deletion mutations on pMM102 (pMM102 is a pBR322 derivative carrying the region encoding microcin B17). Results from these experiments indicate that at least four plasmid genes are required for microcin production.

  15. Plasmid P1 replication: negative control by repeated DNA sequences.


    Chattoraj, D; Cordes, K.; Abeles, A


    The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pi...

  16. [Isolation of the R'his plasmids of Vibrio cholerae]. (United States)

    Rusina, O Iu; Tiganova, I G; Aleshkin, G I; Andreeva, I V; Skavronskaia, A G


    V. cholerae strain VT5104 capable of donor activity in conjugation has been constructed by the genetic technique based on plasmid RP4::Mucts62 integration into V. cholerae chromosome due to plasmid homology with Mucts62 inserted into the chromosome. The gene for histidine synthesis has been mobilized and transferred into the recipient cells from VT5104 donor. The conjugants obtained are able to efficiently transfer his+ gene included into the plasmid structure in conjugation with eltor recipient. Thus, the constructed strain VT5104 generates R' plasmids carrying V. cholerae chromosomal genes.

  17. Degradative Plasmid and Heavy Metal Resistance Plasmid Naturally Coexist in Phenol and Cyanide Assimilating Bacteria

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    Bahig E.  Deeb


    Full Text Available Problem statement: Heavy metals are known to be powerful inhibitors of xenobiotics biodegradation activities. Alleviation the inhibitory effect of these metals on the phenol biodegradation activities in presence of heavy metals resistant plasmid was investigated. Approach: Combination of genetic systems of degradation of xenobiotic compound and heavy metal resistance was one of the approaches to the creation of polyfunctional strains for bioremediation of soil after co-contamination with organic pollutants and heavy metals. Results: A bacterial strain Pseudomonas putida PhCN (pPhCN1, pPhCN2 had been obtained. This bacterium contained two plasmids, a 120 Kb catabolic plasmid that encode for breakdown of phenol (pPhCN1 and pPhCN2 plasmid (100 Kb that code for cadmium and copper resistant. Cyanide assimilation by this bacterium was encoded by chromosomal genes. The inhibitory effect of cadmium (Cd2+ or copper (Cu2+ on the degradation of phenol and cyanide by P. putida strains PhCN and PhCN1 (contained pPhCN1 were investigated. The resistant strain PhCN showed high ability to degrade phenol and cyanide in presence of Cd2+ or Cu2+ comparing with the sensitive strain PhCN1. In addition, Cd2+ or Cu2+ was also found to exert a strong inhibitory effect on the C23O dioxygenase enzyme activity in the presence of cyanide as a nitrogen source. Conclusion: The presence of heavy metal resistance plasmid alleviated the inhibitory effect of metals on the phenol and cyanide assimilation by resistant strain.

  18. Microevolutionary events involving narrow host plasmids influences local fixation of vancomycin-resistance in Enterococcus populations.

    Directory of Open Access Journals (Sweden)

    Ana R Freitas

    Full Text Available Vancomycin-resistance in enterococci (VRE is associated with isolates within ST18, ST17, ST78 Enterococcus faecium (Efm and ST6 Enterococcus faecalis (Efs human adapted lineages. Despite of its global spread, vancomycin resistance rates in enterococcal populations greatly vary temporally and geographically. Portugal is one of the European countries where Tn1546 (vanA is consistently found in a variety of environments. A comprehensive multi-hierarchical analysis of VRE isolates (75 Efm and 29 Efs from Portuguese hospitals and aquatic surroundings (1996-2008 was performed to clarify the local dynamics of VRE. Clonal relatedness was established by PFGE and MLST while plasmid characterization comprised the analysis of known relaxases, rep initiator proteins and toxin-antitoxin systems (TA by PCR-based typing schemes, RFLP comparison, hybridization and sequencing. Tn1546 variants were characterized by PCR overlapping/sequencing. Intra- and inter-hospital dissemination of Efm ST18, ST132 and ST280 and Efs ST6 clones, carrying rolling-circle (pEFNP1/pRI1 and theta-replicating (pCIZ2-like, Inc18, pHTβ-like, two pRUM-variants, pLG1-like, and pheromone-responsive plasmids was documented. Tn1546 variants, mostly containing ISEf1 or IS1216, were located on plasmids (30-150 kb with a high degree of mosaicism and heterogeneous RFLP patterns that seem to have resulted from the interplay between broad host Inc18 plasmids (pIP501, pRE25, pEF1, and narrow host RepA_N plasmids (pRUM, pAD1-like. TAs of Inc18 (ω-ε-ζ and pRUM (Axe-Txe plasmids were infrequently detected. Some plasmid chimeras were persistently recovered over years from different clonal lineages. This work represents the first multi-hierarchical analysis of VRE, revealing a frequent recombinatorial diversification of a limited number of interacting clonal backgrounds, plasmids and transposons at local scale. These interactions provide a continuous process of parapatric clonalization driving a full

  19. Microevolutionary Events Involving Narrow Host Plasmids Influences Local Fixation of Vancomycin-Resistance in Enterococcus Populations (United States)

    Freitas, Ana R.; Novais, Carla; Tedim, Ana P.; Francia, María Victoria; Baquero, Fernando; Peixe, Luísa; Coque, Teresa M.


    Vancomycin-resistance in enterococci (VRE) is associated with isolates within ST18, ST17, ST78 Enterococcus faecium (Efm) and ST6 Enterococcus faecalis (Efs) human adapted lineages. Despite of its global spread, vancomycin resistance rates in enterococcal populations greatly vary temporally and geographically. Portugal is one of the European countries where Tn1546 (vanA) is consistently found in a variety of environments. A comprehensive multi-hierarchical analysis of VRE isolates (75 Efm and 29 Efs) from Portuguese hospitals and aquatic surroundings (1996–2008) was performed to clarify the local dynamics of VRE. Clonal relatedness was established by PFGE and MLST while plasmid characterization comprised the analysis of known relaxases, rep initiator proteins and toxin-antitoxin systems (TA) by PCR-based typing schemes, RFLP comparison, hybridization and sequencing. Tn1546 variants were characterized by PCR overlapping/sequencing. Intra- and inter-hospital dissemination of Efm ST18, ST132 and ST280 and Efs ST6 clones, carrying rolling-circle (pEFNP1/pRI1) and theta-replicating (pCIZ2-like, Inc18, pHTβ-like, two pRUM-variants, pLG1-like, and pheromone-responsive) plasmids was documented. Tn1546 variants, mostly containing ISEf1 or IS1216, were located on plasmids (30–150 kb) with a high degree of mosaicism and heterogeneous RFLP patterns that seem to have resulted from the interplay between broad host Inc18 plasmids (pIP501, pRE25, pEF1), and narrow host RepA_N plasmids (pRUM, pAD1-like). TAs of Inc18 (ω-ε-ζ) and pRUM (Axe-Txe) plasmids were infrequently detected. Some plasmid chimeras were persistently recovered over years from different clonal lineages. This work represents the first multi-hierarchical analysis of VRE, revealing a frequent recombinatorial diversification of a limited number of interacting clonal backgrounds, plasmids and transposons at local scale. These interactions provide a continuous process of parapatric clonalization driving a full

  20. [A novel Salmonella Typhimurium plasmid, pAnkS: an example for plasmid evolution in antibiotic resistance]. (United States)

    Sahin, Fikret; Karasartova, Djursun; Gerçeker, Devran; Aysev, A Derya; Erdem, Birsel


    In this study, a plasmid, carrying ampicillin resistance (ampR) gene, isolated from a clinical isolate of Salmonella enterica serotype Typhimurium presenting ACSSuT (ampicilin, chloramphenicol, streptomycin, sulphonamide, tetracycline) resistance phenotype, was defined. The length of complete sequence of this plasmid was 8271 base pairs (bp), and it was named as pAnkS owing to its isolation place (plasmid-Ankara- Salmonella). The plasmid was analyzed for potential reading frames and structural features indicative of transposons and transposon relics. The Xmnl enzyme restriction fragments of pAnkS were cloned into E. coli plasmid vectors (pBSK), sequenced and analyzed with the BLAST programs. Plasmid pAnkS has contained a previously defined enterohemorrhagic E. coli (EHEC) plasmid p4821 as a core region and also contained a complete Tn3-like transposon of 4950 bp consisting of the left terminal repeat, Tn3-related tnpR and tnpA genes for transposition functions, ampicillin resistance gene bla(TEM), and the right terminal repeats, pAnkS showed strong homology with another Salmonella plasmid, pNTP16, for sequences that belong to p4821 and partial Tn3 segments. It was found that pNTP16 also carries kanamycin resistance gene (kanR) in addition to ampR gene. Plasmid pAnkS is one of the few completely sequenced plasmids from Salmonella Typhimurium and is in the middle of the pathway of evolution of plasmid from p4821 to pNTP16. The identification of pAnkS might help better understanding of plasmid evolution.

  1. Purification process of Industrialization preparation of pesticide residue grade n-hexane%农残级正己烷的产业化制备纯化工艺研究

    Institute of Scientific and Technical Information of China (English)

    鄢雄伟; 全灿; 金君素; 黄挺; 李红梅


    以工业级正已烷为原料,通过脱色-连续精馏-过滤等一系列纯化工艺制备农残级正己烷,用紫外吸光度、纯度( GC-FID)和杂质峰高(GC-ECD)等检测值作为农残质量控制指标.产品质量与占当前国内垄断市场的进口试剂水平相当.通过试验确定了较为合适的工艺条件:二氧化硅为脱色剂,用量是原料质量的2%,然后用10%的蒸馏水水洗一次,连续精馏装置的导热油加热温度为98℃,脱轻塔和成品塔的回流比分别为10∶1和4∶1,重组分放料速率20 mL/min,产率可达70%左右.%Pesticide residue grade solvents have the characteristic of ultra high purity, low U V value, low pH value, low moisture content and low evaporation residue, which do not have those controlled impurity peak in pesticide chromato-graphic detection. This project funded by the China government, in which the industrialization process of pesticide residue grade n-hexane was studied, the industrial n-hexane as crude after purification approaches including decoloration,distillation and filtration. The optimized process parameters are: firstly utilizing 2% of silicon dioxide as decoloration agent then is rinsed with distilled water before distillation. The heating temperature of two distillation towers is 88 ℃ with reflux ratio of 10:1 and 4:1 for both respectively, the discharge rate of heavy component is 20 mLAnin.the yield is approximately 70%. This patented technique will meet pesticide residue grade n-hexane market in China or globally.

  2. Analysis and Comparison of Water Purification Processes and Finished Water Quality for 3 Water Treatment Plants%关于三个水厂净水工艺与供水水质的比较和分析

    Institute of Scientific and Technical Information of China (English)

    岳宇明; 陆茸; 毛丽娜; 沈元静; 何小清


    该文介绍了国内一自来水公司三个水厂的两个原水水质、净水工艺及出厂水水质。结果显示第一水厂的出厂水质较为理想,第二水厂次之,第三水厂为第三。第三水厂由于水源的问题导致出厂氨氮季节性超标,建议采取有效措施改进水源水质,以提高出厂水质。第二水厂需进行工艺改造,实施臭氧活性炭深度处理以进一步提高供水水质。第三水厂一期系统臭氧生物活性炭池置于砂滤池后较二期活性炭滤池置于砂滤池前出水有机物CODMn及TOC略低,但两者基本相近。建议第三水厂采取必要的措施改进水源水质,或再增加一道臭氧生物活性炭工序。%Water plants with two different raw water qualities,purification processes and their effluent water qualities were introduced in this paper. The finished water quality of NO. 1 plant is the best,and the NO. 2 is better. Due to raw water quality problem No. 3 water plant’s finished water ammonia nitrogen hardly meets the standard of GB 5749-2006 seasonally. Effective measures should be taken to improve the effluent water quality of No. 3 water plant No. 2 water plant need technological transformation to implement advanced treatment of O3 actived carbon to improve finished water quality. The organic indexes of effluent water of first-stage system in No. 3 water plant,of which O3 actived carbon filter is located behind the sand filter,is a little better than that of second-stage system, of which O3 actived carbon filter is located before the sand filter. But their finished water qualities are approximately same. Necessary measures to improve raw water quality for No. 3 water plant or to apply an additional ozone BAC process are recommended.

  3. Effects of Two Purification Pretreatments on Electroless Copper Coating over Single-Walled Carbon Nanotubes


    Zhong Zheng; Lianjie Li; Shijie Dong; Anchun Xiao; Shixuan Sun; Sinian Li


    To achieve the reinforcement of copper matrix composite by single-walled carbon nanotubes, a three-step-refluxing purification of carbon nanotubes sample with HNO3-NaOH-HCl was proposed and demonstrated. A previously reported purification process using an electromagnetic stirring with H2O2/HCl mixture was also repeated. Then, the purified carbon nanotubes were coated with copper by the same electroless plating process. At the end, the effects of the method on carbon nanotubes themselves and o...

  4. Transfer of a gonococcal beta-lactamase plasmid to conjugation-deficient Neisseria cinerea strains by transformation. (United States)

    Genco, C A; Clark, V L


    We have previously shown that some strains of Neisseria cinerea can serve as recipients in conjugation (Con+) with Neisseria gonorrhoeae while others cannot (Con-). To determine if a replication defect contributes to the inability of certain strains of N. cinerea to serve as recipients in conjugation, we attempted to introduce a naturally occurring gonococcal beta-lactamase plasmid into N. cinerea by transformation. Various methods were employed, and all proved unsuccessful. Since specific sequences are required for DNA uptake in transformation of N. gonorrhoeae, we constructed a number of hybrid plasmids containing N. cinerea chromosomal DNA inserted into the N. gonorrhoeae/Escherichia coli beta-lactamase shuttle vector, pLES2. When nine randomly selected plasmids with inserts were used to transform an N. cinerea strain which did not accept the gonococcal beta-lactamase plasmid by conjugation, transformants were observed with four of the hybrid plasmids. The presence of one of the hybrid plasmids, pCAG9, in transformants was confirmed by agarose gel electrophoresis, Southern hybridization, and beta-lactamase production. When an N. gonorrhoeae donor strain containing pCAG9 was used in conjugation with several N. cinerea strains, only those strains that were previously shown to act as recipients could accept and maintain pCAG9. The ability of pCAG9 and the other three hybrid plasmids to transform Con- strains demonstrates that the beta-lactamase plasmid can replicate in Con- strains, and, therefore, the Con- phenotype is due to a block in some other stage of the conjugation process.

  5. ParAB Partition Dynamics in Firmicutes: Nucleoid Bound ParA Captures and Tethers ParB-Plasmid Complexes.

    Directory of Open Access Journals (Sweden)

    Virginia S Lioy

    Full Text Available In Firmicutes, small homodimeric ParA-like (δ2 and ParB-like (ω2 proteins, in concert with cis-acting plasmid-borne parS and the host chromosome, secure stable plasmid inheritance in a growing bacterial population. This study shows that (ω:YFP2 binding to parS facilitates plasmid clustering in the cytosol. (δ:GFP2 requires ATP binding but not hydrolysis to localize onto the cell's nucleoid as a fluorescent cloud. The interaction of (δ:CFP2 or δ2 bound to the nucleoid with (ω:YFP2 foci facilitates plasmid capture, from a very broad distribution, towards the nucleoid and plasmid pairing. parS-bound ω2 promotes redistribution of (δ:GFP2, leading to the dynamic release of (δ:GFP2 from the nucleoid, in a process favored by ATP hydrolysis and protein-protein interaction. (δD60A:GFP2, which binds but cannot hydrolyze ATP, also forms unstable complexes on the nucleoid. In the presence of ω2, (δD60A:GFP2 accumulates foci or patched structures on the nucleoid. We propose that (δ:GFP2 binding to different nucleoid regions and to ω2-parS might generate (δ:GFP2 gradients that could direct plasmid movement. The iterative pairing and unpairing cycles may tether plasmids equidistantly on the nucleoid to ensure faithful plasmid segregation by a mechanism compatible with the diffusion-ratchet mechanism as proposed from in vitro reconstituted systems.

  6. A mutational analysis of the ColE1-encoded cell cycle regulator Rcd confirms its role in plasmid stability. (United States)

    Balding, Claire; Blaby, Ian; Summers, David


    Multimers of multicopy plasmids cause instability. They arise by homologous recombination and accumulate by over-replication in a process known as the dimer catastrophe. Dimers are resolved to monomers by site-specific recombination systems such as Xer-cer of plasmid ColE1. In addition, the Rcd checkpoint hypothesis proposes that a short transcript (Rcd) coded within ColE1 cer delays the division of multimer-containing cells. The crucial observation underpinning the checkpoint hypothesis is that when the Rcd promoter (P(cer)) is inactivated by mutation of its invariant T, the plasmid becomes unstable. Recently, we discovered that this mutation also alters a potential Fis binding site in cer. ColE1-like plasmids are less stable in fis mutant hosts and it is conceivable that instability caused by the mutation is due to altered Fis binding, rather than the loss of Rcd expression per se. We have therefore undertaken an independent test of the role of P(cer)-Rcd in multicopy plasmid stability. We have generated a series of loss-of-function mutants of Rcd and detailed analysis of two of these shows that they cause a level of instability indistinguishable from P(cer) inactivation. This result is consistent with the predictions of the checkpoint hypothesis and confirms the role of Rcd in plasmid stability.

  7. Gas purification using membrane gas absorption processes

    NARCIS (Netherlands)

    Dindore, V.Y.


    Owing to the increasing energy demand and the abundance of low quality natural gas reservoirs containing high percentages of CO2, considerable attention is given to the bulk removal of CO2 and upgrading of low quality natural gas. The main goal in doing so is to increase the heating value of natural

  8. Anti-tumor Effects of pNEgr-mIL-12 Recombinant Plasmid Induced by X-irradiation and Its Mechanisms

    Institute of Scientific and Technical Information of China (English)



    Objective To study the effect of gene radiotherapy combining injection of recombinant plasmid pNEgr-mIL-12 with local X-irradiation on cancer growth and to elucidate the mechanisms of tumor inhibition. Methods Alkaline lysis was used to extract the plasmid and polyethylene glycol 8000 (PEG 8000) was applied for further purification of plasmids. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of IL-12 protein. C57BL/6J mice were subcutaneously inoculated with B16 melanoma cells and the plasmid was injected directly into the tumor. Gene-radiotherapy combining pNEgr-mIL-12 recombinant plasmid with X-irradiation was given three times to C57BL/6J mice bearing B16 melanoma. Changes in immunologic parameters of tumor-bearing mice were detected with relevant immunologic assays. Results Results showed a significant decrease in tumor growth rate (P<0.05-0.001) after 3 times of gene-radiotherapy with IL-12 and X-irradiation. Immunologic studies showed a significant increase in CTL and NK cytolytic activity (P<0.05-0.001) and an up-regulated secretion of IFN-γ and TNF-α (P<0.01-0.001). Moreover, the expression of mIL-12 in B16 melanoma cells of the treated tumor-bearing mice was found to be higher than that of control. Conclusion pNEgr-mIL-12 plasmid combined with X-irradiation can increase tumor control and the mechanism of increased tumor inhibition is related to the enhancement of anticancer immunity in tumor-bearing mice.

  9. Purification and properties of dialkylfluorophosphatase

    NARCIS (Netherlands)

    Cohen, J.A.; Warringa, M.G.P.J.


    1. 1. Zone electrophoresis on starch columns of purified preparations of fluorophosphatase resulted in a further purification. The preparations thus obtained differed in various respects from the cruder ones so far described. 2. 2. In the course of this electrophoresis fractions were obtained, which

  10. Quantum entanglement purification in cavities

    CERN Document Server

    Romero, J L; Saavedra, C; Retamal, J C


    A physical implementation of an entanglement purification protocol is studied using a cavity quantum electrodynamic based proposal, where, the quantum information is stored in quantum field sates inside cavities. Also a procedure is given for quantifying the degree of entanglement between quantum fields. (Author)

  11. [Immobilized microorganisms and water purification]. (United States)

    Mogilevich, N F


    Advantages and disadvantages of cells of aerobic microorganisms immobilized by the type of adhesion and incorporation into the gel beads, the amount of retained biomass, limitations of diffusion of oxygen and nutrients, viability, morphology, biochemical properties are described. Immobilized biocatalysts are discussed in the aspect of their use in purification of sewage waters.

  12. Purification and concentration of alphavirus. (United States)

    Lundstrom, Kenneth


    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.

  13. Advanced purification of petroleum refinery wastewater by catalytic vacuum distillation. (United States)

    Yan, Long; Ma, Hongzhu; Wang, Bo; Mao, Wei; Chen, Yashao


    In our work, a new process, catalytic vacuum distillation (CVD) was utilized for purification of petroleum refinery wastewater that was characteristic of high chemical oxygen demand (COD) and salinity. Moreover, various common promoters, like FeCl(3), kaolin, H(2)SO(4) and NaOH were investigated to improve the purification efficiency of CVD. Here, the purification efficiency was estimated by COD testing, electrolytic conductivity, UV-vis spectrum, gas chromatography-mass spectrometry (GC-MS) and pH value. The results showed that NaOH promoted CVD displayed higher efficiency in purification of refinery wastewater than other systems, where the pellucid effluents with low salinity and high COD removal efficiency (99%) were obtained after treatment, and the corresponding pH values of effluents varied from 7 to 9. Furthermore, environment estimation was also tested and the results showed that the effluent had no influence on plant growth. Thus, based on satisfied removal efficiency of COD and salinity achieved simultaneously, NaOH promoted CVD process is an effective approach to purify petroleum refinery wastewater. Copyright 2010 Elsevier B.V. All rights reserved.

  14. High quality protein microarray using in situ protein purification

    Directory of Open Access Journals (Sweden)

    Fleischmann Robert D


    Full Text Available Abstract Background In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC. This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays. Results Two slide surfaces were examined, chelated Cu2+ slides suspended on a polyethylene glycol (PEG coating and chelated Ni2+ slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu2+ slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents

  15. 超声提取白刺中总生物碱及大孔树脂纯化的工艺研究%Ultrasound extraction total alkaloids from Nitraria sibirica Pall and process research of macroporous resin purification

    Institute of Scientific and Technical Information of China (English)

    王玉玲; 曾会明; 张建秋; 马挺军


    为优化西伯利亚白刺种籽中总生物碱的提取工艺,采用超声波提取总碱,以硫酸阿托品为对照品,酸性染料比色法测定白刺中总生物碱的含量,探讨料液比、提取温度、时间、乙醇浓度4个因素的影响,确定最佳提取参数.从7种大孔吸附树脂中筛选出对白刺生物碱有最佳分离纯化效果的一种树脂,研究其对生物碱静态、动态吸附、解析效果.研究结果表明,当料液比为1∶20,温度60℃,时间40 min,乙醇浓度70%时,总生物碱提取率(生物碱/种籽重)最高,217.372 9 μg/g D101对白刺总生物碱的吸附、解析效果最好,对其进行动态吸附,可知最佳的工艺条件为上样液pH值为5,流速为0.75 mL/min,乙醇的洗脱浓度为80%.%In order to optimize the extract condition, total alkaloids in Nitraria sibirica was extracted by ultrasound and the content of total alkaloids was determined by the acidic dye colorimetry, the effects of solid-liquid ratio, temperature, time and ethanol concentration on the optimum extraction parameters were investigated, thereby determining the optimal extraction parameters; the best one of isolation and purification from seven kinds of resin were screened out to research its effects on the alkaloids of static, dynamic adsorption. The results show that with the conditions of solid-liquid ratio of 1:20, temperature 60 ℃, time 40 minutes, ethanol concentration of 70%, the highest total alkaloid extract rate was 217.372 9 μg/g; the adsorption and resolution effects of D101 on N. sibirica's total alkaloids were the best, the dynamic adsorption tests proved that the optimum process conditions were: sample solution pH 5, flow rate 0.75mL/min, ethanol concentration 80%.

  16. Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Faber, Klaas Nico; Swaving, Gert Jan; Faber, Folkert; Ab, Geert; Harder, Willem; Veenhuis, Marten; Haima, Pieter


    Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene we

  17. Linear plasmid in the genome of Clavibacter michiganensis subsp. sepedonicus. (United States)

    Brown, Susan E; Knudson, Dennis L; Ishimaru, Carol A


    Contour-clamped homogeneous electric field gel analysis of genomic DNA of the plant pathogen Clavibacter michiganensis subsp. sepedonicus revealed the presence of a previously unreported extrachromosomal element. This new element was demonstrated to be a linear plasmid. Of 11 strains evaluated, all contained either a 90-kb (pCSL1) or a 140-kb (pCSL2) linear plasmid.

  18. Homology of plasmids in strains of unicellular cyanobacteria

    NARCIS (Netherlands)

    Hondel, C.A.M.J.J. van den; Keegstra, W.; Borrias, W.E.; Arkel, G.A. van


    Six strains of unicellular cyanobacteria were examined for the presence of plasmids. Analysis of lysates of these strains by CsCl-ethidium bromide density centrifugation yielded a major chromosomal DNA band and a minor band containing covalently closed circular plasmid DNA, as shown by electron micr

  19. Examination of uropathogenic Escherichia coli strains conferring large plasmids

    Directory of Open Access Journals (Sweden)



    Full Text Available Suhartono (2010 Examination of uropathogenic Escherichia coli strains conferring large plasmids. Biodiversitas 11: 59-64. Of major uropathogens, Escherichia coli has been widely known as a main pathogen of UTIs globally and has considerable medical and financial consequences. A strain of UPEC, namely E. coli ST131, confers a large plasmid encoding cephalosporinases (class C β-lactamase or AmpC that may be disseminated through horizontal transfer among bacterial populations. Therefore, it is worth examining such large plasmids by isolating, purifying, and digesting the plasmid with restriction enzymes. The examination of the large plasmids was conducted by isolating plasmid DNA visualized by agarose gel electrophoresis as well as by PFGE. The relationship of plasmids among isolates was carried out by HpaI restriction enzyme digestion. Of 36 isolates of E. coli ST 131, eight isolates possessed large plasmids, namely isolates 3, 9, 10, 12, 17, 18, 26 and 30 with the largest molecular size confirmed by agarose gel electrophoresis and PFGE was ~42kb and ~118kb respectively. Restriction enzyme analysis revealed that isolates 9, 10, 12, 17 and 18 have the common restriction patterns and those isolates might be closely related.

  20. Plasmid cloning vehicle for Haemophilus influenzae and Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.; Clayton, N.L.; Setlow, J.K.


    A new plasmid cloning vehicle (pDM2) was used to introduce a library of Haemophilus influenzae chromosomal fragments into H. influenzae. Transformants of the higly recombination-defective rec-1 mutant were more likely to contain exclusively recombinant plasmids after exposure to ligated DNA mixtures than was the wild type. pDM2 could replicate in Escherichia coli K-12.

  1. Functional analysis of three plasmids from Lactobacillus plantarum

    NARCIS (Netherlands)

    Kranenburg, R. van; Golic, N.; Bongers, R.; Leer, R.J.; Vos, W.M. de; Siezen, R.J.; Kleerebezem, M.


    Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. Th

  2. Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme. (United States)

    Hancock, Steven J; Phan, Minh-Duy; Peters, Kate M; Forde, Brian M; Chong, Teik Min; Yin, Wai-Fong; Chan, Kok-Gan; Paterson, David L; Walsh, Timothy R; Beatson, Scott A; Schembri, Mark A


    Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including blaCMY and blaNDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a blaNDM-1-positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of blaNDM-positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this blaNDM-containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies. Copyright © 2017 American Society for Microbiology.

  3. Deciphering conjugative plasmid permissiveness in wastewater microbiomes

    DEFF Research Database (Denmark)

    Jacquiod, Samuel Jehan Auguste; Brejnrod, Asker Daniel; Milani, Stefan Morberg


    Wastewater treatment plants (WWTPs) are designed to robustly treat polluted water. They are characterized by ceaseless flows of organic, chemical and microbial matter, followed by treatment steps before environmental release. WWTPs are hotspots of horizontal gene transfer between bacteria via...... still remains largely uncharted. Furthermore, current in vitro methods used to assess conjugation in complex microbiomes do not include in situ behaviours of recipient cells, resulting in partial understanding of transfers. We investigated the in vitro conjugation capacities of WWTP microbiomes from...... diversity of recipient bacterial phyla for the plasmid was observed, especially in WWTP outlets. We also identified permissive bacteria potentially able to cross WWTPs and engage in conjugation before and after water treatment. Bacterial activity and lifestyle seem to influence conjugation extent...

  4. Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae

    DEFF Research Database (Denmark)

    Johnson, Timothy J.; Bielak, Eliza Maria; Fortini, Daniela;


    and biofilm formation. Previous plasmid-based replicon typing procedures have indicated that the prevalence of IncX plasmids is low among members of the Enterobacteriaceae. However, examination of a number of IncX-like plasmid sequences and their occurrence in various organisms suggests that IncX plasmid...

  5. TcpM: a novel relaxase that mediates transfer of large conjugative plasmids from Clostridium perfringens. (United States)

    Wisniewski, Jessica A; Traore, Daouda A; Bannam, Trudi L; Lyras, Dena; Whisstock, James C; Rood, Julian I


    Conjugative transfer of toxin and antibiotic resistance plasmids in Clostridium perfringens is mediated by the tcp conjugation locus. Surprisingly, neither a relaxase gene nor an origin of transfer (oriT) has been identified on these plasmids, which are typified by the 47 kb tetracycline resistance plasmid pCW3. The tcpM gene (previously called intP) encodes a potential tyrosine recombinase that was postulated to be an atypical relaxase. Mutagenesis and complementation studies showed that TcpM was required for wild-type transfer of pCW3 and that a tyrosine residue, Y259, was essential for TcpM activity, which was consistent with the need for a relaxase-mediated hydrophilic attack at the oriT site. Other catalytic residues conserved in tyrosine recombinases were not required for TcpM activity, suggesting that TcpM was not a site-specific recombinase. Mobilization studies led to the identification of the oriT site, which was located in the 391 bp intergenic region upstream of tcpM. The oriT site was localized to a 150 bp region, and gel mobility shift studies showed that TcpM could bind to this region. Based on these studies we postulate that conjugative transfer of pCW3 involves the atypical relaxase TcpM binding to and processing the oriT site to initiate plasmid transfer.

  6. Advances in host and vector development for the production of plasmid DNA vaccines. (United States)

    Mairhofer, Juergen; Lara, Alvaro R


    Recent developments in DNA vaccine research provide a new momentum for this rather young and potentially disruptive technology. Gene-based vaccines are capable of eliciting protective immunity in humans to persistent intracellular pathogens, such as HIV, malaria, and tuberculosis, for which the conventional vaccine technologies have failed so far. The recent identification and characterization of genes coding for tumor antigens has stimulated the development of DNA-based antigen-specific cancer vaccines. Although most academic researchers consider the production of reasonable amounts of plasmid DNA (pDNA) for immunological studies relatively easy to solve, problems often arise during this first phase of production. In this chapter we review the current state of the art of pDNA production at small (shake flasks) and mid-scales (lab-scale bioreactor fermentations) and address new trends in vector design and strain engineering. We will guide the reader through the different stages of process design starting from choosing the most appropriate plasmid backbone, choosing the right Escherichia coli (E. coli) strain for production, and cultivation media and scale-up issues. In addition, we will address some points concerning the safety and potency of the produced plasmids, with special focus on producing antibiotic resistance-free plasmids. The main goal of this chapter is to make immunologists aware of the fact that production of the pDNA vaccine has to be performed with as much as attention and care as the rest of their research.

  7. Plasmid-borne prokaryotic gene expression: Sources of variability and quantitative system characterization (United States)

    Bagh, Sangram; Mazumder, Mostafizur; Velauthapillai, Tharsan; Sardana, Vandit; Dong, Guang Qiang; Movva, Ashok B.; Lim, Len H.; McMillen, David R.


    One aim of synthetic biology is to exert systematic control over cellular behavior, either for medical purposes or to “program” microorganisms. An engineering approach to the design of biological controllers demands a quantitative understanding of the dynamics of both the system to be controlled and the controllers themselves. Here we focus on a widely used method of exerting control in bacterial cells: plasmid vectors bearing gene-promoter pairs. We study two variants of the simplest such element, an unregulated promoter constitutively expressing its gene, against the varying genomic background of four Escherichia coli cell strains. Absolute protein numbers and rates of expression vary with both cell strain and plasmid type, as does the variability of expression across the population. Total variability is most strongly coupled to the cell division process, and after cell size is scaled away, plasmid copy number regulation emerges as a significant effect. We present simple models that capture the main features of the system behavior. Our results confirm that complex interactions between plasmids and their hosts can have significant effects on both expression and variability, even in deliberately simplified systems.

  8. [Influence of spv plasmid genes group in Salmonella Enteritidis virulence for chickens. I. Occurrence of spv plasmid genes group in Salmonella Enteritidis large virulence plasmid]. (United States)

    Madajczak, Grzegorz; Binek, Marian


    Many Salmonella Enteritidis virulence factors are encoded by genes localized on plasmids, especially large virulence plasmid, in highly conserved fragment, they create spv plasmid gene group. The aims of realized researches were spv genes occurrence evaluation and composition analysis among Salmonella Enteritidis strains caused infection in chickens. Researches were realized on 107 isolates, where in every cases large virulence plasmid 59 kbp size were detected. Specific nucleotides sequences of spv genes (spvRABCD) were detected in 47.7% of isolates. In the rest of examined bacteria spv genes occurred variably. Most often extreme genes of spv group, like spvR and spvD were absent, what could indicate that factors encoded by them are not most important for Salmonella Enteritidis live and their expressed virulence.


    Directory of Open Access Journals (Sweden)

    Yu. P. Sedlukho


    Full Text Available The paper considers problems and features of biochemical removal of hydrogen sulfide from ground water. The analysis of existing methods for purification of ground water from hydrogen sulfide has been given in the paper. The paper has established shortcomings of physical and chemical purification of ground water. While using aeration methods for removal of hydrogen sulfide formation of colloidal sulfur that gives muddiness and opalescence to water occurs due to partial chemical air oxidation. In addition to this violation of sulfide-carbonate equilibrium taking place in the process of aeration due to desorption of H2S and CO2, often leads to clogging of degasifier nozzles with formed CaCO3 that causes serious operational problems. Chemical methods require relatively large flow of complex reagent facilities, storage facilities and transportation costs.In terms of hydrogen sulfide ground water purification the greatest interest is given to the biochemical method. Factors deterring widespread application of the biochemical method is its insufficient previous investigation and necessity to execute special research in order to determine optimal process parameters while purifying groundwater of a particular water supply source. Biochemical methods for oxidation of sulfur compounds are based on natural biological processes that ensure natural sulfur cycle. S. Vinogradsky has established a two-stage mechanism for oxidation of hydrogen sulfide with sulfur bacteria (Beggiatoa. The first stage presupposes oxidation of hydrogen sulphide to elemental sulfur which is accumulating in the cytoplasm in the form of globules. During the second stage sulfur bacteria begin to oxidize intracellular sulfur to sulfuric acid due to shortage of hydrogen sulfide.The paper provides the results of technological tests of large-scale pilot plants for biochemical purification of groundwater from hydrogen sulfide in semi-industrial conditions. Dependences of water quality

  10. Permissiveness of soil microbial communities towards broad host range plasmids

    DEFF Research Database (Denmark)

    Klümper, Uli

    larger than previously assumed. I was able to show abundant plasmid transfer from the Gram negative donor strains to a wide diversity of Gram positive soil bacteria, formerly thought to constitute distinct clusters of gene transfer. Moreover, among the observed transconjugants, I identified a core super...... environmental factors that modulate plasmid transfer in soil microbial communities. In order to attain these goals, I developed a high-throughput method that enabled me to evaluate the permissiveness of bacterial communities towards introduced plasmids. This new approach is based on the introduction...... fraction of soil the bacteria (up to 1 in 10,000) were able to take up any of these broad host range conjugal plasmids. The transconjugal pools comprised 11 bacterial phyla. This finding indicates that the realized transfer range of broad host range plasmids in environmental microbial communities is much...

  11. Performance of photocatalyst based carbon nanodots from waste frying oil in water purification

    Energy Technology Data Exchange (ETDEWEB)

    Aji, Mahardika Prasetya, E-mail:; Wiguna, Pradita Ajeng; Susanto,; Rosita, Nita; Suciningtyas, Siti Aisyah; Sulhadi [Department of Physics, Faculty of Mathematics and Natural Science Universitas Negeri Semarang, Jalan Raya Sekaran Gunungpati 50229 Indonesia (Indonesia)


    Carbon Nanodots (C-Dots) from waste frying oil could be used as a photocatalyst in water purification with solar light irradiation. Performance of C-Dots as a photocatalyst was tested in the process of water purification with a given synthetic sewage methylene blue. The tested was also conducted by comparing the performance C-Dots made from frying oil, waste fryng oil as a photocatalyst and solution of methylene blue without photocatalyst C-Dots. Performance of C-Dots from waste frying oil were estimated by the results of absorbance spectrum. The results of measurement absorbance spectrum from the process of water purification with photocatalyst C-Dots showed that the highest intensity at a wavelength 664 nm of methylene blue decreased. The test results showed that the performance of photocatalyst C-Dots from waste frying oil was better in water purification. This estimated that number of particles C-dots is more in waste frying oil because have experieced repeated the heating process so that the higher particles concentration make the photocatalyst process more effective. The observation of the performance C-Dots from waste frying oil as a photocatalyst in the water purification processes become important invention for solving the problems of waste and water purification.

  12. Application and progress on study of air purification with membrane separation process%膜分离法空气净化的应用与研究进展

    Institute of Scientific and Technical Information of China (English)



    气体膜分离技术已经成为空气净化的重要技术之一。文章全面综述了近年来的膜法空气净化技术的应用及研究进展,介绍了用于空气净化的分离膜及膜分离工艺,展望了膜法空气净化技术的发展趋势。指出,由于膜分离空气净化工艺具有连续操作、低能耗和高效等优点,有望替代或部分替代传统空气的颗粒物分离、脱湿及脱二氧化碳过程。%The gas membrane separation technology becomes one of important air purification technologies. Here, the application and progress on study of membrane air purification technology in the last years, the separation membrane used in air purification and the

  13. Nanocellulose-Based Materials for Water Purification

    Directory of Open Access Journals (Sweden)

    Hugo Voisin


    Full Text Available Nanocellulose is a renewable material that combines a high surface area with high strength, chemical inertness, and versatile surface chemistry. In this review, we will briefly describe how nanocellulose is produced, and present—in particular, how nanocellulose and its surface modified versions affects the adsorption behavior of important water pollutants, e.g., heavy metal species, dyes, microbes, and organic molecules. The processing of nanocellulose-based membranes and filters for water purification will be described in detail, and the uptake capacity, selectivity, and removal efficiency will also be discussed. The processing and performance of nanocellulose-based membranes, which combine a high removal efficiency with anti-fouling properties, will be highlighted.

  14. Antibiotic resistance of vibrio cholerae: special considerations of R-plasmids. (United States)

    Kuwahara, S


    Studies on the transmission of R plasmid by conjugation between enterobacteria and vibrio or related bacteria were reviewed. The majority of the reports confirmed successful transmission from enterobacteria to Vibrio cholerae and related species, although the transmission frequencies were extremely low and the transmitted R plasmid was very unstable except for thermosensitive kanamycin plasmid and usual R plasmid coexisting with P plasmid. Strains of V. cholerae and Aeromonas liquefaciens as well as A. salmonicida bearing R plasmid were detected in nature. R plasmid was relatively unstable in V. cholerae strains with which transmission of R plasmid to enterobacteria was confirmed. At present, only 3 R plasmids have been obtained from naturally occurring strains of V. cholerae. Although the 2 European plasmids belong to the C incompatibility group with 98 megadalton closed covalent circular DNA molecule, one plasmid belongs to the J group with more than 25 megadalton molecular weight, and no CCC of satelite DNA was detected in bacteria harboring this plasmid.



    Gusti Wahdaniyah*, Purnomo Tri Prasetyo, Arif Setiabudi, Totok R. Biyanto


    The purpose of this paper is to describe the filtration or purification of transformer oil. One of the main equipment in coal-fired power generation unit is transformer. When the transformer fail to operate properly, the continuity of distribution system become interrupted. As a part of transformer, transformer oil contribute the failure of transformer due to the aging. To solve this problem, several methods is applied starting from dehydration process, degasification process, oxidation remov...



    Gusti Wahdaniyah*, Purnomo Tri Prasetyo, Arif Setiabudi, Totok R. Biyanto


    The purpose of this paper is to describe the filtration or purification of transformer oil. One of the main equipment in coal-fired power generation unit is transformer. When the transformer fail to operate properly, the continuity of distribution system become interrupted. As a part of transformer, transformer oil contribute the failure of transformer due to the aging. To solve this problem, several methods is applied starting from dehydration process, degasification process, oxidation remov...

  17. Bacterial Mitosis: ParM of Plasmid R1 Moves Plasmid DNA by an Actin-like Insertional Polymerization Mechanism

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette


    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system...

  18. Purification of phage display-modified bacteriophage T4 by affinity chromatography

    Directory of Open Access Journals (Sweden)

    Figura Grzegorz


    Full Text Available Abstract Background Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. Results Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. Conclusions Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity

  19. Study on Purification Process of Ethanol Extract of Fruit of Rosa Xanthina Lindl with ZTC1+1 Ⅱ Natural Clarificant%ZTC1+1Ⅱ天然澄清剂用于黄刺玫果醇提液的纯化研究

    Institute of Scientific and Technical Information of China (English)



    Objective:To study the best technology of the purification of yellow rosa acicularis alcohol extraction liquid with ZTC1+1 Ⅱnatural clarificant. Methods:The best purification process was selected,considering the effects of some factors such as the adding sequence of ZTC elarifiers A and B,the amount,material liquid concentration ratio, temperature,dosage of clarifying agent,and on the basis of single factor analysis of the orthogonal experiment to design optimization clarifying condition,flavonoids content by ultraviolet spectrophotometer detection. Results:The optimum purification process was as follows:the material to solution ratio was 1:2;the amount of ZTC1 +1-Ⅱ clarificant was 2%B+1%A,with the stirring speed 100 r/min,at temperature 50℃ constant. Conclusion:ZTC1 +1-Ⅱ clarificant can be used in purification process of the ethanol extract of fruit of rosa xanthina Lindl.%目的:研究ZTC1+1Ⅱ天然澄清剂纯化黄刺玫果醇提液的最佳工艺。方法:实验通过ZTC1+1Ⅱ澄清剂A、B的加入顺序、澄清剂用量、醇提液料液浓度比、温度、搅拌速度等影响因素,在单因素分析的基础上应用正交试验设计优化澄清条件,通过紫外分光光度计检测总黄酮含量,优选最佳纯化工艺。结果:优选出的最佳工艺条件是:料液浓度比为1:2,在温度恒为50℃时按2%B+1%A澄清剂用量,以先加B组份后加A组份的次序以100 r/min的搅拌速度进行。结论:ZTC1+1Ⅱ天然澄清剂可用于纯化黄刺玫果醇提液。

  20. Extended Function of Plasmid Partition Genes: the Sop System of Linear Phage-Plasmid N15 Facilitates Late Gene Expression▿ (United States)

    Ravin, Nikolai V.; Rech, Jérôme; Lane, David


    The mitotic stability of the linear plasmid-prophage N15 of Escherichia coli depends on a partition system closely related to that of the F plasmid SopABC. The two Sop systems are distinguished mainly by the arrangement of their centromeric SopB-binding sites, clustered in F (sopC) and dispersed in N15 (IR1 to IR4). Because two of the N15 inverted repeat (IR) sites are located close to elements presumed (by analogy with phage λ) to regulate late gene expression during the lytic growth of N15, we asked whether Sop partition functions play a role in this process. In N15, a putative Q antiterminator gene is located 6 kb upstream of the probable major late promoter and two intrinsic terminator-like sequences, in contrast to λ, where the Q gene is adjacent to the late promoter. Northern hybridization and lacZ reporter activity confirmed the identity of the N15 late promoter (p52), demonstrated antiterminator activity of the Q analogue, and located terminator sequences between p52 and the first open reading frame. Following prophage induction, N15 mutated in IR2 (downstream from gene Q) or IR3 (upstream of p52) showed a pronounced delay in lysis relative to that for wild-type N15. Expression of ir3−-p52::lacZ during N15 wild-type lytic growth was strongly reduced relative to the equivalent ir3+ fusion. The provision of Q protein and the IR2 and SopAB proteins in trans to ir3+-p52::lacZ increased expression beyond that seen in the absence of any one of these factors. These results indicate that the N15 Sop system has a dual role: partition and regulation of late gene transcription during lytic growth. PMID:18359814

  1. Extended function of plasmid partition genes: the Sop system of linear phage-plasmid N15 facilitates late gene expression. (United States)

    Ravin, Nikolai V; Rech, Jérôme; Lane, David


    The mitotic stability of the linear plasmid-prophage N15 of Escherichia coli depends on a partition system closely related to that of the F plasmid SopABC. The two Sop systems are distinguished mainly by the arrangement of their centromeric SopB-binding sites, clustered in F (sopC) and dispersed in N15 (IR1 to IR4). Because two of the N15 inverted repeat (IR) sites are located close to elements presumed (by analogy with phage lambda) to regulate late gene expression during the lytic growth of N15, we asked whether Sop partition functions play a role in this process. In N15, a putative Q antiterminator gene is located 6 kb upstream of the probable major late promoter and two intrinsic terminator-like sequences, in contrast to lambda, where the Q gene is adjacent to the late promoter. Northern hybridization and lacZ reporter activity confirmed the identity of the N15 late promoter (p52), demonstrated antiterminator activity of the Q analogue, and located terminator sequences between p52 and the first open reading frame. Following prophage induction, N15 mutated in IR2 (downstream from gene Q) or IR3 (upstream of p52) showed a pronounced delay in lysis relative to that for wild-type N15. Expression of ir3(-)-p52::lacZ during N15 wild-type lytic growth was strongly reduced relative to the equivalent ir3(+) fusion. The provision of Q protein and the IR2 and SopAB proteins in trans to ir3(+)-p52::lacZ increased expression beyond that seen in the absence of any one of these factors. These results indicate that the N15 Sop system has a dual role: partition and regulation of late gene transcription during lytic growth.

  2. Purificação do subproduto do processo de extração de esteviosídeo Purification of the by-product of the stevioside extraction process

    Directory of Open Access Journals (Sweden)

    Denise Maria Malachini Miotto


    , CaX and MgX. Two experiments were accomplished: a test of saturation of the adsorbents to evaluate its adsorption capacity and a test for maximum clarification to determine the maximum purification reached by adsorption in zeolites. Starting from the results, it was verified that CaX is a more effective adsorbent. Zeolites can be reused up to twice, needing regeneration soon after. The test for maximum clarification showed almost clear solutions, with high clarification levels (80-90% of the compounds with larger likeness, but with high retentions of the sweeteners (~70%, because zeolites retain most of the pigments of the caramel solution, but they also drag stevioside and rebaudioside A, resulting in a process with low yield in relation to the recovery of the sweeteners, but considered satisfactory, in view that caramel, in spite of being rich in sweeteners, has no use at present.

  3. Tetanus toxoid purification: chromatographic procedures as an alternative to ammonium-sulphate precipitation. (United States)

    Stojićević, Ivana; Dimitrijević, Ljiljana; Dovezenski, Nebojša; Živković, Irena; Petrušić, Vladimir; Marinković, Emilija; Inić-Kanada, Aleksandra; Stojanović, Marijana


    Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin's biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor.

  4. Development of new plasmid DNA vaccine vectors with R1-based replicons

    Directory of Open Access Journals (Sweden)

    Bower Diana M


    Full Text Available Abstract Background There has been renewed interest in biopharmaceuticals based on plasmid DNA (pDNA in recent years due to the approval of several veterinary DNA vaccines, on-going clinical trials of human pDNA-based therapies, and significant advances in adjuvants and delivery vehicles that have helped overcome earlier efficacy deficits. With this interest comes the need for high-yield, cost-effective manufacturing processes. To this end, vector engineering is one promising strategy to improve plasmid production. Results In this work, we have constructed a new DNA vaccine vector, pDMB02-GFP, containing the runaway R1 origin of replication. The runaway replication phenotype should result in plasmid copy number amplification after a temperature shift from 30°C to 42°C. However, using Escherichia coli DH5α as a host, we observed that the highest yields of pDMB02-GFP were achieved during constant-temperature culture at 30°C, with a maximum yield of approximately 19 mg pDNA/g DCW being observed. By measuring mRNA and protein levels of the R1 replication initiator protein, RepA, we determined that RepA may be limiting pDMB02-GFP yield at 42°C. A mutant plasmid, pDMB-ATG, was constructed by changing the repA start codon from the sub-optimal GTG to ATG. In cultures of DH5α[pDMB-ATG], temperature-induced plasmid amplification was more dramatic than that observed with pDMB02-GFP, and RepA protein was detectable for several hours longer than in cultures of pDMB02-GFP at 42°C. Conclusions Overall, we have demonstrated that R1-based plasmids can produce high yields of high-quality pDNA without the need for a temperature shift, and have laid the groundwork for further investigation of this class of vectors in the context of plasmid DNA production.

  5. Isolation and Purification of Biotechnological Products (United States)

    Hubbuch, Jürgen; Kula, Maria-Regina


    The production of modern pharma proteins is one of the most rapid growing fields in biotechnology. The overall development and production is a complex task ranging from strain development and cultivation to the purification and formulation of the drug. Downstream processing, however, still accounts for the major part of production costs. This is mainly due to the high demands on purity and thus safety of the final product and results in processes with a sequence of typically more than 10 unit operations. Consequently, even if each process step would operate at near optimal yield, a very significant amount of product would be lost. The majority of unit operations applied in downstream processing have a long history in the field of chemical and process engineering; nevertheless, mathematical descriptions of the respective processes and the economical large-scale production of modern pharmaceutical products are hampered by the complexity of the biological feedstock, especially the high molecular weight and limited stability of proteins. In order to develop new operational steps as well as a successful overall process, it is thus a necessary prerequisite to develop a deeper understanding of the thermodynamics and physics behind the applied processes as well as the implications for the product.

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

    Institute of Scientific and Technical Information of China (English)


    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.

  7. Integrated development and industrial application of high sour natural gas purification technology

    Institute of Scientific and Technical Information of China (English)

    Sun Lili


    Aiming at acute characteristics of flammability,explosibility,causticity and leakiness of high sour nat-ural gas,giving full consideration to local natural conditions in Sichuan Province and starting with purification pro-cess,the paper analyzed process configurations,energy utilization and conservation,safety and environment pro-tection measures. The integrated technology and engineering scheme for high sour natural gas purification was es-tablished and successfully used in industrial application.

  8. Rapid Purification and Characterization of Mutant Origin Recognition Complexes in Saccharomyces cerevisiae


    Hironori eKawakami; Eiji eOhashi; Toshiki eTsurimoto; Tsutomu eKatayama


    Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as ...

  9. Characteristics of Microbial Community and Operation Efficiency in Biofilter Process for Drinking Water Purification%水质净化生物滤池工艺的微生物群落特征及运行效果研究

    Institute of Scientific and Technical Information of China (English)

    向红; 吕锡武; 杨飞; 尹立红; 朱光灿


    In order to explore characteristics of microbial community and operation efficiency in biofilter (biologically-enhanced active filter and biological activated carbon filter) process for drinking water purification, Biolog and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) techniques were applied to analyze the metabolic function and structure of microbial community developing in biofilters. Water quality parameters, such as NH4+ -N, NO2- -N, permanganate index,UV254 and BDOC etc,were determined in inflow and outflow of biofilters for investigation of operation efficiency of the biofilters. The results show that metabolic capacity of microbial community of the raw water is reduced after the biofilters, which reflect that metabolically active microbial communities in the raw water can be intercepted by biofilters. After 6 months operation of biofilters, the metabolic profiles of microbial communities are similar between two kinds of biologically-enhanced active filters, and utilization of carbon sources of microbial communities in the two filters are 73.4% and 75.5% , respectively. The metabolic profiles of microbial communities in two biological activated carbon filters showed significant difference. The carbon source utilization rate of microbial community in granuleactivated carbon filter is 79.6% , which is obviously higher than 53.8% of the rate in the columnar activated carbon filter (p < 0. 01 ).The analysis results of PCR-SSCP indicate that microbial communities in each biofilter are variety, but the structure of dominant microorganisms is similar among different biofilters. The results also show that the packing materials had little effect on the structure and metabolic function of microbial community in biologically-enhanced active filters, and the difference between two biofilters for the water purification efficiency was not significant(p >0.05). However, in biological activated carbon filters, granule

  10. Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

    Energy Technology Data Exchange (ETDEWEB)

    Benoit, T.G.; Wilson, G.R.; Bull, D.L.; Aronson, A.I. (Department of Agriculture, College Station, TX (USA))


    Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.

  11. In vitro replication of cyanobacterial plasmids from Synechocystis PCC 6803. (United States)

    Yang, X; Daniell, H; McFadden, B


    Little knowledge of DNA replication in cyanobacteria is available. In this study, we report the development and characterization of an in vitro system for studies of replication of the endogenous plasmids from the unicellular cyanobacterium Synechocystis 6803. This system (fraction III) was isolated at high salt concentrations and partially purified on a heparin-agarose column. DNA polymerases in Synechocystis 6803 appeared to be associated with membranes and could be released by the addition of ammonium sulfate to 20% saturation. DNA synthesis in fraction III was dependent on the addition of cyanobacterial plasmids isolated from the same strain. The in vitro replication products consist mostly of the supercoiled form of the plasmids. Unlike replication of many Escherichia coli plasmids, replication of cyanobacterial plasmids did not require added ATP, was not inhibited by omission of the ribonucleotides, and was insensitive to the RNA polymerase inhibitor rifampicin and the gyrase inhibitor novobiocin, but was inhibited by ethidium bromide. These data suggest that RNA may not be involved in the initiation of replication of cyanobacterial plasmids from Synechocystis 6803. In addition, intermediates of replication have been detected by two-dimensional gel electrophoresis. Density labeling experiments also indicate that cyanobacterial plasmid synthesis in vitro occurs by a semiconservative replication.

  12. Using H2SiF6 to Precipitate Magnesium from Raffinate of Wet-process Phosphoric Acid Purification by Chemical Precipitation%氟硅酸化学沉淀法净化萃余酸中的镁

    Institute of Scientific and Technical Information of China (English)

    杜怀明; 罗容珍; 刘兴勇; 杨虎; 黄家骏; 李春梅; 万霞


    The second-order orthogonal regression design was used to study the process of Mg2+ precipitation from raffinate acid (raffinate of wet-process phosphoric acid purification) which was not concentrated by chemical precipitation,and the factors of H2SiF6 usage,water evaporation amount from raffinate acid,reaction temperature and residence time were studied respectively.The experimental results indicated that the Mg2+ removed ratio would be optimal and reached the demand of superior DAP production when the H2SiF6 usage is 95.71g,water evaporation amount is 80.41g,reaction temperature is 36.02 ℃ and residence time is 45.03 min.The mathematics model to forecast the Mg2+ removed ratio from raffinate acid was obtained among H2SiF6 usage and water evaporation amount,reaction temperature and residence time,and the factors' effects can use below formula to explain.y =59.1785-0.541156z1 + 0.425071z2-0.660951z3-1.635317z12 + 1.768750z1z2-6.392974z22 + 1.011250z1z3-0.978750z2z3-0.611140z32-1.106885z42.%通过二次回归正交设计,实验研究了化学沉淀法净化未经浓缩的萃余酸中镁离子的工艺条件,对氟硅酸用量、萃余酸水去除量、反应温度和反应时间四个因子进行了研究.实验结果表明:当氟硅酸用量为95.71g,除水量为80.41g,反应温度为36.02℃,反应时间为45.03min时,可使镁离子去除率达到最佳值,达到生产优等DAP的磷酸要求;实验得到的镁去除率(y)与氟硅酸用量(z1)、除水量(z2)、反应温度(z3)和反应时间(z4)的模型预测方程为:y=59.1785-0.541156z1+0.425071z2-0.660951z3-1.635317z(12)+1.768750z1z2-6.392974z22+ 1.011250z1z3-0.978750z2z3-0.611140z32-1.106885z24.

  13. Identification of protein interacting partners using tandem affinity purification. (United States)

    Bailey, Dalan; Urena, Luis; Thorne, Lucy; Goodfellow, Ian


    A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification(1). Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast(2,3) but more recently has been adapted to use in mammalian cells(4-8). As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E(9,10).The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation(10). The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence(8). To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter. Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous

  14. Purification of Tetrahymena cytoskeletal proteins. (United States)

    Honts, Jerry E


    Like all eukaryotic cells, Tetrahymena thermophila contains a rich array of cytoskeletal proteins, some familiar and some novel. A detailed analysis of the structure, function, and interactions of these proteins requires procedures for purifying the individual protein components. Procedures for the purification of actin and tubulin from Tetrahymena are reviewed, followed by a description of a procedure that yields proteins from the epiplasmic layer and associated structures, including the tetrins. Finally, the challenges and opportunities for future advances are assessed.

  15. Bicistronic expression plasmid for the rapid production of recombinant fused proteins in Escherichia coli. (United States)

    Yero, Daniel; Pajón, Rolando; Niebla, Olivia; Sardiñas, Gretel; Vivar, Isbel; Perera, Yasser; García, Darien; Delgado, Maité; Cobas, Karem


    In the post-genomic era, every aspect of the production of proteins must be accelerated. In this way, several vectors are currently exploited for rapid production of recombinant proteins in Escherichia coli. N-terminal fusions to the first 47 amino acids of the LpdA (dihydrolipoamide dehydrogenase A) protein of Neisseria meningitidis have been shown to increase the expression of recombinant proteins. Consequently, we have constructed a modified N-terminal LpdA fusion vector, introducing the blue/white colony selection by exploiting a bicistronic gene organization. In the new vector, the sequence encoding the first 47 amino acids of meningococcal LpdA and the alpha-peptide sequence of beta-galactosidase were connected via a ribosome-binding site, and two MCSs (multiple cloning sites) were located surrounding the latter, allowing efficient cloning by colour selection of recombinants. The vector was also improved with the addition of a C-terminal polyhistidine tag, and an EKS (enterokinase recognition sequence) immediately after the LpdA fusion sequence. The new plasmid was employed in the expression and purification of six different bacterial polypeptides. One of these recombinant proteins, P6 protein from Haemophilus influenzae, was used as a model and its N-terminal fusion sequence was totally removed from the recombinant version after incubation with the enterokinase protease, while the polyhistidine tail successfully allowed the purification of the unfused protein from the protease reaction. Two completely new neisserial vaccine candidates, NMB0088 and NMB1126 proteins, were cloned, expressed and purified using this system. To our knowledge, this constitutes the first report of the cloning and expression of these proteins in E. coli.

  16. Comparing Russian and Finnish standards of water purification


    Maria, Pupkova


    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,...

  17. Plasmid-determined resistance to fosfomycin in Serratia marcescens. (United States)

    Mendoza, C; Garcia, J M; Llaneza, J; Mendez, F J; Hardisson, C; Ortiz, J M


    Multiple-antibiotic-resistant strains of Serratia marcescens isolated from hospitalized patients were examined for their ability to transfer antibiotic resistance to Escherichia coli by conjugation. Two different patterns of linked transferable resistance were found among the transconjugants. The first comprised resistance to carbenicillin, streptomycin, and fosfomycin; the second, and more common, pattern included resistance to carbenicillin, streptomycin, kanamycin, gentamicin, tetracycline, chloramphenicol, sulfonamide, and fosfomycin. The two types of transconjugant strains carried a single plasmid of either 57 or 97 megadaltons in size. Both of these plasmids are present in parental S. marcescens strains resistant to fosfomycin. The 57-megadalton plasmid was transformed into E. coli.

  18. [Epidemiologic study of 2 S. typhimurium outbreaks using plasmid fingerprints]. (United States)

    Baumgartner, A; Breer, C; Schopfer, K


    An outbreak of salmonellosis in an old people's home is reported. The infectious agent, S. typhi-murium, was isolated not only from several inmates but also from sick cows of the farm belonging to the home, in animal feed, from employees of the local butcher's shop, and finally in sludge from the local sewage plant. Plasmid analysis provided evidence of a common origin for the isolated S. typhi-murium strains. The incriminated strains harboured, together with two low-molecular-weight plasmids, a plasmid of approximately 50 Mdal, which was also demonstrated in some other S. typhi-murium strains isolated from clinical cases in the area around St. Gallen.

  19. Effect of Plasmid Incompatibility on DNA Transfer to Streptococcus cremoris


    Van Der Lelie, Daniel; Vossen, Jos M.B.M. van der; Venema, Gerard


    Several Streptococcus cremoris strains were used in protoplast transformation and interspecific protoplast fusion experiments with Streptococcus lactis and Bacillus subtilis, with pGKV110, pGKV21, and ΔpAMβ1 as the marker plasmids. ΔpAMβ1 is a 15.9-kilobase nonconjugative, deletion derivative of pAMβ1, which is considerably larger than the pGKV plasmids (approximately 4.5 kilobases). In general, ΔpAMβ1 was transferred more efficiently than the pGKV plasmids. Using electroporation, we were abl...

  20. Separation of plasmid DNA topoisomers by multimodal chromatography. (United States)

    Silva-Santos, A Rita; Alves, Cláudia P A; Prazeres, Duarte Miguel F; Azevedo, Ana M


    The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases. Here, we describe the separation of supercoiled (sc) and open circular (oc) topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid. The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample. Copyright © 2016 Elsevier Inc. All rights reserved.