• Title, Summary, Keyword: anion exchange chromatography

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Purification of Filamentous Bacteriophage M13 by Expanded Bed Anion Exchange Chromatography

  • Tau Chuan;Chee Kin;Wen Siang;Beng Ti;Wan, Wan-Mohammad;Arbakariya
    • Journal of Microbiology
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    • v.42 no.3
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    • pp.228-232
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    • 2004
  • In this paper, we investigated the development of a simplified and rapid primary capture step for the recovery of M13 bacteriophage from particulate-containing feedstock. M13 bacteriophage, carrying an insert, was propagated and subsequently purified by the application of both conventional multiple steps and expanded bed anion exchange chromatography. In the conventional method, precipitation was conducted with PEG/NaCl, and centrifugation was also performed. In the single step expanded bed anion exchange adsorption, UpFront FastLine$\_$TM/20 (20mm i.d.) from UpFront Chromatography was used as the contactor, while 54$m\ell$ (H$\_$o/=15cm) of STREAMLINE DEAE (p=1.2 g/㎤) from Amersham Pharmacia Biotechnology was used as the anion exchanger. The performance of the two methods were evaluated, analysed, and compared. It was demonstrated that the purification of the M13 bacteriophage, using expanded bed anion exchange adsorption, yielded the higher recovery percentage, at 82.86%. The conventional multiple step method yielded the lower recovery percentage, 36.07%. The generic application of this integrated technique has also been assessed.

Screening and Purification of Superoxide Dismutase Producing Marine Bacterium Using Photochemically Generated Superoxide Ion (광화학적으로 제조된 Superoxide Radical을 이용한 Superoxide Dismutase를 생산하는 해양미생물의 탐색 및 효소정제)

  • 조기웅
    • Korean Journal of Microbiology
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    • v.38 no.2
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    • pp.81-85
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    • 2002
  • A marine bacterium producing superoxide dismutase, strain number B446, was screened with nitrite quantitation method using hydroxy amine and photochemically generated superoxide ion, and the superoxide dismutase was purified through 35-75% ammonium sulfate precipitation, DEAE-Sephadex A-25 ion exchange chromatography, Sephadex G-200 gel filtration chromatography, and High-Q anion exchange chromatography to a yield of 6% and purification fold of 32.3.

Studies on the interaction of thiamines and cyclodextrins

  • Im, In-Seon;Lee, Wang-Kyu;Park, Man-Ki;Kim, Bak-Kwang
    • Archives of Pharmacal Research
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    • v.6 no.1
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    • pp.35-44
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    • 1983
  • Interactions between thiamine.HCl and its disulfide derivatives TTFD. TPD and .alpha., .betha. cyclodextrins were investigated. By measuring the H-NMR, C-NMR chemical shifts, the assumption that cyclodextrin may from a inclusion complex with thiamines was supported qualitatively. To calculated the stability constants of them, anion exchange chromatography was applied. The simple, rapid HPLC method was proved to be pertinent thiamine/cyclodextrin system which was chemically unstable and less soluble.

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Purification of Odontoglossum Ringspot Virus by DEAE-Cellulose Chromatography (DEAE 셀루로오즈 컬럼 크로마토그래피 기법에 의한 Odontoglossum 윤문 바이러스의 정제)

  • 이철호;박종오;정효원;나용준
    • Korean Journal Plant Pathology
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    • v.14 no.6
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    • pp.559-562
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    • 1998
  • Odontoglossum ringspot virus (ORSV) was finally purified from ORSV-infected orchid plants by diethylaminoethyl (DEAE) cellulose anion exchange column chromatography. The virus was reliably eluted by potassium chloride at the concentration from 0.1 M to 0.13 M. Partial purification was done by solubilization with Triton X-100 (allkylphenoxypolyethoxy ethanol) and precipitation with polyethylene glycol (PEG; MW 8,000). The finally purified ORSV represented one distinct homogeneous band and the molecular weight of its capsid protein was about 17,500 Dalton in electrophoretic analysis. Electron microscopy showed not only intact particles ranged from 280 nm to 340 nm in length, but also segmented particles that final 140 nm to 220 nm and even disks. Enzyme-linked immunosorbent assay (ELISA) showed that final yield was 12 mg/100 g of the infected leaves. Bioassay demonstrated that the purified ORSV had the normal infectivity to orchid plants and Nicotiana glutionsa. Based on these data, anion exchange column chromatography could be efficiently applied to the purification of ORSV and other viruses similar to ORSV.

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Separation of Burnup Monitors in Spent Nuclear Fuel Samples by Liquid Chromatography

  • Joe, Kih-Soo;Jeon, Young-Shin;Kim, Jung-Suck;Han, Sun-Ho;Kim, Jong-Gu;Kim, Won-Ho
    • Bulletin of the Korean Chemical Society
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    • v.26 no.4
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    • pp.569-574
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    • 2005
  • A coupled column liquid chromatography system was applied for the separation of the burnup monitors in spent nuclear fuel sample solutions. A reversed phase column was studied for the adsorption behavior of uranyl ions using alpha-hydroxyisobutyric acid as an eluent and used for the separation of plutonium and uranium. A cation exchange column prepared by coating 1-eicosylsulfate onto the reversed phase column was used for the separation of the lanthanides. In addition, retention of Np was checked with the reversed phase column and cation exchange column, respectively, according to the oxidation states to observe the interference effect for the separation of burnup monitors. This chromatography system showed a great reduction in separation time compared to a conventional anion exchange method. A good agreement from the burnup data was obtained between for this method and a conventional anion exchange method to within 1% of a difference for the spent nuclear fuel samples of about 40 GWD/MTU.

Overexpression and Purification of Bacillus subtilis Glutamyl-tRNA Synthetase in Escherichia coli (대장균에서 Bacillus subtilis glutamyl-tRNA synthetase의 과발현 및 정제)

  • Oh, Jong-Shin;Yoon, Jang-Ho;Hong, Kwang-Won
    • Applied Biological Chemistry
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    • v.45 no.4
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    • pp.190-194
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    • 2002
  • Expression of Bacillus subtilis glutamyl-tRNA synthetase (GluRS) in Escherichia coli is lethal for the host, probably because this enzyme misaminoacylates ${tRNA_l}^{Gln}$ with glutamate in vivo. In order to overexpress B. subtilis GluRS, encoded by the gltX gene, in E. coli, this gene was amplified from B. subtilis 168 chromosomal DNA using PCR method and the entire coding region was cloned into a pET11a expression vector so that it was expressed under the control or the T7 Promoter. The resulting recombinant pEBER plasmid was transformed into E. coli Novablue (DE3) bearing the T7 RNA polymerase gene for expression. After IPTG treatment, the overproduced enzyme was purified using ammonium sulfate fractionation, Source Q anion exchange chromatography, Superdex-200 gel filtration, and Mono Q anion exchange chromatography. The purified enzyme yielded 18-fold increase in specific activity over the crude cell extract and its molecular weight was approximately 55 kDa on SDS-PAGE.

Quantification of Inulo-oligosaccharides Using High pH Anion Exchange Chromatography with Pulsed Amperometric Detector (HPAEC-PAD)

  • Kang, Su-Il;Chang, Yung-Jin;Kim, Kyoung-Yun;Kim, Su-Il
    • Journal of Applied Biological Chemistry
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    • v.42 no.4
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    • pp.166-168
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    • 1999
  • Inulo-oligosaccharides (IOS, $F_n$, n=2-6) were purified from enzymatic hydrolysates of water-soluble extract of Jerusalem artichoke tubers. Quantification of inulo-oligosaccharides was done using high pH anion exchange chromatography with pulsed amperometric detector (HPAEC-PAD) at the concentration range of 10-100 mg/L, which was compared with that of fructo-oligosaccharides (FOS, $GF_n$, n=1-7). Peak areas per mg IOS were higher than FOS at the same degree of polymerization (DP). Specific peak areas of IOS increased proportionally as DP increased up to six, in contrast to FOS showing no linearity.

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Isolation and Purification of Fibrinolytic Enzyme of Edible Mushroom, Sarcodon aspratus(Berk.)S. Ito (능이버섯으로부터 Fibrin 분해활성이 있는 단백질의 분리 및 정제)

  • 이종호;양정례;정청송;김희숙;조재선
    • Journal of Life Science
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    • v.11 no.6
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    • pp.561-567
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    • 2001
  • To isolate and purify fibrinolytic active substance from Sarcodon aspratus(N $H_4$)$_2$S $O_4$ precipitation, DE52 anion exchange column chromatography, Sephacryl-S 200gel filtration chromatography and Mono S cation FPLC were carried out and the characterizations of the purified enzyme were investigated. The bound active fraction on DE52 anion exchange column chromatography were eluted with 0.2 M NaCI and the fibrionlytic enzyme was purified after following Sephacryl-S200 gel fitration chromatography and Mono S cation EPLC. The specific activity of purified enzyme was 55.2 U/mg protein and increased 11.3 fold comparing crude extract and the yield was 49.5%. 12% SDS-PAGE electrophoresis and gel filtration chromatography revealed that Sarcodon aspratus fibrionloytic enzyme was highly purified and had 29.300 Da molecular weight. Enzyme activity of the purified fibrinolytic enzyme from Sarcodon aspratus was increased on higher pH and was stable until pH 10.5. On temperature dependent stability, the enzyme activity was decrease sharply but remained 25% relative activity on 8$0^{\circ}C$. This enzyme activity was inhibited by heavy metal ion, C $U^{2+}$ and $Co^{3+}$ with 68% and 38%, respectively. And also, the enzyme activity was inhibited with $Ca^{2+}$ chelator EDTA and serine protease inhibitor PMSF. These results from this study suggested that the fibrinolycit enzyme from Sarcodon aspratus is a serine protease and the enzyme activity was increased by $Ca^{2+}$ or $Mg^{2+}$ ion.n.ion.n.

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A study on the separation and recovery of uranium (우라늄의 분리 및 회수에 관한 연구)

  • 노기환;김준태
    • Journal of environmental and Sanitary engineering
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    • v.12 no.1
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    • pp.15-23
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    • 1997
  • The anionic exchange resins with the Dowex-1 and Amberlite CG-400 form were transformed into resin of sulfate and acetate acid form, respectively. The uranyl complex ions with SO$_{4}$$^{2-}$ and CH$_{3}$COO$^{-}$ were adsorbed on the anion exchange resion mentioned above, and these complex ions were eluted as mixture eluents of 0.7M HNO$_{3}$ - 0.5M NH$_{4}$NO$_{3}$ by anion exchange chromatography. The optimum adsorption conditions of uranyl anion complex ions adsorbed on the upper of the resin colmun were 1.5-2.0 ml/min of flow rates at pH 2.0 and adsorptive power of uranyl complex ion of sulfuric acid type were nearly consistent with the Caussion normal distribution curve, whereas the elution state of UO$_{2}$(Ac)$_{2}$$^{4-}$ with acetic acid type was departed. The weighing form obtained from resin of sulfuric acid and aceric acid type was U$_{3}$O$_{8}$ whose recovery was 91.7%. The possibility of recovering uranium from the monazite sulfate solution using a strong base anion resin, Amberlite CG-400(sulfate form), was successfully recovered more than 90%.

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