• Title/Summary/Keyword: Purification method

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Isolation and Purification of Methyl Mercaptan Oxidase from Thiobacillus thiooxidans for Detection of Mercaptasn (머캅탄류 검출을 위한 Thiobacillus thiooxidans가 생산하는 메칠머캅탄 산화효소의 분리 및 정제)

  • 김상준;신현재;이대실;양지원
    • KSBB Journal
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    • v.15 no.2
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    • pp.145-149
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    • 2000
  • Methyl mercaptan oxidase was isolated and purified from Thiobacillus thiooxidans KCTC2505 for the detection of mercaptans. T The produre of purification involved DEAE-Sephacel and Superose 12 column chromatographies with recovery yields of 40 a and 6.3%, and specific activity of 19.7 and 80.1 units/mg-protein, respectively. The molecular weight of purified methyl m mercaptan oxidase was determined to be 68.1 kDa by SDS-PAGE. The extract from DEAE-Sephacel column chromatography h had a high activity in oxidizing methyl mercaptan to produce formaldehyde which can be easily detected by purpald-coloring m method. Optimum temperature for activity was observed at $43^{\circ}C$. This enzyme was activated by $NH_4CI and (NH_4)_2S0_4$, and | inhibited by KCI and NaC!.

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Identification of Histone Deacetylase 1 Protein Complexes in Liver Cancer Cells

  • Farooq, Muhammad;Hozzein, Wael N.;Elsayed, Elsayed A.;Taha, Nael A.;Wadaan, Mohammad A.M.
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.2
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    • pp.915-921
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    • 2013
  • Background: Hepatocellular carcinoma is one of the leading causes of mortalities worldwide. The search for new therapeutic targets is of utmost importance for improved treatment. Altered expression of HDAC1 in hepatocellular carcinoma (HCC) and its requirement for liver formation in zebrafish, suggest that it may regulate key events in liver carcinogenesis and organogenesis. However, molecular mechanisms of HDAC1 action in liver carcinogenesis are largely unknown. The present study was conducted to identify HDAC1 interacting proteins in HepG2 cells using modified SH-double-affinity purification coupled with liquid mass spectrophotemetery. Materials and Methods: HepG2 cells were transfected with a construct containing HDAC1 with a C-terminal strepIII-HA tag as bait. Bait proteins were confirmed to be expressed in HepG2 cells by western blotting and purified by double affinity columns and protein complexes for analysis on a Thermo LTQ Orbitrap XL using a C18 nano flow ESI liquid chromatography system. Results: There were 27 proteins which showed novel interactions with HDAC1 identified only in this study, while 14 were among the established interactors. Various subunits of T complex proteins (TCP1) and prefoldin proteins (PFDN) were identified as interacting partners that showed high affinity with HDAC1 in HepG2 cells. Conclusions: The double affinity purification method adopted in this study was very successful in terms of specificity and reproducibility. The novel HDAC1 complex identified in this study could be better therapeutic target for treatment of hepatocellular carcinoma.

Improved Purification Process for Cholera Toxin and its Application to the Quantification of Residual Toxin in Cholera Vaccines

  • Jang, Hyun;Kim, Hyo-Seung;Kim, Jeong-Ah;Seo, Jin-Ho;Carbis, Rodney
    • Journal of Microbiology and Biotechnology
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    • v.19 no.1
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    • pp.108-112
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    • 2009
  • 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-${\mu}m$ 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-${\mu}m$ 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, pH 7.0, containing 1.0 M 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 $3.1\;EU/{\mu}g$ 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.

The Water Quality and Purification Load Assessment of Drain Water of Facility Horticulture Areas (시설원예 배출 배액의 수질환경 평가 및 정화 부하량 산정)

  • Son, Jinkwan;Choi, Dekkyu;Kong, Minjae;Yun, Sungwook;Park, Minjung;Kang, Donghyeon
    • Journal of Environmental Science International
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    • v.28 no.12
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    • pp.1199-1208
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    • 2019
  • Korea's protected horticulture is rapidly increasing in scale due to various advantages such as year-round harvesting, labor savings through automation and shortened culture period, and greater income generation. This study was conducted to investigate the impact of protected horticulture on water quality. The results of this study are expected to provide basic data contributing to improvements towards sustainable agriculture and eco-friendly design of protected horticulture complex. The average T-N and T-P loads from vinyl greenhouses were 286.55± 143.98 mg/L and 59.14±13.77 mg/L, respectively and those from glass greenhouses 380.68 ± 150.41 mg/L and 61.85±20.72 mg/L. The annual discharge of wastewater derived from the monthly discharge from the horticulture greenhouses were estimated at 2597 ton/ha, with the annual phosphorus load amounting to 155.3 kg/ha. The average T-N and T-P loads in the tested greenhouse effluents were in excess of 8.3- and 13.5-fold the standards for the Korean wastewater plant effluent. The waste nutrient solution discharged from a protected horticulture complex can cause water contamination. Therefore, there is a need to conduct follow-up research using a water purification system or a trench method to develop a eco-friendly protected horticulture complex for sustainable agriculture.

Purification and Characterization of Kiwifruit Protease (키위열매 Protease 의 추출 정제 및 그 특성에 대하여)

  • Kim, Bok-Ja
    • Korean Journal of Food Science and Technology
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    • v.21 no.4
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    • pp.569-574
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    • 1989
  • These studies were conducted to investigate the purification and characterization of Kiwifruit protease, and the results obtained were as follows The protease was purified by ammonium sulfate fractionation, Sephadex G-100 filtration and DEAE-Sephadex A-50 column chromatography and purified enzyme gave a single protein band on polyacrylamide gel electrophoresis The specific activity of purified enzyme was 30,10 units/mg protein and the yield was 7.48. The purified enzyme showed a high affinity for casein and hemoglobin. The optimal pH and temperature for enzyme activity were 7.0 and $45^{\circ}C$, respectively. The enzyme activity was strongly inhibited by $HgCl_2,\;MnSO_4$. However. the enzyme was activated by cysteine and EDTA. The Michaelis constant for casein was calculated to be 50.5mg/ml according to the Line weaver-Burk method, and its molecular weight was determied as 23,500 by polyacrylamide gel electrophoresis.

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Purification of Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) Esters from Squid Oil by Silver Ion Chromatography (은 이온 크로마토그래피에 의한 오징어유로부터 eicosapentaenoic acid(EPA) 및 docosahexaenoic acid(DHA)의 분리농축)

  • Gyoung, Young-Soo;Yu, Ying-Lian;Yoon, Jung-Ro
    • Korean Journal of Food Science and Technology
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    • v.36 no.2
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    • pp.361-364
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    • 2004
  • EPA and DHA extracted from methyl esterified squid oil were purified by silver exchanged resin, silver nitrate-impregnated silica gel, silver exchanged zeolite, and silica gel column chromatography, among which column chromatography using mixture of silver exchanged resin and silica gel (10% by weight) showed the best result. By this simple purification method, EPA and DHA were concentrated from 12.5 to 27.9% (yield, 86,0%) and from 21.7 to 49.5% (yield, 87.3%), respectively. Silver exchanged resin had additional advantages of outstanding reusability and simple recovery of silver.

Investigation on purification of α-Fe2O3 from zinc smelting iron slag by superconducting HGMS technology

  • Zhang, Peng;Li, Su-qin;Guo, Zi-jie;Zhang, Chang-quan;Yang, Chang-qiao;Han, Shuai-shuai
    • Progress in Superconductivity and Cryogenics
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    • v.20 no.2
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    • pp.16-19
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    • 2018
  • Comprehensive utilization of zinc smelting iron slag not only solves environmental problems but also creates huge economic benefits. This study was conducted on the enrichment and recovery of ${\alpha}-Fe_2O_3$ from zinc smelting iron slag by superconducting HGMS technology. Several variables such as slurry flow velocity, slag concentration, magnetic field intensity and the amount of dispersing agent were tested in magnetic separation. In the experiments, obtained optimal magnetic separation parameters were 1.60 T of magnetic flux intensity, 600 mL/min of slurry flow velocity of, 15 g/L of slag concentration of, 0.10 g/L of dispersing agent. Under this condition, the content of ${\alpha}-Fe_2O_3$ was increased from 86.22% to 94.39% that can approach the Chinese national standard requirements (A level) of iron oxide red. It was concluded that using superconducting HGMS technology was an effective method for the purification of ${\alpha}-Fe_2O_3$ from zinc smelting iron slag.

Effect of Surface Modification of the Porous Stainless Steel Support on Hydrogen Perm-selectivity of the Pd-Ag Alloy Hydrogen Separation Membranes (다공성 스테인리스 강 지지체의 표면개질에 따른 팔라듐-은 합금 수소 분리막의 수소 투과 선택도의 변화)

  • Kim, Nak-Cheon;Kim, Se-Hong;Lee, Jin-Beum;Kim, Hyun-Hee;Yang, Ji-Hye;Kim, Dong-Won
    • Journal of the Korean institute of surface engineering
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    • v.49 no.3
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    • pp.286-300
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    • 2016
  • Pd-Ag alloy membranes have attracted a great deal of attention for their use in hydrogen purification and separation due to their high theoretical permeability, infinite selectivity and chemical compatibility with hydro-carbon containing gas streams. For commercial application, Pd-based membranes for hydrogen purification and separation need not only a high perm-selectivity but also a stable long-term durability. However, it has been difficult to fabricate thin, dense Pd-Ag alloy membranes on a porous stainless steel metal support with surface pores free and a stable diffusion barrier for preventing metallic diffusion from the porous stainless steel support. In this study, thin Pd-Ag alloy membranes were prepared by advanced Pd/Ag/Pd/Ag/Pd multi-layer sputter deposition on the modified porous stainless steel support using rough polishing/$ZrO_2$ powder filling and micro-polishing surface treatment, and following Ag up-filling heat treatment. Because the modified Pd-Ag alloy membranes using rough polishing/$ZrO_2$ powder filling method demonstrate high hydrogen permeability as well as diffusion barrier efficiency, it leads to the performance improvement in hydrogen perm-selectivity. Our membranes, therefore, are expected to be applicable to industrial fields for hydrogen purification and separation owing to enhanced functionality, durability and metal support/Pd alloy film integration.

Improved Purification of Thermophilic FoF1-ATP Synthase c-Subunit Rings and Solid-State NMR Characterization of Them in Different Lipid Membranes

  • Bak, Suyeon;Kang, Su-Jin;Suzuki, Toshiharu;Yoshida, Masasuke;Fujiwara, Toshimichi;Akutsu, Hideo
    • Journal of the Korean Magnetic Resonance Society
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    • v.17 no.2
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    • pp.67-75
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    • 2013
  • ATP synthase produces ATP, a major energy source for metabolic processes in organisms, from ADP and inorganic phosphate in cellular membranes. ATP synthase is known as a rotary motor, in which the c-subunit ring functions as a rotor. In this work, we have tried to develop a more general preparation procedure of thermophilic $F_oc$-ring ($TF_oc$-ring) for NMR measurements. The expression of $TF_oF_1$ is easily affected by various experimental conditions such as temperature, shape and size of a flask, a volume of medium, and shaking rate of an incubator. Accordingly, we have tried to optimize the expression conditions of $TF_oF_1$. $TF_oc$-rings were purified from $TF_oF_1$ according to a reported method. We modified purification procedures to improve purity and yield of $TF_oc$. On top of them, we found a new combination of detergents for the purification at anion-exchange column chromatography. To examine the effect of lipid environments on the structure, the $TF_oc$-rings were reconstituted into two kinds of lipid bilayers, namely, saturated and unsaturated lipid ones. Then, we have compared characteristics of the $TF_oc$-ring structures in these membranes with solid-state NMR.

Indole Crystallization in Coal Tar Absorption Oil using Methanol Solvent Extraction (메탄올 용매추출을 이용한 콜타르 흡수유 중의 인돌 결정화)

  • Ryu, Heeyong;Lee, Sangheon;Shin, Sungsoon
    • Applied Chemistry for Engineering
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    • v.33 no.2
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    • pp.166-172
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    • 2022
  • A method of efficiently purifying high value-added indole among components of coal tar absorption oil was studied using a step-by-step process of extraction-distillation-crystallization. The coal tar absorption oil used in this study contains 1.2% naphthalene, 0.1% quinoline, 0.4% isoquinoline, 6.4% indole, 21.0% 1-methylnaphthalene, 48.8% 2-methylnaphthalene, and 11.7% biphenyl as main components. For the separation and purification of indole, methanol was first used as a solvent to separate indole species in the coal tar absorption oil into an extract phase. And then methanol was recovered by distillation. Subsequently, an extraction solution where methanol was removed was mixed with normal hexane, and then crystallized to recover indole having a purity of 99.3%. Based on the experiments of this study, a purification process scheme for indole in coal tar absorption oil was proposed.