• Title/Summary/Keyword: isolation and purification

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Isolation of Microorganism Producing Chitinase for Chitooligosaccharides Production, Purification of Chitinase, and its Enzymatic Characteristics (Chitoologosaccharides 생산에 적합한 Chitinase를 분비하는 균주의 선별, Chitinase의 분리정제 및 반응특성)

  • 정의준;이용현
    • Microbiology and Biotechnology Letters
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    • v.23 no.2
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    • pp.187-196
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    • 1995
  • In order to produce fuctional chitooligosaccharides, a strain excreting mainly endo-type chitinase suitable for chitooligosaccharides production was newly screened and identified as Aspergillus fumigatus JC-19. The chitinase excretion was repressed in nutrient rich medium but stimulated by colloidal chitin indicating that the chitinase is inducible type enzyme. Maximum secretion of the enzyme was observed at pH 7.0 and 37$\circ$C . The growth and chitinase production patterns of Aspergillus fumigatus JC-19 showed that the cell growth reached maximum after 4-5 days with final chitinase concentration of 0.46 unit per ml. Excreted chitinase was purified by ammonium sulfate precipitation, colloidal chitin adsorption, anion exchange chromatography, and gel filtration, respectively, and measured M.W of 50 KDa. The enzyme reaction carried out both by crude and purified chitinase showed that the purified chitinase accumulated more chitooligosaccharides of 1-6 degree of polymerization than that of crude chitinase.

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Purification and Comparison of NADH-Cytochrome $b_5$ Reductase from Mitochondrial Outer Membrane of Bovine Heart and Turnip

  • 이재양;김영호;이상직
    • Bulletin of the Korean Chemical Society
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    • v.19 no.2
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    • pp.160-164
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    • 1998
  • The NADH-cytochrome b5 reductase (NCBR), a mitochondrial external electron carrier, was purified from bovine heart and turnip and their properties were examined. The mitochondrial outer membranes separated were subjected to NCBR isolation through DEAE-Cellulose ion exchange, DEAE-Sephadex gel chromatography, and hydroxyapatite adsorption chromatography. These processes yielded the purification folds of 88 and 42 and the recovery percentages of 0.2%, 5.67% for turnip and bovine heart, respectively. The molecular weight of the NCBR from the two sources was estimated to be 35,000 using SDS polyacrylamide gel electrophoresis. The Michaelis constant Km and maximum velocity Vmax were determined by measuring the NADH-ferricyanide redox system as well as the NADPH-ferricyanide redox system. The kinetics showed that both NCBRs had higher affinities for NADH than artificial electron-acceptor substrate ferricyanide. Although NADPH had a lower affinity for the enzymes than NADH, this study showed the 2'-phosphate dinucleotide could be used as a substrate.

Isolation and Purification of Hantaan Viral Nucleocapsid Protein Expressed in Escherichia coli (대장균에서 발현된 한탄바이러스 뉴클레오캡시드 단백질의 분리 정제)

  • 노갑수;김종완;하석훈;정근택;문상범;최차용
    • KSBB Journal
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    • v.13 no.6
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    • pp.656-661
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    • 1998
  • Hantaan virus belonging to the genus Hantavirus and family Bunyaviridae causes an acute severe illness of human, Haemorrhagic Fever with Renal Syndrome (HFRS). It is a rodent host-borne pathogen and distributed in Asia and Eastern Europe. Hantaviruses have three major antigens, i.e., G1, G2 glycoproteins and nucleocapsid protein (N). Among them, nucleocapsid protein was reported to be the most invaluable antigen as for diagnosis. We have cloned and expressed Hantaan viral nucleocapsid gene in E. coli BL21(DE3). In this study, we have tried to purify the nucleocapsid protein produced by recombinant E. coli, and could attained a purity of >90% by anti-N monoclonal antibody-coupled immunoaffinity chromatography or phenyl sepharose hydrophobic interaction chromatography.

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Purification and Characterization of Haemolymph Ferritin from the Cricket, Gryllus bimaculatus

  • Seo, Dong-Hwan;Nam, Kung-Pil;Kim, Iksoo;Ryu, Kang-Sun;Yun, Chi-Young
    • Proceedings of the Korean Society of Sericultural Science Conference
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    • 2003.04a
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    • pp.80-80
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    • 2003
  • The isolation and purification of ferritin from the cricket, Gryllus bimaculatus haemolymph were accomplished by anion exchange column chromatography using HiTrap Q column (1.6 $\times$ 4 cm, Amersham Phamacia Co.), 7$0^{\circ}C$ heat and acid treatment, and gel filtration column chromatography using G4000SW column (0.75 $\times$ 60cm, TOSOH Co.) by FPLC system. (omitted)

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Induction of Cd-binding High Molecular Weight Protein [Cd-BP(II)] in Rat Tissues (흰쥐 조직에서의 카드뮴 결합 고분자량 단백질 [Cd-BP(II)]의 유도)

  • 천기정;김봉희
    • YAKHAK HOEJI
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    • v.43 no.5
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    • pp.591-597
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    • 1999
  • The study was carried out on the biochemical characters of Cd-BP(II) after isolation and purification of the protein from the liver of rat ip injection with Cd. A continued study has been doing whether Cd-BP(II) could be induced by Cd or by the other metals such as Zn and Cu. Antisera were made against the antigen of Cd-BP(II) from New Zealand white rabbits. We carried out g-globulin purification, then Ouchterlony test and gel immunodiffusion test. Cd-BP(II) was also found in normal tissues of rat. It was induced up to a considerable level by Cd, whose induced level was higher than that Cu or Zn treatment. The level of induction by Cu or Zn pretreatment plus Cd treatment was lower than that by single treatment of Cu or Zn. Such a result was presumably related to the Cd toxicity.

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Induction of Cd-binding High Molecular Weight Protein in Rat Tissues (흰쥐 조직에서의 카드뮴 결합 고분자량 단백질의 유도)

  • Chun, Ki-Jung;Kim, Bong-Hee;,
    • YAKHAK HOEJI
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    • v.41 no.3
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    • pp.352-358
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    • 1997
  • The study was carried out on the biochemical characters of Cd-BP(I) after isolation and purification of the protein from the liver of rat injected intraperitoneally with Cd. A c ontinued study has been doing whether Cd-BP(I) could be induced by Cd or by other metals such as Zn and Cu. Antisera were made against the Cd-BP(I) from NewZealand white rabbits. Carried out were ${\gamma}$-globulin purification, then Ouchterlony test adn gel immunodiffusion test. Cd-BP(I) was also found in normal tissues of rat. It was induced up to a considerable level by Cd, whose induced level was higher than that of Cu or Zn treatment. The level of induction by Cu or Zn pretreatment plus Cd treatment was lower than that by simple treatment of Cu or Zn. Such a result was presumably related to the Cd toxicity.

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Screening of Inhibitory Effect of Edible Mushrooms on Tyrosinase and Isolation of Active Component (한국산 식용버섯류의 Tyrosinase 활성 저해 검색 및 그 유효성분 분리)

  • 박영현;장성근
    • Journal of Food Hygiene and Safety
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    • v.12 no.3
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    • pp.195-199
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    • 1997
  • For the purpose of isolation and screening of tyrosinase inhibitory activity from edible mushrooms, Pleurotus ostreatus, Auricularia auricula-Judae, Umbilicaria esculenta, Agaricus bisporus, Flammuline velutipes, Lentinus edodes, Ganoderma lucidum, and Coriouls versicolor were examined by tracing inhibitory activities against tyrosinase, utilizing L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate. Among the eight edible mushrooms tested, Umbilicaria esculenta showed potent enzyme inhibitory activities above 7804% against tyrosinase in ethylacetate (EtOAc) extracts. Ganoderma lucidum and Agaricus bisporus showed inhibitory activities of 67.3% and 51.5% in water extracts. EtOAc extracts of Umbilicaria esculenta was fractionated from silicagel column chromatography and one fraction showed the most inhibitory activity of 60.9%. The three bands (Rf=0.38, 0.27, 0.19) were isolated from preparative TLC of the fraction for purification and identified as mixtures of orsellinate, methyl orsellinate, methyl lecanorate, and methyl gyrophorate by high pressure liquid chromatography (HPLC), ultravisible spectrophotometer (UV), mass spectrophotometer (Mass), nuclear magnetic resonance spectrometer (NMR).

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Isolation and Identification of Antimicrobial Substance from Canavalia gladiata

  • Lee, Hang-Young;Jeong, Heon-Sang
    • Food Science and Biotechnology
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    • v.14 no.2
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    • pp.268-274
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    • 2005
  • Novel antimicrobial substance was isolated from seed coat of Canavalia gladiata by extraction with 75% methanol. Isolation and purification were conducted with solvent fractionation and chromatography on silica gel and sephadex LH-20 columns. Each fraction of antimicrobial activity was tested by paper disc method. Single compound was obtained from the 4th fraction of sephadex LH-20 column chromatography using chloroform/methanol (1:4, v/v), and identified as 3,4,5-trihydroxybenzoic acid methyl ester (methyl gallate) based on HPLC, GC/MS, FT-IR, $^1H$ NMR, and $^{13}C$ NMR analyses. This is the first report describing the presence of methyl gallate in C. gladiata.

Isolation and Purification of Polysaccharide from Fruiting body and Culture Broth of Agaricus blazei Murill

  • Youm, Yong-Soo;Hong, Eock-Kee
    • 한국생물공학회:학술대회논문집
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    • 2005.04a
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    • pp.343-347
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    • 2005
  • The polysaccharides were extracted from fruiting body, mycelia, and cell-free broth of Agaricus blazei Murill. The crude polysaccharides were obtained by the ethanol addtion. They were further purified using ion-exchange chromatography and gel chromatography. Ion-exchange chromatography using DEAE-cellulose column separated neutral and acidic polysaccharides. Neutral polysaccharides were then purified with gel filtration chromatography. For single peak obtained from gel filtration chromatography was molecular weight was measured with Sepharose CL-6B. The same procedure with acidic polysaccharides were performed to get the purified polysaccharides.

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Isolation and Purification of Lectin from Phaseolus radiatus (녹두로부터 렉틴 성분의 분리 정제)

  • 정시련;홍승수;전경희
    • YAKHAK HOEJI
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    • v.27 no.3
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    • pp.221-227
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    • 1983
  • New lectins, lymphoagglutinating lectins from mung beans (MBLA) have been isolated and purified. Mung beans crude extracts were made with 0.15M NaCl and these were purified through anionic exchange chromatography. Four fractions were obtained from DEAE Sephadex A-50 by salt gradients elution. Lectin activity, enzyme activity, protein assay, identification of purity by polyacrylamide gel electrphoresis and immunochemical studies were carried out with these four fractions. Through these results, it can be suggested that 0.2M fraction is newly found potent MBLA. There were some relationships with MBLA and L-PHA but no similarities were observed between MBLA and E-PHA.

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