• Title/Summary/Keyword: DEAE cellulose

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Partial Purification and Characterization of the Alkaline Protease from Baccillus sp. (Bacillus sp.가 생산하는 호알카리성 Protease의 부분정제 및 특성)

  • 안장우;오태광;박용하;박관하
    • Microbiology and Biotechnology Letters
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    • v.18 no.4
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    • pp.344-351
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    • 1990
  • An alkalophilic microoganism producing a detergent-resistant alkaline protease was isolated from soil and identified as Baeiltus sp. The alkaline protease has been partially purified by ammonium sulfate fractionation, DEAE-Cellulose, CM-Cellulose and Sephdex G-100 column chromatography. The purified alkaline protease was highly active at pH 12-13 toward casein and stable at pH values from 6 to ll. The optimum temperature for the enzyme reaction was $55^{\circ}C$. The enzyme was completely inactivated by diisopropyl fluorophosphate (DFP) indicating that the enzyme was serine protease, but considerabiy stable in the presence of surface active agents.

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Purification of Inositol Triphosphate Kinase from Bovine Brain (소의 뇌로부터 Inositol Triphosphate Kinase의 정제)

  • Kim, Jung-Hye;Lee, Jae-Tae
    • Journal of Yeungnam Medical Science
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    • v.13 no.1
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    • pp.46-58
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    • 1996
  • Inositol 1,4,5-triphosphate($InsP_3$) is a second messenger for mobilizing intracellular $Ca^{2+}$. It can be dephosphorylated by soluble and particulate forms on $InsP_3$ 5-phosphatase, or phosphorylated to produce inositol 1,3,4,5-tetrakisphosphate($InsP_3$) by $InsP_3$ 3-kinase. These enzymes represent possible targets for the regulation of the $InsP_3/InsP_4$ signal. $InsP_3$ 3-kinase which catalyses th ATP-dependent phosphorylation of $InsP_3$ was purified from bovine brain tissue. All operation were carried out at $4^{\circ}C$. Fresh tissure was homogenized and centrifuged. The supernatant was pooled. Proteins were precipitated from 10% polyethylene glycol, and suspended solution was applied to DEAE cellulose column for chromatography. As the result of above procedure, two isozymes of $InsP_3$ 3-kinase, I and II were obtained. Each isozyme was applied to Matriz green gel, Calmodulin-Affigel 15 column and subsequent phenyl-TSK HPLC column. Specific activites(SA) and fold of puriety were observed at each purification step of chromatography. At DEAE cellulose chromatography, SA were I, 0.6 and II, 4.8 nM/min/mg, and folds were I, 17.2 and II, 16.6. At Matrix green gel chromatography, SA were I, 18 and II, 11 nM/min/mg, folds were I, 62.1 and II, 38.0. At calmodulin-Affigel 15 column chromatography, SA were I, 19 and II, 13 nM/min/mg, folds were I, 65.5 and II, 44.8. Finally $InsP_3$ kinase I and II were purified 3,103-fold and 2,310-fold, and SA were I, 900 and II, 670 nM/min/mg, respectively. SDS-polyacrylamide gel electrophoresis elucidated 3 distinct fractions of Mr of 145,000, 85,000 and 69,500 from isozyme I, and 2 distinct fractions of Mr of 79,000 and 57,000 from isozyme II.

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Investigation of Cellulase of Microbial origin (미생물유래의 섬유소 분해효소의 연구)

  • 김은수;이순진
    • Korean Journal of Microbiology
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    • v.14 no.2
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    • pp.65-74
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    • 1976
  • Atternaria sp. was isolated from soil and crude cellulases were prepared from wheat bran culture of the fungus. The activities of the crude enzyme were studied on five different subvstrates and some phsical properties were also examined, crude enzymes were purified by column chromatography on DEAE Sephadex and Sephadex, Isozymes were separated some of which were active specifically on DEAE cellulose and some were primarily active on cellulose and CM-cellulose. The optimal points of pH and temperature for the crude enzyme were varied depending on the substrates ; On cellulose they were at pH 6.0 and 40.deg.C, on CM-cellulose at pH's 4.0 and 6.0 and 60.deg.C, and on DEAW-cellulose at pH 5.0 and 50.deg.C. Two active fractions, F-1 and F-II on Na-CMC was used as substrate the Km values of crude enzyme, F-I and F-II were calculated to be $4{\times}10^{-5}$ , 1.1 * 10$^{-4}$ , and $1.25{\times}10^{-4}mN$ resepctively. The Ki value of $Cu^{++}$ for crude enzyme was$4{\times}^{-4}mN$ , while that of $Nm^{++}$ while in the same concentration of $Mn^{++}$ it reached to 91%. Some 57% activity of F-1 was inhibited in s mN $Cu^{++}$, whereas it was inhibited as much as 81% in the same concentration above the concentration of 0.3 mM with tis activity reaching up to 137% in 2 mM. On the other hand the F-11 was inhibited by the presence of M $n^{++}$ and some 67% activity was inhibited at 2mM.

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Aspartate and Alanine Aminotransferase in Fasciola hepatica (간질(Fasciola hepatica)의 Aspartate 및 Alanine Aminotransferase에 관하여)

  • 박선효;권년수이희성송철용
    • Parasites, Hosts and Diseases
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    • v.21 no.1
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    • pp.41-48
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    • 1983
  • The activity and distribution of aspartate aminotransferase (EC 2.6. 1. 1) and alanine aminotransferase (EC 2.6.1.2) in adult Fascicle hepatica have been studied. Fasciola hepatica was fractionated by differential centrifugation into nuclear, mitochondrial and cytosolic fractions. The activity of GOT and GPT was measured by the method of Reitman and Frankel. Isozyme patterns of those enzyme were also examined by DEAE-cellulose column chromatography. The results obtained were as follows; 1. The activity of aspartate and alanine aminotransferase was about 0.55 unit and 0.92 unit per 1g of Fascicle hepatica, respectively. 2. The activity of those enzymes was relatively low compared with those in mammalian tissues. 3. The distribution of aspartate aminotransferase in the subcellular organelles showed that 71% of the activity was in cytosolic, 24% in mitochondrial and 5% was in nuclear fraction. 4. About 22% of the total alanine aminotransferase activity was found in the mitochondrial fratstion, about 66% in the cytosolic fraction. 5. Aspartate aminotransferase from cytosolic fraction was separated into two types of isozymes, whereas alanine aminotransferase from cytosolic fraction gave only one active peak on DEAE-cellulose column chromatography.

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Studies on the Generation and Application of Monoclonal Antibodies against Tumor Marker Antigen 1. Production and Characterization of Monoclonal Antibodies against Placental Alkaline Phosphatase (Tumor Marker 항원에 대한 단일 클론항체의 생성과 활용에 대한 연구. I. 태반형 Alkaline Phosphatase에 대한 모노클론항체의 생산과 분석)

  • 김한도;강호성
    • The Korean Journal of Zoology
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    • v.31 no.4
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    • pp.300-308
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    • 1988
  • Human placental alkaline phosphatase (PLAP), one of the oncofetal antigen was purified from placentas through the procedures including butanol extraction, concanavalin A-Sephar-ose, DEAE-cellulose and Sephadex G-200 gel chromatography. Monoclonal antibodies (fibs) against human PMP were produced by hybridizing SP 210-Ag 14 mouse myeloma cells with spleen ceils of Balblc mice immunized with PLAP. Six stable monoclones uvere obtained by cloning tuvice in serial dilutions, and the monoclonal speclfidty of these MAbs was confirmed by biochemical and immunonogical criteria. Tumor marker의 하나인 태반형 alkaline phosphatase(PLAP)에 대한 단일 클론항체의 생산과 분석을 위하여, 태반조직을 재료로 butanol 추출법 및 concanavaline A-Sepharose, DEAE-cellulose, Sephadex G-200 gel 크로마토그라피법에 의하여 PLAP를 순수 분리하였다. 이를 항원으로 하여 하이브리도마 방법에 의해 항-PLAP 단일 클론항체를 생산 분비하는 안정된 6클론세포를 얻었으며 생화학적 및 면역학적 분석방법으로 이들의 단일 클론성을 확인하였다.

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Studies on Enzyme of the Thermophilic Mold-(Part 2.) Thermophilic mold alkaline protease- (고온성 사상균의 효소에 관한 연구-(제2보) 고온성사상균의 알카리성 protease-)

  • Chung, Dong-Hyo;Lee, Ke-Ho
    • Applied Biological Chemistry
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    • v.13 no.3
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    • pp.223-229
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    • 1970
  • 1. The preparation and some enzymatic properties of crude alkaline protease from a thermophilic mold, Myriococcum sp. was investigated. 2. Optimum pH for the hydrolysis of casein was 9.0 at $50^{\circ}C$ for 10 minutes. Optimum temperature was $55^{\circ}C$ at pH 9.0 for 10 minutes. The enzyme was highly stable at the range of pH 6.0 to 11.0 at $30^{\circ}C$ 3. The alkaline protease in the culture filterate was isolated two fractions by elution column chromatography on DEAE-Cellulose.

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Purification and Properties of Isocitrate Lyase from Saccharomycopsis lipolytica (Saccharomycopsis lipolytica Isocitrate Lyase의 정제와 성질)

  • 조석금
    • Microbiology and Biotechnology Letters
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    • v.15 no.6
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    • pp.420-424
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    • 1987
  • Isocitrate lyase from crude extract of Saccharomycopsis lipolytica ATCC44601 and MX9-11RX8 temperature-sensitive mutant was purified about 54 times and 87 times, respectively by ammonium sulfate fractionation, Toyo peal HW-55F gel filtration and DEAE-Cellulose ion exchange chromatography, The molecular weight of the purified isocitrate lyase from this yeast was estimated to be 230, 000 by gel filtration on Sephadex G-200, and SDS-polyacrylamide Eel electrophoresis showed that the enzyme consisted of four identical or similar subunits with a molecular weight of 59, 000 and the enzyme showed optimum activity at pH 6.9.

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Affinity Purification of Human Alpha-Fetoprotein (吸着 크로마토그라피法에 의한 사람 ALpha-Fetoprotein의 分離)

  • Kang, Shin-Sung;Bang, Ok-Sun;Park, Tae-Kyu
    • The Korean Journal of Zoology
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    • v.29 no.4
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    • pp.283-293
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    • 1986
  • For the preliminary step to make and characterize the monoclonal antibodies of human alpha-fetoprotein (AFP) was purified from 534g of human fetal tissues through the procedures of tissue extraction, DEAE-cellulose, concanavalin A-Sepharose, Cibacron Blue F3GA-agarose and immunoadsorbent affinity chromatography. The isolated AFP preparation showed a single band on polyacrylamide gel electrophoresis and a single precipitin are against rabbit anti-human cord serum and anti-human AFP on immunoelectrophoresis. Our AFP also displayed a single band on SDS-polyacrylamide gel electrophoresis. The recovery of AFP was 8.76mg total.

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Purification and Characterization of Intracellular Cellulase from Aspergillus oryzae ITCC-4857.01

  • Begum, Ferdousi;Absar, Nurul
    • Mycobiology
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    • v.37 no.2
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    • pp.121-127
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    • 2009
  • Purification and characterization of intracellular cellulase produced by A. oryzae ITCC-4857.01 are reported. The enzyme was purified by ion-exchange chromatography using DEAE-cellulose followed by Gel filtration. The purification achieved was 41 fold from the crude extract with yield of 27%. The purified enzyme showed single band on poly acrylamide gel. The molecular weight as determined by SDS-PAGE and gel filtration was 38 KDa and 38.6 KDa respectively and contained only one subunit. The enzyme is glycoprotien as nature and contained 0.67% neutral sugar. The apparent Km value of the enzyme against cellulose was 0.83%. The enzyme showed the highest relative ativities on CMC followed by avicel, salicin and filter paper. The optimum pH of activity was 5.5 and very slight activity was observed at or above pH 7.5 as well as bellow pH 3.5. The optimum tempreture of the activity was $45^{\circ}C$ and the highest activity was exhibited in 35 to $45^{\circ}C$. The enzyme lost their activities almost completely (95${\sim}$100%) at $80^{\circ}C$ or above and as well as bellow $25^{\circ}C$.