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말론산에서 배양한 Pesudomonas fluorescens 에서 유도된 말산생성효소
채호준,김유삼 ( Ho Zoon Chae,Yu Sam Kim ) 생화학분자생물학회 1987 BMB Reports Vol.20 No.3
Malate synthase (EC 4.1.3.2) was induced in Pseudomonas fluorescens grown on malonate as a sole source of carbon. This enzyme was purified by the combined methods of ammonium sulfate fractionation, DEAE-Sephacel ion exchange chromatography, tartronate ω-aminohexyl Sepharose 4B affinity chromatography and hydroxyapatite chromatography in an electrophoretically homogeneous form. Molecular size of the enzyme was estimated by Sephadex G-150 gel filtration to be 79,000 dalton composed of single polypeptide. pH optimum of the enzyme was 8.5 and the pI was 4.6. The rate of acetyl group incorporation on variable concentration of substrate into malate was well fitted to a typical Michaelis-Menten kinetics. From the Lineweaver-Burk plot, Km and V_(max) for acetyl-CoA were calculated to be 17.5 μM and 21 umoles/min/㎎, respectively and those for glyoxylate to be 7.7 μM and 35.7 μmoles/min/㎎, respectively. The activity of this enzyme was inhibited by glycolate with 16.8 μM of Ki, and oxalate with 25 μM of Ki, competitively. Some metabolic intermediates such as citrate, isocitrate and succinate, also inhibited the enzyme activity. Iodoacetamide and p-chloromercuriphenylsulfonic acid strongly inhibited the enzyme acitivity, suggesting that there may be a reactive thiol group(s) at or near the active site. The purified malate synthase was immunogenic in rabbit and Ouchterlony double diffusion analysis revealed a single precipitation line with the enzyme.
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Induction of Malate Synthase in Pseudomonas fluorescens Grown on Malonate
채호준,김유삼,Chae, Ho-Zoon,Kim, Yu-Sam 생화학분자생물학회 1987 한국생화학회지 Vol.20 No.3
Malate synthase (EC 4.1.3.2) was induced in Pseudomonas fluorescens grown on malonate as a sole source of carbon. This enzyme was purified by the combined methods of ammonium sulfate fractionation, DEAE-Sephacel ion exchange chromatography, tartronate $\omega$-aminohexyl Sepharose 4B affinity chromatography and hydroxyapatite chromatography in an electrophoretically homogeneous form. Molecular size of the enzyme was estimated by Sephadex G-150 gel filtration to be 79,000 dalton composed of single polypeptide. pH optimum of the enzyme was 8.5 and the pI was 4.6. The rate of acetyl group incorporation on variable concentration of substrate into malate was well fitted to a typical Michaelis-Menten kinetics. From the Lineweaver-Burk plot, Km and $V_{max}$ for acetyl-CoA were calculated to be $17.5\;{\mu}M$ and 21 umoles/min/mg, respectively and those for glyoxylate to be $7.7\;{\mu}M$ and $35.7\;{\mu}moles/min/mg$, respectively. The activity of this enzyme was inhibited by glycolate with $16.8\;{\mu}M$ of Ki, and oxalate with $25\;{\mu}M$ of Ki, competitively. Some metabolic intermediates such as citrate, isocitrate and succinate, also inhibited the enzyme activity. Iodoacetamide and p-chloromercuriphenylsulfonic acid strongly inhibited the enzyme acitivity, suggesting that there may be a reactive thiol group(s) at or near the active site. The purified malate synthase was immunogenic in rabbit and Ouchterlony double diffusion analysis revealed a single precipitation line with the enzyme. 말론산을 유일한 탄소원으로 이용하여 자란 Pseudomonas fluorescens에서 말산 생성효소가 유도되었다. 이 미생물로부터 황산암모니움 분별침전, DEAE-Sephacel 이온교환, 타트론산 Sepharose 4B, hydroxyapatite 크로마로그래피를 통하여 이 효소를 정제하였다. 정제된 효소는 단일 소단위로 되어있으며 분자량은 79,000 달톤이었다. 최적 pH는 8.5였고, pI는 4.6이었다. 아세칠 CoA에 대한 Km과 $V_{max}$는 각각 $17.5\;{\mu}M$과 $21\;{\mu}moles/min/mg$ 이었고 글리옥실산에 대하여는 각각 $7.7\;{\mu}$과 $35.7\;{\mu}moles/min/mg$이었다. 옥살산과 글리콜산은 경쟁적으로 효소활성을 방해하였고 이에 대한 Ki는 각각 $25\;{\mu}M$과 $16.8\;{\mu}M$이었다. 대사중간물인 시트르산, 이소시트르산과 숙신산에 의해서도 효소활성이 방해되었다. 또 요드아세트아미드, 클로로머큐리페닐설폰산이 강력하게 효소활성을 억제하는 것으로 보아 활성자리나 그 근처에 -SH기가 존재할 것이라고 예상된다. 정제된 효소는 토끼에 항체를 형성하게 하였고 이 항체와 정제된 효소사이에 단일띠를 보여주는 침전반응을 하였다.
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박재범,채호준,김유삼,Park, Jae-Bum,Chae, Ho-Zoon,Kim, Yu-Sam 생화학분자생물학회 1986 한국생화학회지 Vol.19 No.3
Malonate를 유일한 탄소 및 에너지원으로 이용하는 배지에서 Acinetobacter calcoaceticus를 배양했을 때 malate synthase가 유도된다는 사실을 확인하였다. 이 미생물로부터 DEAE-Sephacel ion exchange chromatography, oxalate-Sepharose 4B affinity chromatography, hydroxyapatite chromatography를 이용하여 malate synthase를 분리 정제하였고, disc polyacrylamide gel electrophoresis를 수행한 결과 하나의 단백질 띠로 나타났다. 그 최적 pH는 8.0이였으며, 정제한 malate synthase가 관여하는 반응은 기질인 glyoxylate나 acetyl CoA에 대해 전형적인 Michaelis-Menten kinetics에 부합했다 Lineweaver-Burk plot을 이용하여 $K_m$과 $V_{max}$를 정한 결과 acetyl CoA에 대해서는 $1.5{\times}10^{-5}\;M$과 $1\;{\mu}mol/min/mg$ protein이였고 glyoxylate에 대해서는 $3.5{\times}10^{-5}\;M$과 $0.8\;{\mu}mol/min/mg$ protein 이었다. Oxalate와 glyoxylate는 competitive inhibitor였으며 $K_i$ 값은 각각 $4{\times}10^{-5}\;M$, $1.4{\times}10^{-4}\;M$이었다. Pyruvate, succinate, malonate 그리고 tartrate도 효소활성을 억제하였다. Pseudomonas fluorescens의 malate synthase를 항원으로 하여 만들어진 항체는 Acinetobacter calcoaceticus 의 malate synthase활성을 저해 하지 않았다. 이러한 사실로 미루어 분류상 유사한 두 미생물의 malate synthase는 면역학적으로 많은 차이가 있음을 알았다. Malate synthase (EC 4.1.3.2) was induced in Acinetobacter calcoaceticus grown on malonate as a sole carbon source. The enzyme was purified by the combined methods of DEAE-Sephacel anion exchange chromatography, oxalate Sepharose 4B affinity chromatography and hydroxyapatite chromatography in an electrophoretically homogeneous form. The molecular size of the enzyme was estimated to be 75,000 dalton consisted of a single polypeptide. Optimum pH for the enzyme was about 8.0. The rate of acetyl group incorporation on variable concentraions of the substrate into malate was well fitted to a typical Michaelis-Menten kinetics. From the Lineweaver-Burk plot, $K_m$ and $V_{max}$ for acetyl-CoA were $1.5{\times}10^{-5}\;M$ and $1\;{\mu}mol/min/mg$ respectively and those for glyoxylate were $3.5{\times}10^{-5}\;M$ and $0.8\;{\mu}mol/min/mg$ respectively. The activity of this enzyme was inhibited by oxalate with $K_i$, $4{\times}10^{-5}\;M$ and by glycolate with $K_i$, $1.4{\times}10^{-4}\;M$ competitively. Pyruvate, succinate, malonate and tartrate also inhibited the enzyme activity. Antibody prepared against malate synthase from Pseudomonas fluorescens did not cross-react with the enzyme from Acinetobacter, suggesting that the enzyme isolated from two different bacteria are immunologocally different.
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Identification of Malonate-specific Enzymes, Malonyl-CoA Synthetase and Malonamidase, in Rhizobia
김유삼,채호준,이은,김용성,Kim, Yu-Sam,Chae, Ho-Zoon,Lee, Eun,Kim, Yong-Sung The Microbiological Society of Korea 1991 미생물학회지 Vol.29 No.1
Two malonate-specific enzymes, malonyl-CoA synthetase and malonamidase, were found in free-living cultures of Rhizobium japonicum, Rhizobium meliloti, and Rhizobium trifolii, that infect plant roots where contain a high concentration of malonate. Malonyl-CoA synthetase catalyzes the formation of malonyl-CoA, AMP, and PPi directly from malonate, coenzyme A, and ATP in the presence of $Mg^{2+}$ Malonamidase is a novel enzyme that catalyzes hydrolysis and malonyl transfer of malonamate, and forms malonohydroxamate from malonate and hydroxylamine. Both enzymes are highly specific for malonate. These results show that Rhizobia have enzymes able to metabolize malonate and suggest that malonate may be used in symbiotic carbon and nitrogen metabolism.
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Malonate 에서 배양한 Acinetobacter calcoaceticus Malate Synthase 의 정제 및 그 성질
김유삼,박재범,채호준 생화학분자생물학회 1991 BMB Reports Vol.19 No.3
Malate synthase (EC 4.1.3.2) was induced in Acinetobacter calcoaceticus grown on malonate as a sole carbon source. The enzyme was purified by the combined methods of DEAE-Sephacel anion exchange chromatography, oxalate Sepharose 4B affinity chromatography and hydroxyapatite chromatography in an electrophoretically homogeneous form. The molecular size of the enzyme was estimated to be 75,000 dalton consisted of a single polypeptide. Optimum pH for the enzyme was about 8.0. The rate of acetyl group incorporation on variable concentraions of the substrate into malate was well fitted to a typical Michaelis-Menten kinetics. From the Lineweaver-Burk plot, K_m and V_(max) for acetyl-CoA were 1.5 × 10^(-5) M and 1 μmol/min/㎎ respectively and those for glyoxylate were 3.5 × 10^(-5) M and 0.8 μmol/min/㎎ respectively. The activity of this enzyme was inhibited by oxalate with K_i, 4 × 10^(-5) M and by glycolate with K_i, 1.4 × 10^(-4) M competitively. Pyruvate, succinate, malonate and tartrate also inhibited the enzyme activity. Antibody prepared against malate synthase from Pseudomonas fluorescens did not cross-react with the enzyme from Acinetobacter, suggesting that the enzyme isolated from two different bacteria are immunologocally different.
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대황의 형질전환된 뿌리로 부터 anthraquinone의 생산
황성진,표병식,채호준,황백 韓國藥用作物學會 2002 한국약용작물학회지 Vol.10 No.2
대황(Rheum undulatum L.)의 형질전환된 뿌리의 기내(in vitro)배양으로부터 quinone계 알칼로이드 물질인 anthraquinone의 생산성을 확인하고자 하였다. 형질전환된 뿌리로부터 anthraquinone의 생산에 있어서 배지, 초기 pH, 탄소원의 농도, 광 그리고 elicitors의 영향을 조사 하였다. 형질전환된 뿌리는 6% sucrose와 0.5 mg/l GA3 그리고 50 mg/l chitosan이 첨가된 WPM 배지에 치상한 후 16/8의 광조건(16μmol m2s1)하에서 배양시 0.18 mg(생중량)의 anthraquinone의 생산성을 보여주었다. 이와같은 함량은 재배근의 약 1.3배에 해당된다. The production of anthraquinone has been dectected in transformed roots of Rheum undulatum L. The effects of medium, initial pH, concentration of sucrose, light irradiation and elicitors on anthraquinone production in transformed roots of Rheum undulatum L. were investigated. The maximum production of anthaquinone was achieved in WPM medium (pH 5.7) supplemented with 6% sucrose, 0.5 mg/l GA3, and 50 mg/l chitosan at 16h light (16μmol m-2s-1) condition. Under the optimum conditions, the production of anthraquinone reached to 0.18 mg/g(F.W.) after 8 weeks. The content was estimated about 1.3 times of the level of native roots.
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