Regeneration of the Monooxygenating Intermediate by $H_2O_2$ in Bacterial Bioluminescent Reaction of Vibrio fischeri

조기웅,이현재,심상철,Cho, Ki-Woong,Lee, Hyun-Jae,Shim, Sang-Chul 생화학분자생물학회 1986 한국생화학회지 Vol.19 No.2

박테리아의 생체 발광반응에서 hololuciferase와 분자 산소간의 반응으로 형성되는 monooxygenating중간체 (complex-I)가 여러가지 luciferase : FMN 복합체로부터 외부에서 가해준 $H_2O_2$에 의해 인위적으로 형성됨을 확인하였다. $H_2O_2$에 의한 complex-I의 형성은 luciferase : FMN복합체가 생성된 방식에 따라 그 효율을 달리하고 있으며 발광반응을 거쳐 생성된 복합체에서 가장 높은 효율을 보였고 과량의 긴사슬 알데히드와 $H_2O_2$ 존재하에서 complex-I의 지속적인 재형성으로 turnover의 성격을 보이는 발광현상을 야기시킬 수 있었으며 원래의 발광반응과 동일한 발광 spectrum을 보였다. The monooxygenating intermediate formed from the reaction of hololuciferase and oxygen is regenerated from the luciferase:FMN complex directly by exogeneous $H_2O_2$. This regeneration is much more efficient when the luciferase:FMN complex is generated from the luminescent reaction than when generated from equilibrium binding. The bioluminescence spectra from this regenerated intermediate are well matched with emission spectra of the normal reaction.

해양방선균으로부터 Haloperoxidase의 검색과 특성

조기웅,Cho, Ki-Woong 한국미생물학회 2008 미생물학회지 Vol.44 No.2

Haloperoxidase를 생산하는 미생물을 분리하기 위하여 국내 연근해와 남북극 등의 해양시료에서 분리된 방선균 균주를 대상으로 탐색을 수행하여 남해 백도 해조류 추출물로부터 분리된 한 종류의 방선균(#1460)에서 높은 haloperoxidase 활성이 확인되었다. 본 균주의 생리.생화학적 특성은 Streptomyces 속과 유사하며 생산되는 haloperoxidase는 세포 조 추출물로부터 ammonium sulfate precipitation, High-Q column chromatography, gel permeation chromatography, Hydroxyapetite chromatography 그리고 hydrophobic interaction chromatography를 통하여 42%의 수율과 purification fold 70으로 정제하였다. 본 효소의 최적 반응 pH는 7이고 pH 8에서 더 높은 안정성을 보여 $60^{\circ}C$에서 1시간 반응에 효소활성의 50%가 생존한다. 또 cyanide와 azide 이온에 의해 강한 저해현상을 보인다. In my search of microbial source of novel enzymes, a marine actinomycetes, A1460, producing haloperoxidase was isolated from macroalgae from south sea, Korea and studied for physiological and biochemical properties. The haloperoxidation reaction was followed by the bromination of phenol red in the presence of hydrogen peroxide and potassium bromide. The haloperoxidase was partially purified from the cell extract with $35\sim75%$ ammonium sulfate precipitation, High-Q anion exchange chromatography, gel filtration chromatography, hydroxyapetite chromatography and hydrophobic interaction chromatography to a yield of 42% and purification fold of 70. This enzyme showed relatively high heat stability without losing 50% of activity after 1 hr incubation at $60^{\circ}C$. The highest activity was found at $45^{\circ}C$, and the optimal pH was about pH 7, but higher stability was observed at pH 8. Azide and cyanide ion showed strong inhibition at less than 1 $\mu M$ level suggesting that the enzyme was Fe ion dependent haloperoxidase.

Nature of High Energy Intermediate Involved in the Bioluminescence Reaction Catalyzed by Bacterial Luciferase

조기웅,이현재,Cho, Ki-Woong,Lee, Hyun-Jae 생화학분자생물학회 1984 한국생화학회지 Vol.17 No.1

해양 미생물인 P. fischeri의 발광효소에 의한 생체 발광반응의 양상을 stopped-flow장치를 이용, 형광도를 측정 비교함으로써 연구를 추진하였으며, 그 결과로부터 다음과 같은 결론을 추정할 수 있었다. 즉 박테리아 luciferase에 의한 생체 발광반응은 적어도 2단계 반응으로 추진되며, 그 첫째는 luciferase에 결합된 환원형 flavin에 의하여 산소분자를 활성화하여 강력한 monooxygenation 반응을 일으킬 수 있는 flavin-hydroperoxy anion, 또는 oxiene 형성과정이며, 둘째 단계는 활성화된 산소에 의한 aldehyde의 monooxygenation 반응으로서 이 결과 고 에너지를 갖는 dioxirane 중간체를 거치게 되며, 이 고 에너지 중간체가 생체내 lumazine을 함유한 형광단백질에 에너지를 전달시킴으로써 해당되는 카르복시산으로 전환되고 아울러 형광 발생도 일으키게 된다고 본다. Reaction mode of bioluminescence mediated by bacterial luciferase from P. fischeri was studied by stopped-flow apparatus measuring the light intensity, and from the results, it was suggested that the bacterial bioluminescence reaction occurs by at least two step mechanism, i.e., the first step is the activation of molecular oxygen to form a flavin-hydroperoxy anion or oxiene by the luciferase bound reduced flavin, and the second step is the monooxygenation of aldehyde by the activated oxygen to form a three membered ring structure of dioxirane as the high energy intermediate, which in turn, transfers its energy intermolecularly to the lumazine containing blue fluorescence protein in vivo resulting the elevation of the lumazine to its electronic excited state coupled with the conversion of dioxirane to the corresponding carboxylic acid.

A New Assay Method for Bovine serum Monoamine Oxidase: Bacterial Luciferase Coupled Reaction

조기웅,심상철,Cho, Ki-Woong,Shim, Sang-Chul 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.1

생리학적으로 매우 중요한 생체 amine의 대사에 관여하는 효소중의 하나인 monomine oxidase (amine; oxygen deaminating oxidase, EC 1.2.3.4)의 활성도 측정을 위해 Bacterial luciferase 과의 연계를 통하여 발광형 측정법을 개발하였다. Nonyl amine과 그 반응산물인 nonyl aldehyde가 이 측정 체계에 가장 적절한 기절임을 확인하였고$(K_{m}=34{\mu}M)$ 이를 기질로 사용하고 해양 발광 세균 Vibrio harveyi로부터 정제된 luciferase를 이용하여 Bovine serum에서 분리된 onoamine oxidase의 반응특성을 조사함으로써 이 방법이 기존의 분광광도법에의한 측정보다 척어도 100배 이상 기질 및 효소활성 측정 강도를 갖고 있음을 확인하였다. A new luminescence based, highly sensitive, rapid, and easy assay method of measuring monoamine oxidase activity was developed using bacterial luciferase coupled reaction. A series of long chain aliphatic amines and bovine serum monoamine oxidase were used for the demonstration. Nonyl amine and nonyl aldehyde, the product, pair were found to be the most suitable substrate for this system. $K_m$ value of nonyl amine was $24{\mu}M$. This luminescent method has at least 100 folds higher detectability of aldehyde product than spectrophotometric assay methods using absorption of benzaldehyde generated from benzyl amine at 250 nm wavelength.

Acetaldehyde-Induced Bacterial Bioluminescence of Photobacterium fischeri Luciferase and Stimulation by Long Chain Alkyl Compounds

조기웅,이현재,심상철,Cho, Ki-Woong,LeeKim, Hyun-Jae,Shim, Sang-Chul Korean Society for Biochemistry and Molecular Biol 1987 한국생화학회지 Vol.20 No.1

세균 루시퍼라아제에 의해 촉매되는 아세트알데히드의 생체 발광은 긴 사슬 화합물에 의해 촉진, 강화되는 것이 발견되었다. 이러한 촉진 정도는 관여하는 긴 사슬 화합물의 사슬 길이 및 그 말단기의 성격에 의존하며 노닐기와 브롬의 경우 그 극대값을 보였다. 아세트알데히드와 긴 사슬 알데히드의 공존 상태에서는 이러한 촉진은 공동 상승 현상을 보였다. 또 긴 사슬 화합물에 의해 촉진되는 아세트알데히드의 발광 반응과 긴 사슬 알데히드에 의한 정상 발광 반응을 상호 비교하였다. The bioluminescence of acetaldehyde catalyzed by bacterial luciferase is stimulated by long chain alkyl compounds. The degree of stimulation is dependent upon the chain length of alkyl group and the properties of the polar head group, showing maximum at nonyl group and bromide. In the presence of the long chain aliphatic aldehydes, a synergistically stimulated luminescence was resulted. The acetaldehyde bioluminescence stimulated by long chain aliphatic compounds is compared with that of corresponding long chain aldehydes.

해양세균의 생체발광 반응을 이용한 Monoamine Oxidase 활성도 측정법

조기웅,심상철 ( Ki Woong Cho,Sang Chul Shim ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.1

A new luminescence based, highly sensitive, rapid, and easy assay method of measuring monoamine oxidase activity was developed using bacterial luciferase coupled reaction. A series of long chain aliphatic amines and bovine serum monoamine oxidase were used for the demonstration. Nonyl amine and nonyl aldehyde, the product, pair were found to be the most suitable substrate for this system. K_m value of nonyl amine was 24 μM. This luminescent method has at least 100 folds higher detectability of aldehyde product than spectrophotometric assay methods using absorption of benzaldehyde generated from benzyl amine at 250 nm wavelength.

제주-대한해협의 표층해양의 용존 탄화수소

조기웅,정경화,신종헌,김영일,정창수,홍기훈,Cho, Ki-Woong,Jung, Kyung-Hwa,Shin, Jung-Hun,Kim, Young-Il,Chung, Chang-Soo,Hong, Gi-Hoon 한국해양학회 2000 바다 Vol.5 No.4

제주-대한해협중 소흑산도에서 부산앞 대마도부근 까지의 제주도 이북의 대륙붕 해역 (33$^{\circ}$30'${\sim}$34$^{\circ}$N 125$^{\circ}$${\sim}$128$^{\circ}$E) 용존탄화수소의 분포와 성인을 조사하였다. 표층 해수중 용존탄화수소 총함량의 시 ${\cdot}$ 공간적 변이는 10배 이상이 대체로 연안에서 높고 외해에서 낮으며, 총함량은 가을에 봄보다 높다. 탄화수소의 성인을 n-alkanes과 pristane함량을 기준으로 보면 해수중 용존 탄화수소는 1998년 4월의 경우, 황해남부(125$^{\circ}$E) 해역에서는 식물플랑크톤기원 및 석유기원 탄화수소가 우세하고, 남해에서는 육상기원과 석유기원이 우세하다. 그리고 1998년 9월에는 남해에 식물플랑크톤과 석유기원이 우세하게 나타났다. Dissolved aliphatic hydrocarbon concentrations in the surface seawater were investigated to describe their distribution and elucidate their sources in the Cheju-Korea Straits region (33$^{\circ}$30‘-34$^{\circ}$N 125$^{\circ}$-128$^{\circ}$E). Seawater sampling was made in spring and autumn in 1998. A large temporal and spatial variability were observed in the dissolved hydrocarbon concentrations in the region. The sources of dissolved hydrocarbons in seawater were elucidated based on the molecular concentrations of n-alkanes and pristane. Dissolved hydrocarbons in the surface water appears to be largely originated from phytoplankton and petroleum in the southern Yellow Sea (125$^{\circ}$), and terrigenous and petrogenic in the Cheju-Korea Straits region in April 1998. In September 1998, dissolved hydrocarbons in the surface waters were largely derived from phytoplanktons and terrestrial material in the Cheju-Korea Soaits region.

Photobacterium fischeri 루시퍼라아제에 의한 아세트알데히드의 생체 발광 반응과 긴 사슬 화합물에 의한 촉진 현상

조기웅,이현재,심상철 ( Ki Woong Cho,Hyun Jae Lee,Sang Chul Shim ) 생화학분자생물학회 1987 BMB Reports Vol.20 No.1

The bioluminescence of acetaldehyde catalyzed by bacterial luciferase is stimulated by long chain alkyl compounds. The degree of stimulation is dependent upon the chain length of alkyl group and the properties of the polar head group, showing maximum at nonyl group and bromide. In the presence of the long chain aliphatic aldehydes, a synergistically stimulated luminescence was resulted. The acetaldehyde bioluminescence stimulated by long chain aliphatic compounds is compared with that of corresponding long chain aldehydes.

박테리아 Lucifease 에 의한 생체 발광 반응에서의 고에너지 중간체에 관한 연구

조기웅,이현재 ( Ki Woong Cho,Hyun Jae Lee ) 생화학분자생물학회 1984 BMB Reports Vol.17 No.1

Reaction mode of bioluminescence mediated by bacterial luciferase from P. fischeri was studied by stopped-flow apparatus measuring the light intensity, and from the results, it was suggested that the bacterial bioluminescence reaction occurs by at least two step mechanism, i.e., the first step is the activation of molecular oxygen to form a flavin-hydroperoxy anion or oxiene by the luciferase bound reduced flavin, and the second step is the monooxygenation of aldehyde by the activated oxygen to form a three membered ring structure of dioxirane as the high energy intermediate, which in turn, transfers its energy intermolecularly to the lumazine containing blue fluorescence protein in vivo resulting the elevation of the lumazine to its electronic excited state coupled with the conversion of dioxirane to the corresponding carboxylic acid.

해수로부터 용존 유기물의 Solid Phase Extraction을 위한 다수 시료 처리 장치

조기웅(Ki Woong Cho),정경화(Kyungwha Jung),신종헌(Jongheon Shin),김석현(Suk Hyun Kim),홍기훈(Gi-Hoon Hong) 한국해양환경·에너지학회 2000 한국해양환경·에너지학회지 Vol.3 No.3