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RISS 인기검색어
- 검색키워드
- 저자 : 최석정(Choi Suk-Jung) (검색결과 19 건)
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Cloning and Overexpression of Escherichia Coli Hydrogenase Operon
최석정,양철학,Choi, Suk-Jung,Yang, Chul-Hak Korean Society for Biochemistry and Molecular Biol 1990 한국생화학회지 Vol.23 No.3
A set of hydrogenase-defective E. coli mutant strains have been isolated. Among them, three mutants, E. coli HD24, 43, and 50, were complemented with pHY1 previously reported (Oh et al., 1987). Another mutant strain, HD6, was transformed with E. coli chromosomal DNA library and a plasmid, pHY3, was isolated which restored hydrogenase activity in this strain. This plasmid was proved to contain two genes essential for hydrogenase activity by the complementation test of various mutants with each fragment of the insert. The two genes are contained in 2.1 kb SalI fragment of pHY3-insert. It seems that this fragment also has its own promoter. When it was expressed under the control of tac promoter, it gave two polypeptides of molecular masses, 31 and 43 kDa with a cellular content of about 10 percent. However, their increase did not correlated with the increase of hydrogenase activity. That is, they are essential components for hydrogenase activity but not sufficient. 수소발생효소의 활성이 결핍된 일련의 대장균 돌연변이체를 얻었다. 그 중에, HD24, 43 그리고 50의 세 돌연변이체는 전에 보고되었던 plasmid, pHY1에 의해 보완되었다. 다른 돌연변이체인 HD6를 대장균 염색체 DNA library로 형질변환 시킨 후, 이 돌연변이체의 수소발생효소 활성을 회복시키는 plasmid, pHY3를 얻었다. 이 plasmid는, 그 insert의 각 fragment를 이용한 보완실험에 의해, 수소 발생효소에 필수적인 두 개의 유전자를 갖고 있는 것으로 밝혀졌다. 그 두 유전자는 2.1 kb인 SalI-fragment에 포함되어 있었다. 또한 이 조각은 그 자체의 promoter를 갖고 있는 것으로 생각되며, tac promoter의 조절하에 형질발현 시켰을 때, 그 분자량이 각각 31과 43 kDa인 두 폴리펩티드를 만들었고, 그 세포내 함량은 대략 10퍼센트 정도였다. 그러나, 그 두 폴리펩티드의 양의 증가로 인해 수소발생효소 활성이 증가하지는 않는 것으로 보아, 그들은 효소활성에 필수적이기는 하나, 충분하지는 않은 것으로 생각된다.
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최석정,양철학 ( Suk Jung Choi,Chul Hak Yang ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.3
A set of hydrogenase-defective E. coli mutant strains have been isolated. Among them, three mutants, E. coli HD24, 43, and 50, were complemented with pHY1 previously reported (Oh et al., 1987). Another mutant strain, HD6, was transformed with E. coli chromosomal DNA library and a plasmid, pHY3, was isolated which restored hydrogenase activity in this strain. This plasmid was proved to contain two genes essential for hydrogenase activity by the complementation test of various mutants with each fragment of the insert. The two genes are contained in 2.1 kb Sall fragment of pHY3-insert. It seems that this fragment also has its own promoter. When it was expressed under the control of tac promoter, it gave two polypeptides of molecular masses, 31 and 43 kDa with a cellular content of about 10 percent. However, their increase did not correlated with the increase of hydrogenase activity. That is, they are essential components for hydrogenase activity but not sufficient.
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최석정,양철학 ( Suk Jung Choi,Chul Hak Yang ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.4
A spectrophotometric assay method for hydrogenase was studied. The sample solution was flushed with nitrogen or hydrogen to remove oxygen, and the reaction started. At the end of the reaction, 2`,3`,5`-tiphenyl tetrazolium chloride was reduced with reduced methyl viologen, and the concentration was determined by monitoring the absorbance at 600 nm. The absorbance increased linearly with increasing amount of enzyme in both cases of uptake and evolution assay. Alternatively, the concentration of reduced MV can be measured directly with autofill or microflow device which avoids a contact of the solution with air. This method also showed good results.
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Molecular Cloning of Hydrogenase Gene in Escherichia coli
오경준,최석정,양철학,Oh, Kyoung-Joon,Choi, Suk-Jung,Yang, Chul-Hak Korean Society for Biochemistry and Molecular Biol 1987 한국생화학회지 Vol.20 No.2
대장균 HB101에 MNNG를 처리하고 methyl viologen 여과지법을 이용하여 hydrogenase defective 변종을 분리하였다. 대장균의 DNA를 제한효소 EcoRI으로 부분가수분해 하고 플라스미드 pBR322의 EcoRI site에 접합 시켰다. 생성된 재조합 플라스미드로 hydrogenase defective 변종 대장균 HY1을 형질전환시켰다. 그 결과 hydrogenase를 생산하는 유전자를 갖는 플라스미드 pHY1 을 얻었다. 이 플라스미드는 대장균 HY2, HY3 및 LCB850을 형질전환시키지 못하였다. 대장균의 hydrogenase 야생종, 변이종 및 pHY1-10으로 형질전환된 변이종의 crude extract를 만들어 폴리아크릴아미드 전기영동법을 이용하여 hydrogenase activity를 분석하였다. Hydrogenase defective mutants of Escherichia coli HB101 were isolated by MNNG-mutagenesis and methyl viologen filter paper screening method. E. coli DNA was digested with restriction endonuclease EcoRI and ligated into the EcoRI site of plasmid pBR322. The resulting recombinant plasmids were used to transform E. coli HY1, a mutant with a defective hydrogenase activity. Complementation of this hydrogenase mutation identified a bacterial clone carrying the gene for a E. coli hydrogenase in plasmid pHY1-10 This plasmid was not able to complement the other mutants, E. coli HY2, HY3, and LCB850. Polyacrylamide gel electrophoresis was used to analyze the hydrogenase(s) of the wild type, mutants, and mutant strains transformed with pHY1-10.
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오경준,최석정,양철학 ( Kyoung Joon Oh,Suk Jung Choi,Chul Hak Yang ) 생화학분자생물학회 1987 BMB Reports Vol.20 No.2
Hydrogenase defective mutants of Escherichia coli HB101 were isolated by MNNG-mutagenesis and methyl viologen filter paper screening method. E. coli DNA was digested with restriction endonuclease EcoRI and ligated into the EcoRI site of plasmid pBR322. The resulting recombinant plasmids were used to transform E. coli HY1, a mutant with a defective hydrogenase activity. Complementation of this hydrogenase mutation identified a bacterial clone carrying the gene for a E. coli hydrogenase in plasmid pHY1-10. This plasmid was not able to complement the other mutants, E. coli HY2, HY3, and LCB850. Polyacrylamide gel electrophoresis was used to analyze the hydrogenase(s) of the wild type, mutants, and mutant strains transformed with pHY1-10.
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Fluorescein 형광의 pH 의존성을 이용한 lipase 활성 측정방법
박종원(Jong-Won Park),최석정(Suk-Jung Choi) 한국생명과학회 2008 생명과학회지 Vol.18 No.8
이 연구의 목적은 물-오일 계면에서 특이적인 lipase 활성을 측정할 수 있는 high-throughput assay 방법을 확립하는 것이다. 이 방법은 pH에 따라 형광의 세기가 변하는 fluorescein의 특성을 이용하여 lipase의 작용으로 방출되는 지방산으로 인한 pH 변화를 fluorescein의 형광 변화로 측정하도록 되어 있다. 활성의 측정은 오일 에멀션과 fluorescein 그리고 효소를 포함하는 반응 용액을 반응시키면서 일정한 간격으로 형광을 측정함으로써 이루어진다. 이 방법을 통해 형광의 세기가 효소의 양에 비례하는 속도로 감소하는 것을 관찰할 수 있었으며 시간에 따른 형광 변화 그래프로부터 계산한 반응 속도가 효소의 양에 선형으로 비례한다는 것을 확인할 수 있었다. 또 한 가지 중요한 사실은 assay를 하는데 있어서 pH 6.0-8.0의 범위에서 다른 pH 조건을 사용할 수 있었다는 점이다. The purpose of this study was to establish a high-throughput assay method capable of estimating specific lipase activity at oil-water interface. The method is based on the fact that fluorescence intensity of fluorescein is affected by pH. The pH-dependence might be used to monitor pH change caused by the release of fatty acid through the action of lipase. Assay was performed by incubating a reaction mixture containing oil emulsion, fluorescein and enzyme and by monitoring fluorescence intensity periodically. Fluorescence intensity decreased linearly with a rate proportional to the enzyme amount. Linear relationship was observed between enzyme amount and reaction rate which was calculated from a graph of fluorescence change against time. The assay was possible at different pH conditions in the range of pH 6.0-8.0.
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넙치와 조기의 원산지 판별을 위한 random amplified polymorphic DNA 패턴 연구
강덕진,이석근,진덕희,최석정,Kang Duk-Jin,Lee Suk-Keun,Jin Deuk-Hee,Choi Suk-Jung 한국생명과학회 2006 생명과학회지 Vol.16 No.1
본 연구에서 넙치와 조기의 원산지를 판별하기 위한 도구로 RAPD PCR 방법의 가능성을 확인하였다. 넙치는 한국의 주문진 자연산, 통영 양식산, 거제 양식산 그리고 북한 자연산을 실험에 사용하였다. 조기는 한국산과 중국산을 사용하였다. 넙치의 RAPD 패턴에서는 뚜렷하고 일관성이 있는 진단용 띠들을 쉽게 찾을 수 있었다. 조기의 경우에는 유전적인 이질성으로 인하여 각 개체의 RAPD 패턴에서는 일관성이 있는 진단용 띠를 찾기 어려웠지만 각 원산지별로 얻은 RAPD 패턴에서는 가능성이 있는 진단용 띠들을 찾을 수 있었다. The random amplified polymorphic DNA (RAPD) technique was investigated as a potential tool for the origin identification of olive flounder (Paralichthys olivaceus) and redlip croaker (Pseudosciaena polyactis). Olive flounder specimens were collected from North Korea and several locations of South Korea (Jumunjin, Tongyoung and Geoje). Fishes obtained from Tongyeong and Geoje were cultured products. Redlip croaker specimens were collected from South Korea and China. Consistent and distinct diagnostic bands were easily identified in the RAPD patterns of the olive flounder specimens. Although consistent diagnostic bands rarely appeared in the RAPD pattern of redlip croaker specimens because of their genetic heterogeneity, we were able to find potential diagnostic bands in the average RAPD pattern of each origin.
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