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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.
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.
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.
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.
이상규,최석정,양철학,Lee, Sang-Kyou,Choi, Suk-Jeong,Yang, Chul-Hak 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.6
산소존재하에서 자란 대장균 HB101로부터 전기이동시 활성이 보이지 않는 하이드로지나아제 동위효소를 부분적으로 분리하였다. 분리과정은 데옥시콜레이트에 의한 세포막의 분산, 암모니움 설페이트 침전, 이온교환, 하이드록시아파타이트, 그리고 gel 여과단계 등을 포함했다. 수소 : 메철바이올로젠의 산화환원 활성도로 효소의 활성을 정량적으로 측정하였고 이온 교환 chromatography 단계부터 약 50배의 분리가 이루어졌다. 마지막으로 얻은 효소의 비활성도는 1분에 mg 단백질당 248.2 nmol(산화된 메칠바이올로젠)이였고, 이 효소는 39 kDa과 30 kDa 두개의 폴리펩타이드로 구성되어 있었다. 또한 효소의 활성이 가장 큰 pH 범위는 7과 8 사이였고 $70^{\circ}C$에서도 최고활성이 52%를 유지하였다. An electrophoretically labile hydrogenase from the membrane fraction of aerobically grown Escherichia coli was partially purified. The enzyme was assayed by quantification of the $H_2$ : methyl viologen oxidoreductase activity and purification was achieved 50-fold by ion exchange chromatography and gel filtration steps. The specific activity of the final preparation was 248.2 nmol methyl viologen oxidized $min^{-1}$ mg $protein^{-1}$. The purified enzyme consists of two subunits whose molecular weights are 39000 D and 30000 D. The enzyme showed a pH optimum between 7 and 8, and retained 52% of the maximal activity at $70^{\circ}C$.
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.
전기이동시 불안정한 하이드로지나아제 동위효소의 산소존재하에 자란 대장균에서의 부분분리 및 효소의 특성
이상규,최석정,양철학 ( Sang Kyou Lee,Suk Jeong Choi,Chul Hak Yang ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.6
An electrophoretically labile hydrogenase from the membrane fraction of aerobically grown Escherichia coli was partially purified. The enzyme was assayed by quantification of the H₂ : methyl viologen oxidoreductase activity and purification was achieved 50-fold by ion exchange chromatography and gel filtration steps. The specific activity of the final preparation was 248.2 nmol methyl viologen oxidized min^(-1)·㎎· protein^(-1). The purified enzyme consists of two subunits whose molecular weights are 39000 D and 30000 D. The enzyme showed a pH optimum between 7 and 8, and retained 52% of the maximal activity at 70℃.