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Heterologous plasmid DNA 의 transformation에 있어서의 細胞膜 蛋白質의 기능에 對하여
徐正塤,洪淳德,李麟九,金英浩 慶北大學校 1983 論文集 Vol.36 No.-
Terramycin resistance plasmid DNA and Streptomycin resistance plasmid DNA have been prepared from Streptomyces rimosus and E. coli KPM 105, respectively, by phenol extraction of lysozyme-lysed cells. And the plasmid DNA was introduced into B. subtilis KPM 73, B. subtilis BD 224, B. subtilis 110, B. subtilis RM 125, B. amyloliquef aciens and B. megaterium IAM B425 by transformation. The variable characters affecting heterologous plasmid transformation, and the functions of competence factor and binding factor existing in the recipient cell surface were futhermore studied. Terramycin resistance plasmid was well transformed into Bacillus subtilis KPM 73 and B. subtilis RM 125, and Streptomycin resistance plasmid was well transfomed into B. subtilis BD 224, B. subtilis 110 and B. megaterium IAM B 425 at high frequency. The high frequency of plasmid transformation was obtained at 4 hr of incubationin growth medium. 20 to 50 min in competence medium, and the optimal pH and temperature for competence were 7.0 and 20C to 30C, respectively. The transformation of B. subtilis KPM 73 reached a maximum level after 20 min of exposure to DNA, B. subtilis BD 224 after 30min, and B. subtilis RM 125 after 10 min. Ca, Cu, Fe, Mg and Mn ions increased the transformation frequency of B. subtilis KPM 73 but Hg, Zn and Co ions decreased the transformation frequency. Ca ions were required for transformation of E. coli C600 and tranrsformation frequency was increased 20 times by addition of 75mM CaCl_2. The competence development of recipient cell was enhanced by addition of lysozyme in the competence medium and lysozyme was more affective in the early stage of competence development than the late stage of competence development, in which the spontaneous competence development reached maximum level. In sight of these observations, competence factor would seem to be a kind of cell wall lytic enzyme such as lysozyme. when the protoplasts of B. subtilis KPM 73 were transformed by terramycin resistance plasmid, the protoplasts were successfully transformed and the transformation frequency was 10^-2. The competent cells were treated with small amount of protein-digesting enzyme such as trypsin, and then exposed to transforming plasmid DNA. The recipient cells that were treated with trypsin were transformed 10 to 100 folds lower than non-trypsin-treated control cells. These results show that the binding factor must be a protein or at least a complex containing protein that is essential to it's activity. And external protein such as bovine albumin was effective on transformation of trypsin treated cell, suggesting that the cell surface-located binding factor did not have a specificity and the binding factor could be replaced by external substance such as bovine albumin. Terramycin resistance plasmid extracted from Ter^R transformant of B. subtilis KPM 73 was well transformed into B. subtilis KPM 73. And the electrophorsis pattern of Ter^R plasmid extracted from Ter^R transformant of B. subtilis KPM 73 was equal to that from St. rimosus.
이인구,서정훈 한국농화학회 1969 Applied Biological Chemistry (Appl Biol Chem) Vol.12 No.1
This glucose isomerizing enzyme, which Actinomyces sp. (strain, K-17) produced, was inhibited by citrate, oxalate, ethylene diamine tetraacetic acid and cysteine on the enzyme reaction. This enzyme isomerized xylose to ketose as well as glucose. The Michaelis constant of this enzyme was 7.2×10^(-1)M. on D-glucose. The enzyme activity of intact cells which were harvested in the none xylose containing medium was very weak. If these intact cells of low activity were treated in the buffered xylose solution, its activity was considerably increased. After fifteen hours at 32℃. on xylose treatment, the enzyme activity was increased to equilibrium and the treating condition was proper at neutral pH and in aerobic state. The adequate concentration of xylose on the treatment was about 0.5 percent.
Xylose isomerase 유전자를 클로닝한 알콜 발효성 효모의 개발:대장균의 xylose isomerase 유전자의 클로닝
이인구,서정훈,박우철,박희동,조진기,배신철 경북대학교 유전공학연구소 1986 遺傳工學硏究所報 Vol.1 No.1
For the purpose of cloning of Escherichia coli D-xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) gene to Saccharomyces cerevisiae, a 12.9Kb DNA fragment of Hind Ⅲ-digested chromosomal DNA of E. coli, containing genes for D-xylose utilization was inserted into the Hind Ⅲ site of YEp13, a shuttle plasmid between E. coli and S. cerevisiae. This recombinant plasmid, pEXI, could genetically complemented various kinds of D-xylose negative mutant in E. coli. The 12.9Kb DNA fragment of pEXI contained not only xylA and xylB genes but also xylR/T gene. The 1.6Kb DNA fragment of Bgl Ⅱ-digested pEXI was isolated and subcloned into Bam HI-digested or Bgl Ⅱ-digested YEp13 plasmid. The 1.6Kb DNA fragment of plasmid contained structural gene and promotor for D-xylose isomerase. Transformant of pEXI in E. coli produced D-xylose isomerase 3.0 times as much as parent strain.
고온성 알콜발효 효모의 Alcohol Dehydrogenase의 특성
예상수,임시규,손호용,진익렬,이인구,김영호,서정훈,박완 한국산업미생물학회 1997 한국미생물·생명공학회지 Vol.25 No.4
고온성 알콜발효효모인 S. cerevisiae RA-74-2와 K. marxianus RA-912의 alcohol dehydrogenase특성을 산업적으로 사용하고 있는 중온성 S. cerevisiae D균과 비교 검토하였다. 30℃, 혐기적인 발효 조건에서 배양된 S. cerevisiae RA-74-2와 D의 ADH의 활성은 비슷하였고 알콜 발효력이 떨어지는 K. marxianus RA-912는 S. cerevisiae RA-74-2와 D에 비해 약 43%의 활성을 나타내었다. 37℃에서 배양한 경우, 고온 발효력이 우수한 S. cerevisiae RA-74-2의 ADH 활성은 30℃와 비슷하였나, S. cerevisiae D균은 30℃에서의 활성에 비해 약 70% 감소하였다. K. marxianus RA-912도 30℃에 비해 약간의 활성감소를 나타내었다. 이는 이들 균주의 알콜발효력과 잘 일치되는 것으로, 온도에 따른 발효력과 ADH 활성의 연관성을 보여주고 있다. 또한 S. cerevisiae RA-74-2의 ADH의 열 안정성도 S. cerevisiae D에 비해 높게 나타나 S. cerevisiae RA-74-2이 고온 알콜발효능을 반영하는 것으로 보였다. 그러나 45℃에서도 발효가 가능한 K. marxianus RA-912의 열 안정성은 예상과는 달리 S. cerevisiae D에 비해 낮게 나타났다. ADH의 isozyme 양상에서는 S. cerevisiae인 D와 RA-74-2의 경우 강한 활성염색을 보이는 두개의 주요 밴드로 나타났으나 아래 밴드의 이동도가 약간의 차이를 보였으며, S. cerevisiae RA-74-2는 D와 달리 30℃와 37℃에서 거의 유사한 활성염색 강도를 보였다. K. marxianus와 RA-912의 단백질 및 활성염색 양상은 S. cerevisiae와 다른 양상을 보여 K. marxianus 특유의 ADH 효소계를 갖고 있는 것으로 생각된다. The charcteristics of alcohol dehydrogenase (ADH, EC 1.1.1.1, alcohol:NAD oxidoreductase) of thermotolerant alcohol-producing yeasts, Saccharomyces cerevisiae RA-74-2 and Kluyveromyces marxianus RA-912, were compared with that of mesophilic S. cerevisiae D, an industrial strain. Under anaerobic culture condition, both S. cerevisiae RA-74-2 and D had similar level of ADH activity at 30℃ and the activity of S. cerevisiae RA-74-2 at 37℃ was the same level at 30℃. However, the level of ADH activity of S. cerevisiae D at 37℃ decreased about 70% of that at 30℃. The level of enzyme activity of K. marxianus RA-912, which showed lower alcohol productivity than S. cerevisiae RA-74-2 and D, was about 43% of those strains at 30℃, and decreased somewhat at 37℃. The results showed a good correlation between tha alcohol productivities and the level of ADH activities of these strains grown at 30℃ and 37℃. And the higher heat stability of ADH of S. cerevisiae RA-74-2 than that of S. cerevisiae D seemed to reflect the ability of high temperature fermentation. Despite of its fermentation ability even at 45℃, however, the ADH of K. marxianus RA-912 showed lower heat stability than that of S. cerevisiae D. Both S. cerevisiae RA-74-2 and D showed similar patterns of two bands of ADH isozyme, and the low band of S. cerevisiae RA-74-2 moved slightly faster than that of S. cerevisiae D. The staining intensity of the bands of S. cerevisiae D at 37℃ was weaker than those at 30℃. However, S. cerevisiae RA-74-2 showed no differences in total intensity of the bands of 30℃ and 37℃. As the patterns of cellular proteins and ADH isozyme of K. marxianus RA-912 were different from S. cerevisiae RA-74-2 and D,K. marxianus might have its own characteristic ADH system.