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배양온도와 시간에 따른 Serratia marcescens 표면의 소수성 성질변화
이상열,신용철,권헌영,조무제,강은경 한국미생물 · 생명공학회 1990 한국미생물·생명공학회지 Vol.18 No.3
Serratia marcescens를 $30^{\circ}C$에서 진탕배양했더니, 적색색소인 prodigiosin이 초로기(senescent phase of growth)에서 생성되었다. 그리고 이조건에서 배양한 세포를 polystyrene dish를 사용하여 세포의 hydrophobicity를 측정한 결과 상당한 소수성 성질이 발현되어 대부분의 세포가 비극성 성질의 polystyrene dish에 흡착되었다. 그러나 이 박테리아를 $37^{\circ}C$에서 배양했더니, 적색 색소인 prodigiosin도 생성되지 않았을 뿐 아니라 소수성 성질도 발현되지 않음으로서 세포가 polysyrene dish에 흡착되지 않고 pre-washing 단계에서 모두 씻겨져 났다. 또한 $30^{\circ}C$와 $37^{\circ}C$에서 배양한 serratia marecescens의 지질성분을 분석한 결과, $30^{\circ}C$에서 배양한 세포의 지질은 phospholipid, glycolipid 및 확인되지 않은 지질 등이 생성되었으나 $37^{\circ}C$에서 배양한 세포의 경우는 주로 양쪽성 성질의 aminolipid인 serratamolide가 생성되어, 배양한 온도조건에 따라 뚜렷한 차이를 보였다. S. marcescens cultured at $30^{\circ}C$ with vigorous shaking was shown to produce red-pigment, prodigiosin, in the senescent phase of growth. Also, it showed many hydrophobic characteristrics, which were tested by the adherence to noncharged surfaces of polystyrene dishes, a typical agent for the binding of hydrophobic cells and molecules. However, when the cell was cultured at $37^{\circ}C$, it no longer produced either red pigment or hydrophobic materials. Therefore, the bacteria cultured at $37^{\circ}C$ was completely washed-out from the polystyrene dishes at the copious washing step with tap water, in contrast to the cell cultured at $30^{\circ}C$ which was sticked onto the polystyrene dishes very tightly. The lipid compositions extracted from the S. marcescens cultured at $30^{\circ}C$ or $37^{\circ}C$ were very different from each other; the phospholipids, glycolipids and unidentified lipids were produced from the cell cultured at $30^{\circ}C$, whereas large amounts of serratamolide, amphipathic compound, were produced from the cell cultured at $37^{\circ}C$. The data suggest that the pronounced cell surface hydrophobicity of the S. marcescens is mediated by a combination of several surface factors that were affected by cultivation time and temperatures.
Serratia marcescens Chitinase 유전자의 대장균에로의 클로닝
장규일,김기석,조무제,이상열,신용철 한국미생물 · 생명공학회 1992 한국미생물·생명공학회지 Vol.20 No.2
본 연구에서는 Serratia marcescens ATCC 27117 균주로부터 키나아제 유전자를 대장균으로 클로닝 하고 발현시켰다. pUC 19 플라스미드를 이용하여 S.marcescens의 genomic library를 만들고 팽화된 키틴이 포함된 한천배지에서 키티나아제 활성을 가지는 클론을 선별하였다. 약 1x10 transformant들 중에서 키티나아제 활성을 보이는 하나의 클론을 선발하였으며 이것은 pUC 19 플라스미드속에 8.9Kb 염색체 DNA 삽입단편을 가지고 있었다. A chitinase gene of Serratia marcescens ATCC 27117 was cloned and expressed in Escherichiu di. A genomic library of S, marcescens was constructed with pUC 19 and screened using the swollen chitin agar plate for chitinolytic clones. A positive clone showing chitinclearance contains a recombinant pCHI 89, composed of 8.9 Kb chromosomal DNA fragment and pUC 19. Plasmid pCHI 89 produced 58 KD chitinase in E. coli, which was coincided with one of five extracellular chitinases produced by S. nzarccscens. Restriction endonuclease cleavage sites of the 8.9 Kb insert DNA fragment were mapped. E. coli JM109 harboring pCHI 89 inhibits the growth of a plant pathogenic fungus, Fusarium oxysporum.
Serratia marcescens 로 부터 5 가지 Chitinase Isozymes 의 동정
황재령,갈상완,이경애,신용철,조무제,이상열 ( Jae Ryoung Hwang,Sang Wan Gal,Kyeong Ai Lee,Yong Chul Shin,Moo Je Cho,Sang Yeol Lee ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.3
S. marcescens KCTC2172 produced five extracellular chitinases that were involved in chitin degradation. These enzymes were identified as chitinase isozymes by substrate activity staining in SDS-PAGE gel after removal of SDS, because chitinases were resistant to SDS and existed as monomeric proteins. Their apparent molecular weights obtained by SDS-PAGE were 58, 47, 43, 37, and 21 kd. The chitinase activities were induced about seven folds by the addition of 1.5% chitin to LB-medium compared with those obtained without chitin, and the induction was dependent on the various forms of chitin. Chitinases were purified using the regenerated chitin column by successive pH changes of elution buffer. When the column was eluted with enough volume of pH 3.3 buffer, five chitinase isozymes were eluted. However, three of them whose molecular weights were 58, 47 and 43 kd were further eluted by the pH changes of elution buffer from pH 3.3 to 2.0. Considering the pH dependent stability of chitinases, it was known that S. marcescens produced two groups of chitinase isozymes; One is resistant and the other is very sensitive to acidic conditions, at pH below 3.3.
김외연,이훈실,서숙재,조무제,이상열,김재원,Kim, Woe-Yeon,Lee, Hoon-Sil,Suh, Sook-Jae,Cho, Moo-Je,Lee, Sang-Yeol,Kim, Jae-Won The Microbiological Society of Korea 1994 미생물학회지 Vol.32 No.2
Serratia marcescens가 세포외로 분비하는 nuclease의 유전자가 발현된 Escherichia coli JM107을 배양하여 다량의 효소를 정제하였다. Matrex green gel과 heparin agarose gel column chromatography법으로 약 50배 정제한 효소는 분자량이 29KDa였으며, 전기영동 상에서 단일 띠를 보였다. 이 단백질을 이용하여 polyclonal antibody를 만들고, 면역조직화학법으로 세포내의 분포를 조사하였다. Nuclease는 주로 세포막에 존재하였고, 이를 토대로 효소가 세포질에서 합성된 후 세포막으로 빠르게 이동함을 알 수 있었다. 이 결과는 세포의 막분획에서 효소의 활성의 대부분이 회수되며, 면역블럿 방법으로 효소의 대부분이 세포막에서 검출된다는 결과와 일치하였다. Nuclease was secreted to the environmental media from the Escherichia coli JM107 tranformant harboring the extracellular nuclease gene of Serratia marcescens in the plasmid of pNUC4. Under the growth conditions, the amount of secreted enzyme was increased in parallel with bacterial growth conditions, the amount of secreted enzyme was increased in parallel with bacterial growth. The enzyme was purified using chromatofraphic procedures of Matrex green gel and heparin agarose affinity gel, resulted in 50-fold purification with 15% recovery of the enzyme. The apparent molecular weight of the enzyme was estimated to be 29Kda by sodium dodecylsulfate denaturing gel electrophoresis. Using the purified enzyme, polyclonal antibody was obtained from the rabbit. The specificity of the antibody was confirmed by immunoblotting and immunoprecipitaion. For the investigation of cellular distribution of the enzyme, cells were fractionated into three fractions; cytoplasm, periplasm and extracellular fluid. While more than 80% of the enzymatic activity was detected in the extracellular fluid and periplasm, a little was found in the cytoplasm, indicating that the enzyme was likely to be immediately exported to the membrane for excretion after biosynthesis. These results were confirmed again by immunocytochemistry technique using the antibody.