• 한국미생물·생명공학회지
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• 제18권4호
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• pp.437-441
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• 1990

근채류 식물의 근부원인이 되는 식물병원균 Fusarium solani의 생육을 강력히 길항하는 생물반제균 Pseudomonas stutzeri YPL-1을 모균주로 하여 UV나 NTG로 돌연변이시킴으로써 길항능이 증강된 강력한 생물방제균을 유전적으로 육종하고자 하였다. 그 결과 길항기작의 원인인 외막가수분해효소 chitinase 생산능이 2.5배, 2.0배 정도로 증강된, 동시에 길항능도 모균주에 비해 1.7배, 1.5배 정도로 비례해서 증강된, 강력한 우수 길항변이주 P.stutzeri YPL-M26(UV)과 P.stutzeri YPL-M178(NTG)을 유전적으로 육종할 수 있었다. 길항종강변이주에 의한 F.solani의 생육억제기작도 모균주에서와 같이 고분자 물질인 chitinase를 주로 하는 외막가수분해효소에 의한 것으로 확인되었고, 균사신장억제율도 조사해 본 결과 조효소액 첨가 경우 24시간째에는 모균주 경우 87.1 정도인데 비해 거의 100의 생육억제율을 나타내는 강력한 생물반제균으로 유전적 육종을 할 수 있었다. 한편 변이주와 모균주의 효소에서도 그 최적반응 pH등 각종 효소학적 특성이 동일하였다.

• 한국미생물·생명공학회지
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• 제18권1호
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• pp.81-88
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• 1990

근채류식물의 근부원인이 되는 토양유래의 식물병원 성진균에 대한 생물학적 방제를 위하여 저병해인삼경작 지토양으로부터 식물근부균 Fusarium solani의 생육을 강력히 길항하는 억제세균 YPL-1을 분리, 선발하였으며 이들 동정한 결과 Pseudomonas stutzeri이거나 그 근연종으로 확인하였다. 선발된 P.stutzeri YPL-1에 의해 생산된 근부균생육억제물질은 열에 민감하고 고분자의 단백질물질로서 chitinase 및 laminarinase 등 F.solani의 외막가수분해효소인 것으로 추정된다. 더욱이 chitinase 생산능과 근부균생육억제능은 정관계로 비례한다는 것도 알았다. 이는 NTG를 이용하여 얻은 chitinase 및 laminarinase 생산불능변이주 P.stutzeri YPL-M122(chi-, lam-), P.stutzeri YPL-M153(chi-)에 의해서도 확인되었다. 그러나 본 P.stutzeri YPL-1은 siderophore를 전혀 생산하지는 못하였다. 이 결과로 미루어 보아 선발된 억제균 P.stutzeri YPL-1 균주에 의한 식물근부균 F.solani의 생육억제기작은 저분자물질인 항생물질이나 siderophore가 아닌 chitinase를 주로 하는 외막가수분해효소에 의한 근부균 F.solani의 세포벽분해에 기인된 것으로 생각된다.

• 미생물학회지
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• 제25권1호
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• pp.1-8
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• 1987

An extracellular $\beta$-1, 3-glucanase from Pseudomonas stutzeri KF 13 was purified about 390 with 26% recovery. The purified enzyme revealed a single band by polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The enzyme was stable in a pH 6.0 to 9.0, and relatively thermostable. The optimal pH and temperature on the enzyme activity were found to be 5.8 and 45.deg.C, respectively. The activation energy was calculated to be 16,130 cal per mole. The Km value for laminarin was found to be 3ng per ml and the molecular weight was determined to be 28,000 by gel filtration and 26,000 daltons by SDS-acrylamide gel electrophoresis. The enzyme was inhibited by 1.0mM of $Hg^{2+}$, and strongly inhibited by 1.0mM of p-chloromercuribenzoic acid.

• Journal of Microbiology and Biotechnology
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• 제4권2호
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• pp.134-140
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• 1994

An antagonistic bacterium Pseudomonas stutzeri YPL-1 liberated extracellular chitinase and $\beta$-1,3-glucanase which are key enzymes in the decomposition of fungal hyphal walls. The lytic enzymes caused abnormal swelling and retreating at the hyphal tips of plant pathogenic fungus Fusarium solani in a dual culture. Scanning electron microscopy revealed the hyphal degradation of F. solani in the regions interacting with P. stutzeri YPL-1. The production of chitinase and properties of a crude preparation of the enzyme from P. stutzeri YPL-1 were investigated. Peak of the chitinase activity was detected after 4 hr of cultivation. The enzyme had optimum temperature and pH of 50$^{\circ}C$ and pH 5.3, respectively. The enzyme was stable in the pH range of 3.5 to 6.0 up to 50$^{\circ}C$. The enzyme was significantly inhibited by metal compounds such as $HgCl_2$, but was stimulated by $CoCl_2$. P. stutzeri YPL-1 produced high levels of the enzyme after 84 hr of incubation. Among the tested carbon sources, chitin was the most effective for the enzyme production, at the concentration level of 3%. As a source of nitrogen, peptone was the best for the enzyme production, at the concentration level of 4%. The maximum amount of enzyme was produced by cultivating the bacterium at a medium of initial pH 6.8.

• Applied Biological Chemistry
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• 제27권2호
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• pp.73-78
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• 1984

Pseudomonas stutzeri IAM 12097의 trypticase 배지(培地)에서 36시간(時間), initial pH는 6.3일때 Exo-maltotetraohydrolase가 최대로 생산되었다. Exo-maltotetraohydrolas, 황산(黃酸)암모니아분획(分劃)과 2회(回)의 DEAE-cellulose column chromatography에 의하여 정제(精製)하였으며 정제효소(精製酵素)의 specific activity는 108.6 u/mg protein, 수율(收率)은 9.4%이었다. 본정제효소(本精製酵素)는 polyacrylamide gel electrophoresis와 SDS-polyacrylamide gel electrophoresis에 의하여 각각(各各) 단일(單一) band를 나타내었다.

• Journal of Microbiology
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• 제33권4호
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• pp.295-301
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• 1995

An antifungal Pseudomonas stutzeri YPL-1 produced extracellular chitinase and .betha.-1, 3-glucanase that were key enzymes in the decomposition of fungal hyphal walls. These lytic extracellular enzymes markedly inhibited mycelial growth of the phytopathogenic fungus Fusarium solani. A chitinase from P. stutzeri YPL-1 inhibited fungal mycelial growth by 87%, whereas a .betha.-1, 3-glucanase from the bacterium inhibited growth by 53%. Furthermore, co-operative action of the enzymes synergistically inhibited 95% of the fungal growth. The lytic enzymes caused absnormal swelling and retreating on the fungal hyphal walls in a dual cultures. Scanning electron microscopy clearly showed hyphal degradation of F. solani in the regions interacting with P. stutzeri YPL-1. In an in vivo pot test, P. stutzeri YPL-1 proved to have biocontrol ability as a powerful agent in controlling plant disease. Planting of kidney bean (Phaseolus vulgaris L.) seedlings with the bacterial suspension in F. solani-infested soil significantly suppressed the development of fusarial root-rot. The characteristics of a crude preparation of .betha.-1, 3-glucanase produced from P. stutzeri YPL-1 were investigated. The bacterium detected after 2 hr of incubation. The enzyme had optimum temperature and pH of 40.deg.C and pH 5.5, respectively. The enzyme was stable in the pH range of 4.5 to 7.0 and at temperatures below 40.deg.C, with a half-life of 40 min at 60.deg.C.

• 미생물학회지
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• 제24권3호
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• pp.295-301
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• 1986

The bacteria, which were capable of producing ${\beta}-1$, 3-glucanase inducibly by utilizing cell wall of Aspergillus fumigatus as a sole carbon source, were isolated from soil in the campus of Kyungpook National University. Among them, the strain which produced the enzyme excellently was selected and identified to be Pseudomonas stutzeri KF 13 by morphological, cultural and physiological examination. The optimal conditions for the enzyme production from Pseudomonas stutzeri KF 13 were investigated. the enzyme production was reached maximum state shen the broth cultured for 72hr at $30^{\circ}C$. And the enzyme showed the highest activity in the medium containing 3.5% cell wall as an inducer, 15% yeast autolysate as a nitrogen source and 0.05% $MnSO_4$ at pH 7.5.

• 한국미생물·생명공학회지
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• 제18권5호
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• pp.454-459
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• 1990

식물근부균 Fusarium solani의 생육을 강력히 길항하는 생물방제균 Pseudomonas stutzeri YPL-1에 외부유전자 도입을 통한 유전공학적 육종방법의 기초를 확립하고자 하였다. 이를 위해 plasmid pKT230을 vector로 하여 형질전환의 가능성을 조사하였으며 이때, 혈질전환에 필요한 최적조건을 조사한 결과 P.stutzeri YPL-1의 형질전환에는 대수증식기 초기의 균체가 가장 적합하였고, 20mM RbCl과 100mM $CaCl_2$를 함유한 냉각용액에 1${\mu}g$/ml의 plasmid DNA를 첨가하였을 때 최대의 형질전환 빈도를 나타내었다. 또한 plasmid DNA와 competent cell를 혼합한 후 $0^{\circ}C$에서 60분간 처리하는 것이 가장 효과적이었으며 이와 같은 조건에서 형질전환 빈도는 2~$6 \times 10^{-6}$으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.335-341
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• 2017

The complexity of the bacterial recombination system is a barrier for the construction of bacterial mutants for the further functional investigation of specific genes. Several protocols have been developed to inactivate genes from the genus Pseudomonas. Those protocols are complicated and time-consuming and mostly do not enable easy construction of multiple knock-ins/outs. The current study describes a single and double crossover-recombination system using an optimized vector-free allele-exchange protocol for gene disruption and gene replacement in a single species of the family Pseudomonadaceae. The protocol is based on self-ligation (circularization) for the DNA cassette which has been obtained by overlapping polymerase chain reaction (Fusion-PCR), and carries an antibiotic resistance cassette flanked by homologous internal regions of the target locus. To establish the reproducibility of the approach, three different chromosomal genes (ncRNA31, rpoN, rpoS) were knocked-out from the root-associative bacterium Pseudomonas stutzeri A1501. The results showed that the P. stutzeri A1501 mutants, which are free of any plasmid backbone, could be obtained via a single or double crossover recombination. In order to optimize this protocol, three key factors that were found to have great effect on the efficiency of the homologous recombination were further investigated. Moreover, the modified protocol does not require further cloning steps, and it enables the construction of multiple gene knock-in/out mutants sequentially. This work provides a simple and rapid mutagenesis strategy for genome editing in P. stutzeri, which may also be applicable for other gram-negative bacteria.

• 한국환경과학회지
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• 제14권10호
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• pp.955-963
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• 2005

From sludge of S municipal wastewater treatment plant in Busan, Korea, we isolated the denitrifier DN-9 which showed the ability of denitrification under aerobic conditionby the color change and gas formation in liquid culture with Giltay medium. The isolated strain was identified as Pseudomonas sp. DN-9 on the basis of the morphological, physiological, biochemical and nucleotide sequence analysis of l6S rRNA. The isolated strain, Pseudomonas sp. DN-9, has cytochrome $cd_1$, nirS of nitrite reductase. By the co-existance of additional ammonium and nitrate ion, the strain was not affected largely on growth in SL series broth. It seemed the result of denitrification. Although Pseudomonas sp. DN-9 has a good nitrate reduction activity under aerobic condition, the activity is less than Pseudomonas stutzeri in same cultivation condition. However, Escherichia coli had little the activity of aerobic denitrification and Pseudomonas putida showed lower activity of aerobic denitrification than Pseudomonas sp. DN-9 and Pseudomonas stutzeri in this study.