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pBRG-4를 이용한 Metarhizium anisopliae의 형질전환
이동규,예완해,황철원,권석태,강선철,Lee, Dong-Gyu,Yeh, Wan-Hae,Hwang, Cher-Won,Kwon, Suk-Tae,Kang, Sun-Chul 한국응용생명화학회 1998 Applied Biological Chemistry (Appl Biol Chem) Vol.33 No.2
작물 병해충에 대한 무공해 미생물 농약 개발의 일환으로 곤충 병원성 곰팡이 Metarhizium anisopliae의 분자생물학적 육종을 위해 이 균주의 형질전환계를 확립하였다. M. anisopliae의 원형질체를 제작하여 benomyl 약제에 대하여 저항성을 나타내는 Aspergillus flavus 유래의 ${\beta}-tubulin$ 유전자를 갖는 pBRG-4 plasmid DNA를 polyethylene glycol 방법으로 형질 전환하였다. 이 형질전환은 pBRG-4 DNA $50\;{\mu}g$당 10개의 효율로 이루어졌으며, 그 결과 야생형 균주들의 $2.5\;{\mu}g/ml$ 농도의 benomyl 존재 하에서도 성장이 억제되는데 반하여 선발된 형질전환체들은 $5.0\;{\mu}g/ml$ 농도의 benomyl 함유 배지에서도 잘 성장하였다. 또한 이 형질전환체들의 chromosomal DNA를 분리하여 Southern 분석한 결과 ${\beta}-tubulin$ 유전자가 homologous recombination에 의하여 M. anisopliae의 genome속에 삽입 되었음을 확인하였다. We have established a transformation system for entomopathogenic fungus, Metarhizium anisopliae, in order to develop mycoinsecticide by recombinant DNA techniques. Protoplasts of M. anisopliae would be transformed to a benomyl-resistant by introducing pBRG-4 plasmid DNA, which contains a ${\beta}-tubulin$ gene of Aspergillus flavus conferring resistance to benomyl and a pyr4 gene of Neurospora crassa, in the presence of 5% polyethylene glycol and 10 mM calcium chloride. Transformants occuring at a frequency of 10 colonies per $50\;{\mu}g$ pBRG-4 DNA grew on the $5\;{\mu}g/ml$ concentrations of benamyl, while the wild type was inhibited by $2.5\;{\mu}g/ml$. From the Southern analysis using genomic DNAs isolated from M. anisopliae transformants, the positive signals suggested that the ${\beta}-tubulin$ gene had integrated in the M. anisopliae genome by homologous recombination.
pBRG-4 를 이용한 Metarhizium anisopliae 의 형질전환
이동규,예완해,황철원,권석태,강선철 ( Dong Gyu Lee,Wan Hae Yeh,Cher Won Hwang,Suk Tae Kwon,Sun Chul Kang ) 한국응용생명화학회 1998 Applied Biological Chemistry (Appl Biol Chem) Vol.41 No.3
We have established a transformation system for entomopathogenic fungus, Metarhizium anisopliae, in order to develop mycoinsecticide by recombinant DNA techniques. Protoplasts of M. anisopliae would be transformed to a benomyl-resistant by introducing pBRG-4 plasmid DNA, which contains a β-tubulin gene of Aspergillus flavus conferring resistance to benomyl and a pyr4 gene of Neurospora crassa, in the presence of 5% polyethylene glycol and 10 mM calcium chloride. Transformants occuring at a frequency of 10 colonies per 50 ㎍ pBRG-4 DNA grew on the 5 ㎍/㎖ concentrations, of benamyl, while the wild type was inhibited by 2.5 ㎍/㎖. From the Southern analysis using genomic DNAs isolated from M. anisopliae transformants, the positive signals suggested that the β-tubulin gene had integrated in the M. anisopliae genome by homologous recombination.
Micrococcus sedentarius KL-37이 생성, 분비하는 Alkaline Proteases의 정제 및 성질에 관한 연구
이동규,강선철 대구대학교 (한사대학) 산업기술연구소 1994 産業技術硏究 Vol.13 No.-
Micrococcus sedentarius KL-37, an alkalophilic bacterium isolated from soil, produces and secretes at least three distinct proteases to the growth medium. Two of them, protease I and II, characterized in more details for their enzymatic properties were homogeneously purified through ammonium sulfate fractionation and CM-cellulose column chromatography but the other, protease III, was only partially purified even after a further purification step like DEAE-cellulo column chromatography. The molecular weights of protease Iandd II were 35,000 and 14,000 daltons, respectively, being judeged by SDS-PAGE. The enzymes revealed their maximum activities at pH 11l0 and 40℃, and were completely inactivated by PMSF and EDTA withil serine-metallo protease. And the effects of divalent ions on hese enzymes showed that the enzyme were activates by Mg_(++), Ca_(++) and Co_(++), while completely inactivated by Fe_(++), in the 10mM concentration of those ion.
이동규,강선철 대구대학교 산업기술연구소 1991 産業技術硏究 Vol.10 No.-
A coccal bacterium excreting a large quantity of proteases to an alkaline growth medium was isolated from soil and throughly identified. The isolate was Gram-positive, coccal, tetrad and strictly aerobic bacterium. And so it is suggested that the bacterium might belong to the genus Micrococcus. Also according to the data of the physiological and biochemical analyses performed by the dichotomous identification scheme, the isolated bacterium could be deduced to be a setrain of Micrococcus sedentarius. And then this strain is designated M. sedentarius KL-37.
Penicillium sp. GL-101 의 액침배양중 Mycelial Pellet 크기에 영향을 주는 배양조건 및 첨가물
이동규,강선철,하철규,이태근 한국농화학회 1999 Applied Biological Chemistry (Appl Biol Chem) Vol.42 No.3
In order to minimize the mycelial pellet formation, one of the critical obstacles during the fermentation processes of filamentous fungi, an investigation was focused on the culture conditions(media and initial inoculum) and additives(soils, surfactants and polyethylene glycol 200) when a high phosphate-dissolving fungus, Penicillium sp. GL-101, was cultured in liquid media. Culturing the strain in PDB, SDB and YPD media, their pellet sizes decreased to the order of YPD $gt; SDB $gt; PDB. And at the high concentrations of the initial inoculum in the range from 1×10³ to 1×10^6 conidia/㎖, the small sizes of pellet were formed in the PDB media. For the initial inoculum between 1x10^7 and 1x10^8 conidia/㎖, however, an amorphous pellet or loose aggregate was formed. The addition of soils, zeolite and diatomite, up to 1.0% decreased the pellet sizes to 3/4 and 1/2, respectively, but the pellet was increased to 2.5 times by the addition of bentonite. Surfactants also affected on the size of pellet; the addition of Triton X-100 and Tween 80 up to 1.0% decreased the pellet sizes maximally to 1/10 and 1/4, respectively, while SDS completely inhibited the fungal growth. Among the four additives tsted, polyethylene glycol 200 was the most effectively reduced the pellet sizes to 0.2±0.1 ㎜ that resulted in about 25- fold reduction compared to the control.
호알칼리성 단백질 분해 균주 Bacillus sp. TUB 25 의 최적성장에 관한 연구
양재섭,최명철,이동규,강선철 대구대학교 기초과학연구소 1989 基礎科學硏究 Vol.6 No.-
The optimal growth conditions for Bacillus sp. TUB 25 to secrete a large amount of alkaline protease were investigated. The bacterium was reached to maximum growth level in the culture medium containing 1.0% glucose, 0.5% yeast extract, 0.5% peptone, 0.1% K₂HPO₄, 0.02% MgSO₄, 1.0% Na₂CO₃ and 1.0% NaCl. By addition of Na+, the growth of the strain TUB 25 was highly stimulated. And the growth curve showed the highest optical density when Bacillus sp. TUB 25 cultivated at 37℃ for 24 hours. It was characteristic of that the growth of Bacillus sp. TUB 25 was very excellent in alkaline media, and the optimal pH for the growth was about 10.0∼11.0. The relationship between growth and extracellular protease production of the strain TUB 25 showed that protease activity was not detected during the lag and the exponential phase, but was detected during the early stationary phase.