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연구논문 : 생명과학 ; 대장균에서 발현시킨 글리포제이트 내성 콩의 5-enolpyruvylshikimate-3-phosphate 합성 효소는 거의 앨러지원성이 없음
장현성 ( Hyun Sung Chang ),김남희 ( Nam Hee Kim ),박명진 ( Myong Jin Park ),임시규 ( Si Kyu Lim ),김성철 ( Sung Chull Kim ),김지영 ( Ji Young Kim ),김정애 ( Jung Ae Kim ),오혜영 ( Hye Young Oh ),이추희 ( Chu Hee Lee ),허근 ( Keun 영남대학교 약품개발연구소 2003 영남대학교 약품개발연구소 연구업적집 Vol.13 No.-
Isolation of Streptomyces sp. YU100 Producing Extracellular Phospholipase D
LIM, SI KYU,CHOI, JAE WOONG,LEE, EUN TAG,KHANG, YONG HO,KIM, SANG DAL,NAM, DOO HYUN 영남대학교 약품개발연구소 2002 영남대학교 약품개발연구소 연구업적집 Vol.11 No.-
Soil samples were screened for actinomucete strains capable of producing phospholipase D, and a strain, Streptomyces sp. YU100, showing a high transphosphatidylation activity was isolated. This strain secreted phospholipase D in a culture broth after 12 h of cultivation, and its productivity continued to increases for 36 h of fermentation. In addituon, its transphosphatidylation rate of phosphatidylcholine to phosphatidylseine was almost 68% within 1 h. The morphological and chemotaxonomical characteristics showed that this strain could be classified as a number of the Streptomycetaceae family, particularly due to the spiral form of its spore chain consisting of 60-70 smooth spores (0.75×1.0??m) on an aerial mycelium, FA-2c type of fatty acid profile in the cell wall, and LL-DAP component in the cell wall peptidoglycan. A phylogenctic analysis of the 16S rDNA provided a clue that the strain YU100 was actually a member of the genus Streptomyces, because the determined sequence exhibited a higher homology with Streptomyes sp. ASB27, S. peucetius JCM9920, and S. griseus ATCC10137. A dendrogram based on the 16S rDNA sequences also showed a phylogenetic relationship between the strain YU100 and these strains. However, the strain YU100 has not yet been assigned to a particular species, because of absence of any other classified species with a high matching score.
Production and Characterization of Extracellular Phospholipase D from Streptomyces sp. YU100
LIM, SI KYU,CHOI, JAE WOONG,CHUNG, MIN HO,LEE, EUN TAG,KHANG, YONG HO,KIM, SANG DAL,NAM, DOO HYUN 영남대학교 약품개발연구소 2002 영남대학교 약품개발연구소 연구업적집 Vol.11 No.-
Using Streptomyces sp. YU100 isolated from Korean soil, the fermentative production of phospholipase D was attempted along with its purification and characterization studies. When different carbon and nitrogen sources were supplemented in the culture medium, glucose and yeast extract were found to be the best. By varying the concentration of nutrients and clacium carbonate, the optimal culture medium was determined as 2.0% glucose, 1.5% yeast extract, 0.5% tryptone, and 0.3% calcium carbonate. During cultivation, the strain secreted most of the phospholipase D in the early stage of growth within 24 h. The phospholipase D in the early stage of growth within 24 h. The phospholipase D produced in the culture broth exhibited hydrolytic activity as well as transphosphatidylation activity on lecithin (phosphatidylcholine). In particular, the culture broth showed 8.7 units/ml of hydrolytic activity when cultivated at 28℃ for 1.5 days. The phospholipase D was purified using 80% ammonium sulfate percipitation and DEAE-Sepharose CL-6B column chromatography,which produced a major band of 57kDa on a 10% SDS-polyacrylamide gel with purity higher than 80%. The enzyme showed an optimal pH of 7 in hydrolytic reaction, and at pH 4 in a transphosphatidylation reaction. The enzyme activity increased until the reacton temperature was elevated to 60℃. The enzyme was relatively stable at high temperatures and neutral pH, but significantly unstable in the alkaline range. Among the detergents tested as cmulsifiers of phospholipids, the highest enzyme activity was observed when 1.5% Triton X-100 was employed. However, no inhibitory effect by metal ions was detected. Under optimized reaction conditions, the purified enzyme not only completely decomposed PC to phosphatidic acid within 1 h, but also exhibited higher than 80% conversion rate of PC to PS by transphosphatidylation within 4 h.
Membrane Transporter Genes in Cephabacin Biosynthetic Gene Cluster of Lysobacter lactamgenus
NAM, DOO HYUN,LIM, SI KYU,CHUNG, MIN HO,LEE, EUNG SEOK,SOHN, YOUNG SUN,RYU, DEWEY D. Y. 영남대학교 약품개발연구소 2001 영남대학교 약품개발연구소 연구업적집 Vol.11 No.-
In order to clone the peptide syntherase gene from Lysobacter lactamgenus IFO 41,288. the gene fragments were amplified using primers for the adenylation domain and the thionylation domain of the peptide synthetase genes in other organisms by polymerase chain reaction (PCR). The resulting 0.5-kb fragment was cloned in a pGEM-T vector, and the nucleotide sequences were determined. Six different PCR products were obtained; three were identified to be a part of L-∝-aminoadipyl-L-cysteinyl-D-valine (ACV) synthetase and three to be other peptide synthetases. Using each of the two different classes of PCR products as mixed probes, a cosmid library of L lactamgenus chromosomal DNA constructed in a pHC79 vector was screened by an in situ hybridization procedure, and one positive clone was selected which was bound by peptide synthetase gene fragments as well as ACV synthetase gene fragments. The partial sequence analysis from the obtained pPTS-5 cosmid showed the presence of more than two open reading frames. These were for two putative membrane transporters, which were homologous with several integral membrane proteins including the ABC transporter ATP-binding protein of E. coli (UbjZ)and the metal ion uptake protein of Bacillus subilis (YvrN). A45% homology was also found between the two transporter proteins at the carboxy terminus. Through a hydropathy analysis and transmembrane analysis, 4-5 transmembrane domains were found in these two proteins. When the genes were expressed in Escherichia coli, the gene products inhibited the host cell growth, probably due to the disturbance of the membrane transport system.
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.
Allergenicity Test of Genetically Modified Soybean in Sprague Dawley Rats
Chang, Hyun Sung,Bae, Youn Kyoung,Lim, Si Kyu,Jeong, Tae Cheon,Kim, Hyung Soo,Chung, Seung Tae,Kim, Dong Sup,Nam, Doo Hyun 영남대학교 약품개발연구소 2001 영남대학교 약품개발연구소 연구업적집 Vol.11 No.-
Allergenicity of genetically-modified (GM) soybean was evaluated in male Sprague Dawley rats. To confirm the GM soybean used in this study, the polymerase chain reaction (PCR) was performed using the chromosomal DNA of soybeans. The PCR result provided the clear discrimination of genetically-modified (GM) soybeans. To evaluate the allergenicity of GM soybean and non-GM control one, the soybean homogenate was sensitized subcutaneously 3 times a week for 3 weeks. The doses of soybean were 0, 2 and 20 mg/kg of soybean homogenate containing 1% Evans blue, no sign of passive cutaneous anaphylaxis reaction was detected. In addition, when the sera were treated in the cultures of peritoneal mast cells, the increase of histamine release by anti-(GM soybean) sera was not observed when compared to that by anti-(non-GM soybean) sera. The present results indicate that the GM soybean might not act as a strong allergen in male Sprague Dawley rats.
전병권,박성대,임시규,박완 한국산업미생물학회 1993 한국미생물·생명공학회지 Vol.21 No.5
대장균의 온도 감수성 형태형성 변이주 TMM 23(mrdAts)와 TMM 24(mrdBts)는 형태 변이가 일어나는 고온조건하에서 특이적으로 sarkosyl, SDS, Na-DOC와 같은 음이온성 계면활성제에 의해 생육이 저해되었으며 그 작용 약식은 살균적이었다. 각 균주의 변이형에 대한 sarkosyl의 최소생육저지종도 및 최소사멸농도는 동일하였으며 TMM 23에 대해서는 0.1%, TMM 24의 경우는 0.3%이었다. 생육저해작용이 없는 중성 계면활성제 Triton X-100에 의해서는 형태변화에 관계없이 생세포로부터 단백질이 거의추출되지 않았으나, sarkosyl에 의해서는 TMM 23(mrdAts), TMM 24(mrdBts) 모두, 변이형인 구균형의 생세포는 야생형인 간균형에 비해 맣은 양의 단백질이 추출되었다. 또, SDS-polyacryamide gel 전기영동에 의한 막단백질의 분석 결과, 간균형 세포의 외막에 다량 존재하는 주요 외막 단백질들이 구균형의 세포에서는 소량 밖에 보이지 않았으며, 내막의 여러 단백질의 조성의 변화도 관찰되었다. 이는 형태형성 변이주의 막구조에 물리 화학적인 성질의 차이가 있음을 나타내는 것으로 대장균 세포의 형태형성 기구는 peptidoglycan 뿐만이 아닌 내, 외막을 포함하는 세포표층의 전 구조체가 관여하고 있을 가능성을 시사하고 있다. Escherichia coli FmrdAts and mrdBts mutants forming spherical cells at 42℃, were employed to investigate the possible role of both inner and outer membrane structures in the determination of cell shape of gram-negative cells. Spherical cells, but not rod-shaped wild types, were specifically killed by anionic detergents, such as sarkosyl, sodium dodecylsulfate and sodium deoxycholate. From the spherical intact cells grown overnight at 42℃, much more proteins were released by sarkosyl. However, nonionic detergent like Triton X-100 which showed no inhibition of growth of spherical cells extracted little amounts of proteins from spherical intact cells as well as from rod-shaped cells. Analyses of membrane fractions of spherical cells by SDS-polyacrylamide gel electrophoresis showed that there were apparently reduced amounts of major outer membrane proteins and also some changes in the pattern of inner membrane proteins. Based on these results showing the differences in physicochemical properties of the membrane structures of spherical and rod-shaped cells, we suggest that the determination of cell shape in E. coli cells might be associated with both outer and inner membrane structures along with peptidoglycan.