낙동강의 조류 및 방선균에 의한 이취 특성 연구

양상용 동국대학교 2005 국내박사

Water serves human and living organisms as a basic constituent. It is also essential material in agricultural, industrial as well as in service areas. Besides, as a key element for the maintenance of biological activity, we cannot think of life without water. Daily average consumption of water in Korea was estimated as 360ℓ per day per person. Common criteria for the water quality standards are color, turbidity, taste and odor. Especially for drinking water, odor criterion should be strictly maintained to eliminate public distrust to drinking water. Some of the biological source of odor from water are algae, actinomycetes, decaying organic matter, and secretion of metabolites. Among them, odors related to the growth of algae has been known for long times. Nackdong river is rich in nutrients which support growth of algae. Therefore, diatom and cyanobacterial bloom recur every year in the Nakdong river. Moreover, there is also potentiality of natural actinomycetes odor which also needs microbiological investigation. In this study, water quality, dominant algae, actinomycetes, geosmin and 2-MIB, the major known odor materials were investigated from January to December 2003 at 6 sites in the Nackdong river which serves as an important source of drinking water around the Youngnam region. Besides, odor producing actinomycetes were isolated from the Nackdong river, identified and their growth and production of geosmin and 2-MIB were characterized. Conclusions derived from the results of the investigation are summarized as follows. 1. Among the algae occurred in the Nackdong river, major species of diatom were in the genus Stephanodiscus, Aulacoseira, Synedra Nitschia, major green algae was Actinastrum, major blue green algae were Microcystis and Lyngbya, and dominant flagellate algae was Cryptomonas. Especially, diatom in the genus Stephanodiscus was dominant during the low temperature drought season in the down stream stations. 2. Counts of actinomycetes in the Pungji site (upstream) were 22 CFU/㎖ on average with maximum value of 60 CFU/㎖. The counts in the Waekwan site (midstream) were 22 CFU/㎖ on average with maximum value of 70 CFU/㎖. As a whole the counts of actinomycetes in the up-and midstreams were higher than those in the downstream sites. In April, June, and September, counts of actinomycetes were increased presumably due to the rainfall which may have washed the soil inhabiting actinomycetes off to the river Little relationship were found between the counts and environmental factors such as temperature, pH, DO, COD, Chlorophyll-a, and nutrients (nitrate, TN, soluble phosphate, TP). 3. The odor materials, geosmin and 2-MIB were mainly detected in mid-and downstream sites, Koryoung, Namji, Mulkeum, and westnackdong-river during summer and autumn seasons along with the occurrence of odor producing algae such as Anabaena, Lyngbya and Oscijlatoria. Highest concentration of geosmin was found at Mulkeum site in September (202.3 ng/ℓ) and highest concentration of 2-MIB was found at Namji site in July(425.2 ng/ℓ). However both compounds were not detected from January to March when the diatom Stephanodiscus was dominant. 4. Odor producing microorganisms were isolated from the Nackdong river and identified according to their morphological (mycelium, spore),biochemical (carbon source assimilation), chemotaxonomical (structure of diaminopimelic acid, cell membrane fatty acid composition) and molecular biological (sequence of 16SrDNA) characteristics. Results of the tests for the taxonomic identification indicate that all 18 isolates belongs to the genus Streptomyces. The isolates were named as Streptomyces sp. YSY01~ YSY18. 5. Optimal pH for the growth of isolates was 7.0~7.9 and optimal temperature for growth was 20~25℃. Growth were severely retarded at temperatures higher than 30℃ and lower than 10℃. 6. When water sampled from Nackdong and Keumho river was inoculated with washed mycelia of the isolates such as Streptomyces sp. YSY01, small amount of geosmin and 2-MIB were detected temporarily. However, the conditions did not support the growth of mycelium. On the contrary, when starch was added to the filtrates of water from Nackdong river, Keumho river and Seungse stream and inoculated with the spores of the isolates, growth of the culture and production of geosmin and 2-MIB were increased in proportion to the amount of added starch. 7. Control solution of geosmin and 2-MIB (1,000 ng/ℓ) did not inhibit the luminescence of luminescent bacteria (Vibrio fischerii NRRL-B-1177). However, culture solution of the isolated actinomycetes exerted inhibition to the luminescent bacteria according to the increase in the amount of the mycelium. The results indicate that the luminescence inhibition toxicity of the Streptomyces culture to Vibrio fischerii NRRL-B-1177 may be caused by unknown metabolites rather than the effect of geosmin or 2-MIB.

湖沼水에서 분리한 放線菌의 異臭味誘發에 관한 硏究

金愛卿 전남대학교 1999 국내석사

최근 수질의 악화에 따른 상수원에서의 곰팡이 냄새발생은 수질관리에 많은 문제가 되고 있다. 호소에서의 이취미의 발생은 대부분 수중미생물에 의한 것으로 곰팡이 냄새의 원인생물인 방선균을 저수지에서 분리, 배양하여 여기서 생성하는 냄새원인물질을 확인, 정량하고 곰팡이 냄새 유발 방선균의 성장조건을 조사하여 방선균에 의한 이취미 유발 정도 등을 알아보는데 본 연구의 목적이 있다. 동복호와 덕남저수지에서 분리한 방선균은 모두 27개로 이중 6개의 菌株에서 곰팡이 냄새를 확인했다. 상수원인 동복호 원수에 곰팡이 냄새를 유발하는 방선균을 배양한 결과, 방선균의 생장 및 냄새강도가 매우 낮았으나, 여기에 탄소, 인, 질소분을 첨가한 배양액에서는 뚜렷한 균의 증식과 강한 냄새를 발생하였다. 방선균에 의한 곰팡이냄새의 원인물질은 2-MIB와 Geosmin인 것으로 확인되었으며, 6개의 균주 모두에서 두가지 물질이 다 검출되었다. 이때 냄새물질의 농도는 DBL-2 배양액에서 2-MIB가 최고 3,823.7 ng/ℓ로, Geosmin의 농도는 최고 4,729 ng/ℓ로 측정되었으며, BS배지에서는 2-MIB가 85,755.4 ng/ℓ, Geosmin은 366 ng/ℓ로 검출되어 방선균에 의한 곰팡이냄새의 유발정도가 매우 높은 것으로 조사되었다. 방선균은 30∼40℃에서 잘 생장하며 pH는 7∼9의 약알카리성에서 잘 자라는 것으로 조사되었다. 뿐만아니라 C, N, P등의 영양염이 방선균의 증식과 냄새유발에 필요한 것으로 밝혀졌으며 탄소나 질소 인 등의 농도에 비례하여 균체량도 증가하는 양상을 나타냈다. Nowadays, the musty smell originated from water pollution in raw water has arisen so many problems in water management. This study was carried out to investigate the growth conditions of actinomycetes inducing the odor, the degree of the odor produced, and the habitual environment in Dongbok and Duknam reservoir. Among 27 strains of Actinomycetes, 6 strains were used to induce the musty smell. The substances causing the musty odor were Geosmin and 2-MIB. They can induce the odor from strains in individual or combination. Using water service in Dongbok and Duknam reservoir, were the cultivate Actinomycetes inducing the odor of the growth and thresholds is very low. When carbon, phosphorous and nitrogen were added into the raw water to make cultivating solution, the growth of Actinomycetes was observed and caused. The substance causing the musty odor was confirmed to be Geosmin and 2-MIB. The highest concentrations of 2-MIB and Geosmin were 3,823.7ng/ℓ and 4,729ng/ℓ respectively in a culture solution of DBL-2. The maximum concentrations of 2-MIB and Geosmin were 85,755.4ng/ℓ and 366ng/ℓ respectively in a culture solution of BS medium. As the results the musty order caused Actinamycetes was significantly high. Actinomycetes were well grown on the condition of pH 7∼9, weak alkaline state, and temperature 30∼40℃. Furthermore, the nutrient containing C, N and P was necessary on the multiplication of Actinomycetes and the induction of the odor.

韓國 논 土壤에서 放線菌의 分布狀態와 生理的特性에 관한 硏究

박경수 朝鮮大學校 大學院 1987 국내박사

Actinomycetes are widely distributed in the soils, and sometimes they occupy larger part of the soil microbial population. This study was conducted to find out distribution of actionomycetes and soil microflora of the paddy soils distributed in Korea. The samples were collected from the different paddy soil areas to find relationships between soil physicochemical properties and soil microorganisms. The seasonal population of actinomycetes in paddy soils with hay compost and inorganic fertilizer has been investigated. Having isolated actinomycetes by using selective media, the population densities of actinomycetes in paddy soils were examined. According to the result of several morphological observations, similar strains were classified into 26 groups, and detailed taxonomic characterization on the isolated strains and antibiotics sensitivity test on actinomycetest isolates have been carried out, respectively. The results are summarized as follows ; The mean number of soil microflora in collected paddy soils were 2.58 × 10^(6) cells/g soil in actinomycetes (A), 1.20 × 10^(7) cells/g soil in bacteria (B), and 3.18 × 10^(4) cells/g in fungi (F). The ratios of B/A, A/F and B/F were 5.1., 84.0 and 387.1, respectively. The numbers of actinomycetes and soil microorganisms were the least in Mountainous area and Gyengi bay, whereas it revealed the most in Honam plain and Southern part area. The significant correlations were obtained among the numbers of actinomycetes, soil microorganisms and soil chemical properties ; available phosphorous, K^(+), Mg^(+), T-C and pH of soil. The seasonal population of actiomycetes reached a range of 2.1 × 10^(6) cells/g soil to 7.4 × 10^(7) cells/g soil. The composition of actinomycetes flora changed considerably after the hay compost had applied. The significant positive correlations between the organic matter contents in paddy soils and the actinomycetes populations were shown at 1.38 to 2.69 level. In the isolated 250 actinomycetes strains, twenty groups of Streptomyces were 87.2% and six groups of Non Streptomycetes were 12.8 %, respectively. Streptomyces with sporophore of the spiral chain form reached to 85 % of all the Streptomyces isolates. Surface morphology of spores has been ditermined with the electron microscope ; one group has a hairy surface, three groups have a spiny surface, and sixteen groups have a smooth surface. Sreptomyces groups were indentified as S. bobilli, S. albus, S. almquisti, S. rimosus, S. sp. , S. nigrifaciens, S. achromogenes, S. grtseosporeus, S. eurythermus, S. sendaiensis, S. chibaensis, S. chromofuscus, S. parvullus, S. arenae, S. albaduncus, S. sp. , paucidiastaticus, S. aurigineus, S. sp. Non-Streptomyces groups of A-1, A-2 and A-3 were identified as Nocardia sp. , and the strains of others groups remained uncertain. Actinomycetes isolates were selected as the strains having predominant antibacterial activities against the test organisms among the 26 groups which have antibacterial activities. Selected actinomycetes isolates showed high antibiotic sensitivity of Streptomyces sendatensis (8.56 r/㎖), Streptomyces achro mogenes (5.23 r/㎖), and Streptomyces albus (7.46 r/ ㎖) against B. subtilis E. coli and S. aureus.

化學肥料 및 有機質肥料施用 時 土壤 중 放線菌의 生育比較에 관한 硏究

홍사현 建國大學校 大學院 1996 국내석사

土壤에 天然有機物, 化學肥料, 그리고 有機物醱酵肥料 施用이 放線菌 및 土壤微生物相의 構成變化에 미치는 影響과 放線菌의 屬 특히, Streptomyces 屬을 觀察하여 다음과 같은 結果를 얻었다. 1. 土壤중 放線菌의 菌數는 크로버施用區, 볏짚施用區, 對照區, 化學肥料施用區 그리고 有機物 醱酵肥料施用區의 順으로 낮았으며,크로버施用區가 化學肥料施用區 보다 約 10배 정도 많았다. 로로버와 볏짚施用區는 7일째, 他施用區는 14일에 最大値에 달하였다. 2. 土壤중 全細菌數는 크로버施用區, 볏짚施用區, 有機物 醱酵肥料施用區, 對照區 그리고 化學肥料施用區 의 順으로 낮았으며, 크로버施用區가 7일까지 增加하다 서서히 減少한 반면 볏짚施用區는 14일째 減少했으나, 다시 增加해 28일까지 增加했다. 3. 土壤중 곰팡이수는 크로버施用區, 볏짚施用區, 對照區, 有機肥物 醱酵肥料施用區 그리고 化學肥料施用區 順으로 낮았으며, 全施用區가 7일까지 增加하다 顯著히 減少하기 始作했다. 4. 土壤중 胞子形成細菌의 數는 크로버施用區, 볏짚施用區, 化學肥料施用區, 對照區 그리고 有機物 醱酵肥料施用區의 順으로 낮았으며, 有機物 醱酵肥料施用區의 數値가 가장 낮다. 크로버施用區는 35일째 增加하였으나, 그후 顯著히 減少했고 볏짚은 계속 增加했다. 5. 土壤중 그람陰性細菌의 數는 볏짚施用區, 크로버施用區, 有機物醱酵肥料施用區, 對照區 그리고 化學肥料施用區 의 順으로 낮았으며, 化學肥料, 有機物 醱酵肥料施用區는 7일 이후부터 顯著한 減少를 보이고, 볏짚施用區은 점진적인 增加를 보여준다. 6. 土壤pH의 數値는 크로버施用區, 化學肥料施用區, 볏짚施用區, 對照區 그리고 有機物 醱酵肥料施用區 의 順으로 낮았으며, 크로버와 化學肥料施用區는 7일 이후부터 점진적으로 減少하며 有機物醱酵肥料施用區의 pH가 가장 낮다. 7. 實驗區로부터 選別한 放線菌의 約 70%는 Streptomyces屬 이었으며 Nocardia屬이 그 다음으로 많았다. 8. 土蘿內 Streptomyces屬의 營養菌絲의 色素分泌 分布는 yellow-brown이 가장 많았고 다음이 red-orange, violet順 이었고氣中菌絲의 色은 흰색과 회색이 대부분이었다. This study has been conducted to investigate the periodic change of soil microflora as well as actinomycetes, particularly Streptomyces spp. when organic materials and chemical fertilizer were treated in soil. The results obtained from this study are as follows: 1. The number of actinomycetes was high in order of clover plot, straw plot, control plot, chemical fertilizer plot and commercial compost plot in incubated soil. The number of actinomycetes in clover plot was increased by 10 times comparing with chemical fertilizer plot at the 7th day. 2. The number of total bacteria was high in order of clover plot, straw plot, commercial compost plot, control plot and chemical fertilizer plot in incubated soil. The number of bacteria in straw plot were not decreased so quickly as that in clover plot. 3. The number of fungi was high in order of clover plot, straw plot, control plot, commercial compost plot and chemical fertilizer plot in incubated soil. The number of fungi in all plots was increased by the 7th day and after that day decreased remarkably. 4. The number of spore-forming bacteria was high in order of clover plot, straw plot, control plot, chemical fertilizer plot and commercial compost plot in incubated soil. Commercial compost plot showed the lowest number. 5. The number of gram-negative bacteria was high in order of straw plot, clover plot, commercial compost plot, chemical fertilizer plot and control plot in incubated soil. The straw plot only showed the steady increasing number by the 49th day. 6. The change of pH was increased in high of clover plot, chemical fertilizer plot, straw plot, control plot and commercial compost plot in incubated soil. Commmercial compost plot had the lowest value and the value of clover and chemical fertilizer were decreased remarkably after the 7th day. 7. The percent of Streptomyces spp. of actinomycetes from each treated soils is about 70% and which is highest value among other genus. Nocardia showed about 10% of distribution. 8. The Distribution of Streptomyces spp. according to substrate mycelium color was high in order of yellow-brown, red-orange, violet and green. The aerial mycelium is mostly white and gray in color.

대나무 군락지 내 방선균 계통학적 다양성 및 신종 방선균 분류·동정

이효진 목원대학교 대학원 2015 국내박사

생명산업분야에 널리 활용되고 있는 방선균 유전자원 다양성 확보를 위하여 차세대 분자기법인 pyrosequencing 염기서열 해석을 통하여 대나무 군락지 내에 분포하는 방선균 군집의 계통학적 다양성을 검토하고 분리된 방선균의 분류학적 특성을 조사함과 동시에 다상분류법에 의거하여 신종 방선균의 분류·동정을 수행하였다. 국내 자생하는 왕대, 분죽, 조릿대, 호마죽과 같은 다양한 대나무 군락지의 낙엽층 및 근권토양 내에 분포하는 방선균 밀도를 측정한 결과 2.7×106~2.7×108 CFU/g로 계수되었다. 특히, 조릿대 대나무 군락지의 낙엽층 내에는 2.7×108 CFU/g로 본 연구에서 조사한 다양한 토양에 비해 10~100배 이상의 높은 밀도를 나타내었다. 조릿대 대나무 군락지 내 방선균 군집의 계통 해석을 위해 454 pyrosequencing 기법으로 염기서열을 분석한 결과, 6목(目) 36과(科) 121속(屬)을 포함하는 다양한 방선균이 분포해 있음을 확인하였다. 방선균 군집 중 Actinomycetales 목에 속하는 계통군이 전체의 80% 이상 높은 분포율을 나타내었다. 우점 방선균군인 Actinomycetales 목에 속하는 방선균은 26과 111속으로 분류되었다. 조릿대 대나무 군락지 내에 분포하는 다양한 방선균을 분리하기 위해 방선균 분리법에 널리 이용되고 있는 매뉴얼법에 따라 총 200균주를 분리하고 방선균 콜로니형태(기균사, 기중균사, 수용성 색소)를 관찰한 결과, 25개 type의 방선균 콜로니 형태그룹으로 분류되었다. 이들 분리균주의 16S rRNA 유전자 염기서열 해석 결과, 모든 균주가 Streptomyces 속에 속하는 방선균으로 나타났다. 다양한 방선균 확보를 위해 토양시료를 습열(55℃, 65℃)전처리 한 후, 4종류의 다양한 배지를 사용하고 배양기간 및 배양조건 등을 고려하여 총 300균주를 분리한 결과, 45개 type의 방선균 콜로니 형태그룹으로 분류되었다. 이들 분리균주의 16S rRNA 유전자 염기서열 해석 결과, Asanoa, Actinomadura, Catenulispora, Dactylosporangium, Gordonia, Kibbella, Microbispora, Micromonospora, Nonomuraea 그리고 Streptomyces 속에 속하는 균주로 확인되었다. 이상, 상기의 비배양법에 의해 확인된 방선균 121속 중 개량화한 배양법으로 분리한 결과, 11 속이 성공적으로 분리되었다. 이들 분리된 11개 속 중 약 83%가 Streptomyces 속에 속하는 균주로, cluster I (20균주), cluster II (294균주), cluster III (102균주) 그리고 cluster IV (56균주)로 세분류되었다. 본 연구를 통해서 분리된 방선균 총 500균주의 16S rRNA 유전자 염기서열 해석 결과, 11속(屬) 56종(種)의 다양한 방선균 중 근연종과 98.7% 이하의 상동성을 나타내는 신종 방선균 20균주가 선발되었다. 이들 신종 방선균은 다상분류법에 의거하여 동정하였으며 국제미생물분류학회지(International Journal of Systematic and Evolutionary Microbiology, IJSEM)에 게재하였다. 신종 방선균 Streptomyces gramineus JR-43T (=KACC 15079T =NBRC 107863T), Catunulispora graminis BR-34T (=KACC 15070T =NBRC 107755T), Streptomyces graminilatus JL-6T (=KACC 16470T =NBRC 108882T), Streptomyces graminisoli JR-19T (=KACC 16472T =NBRC 108883T), Streptomyces rhizophilus JR-41T (=KACC 16580T =NBRC 108885T) 그리고 Streptomyces graminifolii JL-22T (=KACC 17180T =NBRC 109806T)는 국내 농업유전자원센터(KACC)와 일본 Biological Resources Center, Nite (NBRC)에 기탁·등록되었으며, 향후 생명연구자원으로 활용될 것으로 기대한다.

분자계통학적 방법을 이용한 호산성 방선균의 분류학적 연구

전수진 順天大學校 大學院 2004 국내박사

본 연구의 목적은 분자계통학적 분류방법을 이용하여 호산성 방선균의 계통분류학적 위치를 확인하여 Family Streptomycetaceae의 분류체계를 정립하는 기초 자료로 활용하고자 시도되었다. 대상균주는 Seong (1992)이 산성 토양과 광산 폐기물로부터 분리한 호산성 방선균을 수리 분류학적 연구를 하여 분류한 2개의 내중성 호산성 방선균과 4개의 절대적 호산성 방선균 군집의 대표 균주들을 이용하였다. 내중성 방선균군은 형태학적, 화학적 특성을 토대로 Streptomyces 속에 배정되었으며, 165 rBNA 염기서열 분석을 통해서도 확인되었다. CN732는 Streptomyces griseocarneus와 97.2%, Streptomyces malaysiensis 와는 97.1%의 유사도를 보여 이들과 뚜렷한 계통상 차이를 보여 신종으로 인정되어 Streptomyces yeonchonensis sp. nov.로 제안한다. 그러나 JL164는 Streptomyces mirabilis와 99.6%로 아주 유사하였다. 절대적 호산성 방선균군인 JL083, JL206, JL415, CN671은 독특한 pH 양상과 특유의 165 rDNA signature을 나타내고 가수분해 산물 (whole-organism hydrolysates)에서는 L,L-diarninopirnelic acid와 galactose, rhamnose를 함유하고 있었다. 따라서 이들은 유전형과 표현형상의 차이에 기초하여 새로운 종류의 Streptacidiphilus gen. nov.라고 명명한 기존의 제안을 확인하여 주었다. 한편 국내 분리 호산성 방선균인 CN671은 기존에 보고된 종들과 분류학적으로 확연히 구별되는 특징을 보였으므로 이를 Streptacidiphilus sunchonensis sp. nov. 라고 제안한다. Kitasatospora, Streptacidiphilus, Streptomyces 속의 구성원들은 다수의 주요 특징들을 공유하며, 165 rDNA tree에서 안정적인 단 계통 발생학적 가지(stable monophyletic branch)를 형성하였다. 그러므로 Streptomycetaceae 과 (Family Streptomycetaceae)의 기술 (description)은 Kitasatospora와 Streptacidiphilus species가 보여주는 성질을 설명할 수 있게 교정되어져야 한다고 제안한다. 다상적 분류 결과로부터 절대적 호산성 방선균들은 Streptomyces 계통으로부터 산성이라는 환경에 적응하여 분화되어 온 독특한 분류학적 위치를 차지하는 분류군임을 확인할 수 있었다. 한편 내중성 방선균들은 Streptomyces 속에 속한 것으로 나타났으며, 절대적 호산성 방선균과는 상이한 계통학적 분화과정에 의하여 형성된 분류군들로 사료된다. The purpose of this study was to determine the taxonomic positions of acidophilic actinomycetes using molecular systematic methods and to establish their taxonomic relationship among the members of the Family Streptomycetaceae. Six representative strains that grew optimally at pH 4.5 - 5 were chosen from two neutrotolerant clusters and four strictly acidophilic clusters defined by numerical taxonomy (Seong, 1992). The strains were tested against 299 unit characters by Seong (1992), and the phylogenic positions of the strains were investigated using 16S rDNA sequences and several tree-making algorithms. Fatty acid and menaquinone profiles of the strains were also analyzed. All of the tested strains exhibited streptomycete type morphological characteristics, as they formed extensively branching aerial and substrate mycelia and long chains of arthrospores at the tip of the aerial hyphae. In the analysis of membrane lipids, the major fatty acids were found to be 12-methyl tetradecanoic acid (anteiso-C_(15:0)), 14-methyl pentadecanoic acid (iso-C_(16:0)) and hexadecanoic acid (C_(16:0)), comprising 55 - 69 % of the total. The major isoprenoid quinones were menaquinones with hexa- and octahydrogenated nine isoprene Units (MK-9[H]_(6) & MK-9[H]_(8)). The neutrotolerant acidophilic actinomycetes were assigned initially to the genus Streptomyces on the basis of morphological and chernotaxonornic characteristics; this assignment was confirmed by the analysis of 165 rDNA gene sequence data. Strain CN732 formed a distinct phylogenic line within the Streptomyces tree. It was most closely related to Streptomyces griseocarneus DSM40004^(T)(97.2%) and Streptomyces malaysiensis ATB-11^(T)(97.1%) on the basis of 165 rRNA gene sequence data. Therefore, it merits recognition as a novel species, for which the name Streptomyces yeonchonensis sp. nov. is proposed. Strain JL164 was closely related to the type strain of Streptomyces mirabilis, as the two organisms shared 99.6% 165 rRNA gene sequence similarity. Four strictly acidophilic actinomycetes (JL083, JL206, JL415 and CN671) exhibited a distinctive pH profile, an unique 165 rDNA signature and contained major amounts of LL-diaminopimelic acid. This study confirms that the strict acidophilic actinomycetes merit recognition as a new genus, Streptacidiphilus, as proposed by the previous study. In addition, a new species is proposed to accommodate Korean isolate CN671 within the genus, namely Streptacidiphilus sunchonensis sp. nov. The strict acidophilic actinomycetes may have experienced distinct evolutionary processes, in which the major driving forces might be acidic environment and geographic separation.

土壤중의 리그닌分解 放線菌의 分離에 관한 硏究

박연호 建國大學校 大學院 1996 국내석사

각각의 논토양, 밭토양, 삼림토양으로 부터 리그닌分解 放線菌을 分離하기위해 이倂究는 實行되었다. 각각의 土壤에는 重量으로 3%의 리그닌을 添加한 후 30℃에서 80일간 培養하였다. 그리고, 한편으로는 리그닌을 添加한 最少培地에서 液體震湯培養으로 30℃에서 80일간 培養하였다. 각각의 微生物들을 計數하기위해서 平板培養法을 利用하였고, 한편, 炭素原으로서 리그닌을 利用하는 放線菌을 分離하기위해서 실리카 겔을 이용한 平板培養法을 使用하였다, 이 實驗으로 부터 얻은 몇몇의 結果는 다음과 같다. 1. 全菌數는 固體培養과 液體培養 둘다에서 리그닌을 添加한 土壞處理區가 리그닌을 添加하지않은 것보다 더 많았다. 固體培養한 것이 液體培養한 것 보다 全菌數는 더많았다. 2. 絲狀菌數는 固體培養에서 리그닌을 添加한 밭토양과 논토양은 리그닌을 添加하지 않은 것보다 더많았다. 그러나 리그닌을 添加한 삼림토양은 리그닌을 添加하지않은 것보다 絲狀菌數가 더적었다. 液體培養에서는 리그닌을 添加한 삼림토양이 絲狀菌數가 리그닌을 添加하지않은 것보다 더많았다. 그러나 리그닌을 添加한 논토양과 밭토양은 리그닌을 添加하지않은 것보다 80일동안의 대부분 적었다. 固體培養이 液體培養보다 絲狀菌의 數는 더많았다. 3. 放線菌數는 液體培養에서보다 固體培養에서 더많았다. 放線菌數는 80일동안 固體培養에서는 增加하는 傾向이었다. 液體培養에서는 20일 또는 40일에서 最大値였고, 그 이후에는 減少하였다. 放線菌數는 液體培養에서 보다 固體培養에서 더많았다. 4. 리그닌을 分解할 수 있는 放饍菌이 80일동안 固體培養된 후 리그닌을 添加한 밭토양과 리그닌을 添加하지 않은 밭토양에 서 發見되었다. 5. 固體培養과 液體培養에서 유기물의 분해로 생기는 암모니아의 發生에 의하여 pH는 약간씩 增加되는 경향이었다. 6. 固體培養에서 液體培養에서보다 有機物이 더많이 分解되었다. 리그닌을 添加한 것이 添加하지않은 것보다 有機物이 더 많이 分解되었다. This study was conducted to isolate lignin-decomposable actinomycetes from various soil samples ; a paddy soil, an up-land field soil, and a forest soil. After the addition of 3% lignin by weight to the each soil sample, the soil was incubated at 30℃ for 80 days. On the other hand, the minimal medium containing lignin was incubated under the liquid shaking cultural condition at 30℃ for 80 days. The agar plate method was used to count the number of each microorganism and the lignin- contained silica get plate method was used to isolate actinomycetes utilizing lignin as a sole carbon source. Some results obtained from this experiment are as follows : 1. In both solid and liquid cultures, the total number of microorganisms in the soil treated with lignin was more than that in the soil not treated lignin. The total number of microorganisms in solid culture was more than that in liquid culture. 2. In solid culture, the number of fungi in up-land field soil and paddy soil, treated with lignin, was more than that in up-land field soil and paddy soil, not treated with lignin. But the number of fungi in forest soil, treated with lignin, was smaller than that that in forest soil, not treated with lignin. In liquid culture, the number of fungi in forest soil, tredted with lignin, was more than that in forest soil, not tredted with lignin. But the number of fungi in up-land field soil and paddy soil, treated with lignin, was smaller than that in up-land field soil and paddy soil, not treated with lignin, in them, for most of 80 days. The number of fungi in solid culture was more than that in liquid culture. 3. The number of actinomycetes in solid culture was more than that in liquid culture. The number of actinomycetes tended to increase in solid culture for 80 days. The number of actinomycetes in liquid culture was maximum at 20 days or 40days, and, after those days, decreased. The number of actinomycetes in solid culture more than that in liquid culture. 4. After cultivation for 80 days, the lignin-decomposable actionmycetes were found in the up-land field soil of solid cultures treated with and not treated with lignin. 5. A few pHs in solid culture and liquid culture mostly tended to be increased by NH_(3) of decomposed orgarnic (matters). 6. The organic matters in solid culture was decomposed more than that in liquid culture, and was decomposed more in the soils treated with lignin than in them not treated with lignin.

방선균 유전자의 대장균에서의 이종발현에 관한 연구

강경재 인제대학교 대학원 2007 국내석사

메타게놈 숙주인 대장균에서 다른 세균으로부터 유래한 유전자들의 발현 장벽은 기능을 토대로 한 메타게놈학의 발전을 저해하는 가장 큰 문제점 중에 하나이다. G+C 함량이 높은 그람 양성 세균 (High G+C Gram positive bacteria)은 거의 모든 환경에 풍부하게 존재하고, 다양한 생리 활성 물질을 생산하기 때문에 신물질 발굴에 중요한 세균으로 인식됨에도 불구하고, 이들 유전자의 약 70% 정도는 대장균의 전사 단계, 적어도 대장균 주 시그마 인자 (major sigma factor)에 의해 발현되지 않는다고 알려져 있다. 본 연구에서는 G+C 함량이 높은 그람 양성 세균의 모델 유전자로서, 방선균 chiA (Chitinase, SCO5003), chiC (chitinase AAF43629), pro7432 (protease, SCO7432), degP (agarase, NP627674) 유전자의 발현을 대장균에서 각각 전사와 번역 수준에서 분석하였다. 플라스미드 유래의 프로모터의 영향을 최소화하기 위하여 클로닝 자리 양쪽에 전사 종결 신호(transcription terminator)가 삽입된 pUC21을 제조 하고, 각각의 모델 유전자를 이 플라스미드에 클로닝 하였다. 모든 모델 유전자들은 대장균에서 웨스턴 블로팅 상에서 발현이 검출되지 않았다. 하지만, 방선균의 주 시그마 인자인 hrdB와 보조 시그마 인자인 hrdD, sigE를 모델 유전자와 함께 발현시킨 결과, sigE를 동시에 발현시킨 경우, degP 유전자의 발현이 확인되었다. 이 결과로 보아, 대장균에서의 방선균 유전자의 발현 장벽의 한 부분은 대장균에는 방선균의 프로모터 서열을 인식하는 시그마 인자의 결핍에 의한 것으로 추정 된다. Expression barrier of foreign genes in metagenomic host is one of the most challenging obstacles to advance toward function-driven metagenomics. Although high G+C Gram positive bacteria are popularly included in environmental samples due to their ubiquity in virtually all environments and have been given considerable attention as producers of a variety of biological active compounds, about 70 % of their genes, it was predicted, is not transcribed by Escherichia coli transcription machinery, at least major sigma factor. We analyzed expression of the chiA gene coding for chitinase (SCO5003), the pro7432 gene for protease (SCO7432), the degP gene for agarase (NP627674) from Streptomyces coelicolor and the chiC gene for chitinase (AAF43629) from S. peucetius, as model genes of high G+C Gram positive bacteria, in E. coli with respect to transcription and translation levels. To minimize transcription of the model genes from plasmid-born promoters, the models genes were cloned into pUC21 in which two transcription terminator units were inserted on either side of multiple cloning sites. None of the model genes were expressed in E. coli enough to be detected by Western blotting. When the gene encoding major sigma factor, HrdB and genes encoding minor sigma factors, HrdD and SigE of Streptomyces coelicolor were co-expressed with the models genes, sigE induced expression of the degP gene which suggests that a part of expression barrier of streptomyces genes in E. coli results from lack of the sigma factor to recognize the promoter sequence.

제주도 토양방선균의 분리와 항균활성물질

정완석 濟州大學校 大學院 1999 국내박사

방선균은 항생물질의 주요 생산균으로 자연생태계에 널리 분포하며, 제주도의 생물상은 비교적 다양한 것으로 알려져 있다. 제주도내 25개 지점으로부터 토양을 채취하여 방선균의 분리·수집용 시료로 사용하였다. 네 종류의 분리용 배지를 사용하여 총 703주의 방선균을 분리하였다. Soil extract를 함유한 arginine glycerol salts agar 배지를 사용했을 때, 가장 많은 수인 437주의 방선균이 분리되었고, arginine glycerol salts agar 배지를 사용했을 때, 171주, starch-casein-nitrate agar 배지를 사용했을 때, 75주, 그리고 Benedict agar 배지를 사용했을 때 20주가 각각 분리되었다. 제주도 내 지역별로는 목장초지가 방선균의 다양성과 밀도 면에서 다른 지역에 비해서 제일 높은 수치를 보였으며, 그 다음이 감귤원이었다. 분리 수집된 총 703주의 방선균 중에서 526개의 균주에 대하여 항균활성 물질 생산여부를 검정하였다. 검정균으로는 그람 음성세균인 E. coli ATCC 8749와 그람 양성세균인 S. aureur ATCC 6538 그리고 식물 병원균 P. solanacearum ATCC 10692를 사용하였다. 항균활성물질 생산균주의 발생빈도는 분리 및 배양에 사용한 배지의 종류에 따라 0.0∼32.8%로 커다란 차이가 있었다. 공시된 3종의 검정균에 대해서 동시에 항균활성을 나타내는 균주를 1차로 선별하였다. 1차로 선별된 균주 10개 중에서 pH 안정성과 열안정성이 높은 항균활성물질을 생산하는 10-6B-a 균주를 최종 선발·동정하고, 항균환성 물질의 성격을 규명하고자 하였다. International Streptomyces Project의 방법과 Bergey's Manual of Systematic Bacteriology에 수록된 방법을 이용하여 형태학적, 배양학적, 생리학적 특성을 조사한 결과, 10-6B-a는 포자 80∼100개 정도가 나선형으로 연결되어 있는 Streptomyces sp.로 추정되었다. Streptomyces sp. 10-6B-a 균주를 9일 동안 폭기배양하여 얻은 무세포배양액을 항균활성물질의 분리·정제용 시료로 사용하였다. 항균활성물질을 분리·정제하기 위하여 cation exchange chromatography(AG MP-50)와 3차에 걸친 gel permeation chromatography(Sephadex G-10)를 실시하였다. 활성분획을 건조시켜 백색결정의 활성물질을 얻을 수 있었다. 항균활성물질의 물리적, 화학적 특성을 분석하기 위하여 용해도, 아미노산 조성, 자외선 흡광 spectrum, 항균 spectrum, 각종 화학적 발색반응 등을 조사하였다. 이 활성물질은 methanol이나 ethanol보다도 물에 더 잘 용해되는 극성이 강한 화합물로 추정되었다. 산가수분해법으로 활성물질의 구성 아미노산을 분석했을 때, 유리되는 아미노산은 없었다. 활성물질의 자외선 흡광대는 210nm에서 최대 흡수치를 보였으며, 283nm에서 약한 흡수대를 보였다. 항균범위를 조사한 결과 그람 음성균, 양성균 모두에 대해서 활성을 보였으나, 효모에 대해서는 균주에 따라 선택적인 활성을 나타내었다. 항균활성물질을 0.2% ninhydrin 용액, 5% H_(2)5O_(4) ethanol 용액, iodoplatinate (potassium) 용액, orcinol ferric chloride 용액(Bial's reagent) 등을 사용하여 발색을 유도하였으나, 0.2% ninhydrin 용액으로만 분홍색으로 나타났고, 나머지 세 종류의 시약으로는 전혀 발색되지 않았다 이는 항균화합물내에 amino group이 존재함을 제시한다. 활성물질을 thin layer chromatography(TLC)로 분석했을 때, 화합물의 결정은 순수분리가 되지 않은 부분정제된 상태였고, 최소한 4종 이상의 항균 화합물이 혼합되어 있는 것으로 나타났다 활성물질의 high performance liquid chromatography(HPLC)를 이용한 분석에서도, TLC로 분석한 결과와 비슷하게 4개의 항균활성 분획과 기타 미량의 혼합물이 감지되었다. 따라서 항균활성물질을 HPLC로 최종 정제하였으며, 그로부터 얻어진 3개의 화합물에 대해서 분석하였다. 부분정제된 항균활성혼합물의 ^(13)C와 ^(1)H nuclear magnetic resonance (NMR) spectrum과 정제된 항균활성화합물의 ^(1)H NMR spectrum으로부터 그 성격을 추정하였다. 그러나, 이들 항균활성화합물의 구조를 밝히기 위해서는 보다 많은 분석 자료가 필요하다고 하겠다. Actinomycetes are the main producers of antibiotics and found in any natural ecosystem. Cheju Island has been known to have biological diversity even its relatively small size. Soil samples were collected from 25 points in Cheju Island and used for isolation of actinomycetes. Four kinds of isolation media were used for selective isolation of soil actinomycetes that total 703 strains. The greatest number 437 strains were isolated on arginine glycerol salts agar containing soil extract, 171 strains were on arginine glycerol salts agar, 75 strains were on starch-casein-nitrite agar, and 20 strains were on Benedict agar Considering regional distribution, pasture land was the best sources of actinomyceyes in terms of diversity and population density, and then citrus field follows. Only 526 strains out of these 703 collections were screened for antimicrobial activities They were cultivated in a small scale using the isolation media and their cell-free culture filtrates were tested for their antimicrobial activities against three bacterial strains Escherichia coli 3749, Staphyloccus aureus 6538 and Pseudomonos solonacearum 10692. The frequency of bacterial strains producing antimicrobial substances was widely different from 0.0 to 32.8% depending upon the medium used for isolation and cultivation. Ten actinomycete isolates that showed growth-inhibitory effect on the above three test strains were selected after primary screening. Among those, an isolate 10-6B-a was found to produce antimicrobial substances that had strong stability in wide range of pH and at high temperature. The isolate 10-6B-a was chosen for identification and characterization of its antinucrobial substances. Identification of the isolate was peformed according to the methods and keys by International Streptomyces Project and Bergey's Manual of Systematic Bacteriology. From its morphological, cultural, and various physiological characteristics, the isolate 10-6B-a was tentatively identified as Streptomyces sp. that has spirals with 80∼100 spores. Cell-free culture filtrate obtained after 9 days' aerobic cultivation was used as raw material for isolation and purification of antimicrobial compounds. Cation exchange chromatography on AG MP-50 and three times consecutive gel permeation chromatography on Sephadex G-10 were peformed, and then the fractions with antimicrobial activities were dehydrated resulting in white crystals. Such physical and chemical properties of the antimicrobial crystals as solubility, amino acid composition, UV absorption spectrum, antimicrobial spectrum and various coloring reactions were investigated. This biologically active substance was more soluble in water than In methanol or ethanol, suggesting its high polarity. Its hydrolysis by acid revealed no free amino acid, and its UV absorption spectrum showed maximum peak at 210un and minor shoulder at 283nm. The antimicrobial substance inhibited growth of both gram positive and negative bacteria, but showed species-selective inhibition against yeasts. When the active substance was tested for its coloring reaction with ninhydrin, H2s04, lodoplatinate(potassium) and orcinol ferric chloride (Bial's reagent), only ninhydrin made pink color development and the other three did not make any observable color reaction. This coloring reaction suggested the presence of amino group within the substance. At least four different fractions with antimicrobial activity were revealed when the partially purified crystals were analyzed by thin layer chromatography(TLC). Analysis by high performance liquid chromatography(HPLC) also showed the similar fractionation pattern to that by TLC, four different fractions with antimicrobial activity were observed. Thus the antimicrobial crystals were finally further purified by HPLC and the resulting three different compounds were analyzed. The partially purified antimicrobial crystals were analyzed by both ^(13)C and ^(1)H nuclear magnetic resonance(NMR), and the purified compounds were analyzed by IH NMR. Even though their identities were inferred, more analysis data are required for the determination of their structures.

방선균 유전자의 초고속 발현 시스템 개발

정용훈 明知大學校 大學院 2004 국내석사

A new Streptomyces expression system, the pFSE, pFSE-extra vector, was shuttle vector for Escherichia coli and Streptomyces. These vectors contain the replication functions of the Streptomyces wide-host-range multicopy plasmid pIJ101, the tsr gene conferring resistance to thiostrepton in Streptomyces, the fI origin of replication from the pBluescript plasmids for replication in Escherichia coli and the bla gene conferring resistance to amplicillin in Escherichia coli, the amidase region from the pHCE-FOREX plasmids for fast cloning system in Escherichia coli, Six His tag for easily purification in Streptomyces. And the pFSE-extra vector using a secreted expressing SghC promoter derived upstream from the protein C gene of Streptomyces griseus HH1. New replicative vectors have been constructed and used to express independently the sco1551 gene and sco2110(PkaF) gene of Streptomyces coelicolor M145. The putative eukaryotic-type serine/threonine kinase genes(sco1551 and sco2110) from Streptomyces coelicolor, which encodes a 493-amino-acid protein(51kDa) and 667-amino acid protein(70kDa) showing extensive homology to other eukaryotic-type serine/threonine kinases, was cloned by polymerase chain reaction(PCR) using oligonucleotides primers, respectively. For the expression of sco1551 and sco2110 from Streptomyces coelicolor M145 in E. coli BL21(DE3), sco1551 gene and sco2110 gene was subcloned into the Escherichia coli expression vector pET32a(+) and pET28a(+), respectively. To examine activity of this genes by eukaryotic-type kinase, overexpression, purification in S.lividans TK24 and S.coelicolor M145 and kinase assay method will be further studied. As a Result, this convenient plasmid expression system can be readily adapted to study the Streptomyces genes.