KR950012896B1 - Novel microorgamism psendomonas sp - Google Patents
Novel microorgamism psendomonas sp Download PDFInfo
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Description
본 발명은 항진균 물질을 생산하는 미생물 슈도모나스 에스 피. AF-2001 및 이를 이용한 항진균물질의 생산방법에 관한 것이다.The present invention is a microorganism Pseudomonas sp. AF-2001 and a method for producing an antifungal substance using the same.
항진균물질로서 블라스티사이딘(Blasticidin), 카수가마이신(Kasugamycin), 발리다마이신(Validamycin)이 방선균으로부터 발견되어 식물병방제에 사용되었으며, 암포테리신-B(Amphotericin-B), 나이스타틴(Nystatin) 등이 역시 방선균으로부터 발견되어 진균증에 대한 화학요법에 실용화된 이래 수많은 항진균제가 미생물로부터 발견되었고, 또한 유기합성법에 의하여 다양한 항진균제가 개발되었으나 대부분은 약효가 우수하지 못하거나 독성이 문제가 되어 그중 실용화된 것은 얼마되지 않는다. 비록 실용화되고 있는 항진균제라 할지라도 항진균 스펙트럼이 넓지 못하거나 대부분 독성이 강하고 정진균(Fungistatic)효과를 갖는 것들로서 체내 깊숙히 감염된 심재성(Systemic) 진균증은 완치하기 어려운 점을 고려할 때, 앞으로 저독성, 완치성, 속효성 항진균제의 개발이 절실히 요구되고 있다. 이와같은 상기의 문제점을 고려하여 본 발명자들은 낮은 농도에서 효과가 우수하고 단기간에 진균류를 사멸시킬 수 있으며, 항진균 스펙트럼이 광범위한 항진균 물질을 개발하기 위하여 토양미생물 스크리닝(Screening)을 거듭한 결과 이러한 요건을 충족시킬 수 있는 항진균 물질을 생산하는 신규 미생물을 분리하였다.As antifungals, blasticidin, Kasugamycin, and Validamycin have been found from actinomycetes and used in plant disease control.Amphotericin-B, Nystatin Since many antifungal agents have been found in microorganisms since they have been found in actinomycetes and have been applied to chemotherapy for fungal diseases, various antifungal agents have been developed by organic synthesis, but most of them have poor drug efficacy or toxicity problems. It's not long. Although the antifungal agents in practical use are those that have a broad antifungal spectrum or are mostly toxic and have fungalstatic effects, deeply infected systemic fungi are difficult to cure. There is an urgent need for the development of fast-acting antifungal agents. In view of the above problems, the present inventors have excellent effects at low concentrations and can kill fungi in a short period of time. As a result of repeated screening of soil microorganisms in order to develop an antifungal substance with a broad antifungal spectrum, Novel microorganisms were produced that produced antifungal substances that could be met.
이하에서 본 발명을 실시예를 통해 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail with reference to Examples.
[실시예 1]Example 1
토양미생물의 분리는 토양을 멸균수에 현탁하여 NA 배지에 도말함으로써 수행되었다. NA 배지는 pH6.8로 조정하였으며, 121℃에서 15분간 멸균하였다. 토양현탁액이 도말된 배지는 25℃에서 3일간 보존하였으며, 콜로니를 형성한 미생물들을 하기 표 1의 배지에 옮겨 3일간 배양한 다음 각 콜로니를 형성한 미생물들의 항진균 효과를 시험하였다.Separation of soil microorganisms was performed by suspending the soil in sterile water and plating it in NA medium. NA medium was adjusted to pH6.8 and sterilized at 121 ° C. for 15 minutes. The medium in which the soil suspension was smeared was stored at 25 ° C. for 3 days, and the microorganisms that formed colonies were transferred to the media of Table 1, cultured for 3 days, and then tested for the antifungal effect of the microorganisms that formed each colony.
[표 1]TABLE 1
항진균물질 생산 미생물 분리용 배지Antifungal Production Microorganism Separation Medium
이러한 방법으로 다수의 항진균물질 생산 미생물을 분리하였으며, 그중 가장 우수한 항진균 효과를 나타낸 것은 본 발명의 신규 미생물 슈도모나스 에스 피. AF-2001(KFCC10773)이다.In this way, a number of antifungal producing microorganisms were isolated, and among them, the best antifungal effect was shown in the novel microorganism Pseudomonas sp. AF-2001 (KFCC10773).
이 미생물은 운동성이 있으며 0.4-0.6×1.0-1.3(μm) 크기의 그람음성 간상 세균으로서 슈도모나스(Pseudomonas) 속에 포함시킬 수 있는 분류학적 특징을 갖고 있으나, 이미 알려진 슈도모나스속 미생물중에는 본 발명의 미생물과 그 특성이 동일한 것이 없으므로 슈도모나스 에스 피. AF-2001(Pseudomonassp. AF-2001) 이라 명명하고 1992.7.22.자로 KFCC에 기탁하였다. 기탁번호는 KFCC 10773이다.This microorganism is motility and has a taxonomic characteristic that can be included in Pseudomonas as a Gram-negative rod bacteria of 0.4-0.6 × 1.0-1.3 (μm) size, but among the known Pseudomonas genus microorganisms, Pseudomonas sp., Because of the same characteristics. It was named AF-2001 (Pseudomonas sp. AF-2001) and deposited with KFCC as 1992.7.22. The accession number is KFCC 10773.
이 슈도모나스 에스 피. AF-2001의 특성은 버기스 매뉴얼(Bergy's manual of systematic Bacteriology,1984) 에 기재된 슈도모나스 세파시아(Pseudomonas cepacia) 와 대부분 동일하나, 탄소원으로서 말토스(Maltose)를 이용하며 아도니톨(Adonitol)은 이용하지 못한다는 점과 NO3를 환원시키지 못한다는 점이 슈도모나스 세파시아(Pseudomonas cepacia)와 상이하다. 슈도모나스 에스 피. AF-2001의 생리적 특성을 다음의 표 2와 같다This Pseudomonas Esp. The characteristics of AF-2001 are mostly the same as those of Pseudomonas cepacia described in the Bergy's manual of systematic Bacteriology (1984), but it uses maltose as a carbon source and adonitol is used. The difference between Pseudomonas cepacia and its ability to reduce NO 3 is not. Pseudomonas sp. Physiological characteristics of AF-2001 are shown in Table 2 below.
[표 2]TABLE 2
슈도모나스 에스 피. AF-2001의 생리적 특성Pseudomonas sp. Physiological Characteristics of AF-2001
* (+) : 양성반응 (-) : 음성반응* (+): Positive reaction (-): negative reaction
[실시예 2]Example 2
본 발명의 미생물을 배양하여 생산된 항진균 물질을 분리, 정제하였으며, 이 물질의 항진균 효과를 측정하였다. 포도당 30g/l, 펩톤 10g/l, MgSO4·7H2O 0.5g/I, KH2PO40.5g/l, 한천 15g/l로 조성되고 pH가 6.8로 조정된 상기 표 1의 한천배지에 접종하여 1일간 배양한 슈도모나스 에스 피. AF-2001균을 표 3의 액체배지 500ml에 접종하여 25℃에서 1일간 종배양하였다. 종배양된 배양액 500ml을 표 3의 액체배지 10ℓ에 접종하여 25℃에서 2일간 호기적(Aerobic)으로 배양하였고, 이렇게 얻어진 미생물 배양액으로부터 항진균 물질을 분리, 정제하였다.The antifungal substance produced by culturing the microorganism of the present invention was isolated and purified, and the antifungal effect of this substance was measured. Glucose 30g / l, Peptone 10g / l, MgSO 4 · 7H 2 O 0.5g / I, KH 2 PO 4 0.5g / l, agar 15g / l in agar medium of Table 1 above adjusted to pH 6.8 Pseudomonas sp. Inoculated and cultured for 1 day. AF-2001 bacteria were inoculated into 500 ml of the liquid medium of Table 3, and cultured for 1 day at 25 ° C. 500 ml of the cultured culture medium was inoculated into 10 l of the liquid medium of Table 3, and incubated at 25 ° C. for 2 days by aerobic, and the antifungal material was separated and purified from the microbial culture thus obtained.
[표 3]TABLE 3
항진균물질 생산배지Antifungal Production Medium
배양이 완료되면 배양액 10ℓ와 이소프로필 알콜(Isopropyl Alcohol) 10ℓ를 혼합하고 pH4로 조정하였으며, 6시간 경과후 원심분리하여 상등액을 취하였다. 상등액은 감압농축기를 사용하여 이소프로필 알콜을 회수하고, 잔류액을 25℃로 냉각한 다음 원심분리하여 침전물과 상등액을 분리하였다. 먼저 침전물에 이소프로필 알콜 250ml을 가하여 잘 흔들고 원심분리함으로써 가용성분(상등액)과 불용성분을 분리하였다. 이중 상등액은 버리고 침전물(조추출물-A)은 다음 정제를 위하여 보존하였다. 배양액 추출물중 상등액은 알루미나(Alumina)가 충진된 컬럼(Column)을 통과시켜 1차적으로 색소를 제거하였으며, 통과된 액을 취하였다. 알루미나는 사용전 2N-HC1로 수세하고 pH4가 될 매까지 물로 수세하였다. 알루미나 통과액은 pH7로 조정하고 앰버라이트 XAD-2 수지 또는 다이아이온 HP-20(Diaion HP-20) 수지가 충진된 컬럼을 통과시켰으며, 통과액은 버렸다. HP-20 수지에 흡착된 항진균 물질은 50% 이소프로필 알콜로 분리해낸 다음 감압 농축기를 이용, 이소프로필 알콜을 제거하였다. 잔류액은 25℃ 이하로 냉각하고, 형성된 침전물을 원심분리하여 얻었으며, 이 침전물에 250ml의 이소프로필 알콜을 가하여 잘 흔들고 원심분리함으로써 불용성의 침전물(조추출물-B)을 분리해 내었다. 조추출물-A와 조추출물-B를 합하고 pH11로 조정한 50% 이소프로필 알콜 200ml을 가하여 용해한 다음, 이 용액에 2ℓ의 증류수를 가하고 잘 혼합한다.When the culture was completed, 10 l of the culture solution and 10 l of isopropyl alcohol were mixed and adjusted to pH 4, and after 6 hours, the supernatant was taken by centrifugation. The supernatant was recovered using isopropyl alcohol using a vacuum condenser, the residue was cooled to 25 ° C., and centrifuged to separate the precipitate and the supernatant. First, 250 ml of isopropyl alcohol was added to the precipitate, shaken well, and centrifuged to separate the soluble component (supernatant) and the insoluble component. The supernatant was discarded and the precipitate (crude extract-A) was preserved for the next purification. The supernatant in the culture extract was first passed through a column filled with alumina (Alumina) to remove the pigment, and the passed solution was taken. The alumina was washed with 2N-HC1 before use and washed with water until it became pH4. The alumina passthrough was adjusted to pH7 and passed through a column filled with Amberlite XAD-2 resin or Diaion HP-20 resin, and the passthrough was discarded. The antifungal material adsorbed on HP-20 resin was separated with 50% isopropyl alcohol and then isopropyl alcohol was removed using a vacuum concentrator. The residue was cooled to 25 ° C. or lower, and the precipitate formed was centrifuged. 250 ml of isopropyl alcohol was added to the precipitate, followed by well shaking and centrifugation to separate the insoluble precipitate (crude extract-B). Combine crude extract-A and crude extract-B, dissolve with 200 ml of 50% isopropyl alcohol adjusted to pH11, and add 2 liters of distilled water to this solution and mix well.
이렇게 준비된 용액은 HP-20 수지가 충진된 컴럼을 통과시켰으며, 통과액은 버렸다. HP-20 수지에 흡착된 항진균 물질을 50% 이소프로필 알콜로 수세해낸 다음, 이 수세액은 알루이나 컬럼을 통과시켜 통과액을 얻었다. 이러한 과정을 거쳐 정제한 항진균물질의 50% 이소프로필 알콜 용액은, 감압농축기를 사용하여 60℃에서 이소프로필 알콜을 제거하였다. 잔류물을 5℃로 냉각하여 생성된 침전물을 원심분리하여 분리한 다음 건조시켜서 항진균 효과를 측정하는 재료로 사용하였다.The solution thus prepared was passed through a comum filled with HP-20 resin, and the flow was discarded. The antifungal material adsorbed on the HP-20 resin was washed with 50% isopropyl alcohol, and the wash was passed through an aluina column to obtain a pass through. The 50% isopropyl alcohol solution of the antifungal substance purified through this process was removed with isopropyl alcohol at 60 ° C. using a vacuum concentrator. The residue produced by cooling the residue to 5 ℃ was separated by centrifugation and dried to use as a material to measure the antifungal effect.
상기의 방법으로 얻어진 항진균 물질의 항진균 스펙트럼을 조사한 결과 표 4와 같이 나타났다. 표 4에서 볼 수 있는 바와같이 본 발명의 미생물에 의하여 생산된 항진균 물질은 효모균, 사상균, 표재성균, 심재성균 등 모든 진균류에 광범위하게 우수한 항진균효과를 나타내었으며 인체 병원균 뿐만 아니라 농작물 병원성 진균류에도 강한 항진균 효과를 보였다.The antifungal spectrum of the antifungal substance obtained by the above method was examined, and the results were as shown in Table 4. As can be seen in Table 4, the antifungal substance produced by the microorganism of the present invention showed a wide range of excellent antifungal effects on all fungi such as yeast, filamentous fungus, superficial fungi, and heart fungus, and also strong antifungals not only on human pathogens but also on crop pathogenic fungi. It showed an effect.
[표 4]TABLE 4
항진균 스펙트럼Antifungal spectrum
[실시예 3]Example 3
본 발명의 미생물에 의하여 생산된 항진균물질의 정진균(Fungistatic), 또는 살진균(Fungicidal) 효과를 조사한 결과, 최소저지농도(MIC)의 2배 농도로 2일간 처리하면 진균 칸디다 알비칸스(Candia albicans)가 완전 사멸하는 것으로 나타났다. 실시예 2에 기술한 방법에 의하여 균을 배양하고 정제함으로써 얻어진 항진균 물질을 각각 0.05μg/ml, 0.1μg/ml, 0.2μg/ml 씩 함유하는 하기 표 5의 액체배지를 준비하고 여기에 칸디다 알비칸수균을 접종하여 보존하였다. 그리고 시간 경과에 따라 항진균물질의 각 농도별 진균 사멸율을 각각 조사하였다. 시험에 사용할 칸디다 알비칸스 KCTC 1940균은 하기 표 5의 배지를 사용하여 30℃에서 2일간 배양하였으며, 생균수를 측정하여 최종균농도가 105개/ml이 되도록 항진균물질 함유배지에 접종하였다.As a result of investigating the fungicidal or fungicidal effect of the antifungal substance produced by the microorganism of the present invention, the fungus Candida albicans when treated for 2 days at twice the concentration of minimum inhibitory concentration (MIC) Appeared to die completely. Prepare the liquid medium of Table 5 below containing 0.05 μg / ml, 0.1 μg / ml, and 0.2 μg / ml of the antifungal substance obtained by culturing and purifying the bacteria by the method described in Example 2. Khansu was inoculated and preserved. And over time, the fungal death rate of each concentration of antifungal substance was investigated. Candida albicans KCTC 1940 bacteria to be used for the test was incubated at 30 ° C. for 2 days using the medium of Table 5 below. The number of viable cells was measured and inoculated into the antifungal medium containing 10 5 bacteria / ml.
[표 5]TABLE 5
칸디다 알비칸스균 배양용 배지Candida albicans bacteria culture medium
각 농도별로 항진균물질이 처리된 칸디다 알비칸스균은 원심분리하여 균체를 취하였으며, 다시 상기 표 5의 배지로 현탁하고 원심분리하여 균체를 수세하였다. 이와같은 수세를 2회 실시한 다음 2% 한천을 함유하는 표 5의 배지에 수세된 균을 도말하였다. 칸디다 알비칸스균의 사멸율 조사는, 한천배지에 도말한 후 30℃에 2일간 보존함으로써 실시하였다.Candida albicans bacteria treated with the antifungal substance at each concentration were collected by centrifuging the cells, and again suspended in the medium of Table 5 and centrifuged to wash the cells. This washing with water was performed twice, followed by smearing the washed bacteria in the medium of Table 5 containing 2% agar. The killing rate investigation of Candida albicans bacteria was performed by preservation at 30 ° C. for 2 days after plating on agar medium.
하기 표 6에서 볼 수 있는 바와같이 본 발명의 미생물에 의하여 생산된 항진균물질을 최소저지농도의 2배 농도로 2일간 처리하면 칸디다 알비칸스균이 완전 사멸하였으며, 4배 농도로 처리하면 1일후 완전히 사멸하는 것으로 나타났다. 이러한 결과는 이 항진균물질이 강한 살진균(Fungicidal) 효과를 가지고 있음을 시사한다.As can be seen in Table 6, when the antifungal substance produced by the microorganism of the present invention was treated for 2 days at a concentration of 2 times the minimum inhibitor concentration, Candida albicans bacteria were completely killed, and when treated at 4 times the concentration, it was completely lost after 1 day. It appeared to die. These results suggest that this antifungal substance has a strong fungicidal effect.
[표 6]TABLE 6
진균 사멸시험Fungal killing test
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