KR101862993B1 - Burkholderia glumae OK001 strain and a method of toxoflavin production using the strain - Google Patents

Burkholderia glumae OK001 strain and a method of toxoflavin production using the strain Download PDF

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KR101862993B1
KR101862993B1 KR1020170062051A KR20170062051A KR101862993B1 KR 101862993 B1 KR101862993 B1 KR 101862993B1 KR 1020170062051 A KR1020170062051 A KR 1020170062051A KR 20170062051 A KR20170062051 A KR 20170062051A KR 101862993 B1 KR101862993 B1 KR 101862993B1
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김진우
최옥희
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경상대학교산학협력단
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Abstract

The present invention relates to a Burkholderia glumae OK001 strain which produces toxoflavin having a herbicidal activity, an antimicrobial activity and an anticancer activity with high efficiency, a culture thereof, a composition for producing toxoflavin comprising the same, and a high-efficiency method for producing toxoflavin using the same. When the Burkholderia glumae OK001 strain of the present invention is used, toxoflavin can be produced with high efficiency so that the production cost can be remarkably reduced to have a great economic effect.

Description

부르크홀데리아 글루메 OK001 균주 및 이를 이용한 톡소플라빈 제조 방법{Burkholderia glumae OK001 strain and a method of toxoflavin production using the strain}[0001] The present invention relates to a method for producing Burkholderia glumae OK001 strain and toxoflavin production using the same,

본 발명은 톡소플라빈 생산 효율이 높은 부르크홀데리아 글루메(Burkholderia glumae) OK001 균주, 이의 배양물, 이를 포함하는 톡소플라빈 제조용 조성물 및 이를 이용한 톡소플라빈 고효율 제조 방법에 관한 것이다.The present invention relates to a Burkholderia glumae OK001 strain having high toxoflavin production efficiency, a culture thereof, a composition for preparing toxoplasin containing the same, and a method for producing toxoplasin using the same.

톡소플라빈(toxoflavin, PKF118-310)은 잔토트리신(xanthothricin)이라고도 불리는 물질로, IUPAC name은 1,6-Dimethylpyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione이며, 분자식은 C7H7N5O2이고, 분자량은 193.16으로 알려져 있다. 톡소플라빈은 Tcf4/b-catenin 혼합체를 붕괴하여 Tcf4 반응 유전자의 발현을 저해하며 Tcf4/b-catenin 신호전달체계에서 길항작용을 하는 것으로 알려져있다. 또한, 톡소플라빈은 간암(HCC), 대장암(colon tumor), 림프모구백혈병(lymphocytic leukemia) 세포주(cell line)들의 survivin의 발현을 저해하고 이들 암세포주들의 세포자멸사(apoptosis)를 유도하는 것으로 보고된 바 있다. 톡소플라빈은 제초활성을 갖는 것으로도 알려져 있는데, 이는 산소와 빛이 존재할 때 톡소플라빈에 의해 슈퍼옥사이드(superoxide, O2 -) 및 과산화수소(hydrogen peroxide, H2O2)가 생성되어 식물이 죽게된다.Toxoflavin (PKF118-310) is a substance also called xanthothricin, IUPAC name is 1,6-Dimethylpyrimido [5,4-e] [1,2,4] triazine-5,7 1H, 6H) -dione, the molecular formula is C7H7N5O2, and the molecular weight is known as 193.16. Toxoplasin is known to disrupt the Tcf4 / b-catenin mixture to inhibit the expression of the Tcf4 reactive gene and to antagonize the Tcf4 / b-catenin signaling pathway. In addition, toxoplasmin inhibits the expression of survivin in hepatocellular carcinoma (HCC), colon tumor, lymphocytic leukemia cell line and induces apoptosis of these cancer cell lines It has been reported. Toxoflavin is also known to have herbicidal activity, which is formed by toxoplavin in the presence of oxygen and light, producing superoxide (O 2 - ) and hydrogen peroxide (H 2 O 2 ) I will die.

톡소플라빈을 생성하는 미생물로는 부르크홀데리아 글루메(Burkholderia glumae), 부르크홀데리아 글라디올리(Burkholderia gladioli), 부르크홀데리아 글라디올리(Burkholderia gladioli pv. cocovenenans) 등이 보고되어 있으며, 톡소플라빈의 생합성 및 분비체계와 관련하여 5개 유전자(toxABCDE)는 생합성에 관여하고, 4개 유전자(toxFGHI)는 분비에 관여하는 것으로 밝혀진 바 있다. 그러나 톡소플라빈을 고효율로 생산할 수 있는 균주에 대한 연구는 아직까지 이루어지지 않은 상황이다. Burkholderia glumae , Burkholderia gladioli , Burkholderia gladioli pv. Cocovenenans , and the like have been reported as microorganisms that produce toxoplasene, ( ToxABCDE ) are involved in biosynthesis and four genes ( toxFGHI ) are involved in the secretion of the five genes ( toxABCDE ) in relation to the biosynthesis and secretion system of the plant . However, studies on strains capable of producing toxoflavin with high efficiency have not yet been conducted.

대한민국 등록특허 제10-1630211호Korean Patent No. 10-1630211

Journal of Heterocyclic Chemistry, 2014, 51; 594-597. Journal of Heterocyclic Chemistry, 2014, 51; 594-597.

이에 본 발명의 발명자들은 벼이삭으로부터 분리한 부르크홀데리아 글루메(Burkholderia glumae) OK001 균주가 톡소플라빈을 고효율로 생산할 수 있음을 발견하여 본 발명을 완성하였다.Accordingly, the inventors of the present invention have found that Burkholderia glumae OK001 strain isolated from rice paddy can produce toxoplasin with high efficiency, thereby completing the present invention.

따라서, 본 발명의 목적은 톡소플라빈 생산 효율이 높은 부르크홀데리아 글루메(Burkholderia glumae) OK001 균주, 이의 배양물, 이를 포함하는 톡소플라빈 제조용 조성물 및 이를 이용한 톡소플라빈 고효율 제조 방법을 제공하는 것이다.Accordingly, an object of the present invention is to provide a Burkholderia glumae OK001 strain having high tofoflavin production efficiency, a culture thereof, a composition for preparing toxoplasin containing the same, and a method for producing toxoplasin high efficiency using the same will be.

상기와 같은 본 발명의 목적을 달성하기 위하여, 본 발명은 톡소플라빈 생산능을 갖는, 부르크홀데리아 글루메(Burkholderia glumae) OK001 균주(수탁번호: KACC92174P)를 제공한다.According to an aspect of the present invention as described above, the present invention has a Toxoplasma flavin-producing ability, Burkholderia glue methoxy (Burkholderia glumae ) OK001 strain (accession number: KACC92174P).

또한, 본 발명은 본 발명에 따른 부르크홀데리아 글루메 OK001 균주의 배양물을 제공한다.The present invention also provides a culture of the strain Burkholderia glume OK001 according to the present invention.

또한, 본 발명은 본 발명에 따른 부르크홀데리아 글루메(Burkholderia glumae) OK001 균주 또는 이의 배양물을 포함하는 톡소플라빈(toxoflavin) 제조용 조성물을 제공한다.The present invention also provides a composition for the production of toxoflavin comprising Burkholderia glumae OK001 strain or a culture thereof according to the present invention.

또한, 본 발명은 본 발명에 따른 부르크홀데리아 글루메 OK001 균주를 배양하는 단계 및 상기 균주로부터 톡소플라빈을 회수하는 단계를 포함하는, 톡소플라빈 제조방법을 제공한다.The present invention also provides a method for producing toxoplasia comprising culturing the strain Burkholderia glume OK001 according to the present invention and recovering toxoplasin from the strain.

본 발명의 부르크홀데리아 글루메(Burkholderia glumae) OK001 균주는 종래의 부르크홀데리아 글루메 균주보다 300% 이상 높은 톡소플라빈 생산능을 갖는다. 이에, 본 발명의 부르크홀데리아 글루메 OK001 균주는 톡소플라빈을 높은 효율로 제조할 수 있어 생산비용을 현저하게 줄일 수 있는 경제적 효과가 크다. 본 발명의 톡소플라빈 고효율 생산용 부르크홀데리아 글루메 OK001 균주를 이용하여 생산된 톡소플라빈은 식물제초활성, 항균활성 및 항암활성을 가지므로 제초제, 항균제, 항암제, 건강기능식품 등으로 활용할 수 있다.Burkholderia of the invention Syrian glue methoxy (Burkholderia glumae) OK001 strain has a 300% higher Toxoplasma flavin-producing ability than the conventional Burkholderia glue methoxy strain. Thus, the strain Burkholderia glume OK001 of the present invention can produce toxoflavin with high efficiency, thereby significantly reducing the production cost. Toxoflavin produced by using the strain Burkholderia glume OK001 for high-throughput production of the present invention has plant herbicidal activity, antibacterial activity and anticancer activity, and thus can be used as a herbicide, antimicrobial agent, anticancer agent, have.

도 1은 본 발명의 부르크홀데리아 글루메(Burkholderia glumae) OK001 균주의 16S rDNA 염기서열을 이용한 분자계통학적 유연관계 분석 결과를 나타낸 것이다.
도 2는 본 발명의 부르크홀데리아 글루메(Burkholderia glumae) OK001 균주(OK001) 및 대조균 부르크홀데리아 글루메(Burkholderia glumae) BGR1 균주(BGR1)의 배양 결과(콜로니 형태 및 색깔)를 나타낸 것이다.
도 3은 본 발명의 부르크홀데리아 글루메 OK001 균주의 배양물로부터 분리된 독소 물질(아래) 및 톡소플라빈(위)의 ESI-MASS 분석 결과를 나타낸 것이다.
도 4는 본 발명의 부르크홀데리아 글루메 OK001 균주(OK001) 및 대조균 부르크홀데리아 글루메 BGR1 균주(BGR1)에 대하여 28℃ 및 37℃에서의 톡소플라빈(μg/ml) 생산 효율성을 분석한 결과를 나타낸 것이다.Figure 1 of the present invention Burkholderia glue methoxy (Burkholderia glumae ) OK001 strain using the 16S rDNA nucleotide sequence.
Figure 2 is a Burkholderia glue methoxy (Burkholderia of the invention glumae) OK001 strain (OK001) and the control bacteria Burkholderia glue methoxy (Burkholderia glumae ) BGR1 strain (BGR1) (colony shape and color).
FIG. 3 shows the results of ESI-MASS analysis of the toxin material (below) and toxoplasia (above) isolated from the culture of the Burkholderia glume strain OK001 of the present invention.
Figure 4 shows the production efficiency of toxoplasene (μg / ml) at 28 ° C and 37 ° C for the strain Burkholderia glume OK001 (OK001) of the present invention and the control bacterium Burkholderia glume BGR1 (BGR1) .

이하, 첨부한 도면을 참조하여 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 본원의 구현예 및 실시예를 상세히 설명한다.Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings, which will be readily apparent to those skilled in the art to which the present invention pertains.

본 발명은 톡소플라빈 생산능을 갖는, 부르크홀데리아 글루메(Burkholderia glumae) OK001 균주(수탁번호: KACC92174P)를 제공한다.The present invention provides a strain Burkholderia glumae OK001 (accession number: KACC92174P) having toxoplasia production ability.

부르크홀데리아 글루메(Burkholderia glumae)는 부르크홀데리아(Burkholderia)속의 그람음성 토양세균(Gram-negative soil bacterium)이다. 본 발명의 부르크홀데리아 글루메는 벼(Oryza sativa) 이삭으로부터 분리된 부르크홀데리아 글루메 OK001 균주(수탁번호: KACC92174P)로서, 높은 효율의 톡소플라빈(toxoflavin) 생산능을 갖는다. Burkholderia glumae is a gram-negative soil bacterium in the genus Burkholderia. The Burkholderia glume of the present invention is a Burkholderia glume OK001 strain (accession number: KACC92174P) isolated from rice ( Oryza sativa ) sp. And has a high efficiency of producing toxoflavin.

또한, 본 발명은 본 발명에 따른 부르크홀데리아 글루메 OK001 균주의 배양물을 제공한다.The present invention also provides a culture of the strain Burkholderia glume OK001 according to the present invention.

상기 부르크홀데리아 글루메 OK001 균주는 인 비트로(in vitro)에서 세포 성장 및 생존을 지지할 수 있게 하는 배지라면 제한 없이 사용하여 배양할 수 있으며, 세포의 배양에 적절한 당 분야에서 사용되는 통상의 배지를 모두 포함한다. 세포의 종류에 따라 배지와 배양조건을 선택할 수 있다. 세포의 배양에 사용되는 기본배지는 바람직하게는 세포 배양 최소 배지(minimum medium: MM)로, 일반적으로 탄소원, 질소원 및 미량원소 성분을 포함한다. 이런 세포 배양 기본 배지에는 예를들어, Potato Dextrose Broth(PDB), Nutrient Broth(NB), Casamino acid Peptone Glucose(CPG), Tryptic Soybean Broth(TSB), King’s B(KB) 및 Luria Bertani(LB) 등이 있으나, 이로 제한되지 않는다.The strain Burkholderia glume OK001 can be cultured in vitro without limitation as long as it can support cell growth and survival, and can be cultured in a conventional culture medium suitable for culturing cells . Depending on the type of cells, medium and culture conditions can be selected. The basic medium used for culturing the cells is preferably a cell culture minimum medium (MM), and generally includes a carbon source, a nitrogen source and a trace element component. These cell culture basal media include, for example, Potato Dextrose Broth (PDB), Nutrient Broth (NB), Casamino Acid Peptone Glucose (CPG), Tryptic Soybean Broth (TSB), King's B (KB) and Luria Bertani But is not limited to this.

또한, 본 발명은 본 발명에 따른 부르크홀데리아 글루메(Burkholderia glumae) OK001 균주 또는 이의 배양물을 포함하는 톡소플라빈(toxoflavin) 제조용 조성물을 제공한다.The present invention also provides a composition for the production of toxoflavin comprising Burkholderia glumae OK001 strain or a culture thereof according to the present invention.

본 발명의 일실시예에 따르면, 본 발명의 부르크홀데리아 글루메 OK001 균주 및 대조균으로서 부르크홀데리아 글루메 BGR1 균주의 톡소플라빈 생산성을 비교 분석한 결과, 본 발명의 부르크홀데리아 글루메 OK001 균주가 대조균 BGR1 균주보다 300% 이상 톡소플라빈 생성 효율이 높은 것으로 나타났다(실시예 4 및 도 4 참조). 따라서, 본 발명의 부르크홀데리아 글루메 OK001 균주는 톡소플라빈 고효율 생산이 가능한 균주이다.According to one embodiment of the present invention, the productivity of Toxoplasma of the strain Bruchhorferia glume OK001 according to the present invention and the strain Bruchhorferia glume BGR1 as a control strain were compared and found to be as follows: Bruchhorferia glume OK001 The strain showed higher toxoplasia production efficiency than the control strain BGR1 by 300% (see Example 4 and FIG. 4). Therefore, the strain Burkholderia glume OK001 of the present invention is a strain capable of producing toxoplasene with high efficiency.

또한, 본 발명은 본 발명에 따른 부르크홀데리아 글루메 OK001 균주를 배양하는 단계 및 상기 균주로부터 톡소플라빈을 회수하는 단계를 포함하는, 톡소플라빈 고효율 제조방법을 제공한다.The present invention also provides a method for producing high toxoplasin comprising culturing the strain Burkholderia glume OK001 according to the present invention and recovering toxoplasin from the strain.

본 발명의 일 구현예에 있어서, 상기 배양은 Luria Bertani(LB) 배지를 사용하여 수행되는 것일 수 있다. 또한, 부르크홀데리아 글루메의 배양액으로부터 균체를 제거하고 배양 상등액을 얻는 단계를 포함할 수 있다. 구체적으로, 부르크홀데리아 글루메를 LB 배지를 이용하여 약 25℃ 내지 40℃의 온도에서 16 내지 20시간 동안 배양한 후, 원심분리를 이용하여 세포 균체를 제거하고 배양물 상등액을 얻을 수 있다.In one embodiment of the present invention, the culture may be performed using Luria Bertani (LB) medium. Further, it may include the step of removing the cells from the culture medium of Burkholderia glume and obtaining a culture supernatant. Specifically, the bacteriolide glume is cultured in the LB medium at a temperature of about 25 ° C to 40 ° C for 16 to 20 hours, followed by centrifugation to remove cell masses and obtain a culture supernatant.

본 발명의 일 구현예에 있어서, 상기 톡소플라빈의 회수는 클로로포름으로 추출하여 수행되는 것일 수 있다. 구체적으로는 배양 상등액에 1:1 비율의 클로로포름을 첨가하고 흔들면서 톡소플라빈을 추출할 수 있다. 이 때, 톡소플라빈은 클로로포름층으로 이동한다.In one embodiment of the present invention, the recovery of toxoplasmin may be carried out by extraction with chloroform. More specifically, toxoflavin can be extracted by adding chloroform at a ratio of 1: 1 to the culture supernatant and shaking. At this time, toxoplavin migrates to the chloroform layer.

본 발명의 일 구현예에 있어서, 상기 클로로포름 추출 단계 후에, 톡소플라빈을 포함하는 클로로포름 추출물(클로로포름층)로부터 클로로포름을 증류 또는 증발시킨 후 살균수에 녹이는 단계를 추가로 포함할 수 있다. 살균수에 녹인 톡소플라빈은 이용하기에 적당한 농도로 희석하여 사용할 수 있다.In one embodiment of the present invention, after the step of extracting chloroform, chloroform may be distilled or evaporated from a chloroform extract (chloroform layer) containing toxoplasmin, followed by melting in sterilized water. Toxoflavin dissolved in sterilized water may be diluted to a suitable concentration.

본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 상세하게 후술되어 있는 실시예들을 참조하면 명확해질 것이다. 이하, 본 발명을 실시예에 의해 상세히 설명하기로 한다. 그러나 이들 실시예들은 본 발명을 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are for illustrating the present invention specifically, and the scope of the present invention is not limited to these examples.

<실시예 1> 부르크홀데리아 글루메 OK001 균주의 분리 및 동정 &Lt; Example 1 > Isolation and Identification of glue methoxy OK001 strain

벼이삭으로부터 분리한 미생물 가운데 톡소플라빈 생산 효율이 우수한 균주를 선발하였다.Among the microorganisms isolated from rice agar, strains with high toxoflavin production efficiency were selected.

상기 선발된 균주의 분자계통학적 분석을 위하여, 16S rDNA 염기서열 분석을 하였다. 그 결과 서열번호 1로 표시되는 염기서열을 확인할 수 있었다. NCBI BLAST 분석을 통해 해당 염기서열은 부르크홀데리아 글루메(Burkholderia glumae)의 16S rDNA와 100% 일치하였다. 16S rDNA 염기서열의 분자계통학적 유연관계 분석 결과는 도 1에 나타냈다. 도 1에서 볼 수 있는 바와 같이, 상기 선발된 균주는 부르크홀데리아 글루메(Burkholderia glumae)와 동일한 그룹에 속하는 것으로 나타났다. 이로써, 본 발명자들은 상기 선발된 균주를 "부르크홀데리아 글루메(Burkholderia glumae) OK001"이라 명명하고, 이를 2017년 4월 11일자로 국립농업과학원 농업유전자원센터(KACC)에 기탁하여 2017년 5월 1일자로 수탁번호 KACC92174P를 부여받았다.For the molecular phylogenetic analysis of the selected strains, 16S rDNA sequencing was performed. As a result, the nucleotide sequence shown in SEQ ID NO: 1 was confirmed. Through NCBI BLAST analysis, the corresponding nucleotide sequence was 100% identical to the 16S rDNA of Burkholderia glumae . The results of the molecular phylogenetic relationship analysis of the 16S rDNA nucleotide sequence are shown in FIG. As can be seen from Fig. 1, the selected strains were found to belong to the same group as Burkholderia glumae . As a result, the present inventors designated the selected strain as " Burkholderia glumae OK001" and deposited this on the April 11, 2017 issue with the National Institute of Agricultural Science and Technology (KACC) I received the deposit number KACC92174P on the first day of the month.

<< 실시예Example 2>  2> 부르크홀데리아Burg Helderia 글루메Glume OK001 균주의 배양물 제조 Culture of OK001 strain

배양배지로는 Luria Bertani(LB) 배지를 사용하였고 그 조성은 카제인펩톤(casein peptone) 1%, 효모추출물(yeast extract) 0.5%, sodium chloride 0.5%를 함유하고, 고체배양을 위해 한천 1.5%를 사용하였다. 121℃에서 15분간 멸균하여 시용하였다. 배양조건은 28℃ 또는 37℃, 200~220rpm으로 24시간 배양하여 사용하였다. Luria Bertani (LB) medium was used as a culture medium. The composition contained 1% casein peptone, 0.5% yeast extract and 0.5% sodium chloride, and 1.5% agar for solid culture Respectively. And sterilized at 121 DEG C for 15 minutes. Culture conditions were incubated at 28 ° C or 37 ° C, 200-220 rpm for 24 hours.

대조균으로서 Jeong 등(Plant Disease 87:890-895)이 사용한 부르크홀데리아 글루메(Burkholderia glumae) BGR1과 상기 <실시예 1>에서 분리된 부르크홀데리아 글루메 OK001 균주를 각각 배양하여 배양물을 비교해 본 결과, 상기 부르크홀데리아 글루메 OK001 균주는 배양배지에서 노란색 색소를 현저히 많이 생산하는 것을 확인하였다(도 2).The Burkholderia glumae BGR1 used as a control strain by Jeong et al. (Plant Disease 87: 890-895) and the Burkholderia glume OK001 strain isolated from Example 1 were cultured, As a result of comparison, it was confirmed that the strain Burkholderia glume OK001 produced a considerable amount of yellow pigment in the culture medium (FIG. 2).

<< 실시예Example 3>  3> 부르크홀데리아Burg Helderia 글루메Glume OK001 균주의  Of the OK001 strain 톡소플라빈Toxoplavin 생산능Production capacity 분석 analysis

상기 <실시예 2>에서 제조된 부르크홀데리아 글루메 OK001 균주의 배양물의 배양액을 10000 rpm에서 10분 동안 원심분리하여 상등액을 취하였다. 1:1 비율의 클로로포름을 넣어 잘 섞이도록 흔들어 주었다. 실온에서 방치하여 클로로포름층과 수층이 분리되게 한 뒤, 클로로포름층만 따로 분리해 내었다. 같은 방법으로 3번 반복하여 총 300 ml의 클로로포름층을 받아 내었으며, 클로로포름층(클로로포름 추출물)을 증류하여 독소 물질을 회수하여 수득하였다.The supernatant was centrifuged at 10,000 rpm for 10 minutes to obtain a culture solution of the culture of the strain Burkholderia glume OK001 prepared in Example 2 above. Add 1: 1 chloroform and shake well. The mixture was allowed to stand at room temperature to separate the chloroform layer and the aqueous layer, and then the chloroform layer alone was separated. The same procedure was repeated three times to obtain a total of 300 ml of chloroform layer. The chloroform layer (chloroform extract) was distilled to recover the toxin substance.

부르크홀데리아 글루메 OK001 균주의 배양물로부터 수득된 상기 독소 물질에 대한 정성 검정을 위하여, Jeong 등(Plant Disease 87:890-895)이 사용한 일반적인 방법에 따라 ESI-MASS(electrospray ionization mass spectrometry)를 이용하여 질량 분석을 실시하였다. 그 결과, 부르크홀데리아 글루메 OK001 균주가 생성한 독소 물질은 합성 톡소플라빈의 ESI-MASS(Plant Disease 87:890-895) 분석자료와 일치하였다. 따라서, 부르크홀데리아 글루메 OK001 균주가 생산한 독소 물질이 톡소플라빈인 것을 확인하였다(도 3).ESI-MASS (electrospray ionization mass spectrometry) was performed according to the general method used by Jeong et al. (Plant Disease 87: 890-895) for the qualitative assay of the toxin substance obtained from the culture of the strain Burkholderia glume OK001 Mass spectrometry was performed. As a result, the toxin substance produced by the strain Burkholderia glume OK001 was consistent with the ESI-MASS (Plant Disease 87: 890-895) analysis data of synthetic toxoplasia. Therefore, it was confirmed that the toxin substance produced by the strain Burkholderia glume OK001 was toxoplasin (Fig. 3).

<< 실시예Example 4>  4> 부르크홀데리아Burg Helderia 글루메Glume OK001 균주의  Of the OK001 strain 톡소플라빈Toxoplavin 생산 효율성 분석 Production efficiency analysis

부르크홀데리아 글루메 OK001 균주의 톡소플라빈 생산성 효율은 Kim 등(Molecular Microbiology, 54:921-934)이 사용한 일반적인 방법에 따라 실시하였다. 톡소플라빈 생성 대조균으로 Jeong 등(Plant Disease 87:890-895)이 사용한 부르크홀데리아 글루메(Burkholderia glumae) BGR1을 사용하였다. 부르크홀데리아 글루메 OK001 및 BGR1 균주를 각각 28℃ 또는 37℃, 200~220rpm으로 24시간 배양하였고, 배양액의 클로로포름 추출물을 증류하여 얻은 물질을 살균수에 녹여 스펙트로 포토미터(260 nm)에서 정량하였다. 그 결과 28℃에서 부르크홀데리아 글루메 OK001 균주가 대조균 부르크홀데리아 글루메 BGR1보다 톡소플라빈 생산성이 300%이상 높은 것으로 나타났다(도 4). 따라서, 본 발명의 부르크홀데리아 글루메 OK001 균주는 톡소플라빈을 매우 높은 효율로 생산할 수 있음을 확인하였다.Toxoplasma productivity efficiency of the strain Burkholderia glume OK001 was determined according to the general method used by Kim et al. (Molecular Microbiology, 54: 921-934). As Toxoplasma flavin generation control bacteria Jeong etc. (Plant Disease 87: 890-895) is Burkholderia glue methoxy (Burkholderia used glumae ) BGR1 was used. Bruchholderia glume OK001 and BGR1 were cultured at 28 ° C or 37 ° C for 24 hours at 200-220 rpm. The chloroform extract of the culture was distilled and dissolved in sterilized water and quantitated in a spectrophotometer (260 nm) . As a result, the bacterial OK001 strain at 28 ° C showed toxoplavene productivity of 300% or more higher than that of the control bacteria BGR1 (FIG. 4). Therefore, it was confirmed that the strain Burkholderia glume OK001 of the present invention can produce toxoplasin with a very high efficiency.

이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

국립농업과학원 농업유전자원센터National Institute of Agricultural Science KACC92174PKACC92174P 2017050120170501

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Burkholderia glumae OK001 strain and a method of toxoflavin production using the strain <130> PN1703-124 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1493 <212> DNA <213> Burkholderia glumae <400> 1 agagtttgat gtggctcaga ttgaacgctg gcggcatgcc ttacacatgc aagtcgaacg 60 gcagcacggg tgcttgcacc tggtggcgag tggcgaacgg gtgagtaata catcggaaca 120 tgtcctgtag tgggggatag cccggcgaaa gccggattaa taccgcatac gatctacgga 180 agaaagcggg ggaccttcgg gcctcgcgct atagggttgg ccgatggctg attagctagt 240 tggtggggta aaggcctacc aaggcgacga tcagtagctg gtctgagagg acgaccagcc 300 acactgggac tgagacacgg cccagactcc tacgggaggc agcagtgggg aattttggac 360 aatgggcgaa agcctgatcc agcaatgccg cgtgtgtgaa gaaggccttc gggttgtaaa 420 gcacttttgt ccggaaagaa atcctgaggg ctaatatcct tcggggatga cggtaccgga 480 agaataagca ccggctaact acgtgccagc agccgcggta atacgtaggg tgcgagcgtt 540 aatcggaatt actgggcgta aagcgtgcgc aggcggtttg ctaagaccga tgtgaaatcc 600 ccgggctcaa cctgggaact gcattggtga ctggcaggct agagtatggc agaggggggt 660 agaattccac gtgtagcagt gaaatgcgta gagatgtgga ggaataccga tggcgaaggc 720 agccccctgg gccaatactg acgctcatgc acgaaagcgt ggggagcaaa caggattaga 780 taccctggta gtccacgccc taaacgatgt caactagttg ttggggattc atttccttag 840 taacgtagct aacgcgtgaa gttgaccgcc tggggagtac ggtcgcaaga ttaaaactca 900 aaggaattga cggggacccg cacaagcggt ggatgatgtg gattaattcg atgcaacgcg 960 aaaaacctta cctacccttg acatggtcgg aatcctgagg agactcggga gtgctcgaaa 1020 gagaaccgat acacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt 1080 aagtcccgca acgagcgcaa cccttgtcct tagttgctac gcaagagcac tctaaggaga 1140 ctgccggtga caaaccggag gaaggtgggg atgacgtcaa gtcctcatgg cccttatggg 1200 tagggcttca cacgtcatac aatggtcgga acagggggtt gccaacccgc gagggggagc 1260 taatcccaga aaaccgatcg tagtccggat tgcactctgc aactcgagtg catgaagctg 1320 gaatcgctag taatcgcgga tcagcatgcc gcggtgaata cgttcccggg tcttgtacac 1380 accgcccgtc acaccatggg agtgggtttt accagaagtg gctagtctaa ccgcaaggag 1440 gacggtcacc acggtaggat tcatgactgg ggtgaagtcg taacaaggta gcc 1493 <110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Burkholderia glumae OK001 strain and a method of toxoflavin          production using the strain <130> PN1703-124 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1493 <212> DNA <213> Burkholderia glumae <400> 1 agagtttgat gtggctcaga ttgaacgctg gcggcatgcc ttacacatgc aagtcgaacg 60 gcagcacggg tgcttgcacc tggtggcgag tggcgaacgg gtgagtaata catcggaaca 120 tgtcctgtag tgggggatag cccggcgaaa gccggattaa taccgcatac gatctacgga 180 agaaagcggg ggaccttcgg gcctcgcgct atagggttgg ccgatggctg attagctagt 240 tggtggggta aaggcctacc aaggcgacga tcagtagctg gtctgagagg acgaccagcc 300 acactgggac tgagacacgg cccagactcc tacgggaggc agcagtgggg aattttggac 360 aatgggcgaa agcctgatcc agcaatgccg cgtgtgtgaa gaaggccttc gggttgtaaa 420 gcacttttgt ccggaaagaa atcctgaggg ctaatatcct tcggggatga cggtaccgga 480 agaataagca ccggctaact acgtgccagc agccgcggta atacgtaggg tgcgagcgtt 540 aatcggaatt actgggcgta aagcgtgcgc aggcggtttg ctaagaccga tgtgaaatcc 600 ccgggctcaa cctgggaact gcattggtga ctggcaggct agagtatggc agaggggggt 660 agaattccac gtgtagcagt gaaatgcgta gagatgtgga ggaataccga tggcgaaggc 720 agccccctgg gccaatactg acgctcatgc acgaaagcgt ggggagcaaa caggattaga 780 taccctggta gtccacgccc taaacgatgt caactagttg ttggggattc atttccttag 840 taacgtagct aacgcgtgaa gttgaccgcc tggggagtac ggtcgcaaga ttaaaactca 900 aaggaattga cggggacccg cacaagcggt ggatgatgtg gattaattcg atgcaacgcg 960 aaaaacctta cctacccttg acatggtcgg aatcctgagg agactcggga gtgctcgaaa 1020 gagaaccgat acacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt 1080 aagtcccgca acgagcgcaa cccttgtcct tagttgctac gcaagagcac tctaaggaga 1140 ctgccggtga caaaccggag gaaggtgggg atgacgtcaa gtcctcatgg cccttatggg 1200 tagggcttca cacgtcatac aatggtcgga acagggggtt gccaacccgc gagggggagc 1260 taatcccaga aaaccgatcg tagtccggat tgcactctgc aactcgagtg catgaagctg 1320 gaatcgctag taatcgcgga tcagcatgcc gcggtgaata cgttcccggg tcttgtacac 1380 accgcccgtc acaccatggg agtgggtttt accagaagtg gctagtctaa ccgcaaggag 1440 gacggtcacc acggtaggat tcatgactgg ggtgaagtcg taacaaggta gcc 1493

Claims (7)

톡소플라빈 생산능을 갖는, 부르크홀데리아 글루메(Burkholderia glumae) OK001 균주(수탁번호: KACC92174P).Toxoplasma Plata with an empty production capacity, Burkholderia glue-mail (Burkholderia glumae ) OK001 strain (accession number: KACC92174P). 제1항에 있어서,
상기 부르크홀데리아 글루메 OK001 균주는 벼(Oryza sativa) 이삭에서 분리된 것인 부르크홀데리아 글루메 OK001 균주.The method according to claim 1,
The Burkholderia glume OK001 strain was isolated from rice ( Oryza sativa ) sp., A strain of Burkholderia glume OK001. 제1항에 따른 부르크홀데리아 글루메 OK001 균주의 배양물.A culture of a strain of Burkholderia glume OK001 according to claim 1. 제1항에 따른 부르크홀데리아 글루메 OK001 균주 또는 제3항에 따른 부르크홀데리아 글루메 OK001 균주의 배양물을 포함하는 톡소플라빈(toxoflavin) 제조용 조성물.A composition for the production of toxoflavin comprising a strain of Burkholderia glume OK001 according to claim 1 or a strain of Burkholderia glume OK001 according to claim 3. 제1항에 따른 부르크홀데리아 글루메 OK001 균주를 배양하는 단계 및 상기 균주의 배양물로부터 톡소플라빈을 회수하는 단계를 포함하는, 톡소플라빈 제조방법.A method for producing toxoplasia comprising culturing a strain of Burkholderia glume OK001 according to claim 1 and recovering toxoplasia from the culture of said strain. 제5항에 있어서,
상기 배양은 Potato Dextrose Broth(PDB), Nutrient Broth(NB), Casamino acid Peptone Glucose(CPG), Tryptic Soybean Broth(TSB), King’s B(KB) 및 Luria Bertani(LB)으로 이루어진 군으로부터 선택되는 배지를 사용하여 수행되는 것인 톡소플라빈 제조방법.6. The method of claim 5,
The culture was performed on a medium selected from the group consisting of Potato Dextrose Broth (PDB), Nutrient Broth (NB), Casamino acid Peptone Glucose (CPG), Tryptic Soybean Broth (TSB), King's B (KB) and Luria Bertani &Lt; / RTI &gt; 제5항에 있어서,
상기 톡소플라빈의 회수는 클로로포름으로 추출하여 수행되는 것인 톡소플라빈 제조방법.6. The method of claim 5,
Wherein the recovery of the toxoplasmin is carried out by extracting with chloroform.
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KR101630211B1 (en) 2014-07-21 2016-06-16 한국화학연구원 Toxoflavin- degrading enzymes derived from metagenome and the use thereof

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