KR20130124018A - Method for differentiating from enterobacter cloacae and bacillus subtilis using dihydrogenase expressed from yoga gene - Google Patents
Method for differentiating from enterobacter cloacae and bacillus subtilis using dihydrogenase expressed from yoga gene Download PDFInfo
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Abstract
Description
본 발명은 엔테로박터 클로아세의 yogA 유전자로부터 발현되는 디히드로게나아제와 특이적으로 결합하는 형광 화합물을 포함하는 엔테로박터 클로아세 검출 키트 및 이를 이용한 엔테로박터 클로아세 검출 방법에 관한 것이다.The present invention relates to an Enterobacter cloase detection kit comprising a fluorescent compound that specifically binds to a dehydrogenase expressed from the yogA gene of Enterobacter cloase, and an enterobacter cloase detection method using the same.
세균 동정은 감염성 질병 진단의 가장 중요한 과정 중의 하나이다. 기존의 세균 동정 방법은 많은 검사 종목과 특수 배지나 시약을 필요하고, 상용화된 동정 키트는 간단하고 비교적 빠른 결과를 얻을 수 있으나, 키트의 가격이 비싸며 제품에 따라서는 정확한 동정이 이루어지지 않을 수도 있다. Bacterial identification is one of the most important processes of infectious disease diagnosis. Conventional bacterial identification methods require many test items and special media or reagents, and commercially available identification kits can achieve simple and relatively fast results, but the kits are expensive and may not be identified correctly depending on the product. .
바실러스 서브틸리스와 엔테로박터 클로아세를 구별하는데 있어, 유전자 서열법과 같은 거대 장비를 이용하지 않고, 균주 사이의 단백체 차이를 실험실 수준에서 확인 가능한 수단을 제공하는 방법은 기존에 알려져 있지 않다.In distinguishing Bacillus subtilis from Enterobacter cloase, it is not known how to provide a means of identifying protein differences between strains at the laboratory level without using large equipment such as gene sequencing.
따라서, 본 발명에서는 yogA 유전자 발현 산물을 형광 화합물로 검출하여 간단하고, 빠르며, 정확하게 Enterobacter cloacae와 Bacillus subtilis의 구별 방법을 제공하고자 한다.Accordingly, the present invention is to provide a method for distinguishing Enterobacter cloacae and Bacillus subtilis by detecting yogA gene expression products with fluorescent compounds.
일 구체예는 형광 화합물을 이용하여 바실러스 서브틸리스와 엔테로박터 클로아세를 구별하는 방법에 관한 것이다.One embodiment relates to a method of distinguishing Bacillus subtilis from Enterobacter cloace using fluorescent compounds.
다른 구체예는 엔테로박터 클로아세의 yogA 유전자로부터 발현되는 디히드로게나아제와 특이적으로 결합하는 형광 화합물을 포함하는 엔테로박터 클로아세 검출 키트를 제공하는 것이다.Another embodiment is to provide an Enterobacter cloase detection kit comprising a fluorescent compound that specifically binds to a dehydrogenase expressed from the yogA gene of Enterobacter cloase .
일 양상은 엔테로박터 클로아세(Enterobacter cloacae) 및 바실러스 서브틸리스(Bacillus subtilis)로부터 세균 추출물을 수득하는 단계;One aspect includes obtaining bacterial extracts from Enterobacter cloacae and Bacillus subtilis ;
상기 세균 추출물과 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온을 접촉시키는 단계; 및Contacting the bacterial extract with 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'-(9H) xanthene) -3-one ; And
상기 세균 추출물로부터 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온이 표지된 디히드로게나아제(dehydrogenase)를 검출하는 단계를 포함하는 바실러스 서브틸리스와 엔테로박터 클로아세를 구별하는 방법을 제공한다.3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'-(9H) xanthene) -3-one labeled from the bacterial extract It provides a method of distinguishing Bacillus subtilis and Enterobacter cloase comprising the step of detecting the dehydrogenase (hydrogenase).
본 발명자들은 바실러스 서브틸리스와 엔테로박터 클로아세를 쉽게 구별할 수 있는 방법을 연구한 결과, 특정 형광 화합물이 엔테로박터 클로아세의 yogA 유전자로부터 발현되는 디히드로게나아제와 특이적으로 결합한다는 사실을 확인함으로서 본 발명을 완성하였다.The present inventors studied a method for easily distinguishing Bacillus subtilis from Enterobacter cloase , and found that a specific fluorescent compound specifically binds to dehydrogenase expressed from the yogA gene of Enterobacter cloase. By completing the present invention.
상기 방법은 먼저, 바실러스 서브틸리스와 엔테로박터 클로아세로부터 세균 추출물을 수득하는 단계를 포함할 수 있다.The method may first comprise obtaining a bacterial extract from Bacillus subtilis and Enterobacter cloase.
본 명세서에서 용어, "세균 추출물"은 구별하고자 하는 대상의 세균 내에 포함되어 있는 단백체 전체 집단을 의미하는 것으로 해석될 수 있다. 세균 추출물은 당업계에 널리 알려진 방법에 따라, 세균을 용해할 수 있는 키트를 이용하거나, 초음파를 이용하여 세균을 분쇄하고 원심분리를 통해 세균 내 단백체만 얻는 방법 등을 통해 수득할 수 있다.As used herein, the term "bacterial extract" may be interpreted to mean the entire protein group contained in the bacteria of the subject to be distinguished. The bacterial extract may be obtained by using a kit capable of dissolving the bacteria, or by pulverizing the bacteria using ultrasonic waves and obtaining only protein in the bacteria by centrifugation according to methods well known in the art.
본 발명은 엔테로박터 클로아세의 yogA 유전자로부터 발현되는 디히드로게나아제에 특이적으로 결합하는 형광 화합물인 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온을 이용하는 것으로, 바실러스 서브틸리스와 엔테로박터 클로아세를 구별할 수 있다.The present invention relates to 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran), a fluorescent compound that specifically binds to a dehydrogenase expressed from the yogA gene of Enterobacter cloase. By using -1 (3H), 9 '-(9H) xanthene) -3-one, Bacillus subtilis and Enterobacter cloase can be distinguished.
다음으로, 상기 세균 추출물과 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온을 접촉시키는 단계를 거치게 된다.Next, the bacterial extract and 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'-(9H) xanthene) -3-one The contacting step is performed.
본 발명에서 사용되는 형광 화합물인 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온은 Cell TrackerTM Green CMFDA(Invitrogen, inc. Cat# C2925)의 이름으로 판매되는 화합물로서, 하기 화학식 I의 화학식으로 나타낼 수 있다. 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'-(9H) xanthene) -3-one, the fluorescent compound used in the present invention Is a compound sold under the name of Cell Tracker ™ Green CMFDA (Invitrogen, inc. Cat # C2925), which may be represented by the formula of Formula I below.
화학식 IFormula I
일 구체예에 따르면, 상기 형광 화합물을 상기 수득한 세균 추출물에 직접 접촉시킨 이후, 상기 접촉된 결과물을 전기영동하여 상기 세균 추출물의 단백질 패턴을 확인하는 단계를 더 포함할 수 있다. 상기 전기영동은 당업계에 잘 알려진 SDS-PAGE 방법을 통해 이루어질 수 있다.According to one embodiment, after directly contacting the fluorescent compound with the obtained bacterial extract, it may further comprise the step of confirming the protein pattern of the bacterial extract by electrophoresis of the contacted result. The electrophoresis may be performed through the SDS-PAGE method well known in the art.
마지막으로, 상기 방법은 상기 세균 추출물로부터 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온이 표지된 디히드로게나아제를 검출하는 단계를 거치게 된다.Finally, the method is a 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'- (9H) xanthene) -3 from the bacterial extract Detecting labeled dehydrogenase
일 구체예에 따르면, 상기 디히드로게나아제는 엔테로박터 클로아세의 yogA 유전자로부터 발현되는 것일 수 있으며, 상기 yogA 유전자로부터 발현되는 디히드로게나아제는 서열번호 1에 나타내었다.According to one embodiment, the dehydrogenase may be expressed from the yogA gene of Enterobacter cloase , and the dehydrogenase expressed from the yogA gene is shown in SEQ ID NO: 1.
상기 형광 화합물은 상기 접촉된 결과물을 전기영동한 후, 상기 형광 화합물을 검출할 수 있는 스캐너를 이용하여 검출할 수 있으며, 상기 형광 화합물이 결합된 단백질 또는 상기 단백질의 패턴을 비교함으로써, 바실러스 서브틸리스와 엔테로박터 클로아세를 구별할 수 있다.The fluorescent compound may be detected by using a scanner capable of detecting the fluorescent compound after electrophoresis of the contacted product, and by comparing the protein to which the fluorescent compound is bound or the pattern of the protein, Bacillus subtilis It is possible to distinguish between and Enterobacter cloase.
다른 양상은 서열번호 1의 아미노산 서열로 표시되는 엔테로박터 클로아세의 yogA 유전자로부터 발현되는 디히드로게나아제를 포함하는 엔테로박터 클로아세 검출용 바이오 마커를 제공한다.Another aspect provides a close-acetoxy Enterobacter bakteo for detecting a biomarker comprising the dehydrogenase is expressed from a gene of the Enterobacter yogA bakteo claw acetate represented by the amino acid sequence of SEQ ID NO: 1.
또 다른 양상은 엔테로박터 클로아세의 yogA 유전자로부터 발현되는 디히드로게나아제와 특이적으로 결합하는 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온을 포함하는 엔테로박터 클로아세 검출용 키트를 제공한다.Another aspect is 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1) that specifically binds to dehydrogenase expressed from the yogA gene of Enterobacter cloase. Provided is a kit for detecting enterobacter cloase comprising (3H), 9 '-(9H) xanthene) -3-one.
일 구체예에 따른 엔테로박터 클로아세 검출용 키트는 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온 및 이를 안정화시킬 수 있는 버퍼 등을 포함할 수 있으며, 엔테로박터 클로아세의 yogA 유전자로부터 발현되는 디히드로게나아제를 검출하는 용도로서 사용될 수 있다.Enterobacter cloase detection kit according to one embodiment is 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'- (9H) xanthene ) -3-one, a buffer capable of stabilizing the same, and the like, and may be used as a detection of dehydrogenase expressed from the yogA gene of Enterobacter cloase .
한편, 상기 키트는 바실러스 서브틸리스와 엔테로박터 클로아세를 구별하는 데 사용될 수 있다.On the other hand, the kit can be used to distinguish between Bacillus subtilis and Enterobacter cloase.
일 구체예에 따르면, 본 발명의 방법 및 키트는 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온에 특이적으로 결합하는 엔테로박터 클로아세의 yogA 유전자로부터 발현되는 디히드로게나아제를 검출하여 Enterobacter cloacae와 Bacillus subtilis를 구별하므로, 쉽고 빠르며 정확한 동정 결과를 제공할 수 있다.According to one embodiment, the methods and kits of the invention comprise 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'-(9H) xanthene Enterobacter by Dehydrogenase Detection from Enterobacter Cloase YogA Gene cloacae and Bacillus By distinguishing subtilis , it is possible to provide easy, fast and accurate identification results.
도 1은 일 구체예에 따른 방법에 의해 Enterobacter cloacae ATCC 27508와 Bacillus subtilis ATCC 6633의 단백질 패턴을 비교한 결과이다.1 is a result of comparing the protein pattern of Enterobacter cloacae ATCC 27508 and Bacillus subtilis ATCC 6633 by the method according to one embodiment.
이하 하나 이상의 구체예를 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.
Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.
1. 실험 방법 1. Experimental Method
(1) 세균의 배양 및 단백체의 준비(1) Cultivation of bacteria and preparation of protease
nterobacter cloacae ATCC 27508와 Bacillus subtilis ATCC 6633의 단백체를 준비하기 위하여, 상기 세균을 ATCC로부터 구매하였다. Enterobacter cloacae와 Bacillus subtilis는 각각 1 ml의 LB broth(DifcoTM LB Agar, Becton, Dickinson and Company, Lot#: 0049528)에 접종하여 37℃에서 2시간 동안 배양한 후, 20 ul를 LB Agar plate(DifcoTM LB Agar, Becton, Dickinson and Company, Lot#: 0137418)에서 배양하여, 단일 콜로니를 얻은 다음, 100 ml의 LB 배지에 배양하였다. 배양한 세균을 원심분리 하여 상층액을 버리고, PBS 용액(pH 7.4)으로 2번 씻어 준 후, Genlantis 사의 SoluLyse kit(Cat #: L200125)를 이용하여 최종적으로 20 mM Tris-HCl, pH7.5에 용해된 상기 세균들의 단백질 혼합물 용액을 얻었다.
To prepare the proteins of nterobacter cloacae ATCC 27508 and Bacillus subtilis ATCC 6633, the bacteria were purchased from ATCC. Enterobacter cloacae and Bacillus subtilis were inoculated in 1 ml of LB broth (Difco TM LB Agar, Becton, Dickinson and Company, Lot #: 0049528) and incubated for 2 hours at 37 ° C. TM LB Agar, Becton, Dickinson and Company, Lot #: 0137418) to obtain a single colony and then incubated in 100 ml of LB medium. The cultured bacteria were centrifuged, and the supernatant was discarded. The supernatant was discarded and washed twice with PBS solution (pH 7.4). The cells were finally washed with 20 mM Tris-HCl, pH 7.5 using a SoluLyse kit (Cat #: L200125) To obtain a protein mixture solution of the dissolved bacteria.
(2) 형광 화합물 처리 및 SDS-PAGE 이미지 분석(2) Fluorescent compound treatment and SDS-PAGE image analysis
20 mM Tris-HCl, pH 7.5의 조건에서, 동일한 농도의 상기 세균 단백질 혼합물 용액에 DMSO 1%에 포함된 20 uM의 상기 형광 화합물을 처리하고, 25℃에서 1시간 동안 인큐베이션 하였다. 반응을 마친 후, Laemmli 샘플 버퍼를 넣고, 95℃에서 2분 동안 가열한 다음, 15% SDS가 포함된 폴리아크릴아미드 겔에 로딩하여 전기영동(120V 20분, 이어서 180V 60분)한 후, 상기 Enterobacter cloacae 및 Bacillus subtilis의 단백질의 패턴을 형광 SDS-PAGE Scanner(TyphoonTM 9400, GE Health Science)로 확인하였다. 이때, Cell TrackerTM Green CMFDA의 표지 단백질 이미지는 TyphoonTM 9400 기기의 ex: 488 nm, em: 526-SP 조건에서 얻었다.Under the conditions of 20 mM Tris-HCl, pH 7.5, the same concentration of the bacterial protein mixture solution was treated with 20 uM of the fluorescent compound contained in 1% DMSO, and incubated at 25 ° C for 1 hour. After completion of the reaction, the Laemmli sample buffer was added, heated at 95 ° C. for 2 minutes, loaded onto a polyacrylamide gel containing 15% SDS, followed by electrophoresis (120 V 20 minutes, followed by 180 V 60 minutes). Enterobacter cloacae And the pattern of the protein of Bacillus subtilis was confirmed by the fluorescent SDS-PAGE Scanner (Typhoon TM 9400, GE Health Science). At this time, the label protein image of the Cell Tracker ™ Green CMFDA was obtained under ex: 488 nm, em: 526-SP of the Typhoon ™ 9400 instrument.
2. 실험 결과2. Experimental results
도 1에서 보는 바와 같이, SDS-PAGE 결과로부터 Bacillus subtilis의 단백질 패턴에서는 나타나지 않지만, Enterobacter cloacae의 단백질 패턴에서 보이는 36kDa 정도의 단백질이 Cell TrackerTM Green과 상호 작용함을 알 수 있었다. 이후, 상기 36kDa의 단백질을 확인하기 위하여 다음과 같은 생물정보학적 방법을 사용하였다.As shown in FIG. 1, the SDS-PAGE showed no protein pattern of Bacillus subtilis , but the protein of about 36 kDa shown in the protein pattern of Enterobacter cloacae interacted with Cell Tracker TM Green. Then, the following bioinformatics method was used to identify the 36kDa protein.
상기 형광 화합물과 36kDa 단백질의 상호 작용을 예측하기 위해서는 각각의 3차원 구조가 필요하다. 이를 위해, 단백질 구조 모델링 프로그램을 사용하였으며, 본 실시예에서는 호몰로지 모델링(homology modeling) 프로그램인 'MODELLER'(University of California San Francisco)를 이용하여 상기 36kDa 단백질 3차원 구조를 예측하였다. 또한, 상기 36kDa 단백질과 Cell TrackerTM Green과의 상호작용을 예상하기 위하여 단백질과 화합물의 3차원 구조를 바탕으로 상호작용을 예측할 수 있는 도킹(docking) 프로그램을 사용하였다. 본 실시예에서는 'AutoDock'(The Scripps Research Institute) 프로그램을 이용하여 상기 36kDa 단백질과 상기 형광 화합물의 결합을 예측하였다. 그 결과, 상기 36kDa 단백질은 Enterobacter cloacae의 yogA 유전자로부터 발현되는 디히드로게나아제(dehydrogenase)임을 확인할 수 있었다. yogA 유전자는 Enterobacter cloacae와 Bacillus subtilis에 모두 존재하는 유전자이지만, 상기 결과로부터 Cell TrackerTM Green은 yogA 유전자로부터 발현되는 디히드로게나아제와 상호 작용함을 확인할 수 있었다. 따라서, Cell TrackerTM Green을 사용하여 yogA 유전자로부터 발현되는 디히드로게나아제의 존재 여부를 확인함으로써, Enterobacter cloacae와 Bacillus subtilis를 구별할 수 있음을 확인할 수 있었다.Each three-dimensional structure is required to predict the interaction between the fluorescent compound and the 36kDa protein. To this end, a protein structure modeling program was used, and in this embodiment, the 36kDa protein 3D structure was predicted using a homology modeling program 'MODELLER' (University of California San Francisco). In addition, the 36kDa protein and Cell Tracker TM To predict the interaction with Green, we used a docking program to predict the interaction based on the three-dimensional structure of proteins and compounds. In this example, the binding of the 36kDa protein and the fluorescent compound was predicted using 'AutoDock' (The Scripps Research Institute) program. As a result, the 36kDa protein was confirmed to be a dehydrogenase (dehydrogenase) expressed from the yogA gene of Enterobacter cloacae . The yogA gene is a gene present in both Enterobacter cloacae and Bacillus subtilis , but from the above results, it was confirmed that Cell Tracker TM Green interacted with the dehydrogenase expressed from the yogA gene. Therefore, Enterobacter cloacae and Bacillus subtilis could be distinguished by checking the presence of dehydrogenase expressed from the yogA gene using Cell Tracker TM Green.
<110> Korea Institute of Science and Technology <120> Method for differentiating from Enterobacter cloacae and Bacillus subtilis using dihydrogenase expressed from yogA gene <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 202 <212> PRT <213> Enterobacter cloacae <400> 1 Met Asn Arg Phe Val Ile Ala Asp Ser Thr Val Cys Ile Gly Cys Arg 1 5 10 15 Thr Cys Glu Ala Ala Cys Ser Glu Thr His Arg Gln His Gly Leu Gln 20 25 30 Ser Met Pro Arg Leu Arg Val Met Arg Asn Glu Lys Glu Ser Ala Pro 35 40 45 Gln Leu Cys His His Cys Glu Asp Ala Pro Cys Ala Gly Val Cys Pro 50 55 60 Val Asn Ala Ile Thr Arg Val Glu Gly Ala Val Gln Leu Asn Glu Ser 65 70 75 80 Leu Cys Val Ser Cys Lys Leu Cys Gly Ile Ala Cys Pro Phe Gly Ala 85 90 95 Ile Glu Phe Ser Gly Ser Arg Pro Leu His Ile Pro Ala Asn Ala Asn 100 105 110 Ser Pro Lys Ala Pro Pro Ala Pro Pro Ala Pro Ala Lys Val Ser Thr 115 120 125 Leu Leu Asp Trp Val Pro Gly Val Arg Ala Val Ala Val Lys Cys Asp 130 135 140 Leu Cys Ser Phe Asp Glu Gln Gly Pro Ala Cys Val Arg Thr Cys Pro 145 150 155 160 Thr Lys Ala Leu Ile Leu Val Asn Ile Arg Asp Ile Val Arg Thr Ser 165 170 175 Lys Arg Lys Arg Glu Leu Thr Ile Asn Ser Asp Phe Gly Asp Leu Ser 180 185 190 Leu Leu Gln Ala Leu Asn Glu Gly Ala Lys 195 200 <110> Korea Institute of Science and Technology <120> Method for differentiating from Enterobacter cloacae and Bacillus subtilis using dihydrogenase expressed from yogA gene <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 202 <212> PRT <213> Enterobacter cloacae <400> 1 Met Asn Arg Phe Val Ile Ala Asp Ser Thr Val Cys Ile Gly Cys Arg 1 5 10 15 Thr Cys Glu Ala Ala Cys Ser Glu Thr His Arg Gln His Gly Leu Gln 20 25 30 Ser Met Pro Arg Leu Arg Val Met Arg Asn Glu Lys Glu Ser Ala Pro 35 40 45 Gln Leu Cys His His Cys Glu Asp Ala Pro Cys Ala Gly Val Cys Pro 50 55 60 Val Asn Ala Ile Thr Arg Val Glu Gly Ala Val Gln Leu Asn Glu Ser 65 70 75 80 Leu Cys Val Ser Cys Lys Leu Cys Gly Ile Ala Cys Pro Phe Gly Ala 85 90 95 Ile Glu Phe Ser Gly Ser Arg Pro Leu His Ile Pro Ala Asn Ala Asn 100 105 110 Ser Pro Lys Ala Pro Pro Ala Pro Pro Ala Pro Ala Lys Val Ser Thr 115 120 125 Leu Leu Asp Trp Val Pro Gly Val Arg Ala Val Ala Val Lys Cys Asp 130 135 140 Leu Cys Ser Phe Asp Glu Gln Gly Pro Ala Cys Val Arg Thr Cys Pro 145 150 155 160 Thr Lys Ala Leu Ile Leu Val Asn Ile Arg Asp Ile Val Arg Thr Ser 165 170 175 Lys Arg Lys Arg Glu Leu Thr Ile Asn Ser Asp Phe Gly Asp Leu Ser 180 185 190 Leu Leu Gln Ala Leu Asn Glu Gly Ala Lys 195 200
Claims (6)
상기 세균 추출물과 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온을 접촉시키는 단계; 및
상기 세균 추출물로부터 3',6'-비스(아세틸옥시)-5-(클로로메틸)-스피로(이소벤조퓨란-1(3H),9'-(9H)잔텐)-3-온이 표지된 디히드로게나아제를 검출하는 단계를 포함하는 바실러스 서브틸리스와 엔테로박터 클로아세를 구별하는 방법.Obtaining a bacterial extract from Bacillus subtilis and Enterobacter cloase;
Contacting the bacterial extract with 3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'-(9H) xanthene) -3-one ; And
3 ', 6'-bis (acetyloxy) -5- (chloromethyl) -spiro (isobenzofuran-1 (3H), 9'-(9H) xanthene) -3-one labeled from the bacterial extract A method for distinguishing Bacillus subtilis from Enterobacter cloase, comprising detecting a hydrogenase.
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