보고서 정보
주관연구기관 |
(재)경북해양바이오산업연구원 |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2014-01 |
과제시작연도 |
2012 |
주관부처 |
해양수산부 Ministry of Oceans and Fisheries |
등록번호 |
TRKO201400020228 |
과제고유번호 |
1615004993 |
사업명 |
해양생명공학기술개발 |
DB 구축일자 |
2014-11-10
|
키워드 |
다환방향족탄화수소.총단백질체.PAH 분해효소.항체.바이오센서.Polycyclic aromatic hydrocarbon(PAH).Metaproteome PAH-degrading enzyme (Dioxygenase).Anti-body.Biosensor.
|
DOI |
https://doi.org/10.23000/TRKO201400020228 |
초록
▼
- 해양 유류오염지역, 여수(광양), 영광(변산), 군산, 태안에서 해양시료 확보
- 해양환경 시료로부터 총단백질체 추출 방법 확립 및 분석(재현성의 어려움 제기)
- PAH 분해미생물 130종 확보 (PAH 분해활성: 57종, PAH 분해활성 및 PAH 분해 효소(dioxygenase) 활성: 73종) 및 동정완료
- 우수 활성 미생물 48종 선별 (PAH 분해활성: 28종, PAH 분해활성 및 PAH 분해 효소(dioxygenase) 활성: 20종)
- 지금까지 보고되지 않은 신규 5속(18균주))의 Dio
- 해양 유류오염지역, 여수(광양), 영광(변산), 군산, 태안에서 해양시료 확보
- 해양환경 시료로부터 총단백질체 추출 방법 확립 및 분석(재현성의 어려움 제기)
- PAH 분해미생물 130종 확보 (PAH 분해활성: 57종, PAH 분해활성 및 PAH 분해 효소(dioxygenase) 활성: 73종) 및 동정완료
- 우수 활성 미생물 48종 선별 (PAH 분해활성: 28종, PAH 분해활성 및 PAH 분해 효소(dioxygenase) 활성: 20종)
- 지금까지 보고되지 않은 신규 5속(18균주))의 Dioxygenase 활성 미생물 확보
- 지금까지 보고되지 않은 9속(15균주)의 새로운 PAH 분해 미생물 확보
- 신규 PAH dioxygenase 균주 6종 확보 및 동일 균주에서 신규 PAH dioxygenase 일부서열 7개 확보
- H37균주에서 신규 PAH dioxygenase alpha subunit 확보(ORF: 1482bp, 아미노산: 494aa , 분자량: 55,453 KDa
- PAH Dioxygenase 검출용 항체 제작 (그람 음성균용과 그람양성균용 두 패턴)
- 그람 양성균 시료는 dioxygenase 존재 균주와 dioxygenase 없는 균주가 뚜렷하게 구별되는 것을 QCM 센서로 보여짐 (그람 양성균의 항체는 dioxygenase를 가진 박테리아를 screening 할 수 있는 바이오센서 개발 접근을 시사함)
Abstract
▼
Ⅳ. Results
1. Analysis of marine metaproteome
- The collected sediment from south coast, west coast, Y eosu(kwangyang bat area), Y eonggwang gun, Byeonsan peninsula, Gunsan port, Taean gun that oil contaminated soil area in may 2011.
- Targeting marine sample, there is no way to extract tot
Ⅳ. Results
1. Analysis of marine metaproteome
- The collected sediment from south coast, west coast, Y eosu(kwangyang bat area), Y eonggwang gun, Byeonsan peninsula, Gunsan port, Taean gun that oil contaminated soil area in may 2011.
- Targeting marine sample, there is no way to extract total proteome, was extracted by a modification of the extraction method a soil sample first.
- Utilizing extraction methods of Benndorf from 5 g soil samples and 100 g marine sample were extracted total proteome, the results of SDS-PAGE and Silver staining analysis, in comparison to the soil, the amount of protein present in the marine sample that very little has been confirmed.
- Increased the amount of marine samples have been extracted the total proteome with 100 g and 300 g respectively. However, only the increased of impurities(such as humic acid) which could not be confirmed band of protein was confirmed.
- Using TCA and PVPP by methods for extraction of total protein, but was not observed clear result.
2. PAH-degrading and dioxygenase activity microorganism
- 4 kinds of microorganisms (H37, S47, S49, TA24) of PAH degrading activity from commercial gasoline and sediment of Taean area was secured.
- Degrading activity of PAH of TA24 stain was confirmed. (by using HPLC)
- After enrichment culture of sediment and PAHs (Naphthalene [NA], Phenanthrene [PH], Pyrene [PY ], Benzopyrene [BPY ], concentration of 50 ppm), isolated 670 strain by using marine agar
- By using sublimation method, the PAH degradation activity of 271 strains were isolated. Also, 73 strain of dioxygenase activity through indole test were isolated.
- Finally, selected of 28 strain(PAH degradation activity) and 20 strain (PAH degradation and dioxygenase activity)
- Results of identification of selected strains, found 33 strains a new mcriobial has not been reported at NCBI.
- Selected strains of 8-NA-5 is phenanthrene and pyrene degradation activity
- Selected strains of 8-NA-5 showed that degradation activity of Phenanthrene(80%, at 6 hour) and Pyrene(50%, at 24 hour)
3. PAH-degrading enzyme
- As a result of utilizing the PCR, it was analyzed the alpha subunit of 48 strains(PAH degrading activity), to obtain 6 strains with dioxygenase gene sequence (H37, TA24, 1-Mixs-4, 1-NA-3-2, 2-NA-1, and 11-BPY -1-2).
- Gram negative bacteria of 2-NA-1 strain and 11-BPY -1-2 strain were succeed as N2 primer and Nid primer. Also, gram positive bacteria ( H37, TA24, 1-Mixs- 4, and 1-NA-3-2 strain) gene sequences were found only in Nid primer.
- Results found rieske center section nucleotide sequence in the dioxygenase alpha subunit in seven of all six strains, were compared phylogenetically with this sequence, a sequence of existing, in view of that show a difference in the sequence of existing, it is determined to be a new gene.
- Fosmid genomic DNA library to TA24 and H37 strain of the six strains were managed to build.
- As a result of exploring the colony nucleotide sequence of dioxygenase gene is arranged through the fosmid library colony and colony hybridization of H37 and TA24, TA24 strain had no confirmed but, H37 strain was confirmed hybridized colony
- Confirmed the amino acid sequence of rieske center and iron binding site which is present in alpha subunit within the conserved sequence of dioxygenase (open reading frame (ORF): 1482bp, amino acid: 494aa, molecular weight: 55,453 KDa).
4. Detection of PAH-degrading enzyme
- By using the PAH dioxygenase, to produce a two PAH dioxygenase antibody that Gram-negative bacteria (18 peptides) and gram-positive bacteria (17 peptides).
- After that by using two antibodies that are produced and to fix the antibody to the Quartz Crystal Microbalance (QCM) sensor chip, to find microorganisms that have PAH dioxygenase has tested.
- The positive to the strains showed dioxygenase activity strongly in the Indole test, was ready to distinguish between gram-positive bacteria and gram negative bacteria.
- Gram-positive bacteria samples were observed in the QCM sensor, it is clearly distinguishable dioxygenase presence strain (positive strain) and without dioxygenase strain (negative strain).
- However, it shows the results of the jagged, Gram-positive bacteria sample is judged to have come out on the results the antibody not specific or, samples of strains of gram-negative bacteria, such as binding to nonspecific to the sensor chip surface.
- In conclusion, antibody [Gram (+)] of Gram-positive bacteria to be possible to develop of biosensor which can be screening dioxygenase bacteria.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.