초록 ▼

◦연구목표
극지고유 저온적응 생물의 대사체 라이브러리 확보, 생명기작 규명, 바이오신소재 발굴을 통한 극지 바이오 연구의 활성화

◦연구내용 및 결과
- 극지 고유 생물 유래 대사체를 확보하기 위하여 남극 로스해 유래 154종 미생물 균주의 추출물 및 52종의 진균 추출물을 확보함.
- 극지고유 생물 유래 MS-라이브러리는 600개의 화합물 정보를 입력 완료함 (화합물 번호, 이름, 화학식, 분자량 등의 기본적이 정보가 포함됨).
- 7종의 극지 대사체를 분리하였으며 그 중 3종의 구조 분석을 완료함

◦연구목표
극지고유 저온적응 생물의 대사체 라이브러리 확보, 생명기작 규명, 바이오신소재 발굴을 통한 극지 바이오 연구의 활성화

◦연구내용 및 결과
- 극지 고유 생물 유래 대사체를 확보하기 위하여 남극 로스해 유래 154종 미생물 균주의 추출물 및 52종의 진균 추출물을 확보함.
- 극지고유 생물 유래 MS-라이브러리는 600개의 화합물 정보를 입력 완료함 (화합물 번호, 이름, 화학식, 분자량 등의 기본적이 정보가 포함됨).
- 7종의 극지 대사체를 분리하였으며 그 중 3종의 구조 분석을 완료함.
- 양극해 해양 진균 SF6796V 균주의 배양추출물이 항염증효과가 있음을 밝힘.
- 로바릭산과 로바스틴의 염증질환 억제 작용기전을 연구하였고 라말린과 로바릭산이 항아토피활성이 있음을 확인함.
- 극지 추출물 10종이 병행처리시 TMZ의 항암활성을 제고하는 효과가 있음을 밝힘.
- 저온성 단백질 분해효소 P66의 대량생산 시스템을 구축하여 원가 절감효과를 도출함.
- 항동결물질 p-CY01 LM이 세포외 수분과 결합하여 얼음 결정성장을 억제하고 세포내 수분함량을 감소시켜 세포의 동결보호를 유도한다는 기작을 제안함.
- 주석을 함유하지 않는 라말린을 합성하였고 라말린의 치매억제 기작을 연구함.
- 극지유래 리파아제 LipBL을 대장균에서 생산하여 높은 활성을 얻었음.
- 극지 해양미생물의 생산조건을 모델링을 이용하여 최적화 하였고 산호 공생미생물을 확보함.
- 극지 요각류를 활용하여 자외선에 따른 영향연구 및 분자기작을 규명함.

◦연구결과의 활용
이와 같은 연구결과는 극지 생물자원 활용연구 및 관련 산업 활성화에 기여할 것임.

(출처:보고서 초록)

Abstract ▼

IV. R&D Results

The results of this research are as follows.
1. Securement of metabolites derived from polar organisms

A. Acquisition of marine life extracts from Antarctic Ross Sea
Extracts of 154 microbial strains isolated from Antarctic Ross Sea in 2015 and 2016 were acquired.

IV. R&D Results

The results of this research are as follows.
1. Securement of metabolites derived from polar organisms

A. Acquisition of marine life extracts from Antarctic Ross Sea
Extracts of 154 microbial strains isolated from Antarctic Ross Sea in 2015 and 2016 were acquired. In addition, we obtained additional 52 ethyl acetate extracts from fungi.

B. Obtaining MS library derived from polar organisms
ESI Q-TOF MS analysis was performed on the samples of the Antarctic marine fungus extracts, and 600 significant items were entered into the library. The library information is stored in an excel database, and the input information contains basic information such as compound ID, name, formula, and molecular weight.

C. Metabolism analysis and structure identification
The conditions for SPE, filtration, solvent extraction, and solvent partition were established for the analysis of polar metabolites. Optimal conditions of analysis were established through various analysis conditions such as LC mobile phase, stationary phase condition, and MS ionization condition. By analyzing the samples under established conditions, the UV and MS spectra of the metabolites were obtained and the metabolic database was constructed by comparing with the literature. Seven kinds of metabolites were isolated from the two fractions derived from the extracts, and the structures of three kinds of metabolites were analyzed.

2. Research on commercialization of useful metabolites

A. Ensuring availability of new metabolites
Lobaric acid and Lobastin were found to inhibit the inflammation of macrophages.
Lobaric acid and lobastin inhibited the expression of inflammatory mediators in RAW264.7 cells. Based on these results, Lobaric acid and Lobastin in the immune function confirmed the molecular mechanism of immunological function in inflammation control. In RAW264.7 cells, the macrophage cell line, immunoregulatory activities of various substances extracted from polar organisms were confirmed, and immunomodulatory effects were observed in some substances. It was confirmed that Ramalin and Lobaric acid had anti-atopic effect in HaCaT cells, keratinocytes.
An anti-inflammatory activity of the metabolite 6,8,1'-tri-O-methyl averantin isolated from the culture extract of SF6796V strain was confirmed. Compound 6796V-53 (compound 1) inhibited MAPK and NF-κB pathway in BV2 cells derived from mouse microglial and showed excellent anti-inflammatory activity. It was also found that expression of HO-1 through PI3K / Akt and Nrf2 was involved in anti-inflammatory activity. In addition, HO-1 expression was also confirmed in microglial cells isolated from mouse brain, suggesting that HO-1-induced anti-inflammatory activity may also act on primary cultured cells of microglial cells.
As a result of the anticancer activity test, 12 extracts were simultaneously treated with arsenic trioxide (ATO) alone or in combination with existing anticancer agent NB4, but no extracts were found to be equal to or higher than the standard antitumor activity of ATO alone. In T98G, 10 extracts were identified that they did not have the same or higher activity than the conventional anticancer agent temozolomide (TMZ). About 10 extracts were identified to enhance the anticancer activity of TMZ during concurrent treatment.

B. Mass production system of cold-active protease P66
Cold activity of P66, produced larger than molecular weight of monomer, was confirmed by comparing its mutants. P66 inclusion body productivity and active-P66 recovery were increased 5.7 and 2.8 fold, respectively, and method for protein concentration was set-up. It was confirmed that P66 could be applied to various industrial fields such as medical cleaners, dishwsahing detergents, and feed additives.

C. Identification of cryopreservation mechanism of anti-freezing agent p-CY01
In order to establish the cryopreservation mechanism of p-CY01 LM, various experimental results were analyzed and it was expected that it would have a complex cryoprotecting mechanism more than one during the freezing process. In the case of p-CY01 LM, it first binds to extracellular water and induces ice crystal growth inhibition, which firstly reduces cell membrane level damage, and at the same time reduces water content in cells through dehydration. It also reduces the ice crystal content of water to be formed. This means that the total amount of ice crystallization that can be ice-crystallized in the freezing and thawing cycles is reduced by decreasing the enthalpy change level.

D. Identification of dementia inhibition mechanism of Ramalin
No tin was detected in both tin-removing and tin-free synthesis using acidic HCl gas.
Ramalin, which has already been synthesized by the conventional synthesis method, can be used as a sample of the dementia inhibition mechanism after removal of tin using acid.
Ramalin indirectly confirmed the lack of BACE1 promoter regulatory ability through luciferase assay. Ramalin demonstrated that the ability to regulate BACE1 protein was not due to the mRNA change by the promoter. Ramalin was found to be low in BACE1 protease inhibitory ability, and Ramalin was not involved in UTR stability regulation. Ramalin was confirmed to have no effect on mRNA by three experiments, and it was confirmed that there is possibility of new drug screening using UTR regulation function. Ramalin showed that the inhibitory effect on HDAC6 with an IC50 value of about 15.4 uM to 25 uM. In the case of Ramalin, the PK results of the animal test showed that the HDAC6 could be inhibited by the exposure. Ramalin is also thought to have an anti-inflammatory effect and a microtubule-stabilizing effect on HDAC6 inhibitors.

E. Excavation of lipase enzyme
From the 46 strains producing lipase from polar regions, strains with excellent activity were selected. The enzyme LipBL was produced in Escherichia coli and its activity was found to be 923 U / mL. After the protein was identified by SDS-PAGE zymogram, the enzyme reaction characteristics of LipBL were investigated.

F. Isolation of useful microorganisms and validation of their activities
Through production modeling, we identified production conditions that are more reproducible than those using seawater. We need to explore more factors by applying various production model techniques, and substitute this optimized production medium condition with the existing production conditions.
When symbiotic microorganisms were collected from red coral from Antarctica, a total of 56 strains were obtained. As for the 49 strains except 7 strains, it was confirmed that they had more than 98% agreement with the existing strains. In order to know the characteristics of cultivated symbiotic microorganisms, temperature effect on the growth was confirmed.

G. Changes in the protein structure of polar copepod
Large-scale genomic information was obtained from polar copepod, and the changes in the protein body and genome by ultraviolet exposure were analyzed. Polar copepod was used to investigate the effects of ultraviolet rays and molecular mechanisms. The expression patterns of stress and antioxidant response related genes were analyzed.

(출처:SUMMARY)

목차 Contents

• 표지 ... 1제 출 문 ... 2보고서 초록 ... 3요 약 문 ... 4SUMMARY ... 7CONTENTS ... 11목차 ... 14그림목차 ... 17표목차 ... 22제 1 장 서론 ... 24 제 1 절 연구개발의 목적 ... 24 제 2 절 연구개발의 필요성 및 범위 ... 24제 2 장 국내외 기술개발 현황 ... 29 제 1 절 국외 연구수준 ... 29 제 2 절 국내 연구수준 ... 32제 3 장 연구개발수행 내용 및 결과 ... 33 제1절 극지 고유생물 유래 대사체 확보 ... 33 1. 남극 로스해 해양생물 추출물 확보 ... 33 가. 연구 목표 ... 33 나. 연구 수행방법 ... 33 다. 연구 결과 ... 39 라. 결론 및 제언 ... 49 2. 극지 고유생물 유래 MS-라이브러리 확보 ... 49 가. 연구 목표 ... 49 나. 연구 수행방법 ... 49 다. 연구 결과 ... 52 라. 결론 및 제언 ... 52 3. 대사체 분석 및 구조 규명 ... 53 가. 연구 목표 ... 53 나. 연구 수행방법 ... 53 다. 연구 결과 ... 54 라. 결론 및 제언 ... 85 제2절 유용 대사체 활용화 기반 연구 ... 86 1. 신규 대사체 유용성 확보 ... 86 가. 연구 목표 ... 86 나. 연구 수행방법 ... 86 다. 연구 결과 ... 89 라. 결론 및 제언 ... 107 2. 저온성 단백질분해효소 P66의 대량생산 시스템 확보 ... 108 가. 연구 목표 ... 108 나. 연구 수행방법 ... 109 다. 연구 결과 ... 113 라. 결론 및 제언 ... 123 3. 항동결물질 p-CY01의 줄기세포 동결보존 기작 규명 ... 124 가. 연구 목표 ... 124 나. 연구 수행방법 ... 125 다. 연구 결과 ... 127 라. 결론 및 제언 ... 132 4. 항치매치료제 라말린의 치매억제 기작 규명 ... 133 가. 연구 목표 ... 133 나. 연구 수행방법 ... 134 다. 연구 결과 ... 136 라. 결론 및 제언 ... 147 5. 리파아제 효소 발굴 ... 148 가. 연구 목표 ... 148 나. 연구 수행방법 ... 148 다. 연구 결과 ... 149 라. 결론 및 제언 ... 159 6. 유용 미생물 분리 및 활성 검증 ... 159 가. 연구 목표 ... 159 나. 연구 수행방법 ... 160 다. 연구 결과 ... 165 라. 결론 및 제언 ... 179 7. 극지 요각류의 단백질체 변화 양상 ... 180 가. 연구 목표 ... 180 나. 연구 수행방법 ... 180 다. 연구 결과 ... 184 라. 결론 및 제언 ... 191제 4 장 연구개발목표 달성도 및 대외기여도 ... 193 제1절 연구개발목표 달성도 ... 193 제2절 대외기여도 ... 194제 5 장 연구개발개발결과의 활용계획 ... 197제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 198제 7 장 참고문헌 ... 199끝페이지 ... 206