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Characterization of Cold-Adapted Alginate Lyase from Pseudoalteromonas sp. BSi20429 : Pseudoalteromonas sp. BSi20429에서 추출된 저온 활성 알긴산 분해 효소의 특성 조사
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 김효진 | - |
| dc.contributor.author | 구단열 | - |
| dc.date.accessioned | 2022-12-29T07:55:57Z | - |
| dc.date.available | 2022-12-29T07:55:57Z | - |
| dc.date.issued | 2022 | - |
| dc.identifier.other | 000000172959 | - |
| dc.identifier.uri | https://hdl.handle.net/10371/187955 | - |
| dc.identifier.uri | https://dcollection.snu.ac.kr/common/orgView/000000172959 | ko_KR |
| dc.description | 학위논문(석사) -- 서울대학교대학원 : 농업생명과학대학 농생명공학부(바이오모듈레이션전공), 2022. 8. 김효진. | - |
| dc.description.abstract | Alginates have high nutritional value due to their complex polysaccharide content. Alginate lyases catalytically degrade an alginate into alginate oligosaccharide (AOS). AOS is reported to have antioxidant, anti-tumor, anti-bacterial, and anti-hyperglycemic properties. When we want to produce AOS from an alginate, we have to choose an appropriate alginate lyase for our demand. Among many alginate lyases, cold-adapted alginate lyases are promising because heating energy can be saved due to their low optimal temperatures. But cold-adapted alginates are susceptible to temperature changes and cannot be kept in storage for an extended period of time. In this study, AlySNU, a novel alginate lyase was discovered from sea cucumbers intestine metagenome Pseudoalteromonas sp. BSi20429. The gene encoding the alginate-degrading enzyme was cloned from Pseudoalteromonas sp. BSi20429 and then expressed in Escherichia coli. After expression, AlySNU was tested through DNS, HPLC, TLC and ESI-MS. AlySNU was characterized as low temperature activating, temperature-stable, cold-adapted alginate lyase, producing monomers and variety of oligomers, and was effective in directly degrading alginates in brown algae. This AlySNU can be useful as an alginate lyase in the industry to lower energy cost and produce variety of AOS. | - |
| dc.description.abstract | 알긴산은 복합적인 다당류 구조를 통하여 높은 영양적 가치를 가지고 있는 유용한 고분자 물질이다. 알긴산 분해효소는 이러한 알긴산을 알긴산 올리고당(AOS)으로 효소적으로 분해하는 물질이다. AOS는 항산화, 항암, 항균, 항혈당 물질로서 작용한다고 알려진 유용 물질이다. AOS를 알긴산에서 생산할 때 각자의 요구조건에 맞는 알긴산 분해효소를 선택하여야 하며 이러한 이유로 다양한 종류의 알긴산 분해효소가 연구되고 상용화 되어 있다. 많은 종류의 알긴산 분해효소 중 저온 활성 알긴산 분해효소는 저온에서 활성 된다는 특징을 통하여 반응을 시킬 때 적은 에너지가 소모되기에 가치가 있지만 일반적으로 열에 취약하여 고온에서 빠르게 그 활성을 잃는다는 단점이 있다. 이번 연구에서 우리는 AlySNU라고 명명한 고유의 알긴산 분해효소를 해삼의 장내 균총에 있는 Pseudoalteromonas sp. BSi20429에서 DNA 염기 서열을 획득하였으며 대장균에 해당 DNA 염기 서열을 삽입하여 대장균이 AlySNU를 발현하도록 하였다. 이후 대장균에서 추출한 AlySNU를 DNS, HPLC, TLC, ESI-MS를 통하여 그 특징을 연구하였다. 그 결과 AlySNU는 저온 활성 알긴산 분해효소이며 고온에서도 활성을 오래 유지한다는 것이 확인 되었다. 또한 그 최종 분해 산물로서 다양한 종류의 단당류와 올리고당을 생산하며, 해조류 내부에 존재하는 알긴산을 직접적으로 분해 가능함이 확인되었다. 우리의 AlySNU는 산업적으로 이용할 때 높은 저장 안정성과 낮은 활성 온도를 통한 에너지 절약, 다양한 AOS 생산을 기대 가능한 알긴산 분해 효소이다. | - |
| dc.description.tableofcontents | Abstract i
Contents ii List of tables v List of Figures vi I. Introduction 1 1. Characteristics of alginate 1 2. Structure of alginate 3 3. Characteristics of alginate lyase 5 4. Cold-adapted alginate lyases 9 5. Alginate lyase from Pseudoalteromonas sp. BSi20429 12 II. Materials and methods 13 1. The sequence analysis of AlySNU 13 1.1. Cloning of AlySNU 15 1.2. Gene expression in Escherichia coli 15 2. Purification of protein 17 2.1. SDS-Polyacrylamide Gel Electrophoresis 17 3. Enzymatic activity assay of the alginate lyase 18 3.1. Enzyme functionality test 18 3.2. Effects of temperature on AlySNU activity and stability 18 4. Alginate degradation products of AlySNU 19 4.1. Collection of alginate degradation products by AlySNU 19 4.2. Analysis of the products of alginate degradation by AlySNU through HPLC 19 4.3. Analysis of the products of alginate degradation by AlySNU through TLC 20 4.4. Analysis of the products of alginate degradation by AlySNU through ESI-MS 20 5. Analysis of the products of seaweed degradation by AlySNU 21 III. Results 22 1. The sequence analysis of the AlySNU 22 2. Gene expression and purification of recombinant AlySNU 24 3. Effects of temperature on AlySNU activity 29 3.1. The optimal temperature of AlySNU 29 3.2. Effects of temperature on AlySNU stability 31 4. Alginate degradation products of AlySNU 33 4.1. Analysis of the products of alginate degradation by AlySNU through HPLC 33 4.2. Analysis of the products of alginate degradation by AlySNU through TLC 36 4.3. Alginate degradation products analysis of AlySNU by ESI-MS 38 5. Seaweed degradation products of AlySNU 43 5.1. Analysis of the products of seaweed degradation by AlySNU through HPLC 43 5.2. Seaweed degradation products analysis of AlySNU by TLC 51 IV. Conclusions 57 V. Discussion 58 References 61 Abstract in Korean 66 Contents of table Table 1. Alginate lyases used for phylogenetic tree 14 Contents of Figures Fig. 1. Structure of alginate 4 Fig. 2. Types of alginate lyase 6 Fig. 3. Benefits of alginate oligosaccharide (AOS) 8 Fig. 4. Types of alginate lyase (optimal temperature) 10 Fig. 5. Types of alginate lyase (thermal stability) 11 Fig. 6. pGST-parallel-1 alginate lyase-His tag vector 16 Fig. 7. The phylogenetic tree of AlySNU 23 Fig. 8. SDS page results of different sonification amplitudes 25 Fig. 9. SDS page results of different IPTG induction times 26 Fig. 10. SDS page results of different IPTG induction temperature 27 Fig. 11. DNS results of purified AlySNU 28 Fig. 12. Effects of temperature on AlySNU activity 30 Fig. 13. Effects of temperature on AlySNU stability 32 Fig. 14. HPLC results of Guluronic acid and Sodium Alginate 34 Fig. 15. HPLC results of 1 min incubation and 3 hr incubation 35 Fig. 16. TLC result of alginate degradation 37 Fig. 17. ESI-MS results of enzyme only solution 39 Fig. 18. ESI-MS results of sodium alginate 40 Fig. 19. ESI-MS results of 3hr incubation with AlySNU and sodium alginate 41 Fig. 20. ESI-MS results of 12hr incubation with AlySNU and sodium alginate 42 Fig. 21. HPLC results of Ecklonia cava degradation 44 Fig. 22. HPLC results of Omphalodes krameri degradation 45 Fig. 23. HPLC results of Pyropia teneta degradation 46 Fig. 24. HPLC results of Undaria pinnatifida degradation 47 Fig. 25. HPLC results of Undaria pinnatifida sporophyll degradation 48 Fig. 26. HPLC results of Saccharina crassifolia degradation 49 Fig. 27. HPLC results of Hizikia fusiforme degradation 50 Fig. 28. TLC result of Ecklonia cava degradation through AlySNU 52 Fig. 29. TLC result of Pyropia teneta degradation through AlySNU 53 Fig. 30. TLC result of Undaria pinnatifida Sporophyll degradation through AlySNU 54 Fig. 31. TLC result of Saccharina crassifolia degradation through AlySNU 55 Fig. 32. TLC result of Hizikia fusiforme degradation through AlySNU 56 | - |
| dc.format.extent | 75 | - |
| dc.language.iso | eng | - |
| dc.publisher | 서울대학교 대학원 | - |
| dc.subject | alginate | - |
| dc.subject | alginatelyase | - |
| dc.subject | cold-adapted | - |
| dc.subject | Pseudoalteromonassp.BSi20429 | - |
| dc.subject | degradation | - |
| dc.subject | alginateoligosaccharide | - |
| dc.subject.ddc | 571 | - |
| dc.title | Characterization of Cold-Adapted Alginate Lyase from Pseudoalteromonas sp. BSi20429 | - |
| dc.title.alternative | Pseudoalteromonas sp. BSi20429에서 추출된 저온 활성 알긴산 분해 효소의 특성 조사 | - |
| dc.type | Thesis | - |
| dc.type | Dissertation | - |
| dc.contributor.AlternativeAuthor | Danyeol Ku | - |
| dc.contributor.department | 농업생명과학대학 농생명공학부(바이오모듈레이션전공) | - |
| dc.description.degree | 석사 | - |
| dc.date.awarded | 2022-08 | - |
| dc.identifier.uci | I804:11032-000000172959 | - |
| dc.identifier.holdings | 000000000048▲000000000055▲000000172959▲ | - |
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