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Comparative study on the expression and characteristics of lipase genes from entomopathogenic fungi : 곤충병원성 곰팡이 유래 지방질 효소 유전자의 발현과 특성 구명에 대한 비교 연구
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 제연호 | - |
| dc.contributor.author | 김현지 | - |
| dc.date.accessioned | 2022-06-08T06:34:05Z | - |
| dc.date.available | 2022-06-08T06:34:05Z | - |
| dc.date.issued | 2022 | - |
| dc.identifier.other | 000000169768 | - |
| dc.identifier.uri | https://hdl.handle.net/10371/181091 | - |
| dc.identifier.uri | https://dcollection.snu.ac.kr/common/orgView/000000169768 | ko_KR |
| dc.description | 학위논문(석사) -- 서울대학교대학원 : 농업생명과학대학 응용생물화학부, 2022.2. 제연호. | - |
| dc.description.abstract | Lipase (triacylglycerol acyl hydrolase, EC 3.1.1.3)의 ester결합을 가수분해하여 glycerol과 fatty acid의 생성을 촉진하는 효소로서, 동물과 식물, 미생물 등 생물계 전반에 널리 분포되어 있다. 산업적으로 응용되고 있는 많은 효소들 가운데 지방질을 지방산과 monoglyceride 로 분해시키는 lipase가 차지하는 역할은 매우 중요하며, 급속히 성장하고 있는 생물 산업에서 주목 받고 있는 효소 중 하나이다. 곤충병원성 곰팡이인 동충하초 유래 lipase에서 lipase의 가장 큰 특징 중 하나인 위치 특이성이 triacylglycerol의 1(3)번 위치에 특이적으로 작용하는 것으로 판명되면서 많은 연구가 요구되었다. 따라서 본 연구를 통하여 곤충병원성 곰팡이로부터 lipase를 분리하여 그 특성을 규명하고, baculovirus expression system을 이용하여 대량발현 할 수 있는 조건을 구축하고자 하였다.
국내에서 분리한 곤충 병원성 곰팡이 중 lipase 활성이 가장 높은 균주를 선발하였다. 선발한 균주의 lipase와 위치특이성이 있다고 알려진 동충하초 유래 lipase를 베큘로바이러스 발현 벡터계 (baculovirus expression vector system)를 이용하여 재조합 베큘로바이러스를 만들었다. 비분비단백질과 분비단백질로 각각 베큘로바이러스를 발현시켰다. lipase활성을 비교한 결과 분비단백질 형태일 때 활성이 더 높았다. 곰팡이 배양액, 비분비단백질 그리고 분비단백질 3가지의 조건으로 lipase 활성을 측정을 해보았는데 모든 결과값에서 동충하초 유래 lipase보다 Beauveria bassiana JEF-351 균주 유래 lipase의 활성이 높게 측정되는 것으로 보아 Beauveria bassiana JEF-351 균주 유래 lipase가 매우 유용하게 이용이 될 수 있는 효소인 것으로 판단되었다. | - |
| dc.description.abstract | Lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is a class of enzymes that catalyze the hydrolysis of triacylglycerol and/or esterification between glycerol and fatty acid. It is widely distributed throughout the living world, including animals, plants, and microorganisms. Among many industrially applied enzymes, the role of lipase that converts fats into fatty acids and monoglycerides is very important, and it is one of the enzymes attracting attention in the rapidly growing biological industry. Lipase derived from an entomopathogenic fungi, Cordyceps militaris, was found to act specifically at position 1(3) of triacylglycerol. In this study, characteristics of lipases isolated from entomopathogenic fungi were investigated and conditions for mass expression using the baculovirus expression system were established.
Among the entomopathogenic fungi isolated in Korea, Beauveria bassiana JEF-351 strain, which showed the highest enzyme activity, was selected. Lipase genes of the selected strain (BBL351) and Cordyceps militaris (CML), which is known to have stereospecificity, were introduced into the genome of Autographa californica nucleopolyhedrovirus (AcMNPV), respectively, to express corresponding genes using the baculovirus expression system. Recombinant lipases were expressed as non-secreted protein and secreted protein, respectively. In both BBL351 and CML, their enzyme activity was higher when they expressed as secreted protein form, demonstrating that post-translational modification such as glycosylation is crucial for their activity. In addition, the enzyme activity of BBL351 was higher than that of the CML, suggesting that the lipase derived from the B. bassiana JEF-351 strain could be useful as biocatalyst in the biotechnological applications. | - |
| dc.description.tableofcontents | ABSTRACT i
TABLE OF CONTENTS ⅱi LIST OF TABLES vi LIST OF FIGURES ⅴⅰi INTRODUCTION 1 LITERATURE REVIEW 3 1. Lipase 3 2. Entomopathogenic fungi 4 3. Baculovirus expression vector system 5 METERIAL AND METHODS 7 1. Entomopathogenic fungi (EPF) 7 2. Preparation of lipase from EPF culture broth of entomopathogenic fungi 9 3. Bacterial strains and transformation 10 4. Insect cells and baculoviruses 10 5. RNA and reverse transcription PCR (RT-PCR) 10 6. DNA synthesis 11 7. Construction of baculovirus donor vectors 11 8. in vitro transposition 18 9. Transfection 24 10. Infection of cells with baculoviruses 24 11. Extraction of viral DNA 25 12. Purification of non-secretory recombinant protein 25 13. Precipitation of secretory recombinant protein using ammonium sulfate 25 14. Lipase activity assay 26 RESULTS 27 1. Lipase genes from entomopathogenic fungi 27 2. Cloning and sequencing of lipase genes from EPF strains 27 3. Expression of lipase genes in the baculovirus expression system 33 3.1 Construction of the recombinant bacmid bEasyBac 33 3.2 Purification of the non-secretory recombinant baculovirus 33 4. Confirmation of enzyme activity through lipase assay 45 4.1 Non-secretory protein 45 4.2 Secretory protein 45 DISCUSSION 50 LITERATURE CITED 53 ABSTRACT IN KOREAN 59 LIST OF TABLES Table 1. List of entomopathogenic fungal strains used in this study. 8 Table 2. Primers used for construction of baculovirus donor vectors containing non-secretory lipase genes 15 Table 3. Primers used for construction of baculovirus donor vectors containing His-tagged form of lipase genes from entomopathogenic fungi 16 Table 4. Oligonucleotides used for construction of baculovirus donor vectors containing His-tagged form of lipase genes from entomopathogenic fungi 21 LIST OF FIGURES Fig. 1. Liquid and solid culture conditions of selected entomopathogenic fungi 9 Fig. 2. Construction map of the vectors harboring BBL344 and BBL351 genes 12 Fig. 3. Construction map of the vector harboring IFL gene 13 Fig. 4. Construction map of baculovirus donor vectors expressing non-secretory form of BBL344 and BBL351 genes under the control of polyhedron promoter 14 Fig. 5. Construction map of baculovirus donor vectors expressing N-terminally His-tagged lipase genes under the control of polyhedron promoter 19 Fig. 6. Construction map of baculovirus donor vectors expressing C-terminally His-tagged lipase genes under the control of polyhedron promoter 20 Fig. 7. Construction map of baculovirus donor vectors expressing secretory form of CML and BBL351 genes with native signal peptides under the control of polyhedron promoter 22 Fig. 8. Construction map of baculovirus donor vectors expressing secretory form of CML and BBL351 genes with melittin signal peptides under the control of polyhedron promoter 23 Fig. 9. Detection of lipase derived from insect pathogenic fungi in NCBI. 28 Fig. 10. A phylogenetic tree displaying the relationship of lipase genes 29 Fig. 11. Lipase assay of EPF strains 30 Fig. 12. RT-PCR of lipase 31 Fig. 13. Confirmation of the internal structure of the transfer vector, pGEM-T BBL344 and pGEM-T BBL351 by restriction endonuclease digestion pattern 32 Fig. 14. Amino acid sequence analysis of lipases from EPF strains. 34 Fig. 15. Confirmation of EPF lipase genes into baculovirus donor vector by restriction endonuclease digestion pattern 36 Fig. 16. Sf9 cells were infected with recombinant baculovirus 38 Fig. 17. Confirmation genome structure of recombinant baculoviruses expressing lipase genes under the control of polyhedrin promoters by RT-PCR using specific primer sets 40 Fig. 18. Transcription of lipase genes from recombinant baculoviruses expressing lipase genes under the control of polyhedrin promoters by PCR using specific primer sets 42 Fig. 19. Expression of lipases protein by recombinant baculoviruses 44 Fig. 20. Gel electrophoresis of HisPurTM Ni-NTA Spin Column purification of Hislipase genes 46 Fig. 21. Expression of non-secretory protein in insect cells infected with recombinant baculoviruses 47 Fig. 22. Expression of secretory protein in insect cells infected with recombinant baculoviruses 48 | - |
| dc.format.extent | 60 | - |
| dc.language.iso | eng | - |
| dc.publisher | 서울대학교 대학원 | - |
| dc.subject | Lipase | - |
| dc.subject | Entomopathogenic fungi | - |
| dc.subject | Beauveria bassiana | - |
| dc.subject | Baculovirus expression system | - |
| dc.subject | Secreted protein | - |
| dc.subject.ddc | 630.24 | - |
| dc.title | Comparative study on the expression and characteristics of lipase genes from entomopathogenic fungi | - |
| dc.title.alternative | 곤충병원성 곰팡이 유래 지방질 효소 유전자의 발현과 특성 구명에 대한 비교 연구 | - |
| dc.type | Thesis | - |
| dc.type | Dissertation | - |
| dc.contributor.AlternativeAuthor | Hyun Ji Kim | - |
| dc.contributor.department | 농업생명과학대학 응용생물화학부 | - |
| dc.description.degree | 석사 | - |
| dc.date.awarded | 2022-02 | - |
| dc.contributor.major | 곤충미생물 | - |
| dc.identifier.uci | I804:11032-000000169768 | - |
| dc.identifier.holdings | 000000000047▲000000000054▲000000169768▲ | - |
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