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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳昭瑩 | |
| dc.contributor.author | Chien-Jui Huang | en |
| dc.contributor.author | 黃健瑞 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:12:46Z | - |
| dc.date.copyright | 2006-07-19 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-18 | |
| dc.identifier.citation | References
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23937 | - |
| dc.description.abstract | 利用幾丁質培養基自百合根圈土壤分離出一幾丁質分解性臘狀芽胞桿菌菌株28-9，經對峙培養、切離葉等防治試驗得知此菌株具有防治百合灰黴病的潛力。為了解幾丁質水解能力與拮抗百合灰黴病菌的關係，分別從蛋白質與基因組方向進行，得到兩個幾丁質水解酵素基因，命名為chiCH及chiCW。其演譯之蛋白質除訊息序列外，ChiCH具有一催化功能區，ChiCW則具一催化功能區，一fibronectin type III相似功能區及一幾丁質結合功能區。分別自大腸桿菌表現及純化出ChiCH和ChiCW兩蛋白質，經抑菌試驗分析得知ChiCH僅輕微地抑制百合灰黴病菌分生孢子萌芽，而ChiCW可有效地抑制百合灰黴病菌分生孢子萌芽，推測ChiCW是菌株28-9拮抗百合灰黴病菌之重要因子。此外，本研究亦發展出利用融合glutathione-S-transferase的重組幾丁質水解酵素可在大腸桿菌中大量表現幾丁質水解酵素，經動力學、水解產物及基質結合力分析可知ChiCW為內切型而ChiCH為外切型幾丁質水解酵素。最後，本研究對ChiCW的C端區域在酵素功能上的影響進行分析，相對於ChiCW，在高溫及高酸鹼值的條件下，
ChiCWΔFC對醣酐幾丁質有較高之活性，動力學分析結果則顯示ChiCW較ChiCWΔFC容易水解寡分子及聚分子基質，推測菌株28-9將ChiCW修飾成ChiCWΔFC可以提升自身在不同環境下分解幾丁質的效率，效地將大分子基質水解成更小的寡分子基質。 | zh_TW |
| dc.description.abstract | Bacillus cereus 28-9 is a chitinolytic bacterium isolated from rhizosphere of lilies. This bacterium showed antagonistic effect against Botrytis elliptica, the pathogen of lily leaf blight, by detached leaf assays. To investigate the involvement of the chitinolytic activity in
antagonism, two chitinase-encoding genes, chiCH and chiCW, have been cloned. ChiCH consists of a signal peptide followed by a catalytic domain. ChiCW consists of a signal peptide, a catalytic domain, a fibronectin type III-like domain, and a chitin-binding domain. ChiCH and ChiCW are slightly and effectively inhibitory to conidial germination of B. elliptica, respectively, indicating that ChiCW may contribute to the antagonistic activity of B. cereus 28-9 against B. elliptica. In addition, an ideal method for high-level expression of chitinases in E. coli as glutathione-S-transferase fusion proteins was established in this study. According to the results of kinetics, hydrolysis products, and binding activities, ChiCW is an endo-chitinase and ChiCH is an exo-chitinase. Finally, the functions of the C-terminal region on ChiCW activity were investigated. Compared with ChiCW, ChiCWΔFC exhibited higher activity at high temperature and pH although both enzymes had the same optimal temperature and pH for enzyme activities. The kinetic properties of ChiCW and ChiCWΔFC indicate that ChiCW hydrolyzes oligomeric and polymeric substrates more efficiently than ChiCWΔFC. Based on the results, we suggest that B. cereus 28-9 proteolytically modifying ChiCW to ChiCWΔFC could improve efficiency of chitin degradation in different environments. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T05:12:46Z (GMT). No. of bitstreams: 1 ntu-95-F89633001-1.pdf: 2574473 bytes, checksum: 50b8744a98b0d3cf4d122c03d0d46b72 (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | 目 錄
中文摘要 1 ABSTRACT 2 CHAPTER 1: GENE CLONING AND BIOCHEMICAL CHARACTERIZATION OF CHITINASE CH FROM BACILLUS CEREUS 28-9 3 ABSTRACT 3 INTRODUCTION 4 MATERIALS AND METHODS 5 Identification of a bacterial strain 5 Chitinase activity measurements 5 Purification of chitinase CH from the culture supernatnat of B. cereus 28-9 5 SDS-PAGE and zymogram analysis 6 Peptide N-terminal sequencing 6 Cloning of ChiCH-encoding gene 7 Expression and purification of ChiCH from E. coli cells 8 Biochemical characterization of ChiCH 8 Antifungal assay of ChiCH 9 Nucleotide sequence accession number 9 RESULTS AND DISCUSSION 10 Purification and N-terminal sequence of ChiCH 10 ChiCH-encoding gene 10 Analysis and comparison of the amino acid sequence of ChiCH with other chitinases 11 Biochemical properties of ChiCH 13 Inhibition by ChiCH on conidial germination of B. elliptica 13 Figure 1. SDS-PAGE and zymogram analysis. 15 Figure 2. Southern blot analysis of the genomic DNA of B. cereus 28-9. 16 Figure 3. Nucleotide and the deduced amino acid sequences of chiCH. 17 Figure 4. Sequence comparison of three chitinases from B. cereus strains. 18 Figure 5. Effects of pH on the chitinase activity and stability of ChiCH. 19 Figure 6. Effects of temperature on the chitinase activity and stability of ChiCH. 20 CHAPTER 2: IDENTIFICATION OF AN ANTIFUNGAL CHITINASE FROM A POTENTIAL BIOCONTROL AGENT, BACILLUS CEREUS 28-9 21 ABSTRACT 21 INTRODUCTION 22 MATERIALS AND METHODS 23 Bacterial strains and culture conditions 23 Preparation of colloidal chitin 23 Expression and purification of chitinase ChiCW from recombinant E. coli cells 23 Southern blot analysis 24 SDS-PAGE and zymogram analysis 25 Chitinase activity measurements 25 Antifungal assay of ChiCW 26 Nucleotide sequence accession number 26 RESULTS 27 Sequence anlysis of ChiCW-encoding gene 27 Expression and purification of ChiCW from recombinant E. coli 27 Analysis and comparison of the amino acid sequence of ChiCW with other chitinases 28 Inhibition by ChiCW on conidial germination of B. elliptica 29 DISCUSSION 31 Table 1. Purification of ChiCW from periplasmic fraction of E. coli DH5 (pNTU55) 33 Table 2. Effect of ChiCW on conidial germination of B. elliptica 34 Figure. 1. Nucleotide sequence of the chiCW gene of B. cereus 28-9 and the deduced amino acid sequence of the gene product. 36 Figure 2. Southern blot analysis of the genomic DNA of B. cereus 28-9. 37 Figure 3. SDS-PAGE and in-gel activity assay. 38 Figure 4. Alignment of the peptide sequences of the catalytic domains of chitinases from different Bacillus spp. 39 Figure 5. Alignment of the peptide sequences of the fibronectin type III-like domains of chitinases from different Bacillus spp. 40 Figure 6. Alignment of the peptide sequences of the chitin- and cellulose-binding domains of chitinases from different Bacillus spp. 41 Figure 7. Effect of ChiCW on conidial germination of B. elliptica. 42 CHAPTER 3: HIGH-LEVEL EXPRESSION AND CHARACTERIZATION OF TWO CHITINASES, CHICH AND CHICW, OF BACILLUS CEREUS 28-9 IN ESCHERICHIA COLI 43 ABSTRACT 43 INTRODUCTION 44 MATERIALS AND METHODS 46 Bacterial strains, plasmid, and media 46 Construction of expression vectors 46 Bacterial expression of recombinant chitinases 46 Purification of recombinant chitinases 47 PreScission protease cleavage of GST-chitinases 48 SDS-PAGE and chitinolytic zymography assay 48 Chitinase activity measurements and protein concentration determination 49 Kinetic properties characterization 49 Thin-layer chromatography 50 Substrate-binding assay 50 Affinity electrophoresis 51 RESULTS 52 Vector Construction and expression of GST-chitinase fusion proteins in E. coli 52 Purification of recombinant chitinases 52 Kinetics of chitinases 53 Hydrolysis products of chitinases 54 Binding activity of chitinases to polysaccharides 54 DISCUSSION 56 Table 1. Primers used 62 Table 2. Purification of GST-chitinase fusion proteins 63 Table 3. Kinetics of chitinases and GST-chitinase fusion proteins 64 Figure 1. Schematic diagrams of constructed plasmids, pGW59 (A) and pGH60 (B), for overexpression of ChiCW and ChiCH. 65 Figure 2. Expression of GST-ChiCH fusion protein in E. coli cells. 66 Figure 3. Purification of GST-ChiCH and recombinant ChiCH after cleavage by PreScission protease. 67 Figure 4. Purification of GST-ChiCW and recombinant ChiCW after cleavage by PreScission protease. 68 Figure 5. Thin- layer chromatography of hydrolytic products from various N-acetylchitooligosaccharides. 69 Figure 6. Domain structures of ChiCH and ChiCW from B. cereus 28-9 70 Figure 7. Affinity electrophoresis of chitinases in the presence (+) and absence (-) of glycol chitin (A), CM-cellulose (B), and laminarin (C). 71 CHAPTER 4: FUNCTIONS OF THE C-TERMINAL REGION OF CHITINASE CHICW FROM BACILLUS CEREUS 28-9 IN SUBSTRATE-BINDING AND HYDROLYSIS OF CHITIN 72 ABSTRACT 72 INTRODUCTION 73 MATERIALS AND METHODS 74 Bacterial strains, plasmid, and media 74 Construction of expression vectors 74 Bacterial expression of truncated chitinases 74 Purification of ChiCW FC 75 SDS-PAGE and chitinolytic zymography assay 75 Chitinase activity measurement and protein concentration determination 76 Optimal condition for enzyme activity 76 Substrate specificity and binding assay 76 Kinetic properties characterization 77 RESULTS 78 Expression of ChiCW derivatives 78 Purification of ChiCW FC 78 Optimal conditions for enzyme activity 78 Substrate specificity and binding activity 79 Kinetics of ChiCW and ChiCW FC 80 DISCUSSION 81 Table 1. Primers used in this study 85 Table 2. Purification of ChiCW FC 86 Table 3. Substrate specificity of ChiCW and ChiCW FC 87 Table 4. Kinetics of ChiCW and ChiCW FC 88 Figure 1. Schematic diagram of the domain structures of ChiCW and two derivatives. 89 Figure 2. Elution profile of ChiCW FC purification by anion exchange chromatography. 90 Figure 3. SDS-PAGE and zymography assay of ChiCW and ChiCW FC. 91 Figure 4. Effect of temperature on the chitinase activity of ChiCW and ChiCW FC. 92 Figure 5. Effect of pH on the chitinase activity of ChiCW and ChiCW FC. 93 Figure 6. Insoluble substrate binding ability of ChiCW and ChiCW FC. 94 FUTURE ASPECT 95 | |
| dc.language.iso | en | |
| dc.subject | 蛋白質大量表現 | zh_TW |
| dc.subject | 幾丁質水解酵素 | zh_TW |
| dc.subject | 臘狀芽胞桿菌 | zh_TW |
| dc.subject | 百合灰黴病菌 | zh_TW |
| dc.subject | Protein overexpression | en |
| dc.subject | Chitinase | en |
| dc.subject | Bacillus cereus | en |
| dc.subject | Botrytis elliptica | en |
| dc.title | 拮抗性臘狀芽孢桿菌菌株28-9幾丁質水解酵素系統研究 | zh_TW |
| dc.title | Chitinase system of antagonistic Bacillus cereus 28-9 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 劉世東,蔡珊珊,徐源泰,吳蕙芬,賴吉永 | |
| dc.subject.keyword | 幾丁質水解酵素,臘狀芽胞桿菌,百合灰黴病菌,蛋白質大量表現, | zh_TW |
| dc.subject.keyword | Chitinase,Bacillus cereus,Botrytis elliptica,Protein overexpression, | en |
| dc.relation.page | 102 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2006-07-18 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 植物病理與微生物學研究所 | zh_TW |
| 顯示於系所單位： | 植物病理與微生物學系 | |
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