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研究生: 陳潔西
Jesse
論文名稱: 液化芽孢桿菌具抗菌活性之硫氧化還原家族蛋白的表現與功能鑑定
Expression and functional characterization of thioredoxin family protein with antimicrobial activity from Bacillus amyloliquefaciens
指導教授: 胡紹揚
Hu, Shao-Yang
學位類別: 碩士
Master
系所名稱: 農學院 - 生物科技系
Department of Biological Science and Technology
畢業學年度: 109
語文別: 英文
論文頁數: 81
中文關鍵詞: 抗菌物質液化芽孢桿菌硫氧化還原家族蛋白
外文關鍵詞: antimicrobial substance, Bacillus amyloliquefaciens, thioredoxin family protein
DOI URL: http://doi.org/10.6346/NPUST202100294
相關次數: 點閱:24下載:0
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  • Contents
    摘要.................. I
    Abstract ................... III
    Acknowledgement .........V
    Contents ............. VIII
    1. Introduction .............. 1
    2. Literature Review....... 2
    2.1. Probiotics ............ 2
    2.1.1. Definition of probiotics..... 2
    2.1.2. Benefits of probiotics ....... 3
    2.2. Bacillus as probiotics ........... 6
    2.2.1. Bacillus amyloliquefaciens...... 8
    2.3. Antimicrobial substances from probiotics ........ 9
    2.3.1. Bacteriocin............ 9
    2.3.2. Extracellular polysaccharide ....... 13
    2.3.3. Antimicrobial proteins ........... 14
    2.4. Escherichia coli prokaryotic protein expression system ...... 16
    2.5. Research purpose............. 17
    3. Materials and Methods ........ 18
    3.1. Experimental Steps ............. 18
    3.2. Bacterial strains .............. 19
    3.2.1. Bacterial strains and culture conditions ........ 19
    3.3. Enzyme activity............. 19
    3.3.1. Amylase............ 19
    3.3.2. Cellulase.............. 19
    3.3.3. Lipase ................... 20
    3.3.4. Protease ............. 20
    3.3.5. Xylanase............... 20
    3.3.6. Bacteriocin ........... 21
    3.4. Physiological and biochemical characteristics of B. amyloliquefaciens ............... 21
    3.4.1. pH stress................. 21
    3.4.2. Heat shock ................ 22
    3.4.3. Enzyme activity........... 22
    3.4.4. Inhibitory effects of antimicrobial substance against various pathogens .............. 23
    3.4.5. Inhibitory effects of B. amyloliquefaciens against antibiotics ... 23
    3.5. Protein purification and identification.............. 24
    3.5.1. Ammonium sulfate precipitation....................... 24
    3.5.2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) .............. 24
    3.5.3. In gel digestion and protein identification ......... 25
    3.6. Recombinant protein expression and purification ........ 26
    3.6.1. Confirmation of cloned E. coli BL21 (DE3) protein expression and purification............... 26
    3.6.2. Western blot analysis........... 27
    3.6.3. Growth curve ............. 28
    3.7. 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) antioxidant activity assay 31 3.8. Statistical analysis .............. 31
    4. Results............. 32
    4.1. Partial purification, physiological and biochemical characteristics of bacterial strain .................. 32
    4.2. Purification of the antimicrobial substance from B. amyloliquefaciens.......... 44
    4.3. Protein expression of E.coli BL21 (DE3) Transformation ........ 47
    4.4. DPPH antioxidant activity assay......... 57
    5. Discussion ......... 60
    6. Conclusion........ 69
    7. Reference ......... 70

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