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研究生：	張可冀 Chang, Ke-Ji
論文名稱：	混合堆肥巨基因庫中篩選出之新穎生質能源纖維素分解酶 A novel β-endoglucanase from a variety of manure microorganism mategenome for biomass conversion
指導教授：	張敏政 Chang, Ming-Chung
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學暨分子生物學研究所 Department of Biochemistry and Molecular Biology
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	79
中文關鍵詞：	巨基因庫、纖維素分解酶、混合堆肥
外文關鍵詞：	mategenome, dinitrosalicylic acid, p-NPC, endoglucanase, zymography, defibrillation
相關次數：	點閱：66 下載：0
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發展可靠的並且是環保的替代性生質能源是一項重要的議題。尋找新穎的纖維酵素對生質能源的製程是十分重要的。在本篇研究中，本實驗室利用混合堆肥為來源建立一個涵蓋959 Mbps的微生物巨基因庫，並自其中篩選出三個具有纖維素分解活性的重組基因菌株。經由生物資訊的方式進行去氧核醣核酸序列與胺基酸序列的比對，發現三者與目前已發表的纖維酵素相似度低，係為新穎的纖維酵素。並將轉殖菌株進一步做演化樹譜系分析，發現其中一個候選菌株中的纖維素活性是來屬於Glycoside Hydrolase Family 9 (GH 9)的纖維酵素家族，將之命名為htp5；再將htp5構築到表現載體上生產纖維酵素HTP5，利用液相層析管柱將HTP5純化；並進一步的以Dinitrosalicylic acid (DNS)方式分析酵素活性，發現這個新穎的纖維酶在酸鹼值7.0和溫度55℃時，具有最佳的分解能力。並且在55 ℃放置五個小時後，測量其酵素殘存的相對活性仍然接近100 %；在鈣離子的存在下可提升酵素的活性達1.8倍，並在酵素動力學的分析後發現HTP5對於合成的纖維雙醣分子p-nitrophenyl beta-D-cellobioside (p-NPC)具有高度的轉化能力。HTP5 對於可溶性的纖維素分子(例如:carboxymethyl cellulose)具有高度的分解能力，可用於生質能後段的裂解纖維素醣化過程；而且發現HTP5對於累積在纖維布料上的微細纖維(microfibrils)也具有清除的洗滌能力，並且具備抵抗隂離子介面活性劑與抗氧化劑的功能。

Development of renewable fuel alternative with a reduced environment pollution footprint is a critical issue. Discovery of novel cellulolytic enzymes for enhancing the biomass conversion is important. In this study, the metagenomic library was established from a variety of manure microorganisms. The constructed metagenome coverage is approximately 959 Mbps, and three novel clones exhibiting cellulolytic activity were isolated. One of the clones was selected for further investigation. The resulting database and the phylogenetic analysis revealed that the cloned recombinant htp5 expressed in E.coli is a novel cellulase which belongs to Glycoside Hydrolase Family 9 (GH 9). The purified enzyme from E. coli containing HTP5 exhibited optimal cellulolytic activity with pH=7.0 and 55 °C base on Dinitrosalicylic acid (DNS) method. HTP5 exhibited thermal stability at 55 °C after 5 hours incubation and increased 1.8-fold of the catalytic ability by adding 10 mM calcium. It displays high enzymatic activity for its synthetic substrate p-nitrophenyl beta-D-cellobioside (p-NPC; kcat = 94382 min-1). The aforementioned endocellulase HTP5 belongs to GH 9 could degrade the soluble cellulase such as carboxymethyl cellulose (CMC) and equips with defibrillation activity to cotton and lyocell knit fabric. HTP5 was highly resistant to an anionic surfactant and an oxidizing agent. This result indicated that HTP5 may be suitable for last-stage processing of biomass conversion and the microfibrils polishing from fabric.

中文摘要	I
ABSTRACT	II
誌  謝	III
CONTENTS OF THE TABLES	VII
CONTENTS OF THE FIGURES	VIII
CHAPTER 1 INTRODUCTION	1
1-1. THE GLOBAL ENERGY CRISIS AND ENVIRONMENT POLLUTION	1
1-2. BIOMASS IS THE ONLY DOMESTIC, SUSTAINABLE AND RENEWABLE PRIMARY ENERGY RESOURCE	1
1-3. THE PROCESSION FOR BIOLOGICAL CONVERSION OF CELLULOSIC BIOMASS TO ETHANOL	2
1-4. THE DEVELOPMENT OF BIOFUEL	2
1-5. ADVANCED BIOLOGICAL PROCESSING IN BIOMASS CONVERSION	3
1-6. ENZYMES FOR HYDROLYSIS OF LIGNOCELLULOSE	3
1-7. THE MAJOR INDUSTRIAL SOURCES OF CELLULASES AND HEMICELLULASES	4
1-8. DISCOVER THE UNCULTURED MICROORGANISMS BY GENETIC DIVERSITY METAGENOME	4
1-9. DISRUPTION OF FIBER STRUCTURE BY THE BINDING DOMAIN OF BACTERIA	5
CHAPTER 2 MATERIALS AND METHODS	6
2-1. ENVIRONMENTAL METAGENOMIC DNA LIBRARY CONSTRUCTION	6
2-1-1. Genomic DNA source, bacteria strain and mediums	6
2-1-1-1. Bacteria strain	6
2-1-1-2. Vectors	6
2-1-1-3. Antibiotic dosage and IPTG concentration	7
2-1-1-4. Medium	7
2-1-2. Genomic DNA extracted	9
2-1-3. Preparation of the Genomic DNA fragment	12
2-1-4. Separation of the DNA fragment by sucrose density gradient centrifugation	12
2-1-5. Construction of the genomic DNA fragments with the plasmid fragments	17
2-1-6. Transformation of the original Genbank plasmid by heat shock method	18
2-1-7. Constructed Genbank plasmid DNA extraction	18
2-1-8. Estimation of the integrity of metagenomic library	19
2-2. SELECTION OF FUNCTIONAL GENETIC ELEMENTS FROM METAGENOMIC LIBRARY	20
2-2-1. Preparation of the transgenic clones from metagenomic library for screening	20
2-2-2. Screening clones exhibiting cellulolytic activity by Congo red stain	20
2-2-3. Purification of the genetic material of clones exhibiting cellulolytic activity	21
2-4. SEQUENCING AND ANALYZED OF METAGENOMIC INSERTS	21
2-5. SUB-CLONING CELLULOLYTIC ACTIVITY GENE HTP5 INTO EXPRESSION PLASMID BY E.COLI STRAIN BL21	22
2-6. PRODUCTION AND PURIFICATION OF THE RECOMBINANT HTP5	22
2-6-1. Production of the recombinant HTP5	22
2-6-2. Quantity of protein concentration by Bradford dye-binding method	23
2-7. DETERMINATION OF THE MOLECULAR MASS OF HTP5 BY SDS-PAGE	24
2-7-1. Gels casting	24
2-7-1-1. Resolving gel (running gel or separation gel)	24
2-7-1-2. Stacker gel	25
2-7-2. SDS-PAGE analysis	25
2-7-3. PlusOne Coomassie tablets PhastGel Blue R-350 stain	26
2-7-4. Cellulolytic activity of HTP5 investigated by Zymography	27
2-7-4-1. Resolving gel (running gel or separation gel) 10% SDS-PAGE	27
2-7-4-2. Stacker gel	28
2-7-4-3. SDS-PAGE Zymography (Satoshi Nakamura, 1993)	28
2-7-5. Western blotting	29
2-7-5-1. Protein Electro transfer (Semi-dry transfer)	29
2-7-5-2. Blocking and Hybridization	31
2-7-5-3. Chemiluminescent Detection	31
2-8. PURIFICATION OF THE RECOMBINANT HTP5	32
2-8-1. Packing the Sepharose Fast Flow gels	32
2-8-2. Preparation the liquid chromatograpry and enzyme purification	33
2-9. DETERMINATION OF CATALYTIC ACTIVITY OF HTP5	34
2-9-1. Analysis of enzyme activity by Reducing Sugar Estimation by Dinitrosalicylic Acid (DNS) Method Carboxymethyl cellulose (CMC) assay for endo-1, 4-glucanase (T. K. GHOSE Biochemical Engineering Research Centre, Indian Institute of Technology, New Delhi-110016, India)	34
2-9-1-1. Investigation of the optimal reaction temperature for the crude extracted	34
2-9-1-2. Investigation of the optimal reaction pH value for the crude extracted	35
2-9-1-3. Construction of the standard curve for calculation	36
2-9-1-4. Unit calculation of the purified enzyme	37
2-9-1-5. Investigation of the optimal reaction temperature of the purified enzyme	37
2-9-1-6. Investigation of the optimal reaction pH value of the purified enzyme	38
2-9-1-7. Calculation of the enzyme specific activity	38
2-9-2. Enzymatic Assay of cellulase by p-nitrophenyl beta-D-cellobioside	39
2-9-2-1. Investigation of the optimal reaction temperature for the crude extracted	39
2-10. THE EFFECTS OF DIFFERENT CHEMICAL REAGENTS ON THE PURIFIED RECOMBINANT HTP5	40
2-11. THERMAL STABILITY STUDIES OF THE HTP5	40
2-12. SUBSTRATE SPECIFICITY AND KINETIC STUDIES OF THE HTP5	40
2-13. ESTIMATION OF DEFIBRILLATION ACTIVITY OF ENDOGLUCANASE	41
CHAPTER 3 RESULTS	42
3-1. ENVIRONMENTAL METAGENOMIC DNA LIBRARY CONSTRUCTION	42
3-2. SEQUENCE ANALYSIS OF SCREENED CELLULASE GENE	43
3-3. CONSTRUCTION, OVEREXPRESSION AND PURIFICATION OF RECOMBINANT HTP5	43
3-4. EFFECTS OF PH AND TEMPERATURE ON ENZYMATIC ACTIVITY AND STABILITY	44
3-5. ACTIVITY ANALYSIS OF PURIFIED HTP5 WITH VARIOUS SUBSTRATES	44
3-6. THE EFFECT OF METAL IONS AND CHEMICALS ON ENZYMATIC ACTIVITY	45
3-7. THE KINETIC PROPERTIES OF HTP-5	45
3-8. OTHER APPLICATIONS OF THE NOVEL CELLULASE HTP5	45
CHAPTER 4 DISCUSSIONS	47
4-1. ENVIRONMENTAL METAGENOMIC DNA LIBRARY CONSTRUCTION	47
4-2. GENERAL CONCEPTS OF CELLULASES HTP5	47
4-3. EFFECTS OF PH AND TEMPERATURE ON ENZYMATIC ACTIVITY AND STABILITY	47
4-4. THE EFFECT OF METAL IONS AND CHEMICALS ON ENZYMATIC ACTIVITY	48
4-5. ESTIMATION OF DEFIBRILLATION ACTIVITY OF ENDOGLUCANASE HTP5 BELONG TO GH 9	48
REFERENCES              69     
APPENDIX	        72
自述                        79
                                    

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