臺灣博碩士論文加值系統

於本研究室先前的研究中發現，嗜熱菌 Caldimonas manganoxidans JCM 10698（BCRC 17858）有良好生產及降解PHB的能力。在本研究中,將來自 C. manganoxidans 的 phaZCma (1355-2842 of accession no. AB038647) 及開放讀序框架（ORFCma, 1-1233 of AB038647）在大腸桿菌 BL21（DE3）中共同表現。ORFCma 作為ABC Transporter protein 助於 PHB 解聚酶 PhaZCma 的分泌及幫助細胞生長。利用全細胞的大腸桿菌其包含 pORFPHAZ 質體來探討降解 PHB 的效率和特異性。結果顯示，在81小時內 PHB 薄膜可以在 C. manganoxidans 的胞外被分解，而大腸桿菌BL21（DE3）/pPHAZ只需35小時就可以在胞外分解PHB。在28小時內，大腸桿菌BL21（DE3）/pPHAZ降解80％的PHB薄膜，而當PhaZCma 和 ORFCma一起表現時僅需18小時就可以達到相同的降解效果。在大腸桿菌中PhaZCma 被發現在細胞外異源表現，表示大腸桿菌中的蛋白質分泌系統是透過 PhaZCma。從3-羥基丁酸酯濃度和降解溶液的濁度可以得出結論，PhaZCma具有外切酶和內切酶兩種活性。重組PhaZCma已經證明能夠降解至少四種不同的聚酯，即聚 poly(3-hydroxybutyrate) (PHB)，poly(lactic acid) (PLA)，polycaprolactone (PCL)，poly(butylene succinate-co-adipate) (PBSA)，和 PBSA-flax seed blends。

In this study, phaZCma (1355-2842 of accession no. AB038647) and an open reading frame (ORFCma, 1-1233 of AB038647) from Caldimonas manganoxidans were co-expressed in E. coli BL21(DE3). ORFCma is proposed to be an periplasmic binding protein from the ABC transporter superfamily and that may assist the secretion of the PHB depolymerase PhaZCma as well as cell growth. The degradation rate and the substrate specificity of whole-cell E. coli/pORFPHAZ were investigated. The results showed that while PHB films can be completely degraded by extra-cellular fraction of C. manganoxidans in 81 h, only 35 h was required for extra-cellular fraction of E. coli BL21(DE3)/pPhaZ. Further improvement was achieved as 80% of the film was degraded by E. coli BL21(DE3)/pPHAZ in 28 h while only 18 h was needed by co-expressing ORFCma with PhaZCma. PhaZCma that was heterologously expressed in E. coli was found in the extra-cellular compartment, indicating that the protein secretion system in E. coli can be employed by PhaZCma. By testing the time course of 3-hydroxybutyrate concentration and the turbidity of degradation solutions, it can be concluded that PhaZCma had both exo- and endo-enzymatic activities. Recombinant PhaZCma has demonstrated the ability to degrade at-least four different polyesters, i.e., poly(3-hydroxybutyrate) (PHB), poly(lactic acid) (PLA), polycaprolactone (PCL), poly(butylene succinate-co-adipate) (PBSA), and PBSA-flax seed blends.

中文摘要 i
Abstract ii
Table of Contents iii
List of Tables v
List of Schemes vi
List of Figures vii
List of Abbreviations ix
Chapter 1: Introduction 1
1.1 Background 1
1.2 Motivation 2
Chapter 2: Past Literatures 3
2.1 Biodegradable plastics 3
2.2 Biodegradation of polyesters and PHB 4
2.3 PHB depolymerase (PhaZ) 6
2.4 Recycled monomer applications 7
2.5 ABC Transport system 9
Chapter 3: Materials and Methods 10
3.1 Bacterial strains and plasmids used in this study 10
3.2 Medium and growth conditions 10
3.3 DNA Purification 10
3.4 Gel electrophoresis 10
3.5 Plasmid construction 11
3.6 SDS-PAGE analysis 12
3.7 PHB, PCL, PLA, PBSA, and PET degradation sample preparation 13
3.8 Biodegradation experiments 13
3.9 Analytical methods 15
3.10 ORFCma analysis 15
3.11 Growth curve of recombinant strains 16
Chapter 4: Results 17
4.1 Intracellular and extracellular degradation of PHB films by C. manganoxidans 17
4.2 Cloning of pORFPHAZ and construction of pORF and pPHAZ 17
4.3 SDS-PAGE analysis 17
4.4 Intra- and extra- cellular degradation experiments on PHB film 18
4.5 ORFCma analyzation 18
4.6 Whole cell degradation experiments on PHB film 19
4.7 PhaZCma exhibits both endo- and exo- enzymatic degradation 20
4.8 Whole cell degradation experiments on various polyesters and PET samples 21
4.9 Whole cell degradation experiments on modifed PBSA film by recombinant strains 22
Chapter 5: Discussion 23
Chapter 6: Future Prospect 29
References 30
Tables 38
Schemes 42
Figures 52
Appendix I: List of chemicals 68
Appendix II: List of instruments 70
Appendix III: List of calibration curves 71
Appendix IV: Sequencing and Alignment 72
pPHAZORF 72
pORF 76
pPHAZ 79

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