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研究生： 英格麗
Magdiel - Inggrid Setyawati
論文名稱： Genetically Engineered Acetobacter xylinum: Bacterial Cellulose and Self Immobilization System Production
Genetically Engineered Acetobacter xylinum: Bacterial Cellulose and Self Immobilization System Production
指導教授： 李振綱
Cheng-Kang Lee
口試委員: 王孟菊
Meng-Jiy Wang
劉懷勝
Hwai-Shen Liu
學位類別： 碩士
Master
系所名稱： 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年： 2007
畢業學年度： 95
語文別： 英文
論文頁數： 140
中文關鍵詞： Bacterial cellulose 、DAAO 、Acetobacter xylinum 、immobilization
外文關鍵詞： Bacterial cellulose, DAAO, Acetobacter xylinum, immobilization
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    • Gram-negative, rod-shaped, obligate aerobic bacterium, Acetobacter xylinum is the most productive bacterial cellulose (BC) producer. In this study the A. xylinum host strain were genetically engineered for further enhancement of bacterial cellulose production and generate self immobilized cell system.
    Vitreoscilla hemoglobin was constitutively expressed in Acetobacter xylinum to enhance bacterial cellulose (BC) production. The enhancement was much pronounced in static culture than in shaken culture. Reducing O2 tension in gaseous phase of the culture by tightly sealing the culture tube could also enhance BC production by 70 %. O2 tension reduced from 21 % to 15 % in the sealed and static culture of VHb-expressing A. xylinum after 7 days cultivation, while 7.36 g/l of BC with yield of 0.44 were obtained. BC pellicle production was successfully scaled-up in a sealed 4 l disposable zip lock plastic bag with BC yield of 0.35 and concentration of 6.31 g/l.
    Along with the production of BC, the cell became entrapped inside the BC fibers produced. In order to take the advantage of these cells’ self-immobilization phenomenon for biotransformation, D-amino acid oxidase (DAAO) was chosen to be expressed in A. xylinum along with the BC production. A plasmid bearing the Rhodosporidium toruloides daao gene, placP-DAAO-122 was constructed and transformed into A. xylinum to enable the IPTG-induced A. xylinum’s DAAO production. The highest DAAO activity (~1.5 U/ml) was detected when 4% glycerol was supplemented in HS medium (HSG4). The detected DAAO activity was in the order of free enzyme > free cell > immobilized cell. Cell cultured in shaking condition produce more DAAO than in stationary condition. The operational stability of DAAO activity was in the order of free enzyme < free cell < immobilized cell.

    Gram-negative, rod-shaped, obligate aerobic bacterium, Acetobacter xylinum is the most productive bacterial cellulose (BC) producer. In this study the A. xylinum host strain were genetically engineered for further enhancement of bacterial cellulose production and generate self immobilized cell system.
    Vitreoscilla hemoglobin was constitutively expressed in Acetobacter xylinum to enhance bacterial cellulose (BC) production. The enhancement was much pronounced in static culture than in shaken culture. Reducing O2 tension in gaseous phase of the culture by tightly sealing the culture tube could also enhance BC production by 70 %. O2 tension reduced from 21 % to 15 % in the sealed and static culture of VHb-expressing A. xylinum after 7 days cultivation, while 7.36 g/l of BC with yield of 0.44 were obtained. BC pellicle production was successfully scaled-up in a sealed 4 l disposable zip lock plastic bag with BC yield of 0.35 and concentration of 6.31 g/l.
    Along with the production of BC, the cell became entrapped inside the BC fibers produced. In order to take the advantage of these cells’ self-immobilization phenomenon for biotransformation, D-amino acid oxidase (DAAO) was chosen to be expressed in A. xylinum along with the BC production. A plasmid bearing the Rhodosporidium toruloides daao gene, placP-DAAO-122 was constructed and transformed into A. xylinum to enable the IPTG-induced A. xylinum’s DAAO production. The highest DAAO activity (~1.5 U/ml) was detected when 4% glycerol was supplemented in HS medium (HSG4). The detected DAAO activity was in the order of free enzyme > free cell > immobilized cell. Cell cultured in shaking condition produce more DAAO than in stationary condition. The operational stability of DAAO activity was in the order of free enzyme < free cell < immobilized cell.

    ABSTRACT i ACKNOWLEDGEMENT ii TABLE OF CONTENTS iii LIST OF FIGURES viii LIST OF TABLES xii CHAPTER 1 INTRODUCTION 1.1. Bacterial cellulose production enhancement 1 1.2. Self immobilization of DAAO expressing cells 3 CHAPTER 2 LITERATURE STUDY 2.1. Bacterial cellulose 5 2.1.1. Acetobacter xylinum as bacterial cellulose producer 6 2.1.2. Unique bacterial cellulose features 10 2.1.3. Bacterial cellulose applications 11 2.2. Bacterial Hemoglobin 13 2.2.1. Identification of Vitreoscilla hemoglobin (VHb) 14 2.2.2. Regulation of vgb expression 14 2.2.3. Biochemical function of VHb 15 2.2.4. VHb in biotechnology 17 2.3. Immobilization 18 2.3.1. Immobilized viable cells as an economical source of enzyme 20 2.3.2. Gel entrapment as a way of living immobilization 20 2.3.3. Physiology of gel immobilized cells 23 2.4. D-Amino Acid Oxidase 24 2.4.1. The Rhodosporidium toruloides DAAO structure 25 2.4.2. Substrate specificity of RgDAAO 26 2.4.3. Stability of RgDAAO 26 2.4.4. Practical applications of DAAO 27 CHAPTER 3 EXPERIMENT MATERIALS AND METHODS 3.1. Experiments Principles 29 3.1.1. Enhancing Bacterial Cellulose Production in recombinant A. xylinum 29 3.1.2. Construction of placP-DAAO-122 for expression of R. toruloides DAAO in recombinant A. xylinum 30 3.1.3. The R. toruloides DAAO Production in A. xylinum 35 3.1.4. Self immobilization of DAAO expressing A. xylinum 35 3.2. Experiments Materials 3.2.1. Bacterial strains 36 3.2.2. Plasmid vectors 37 3.2.3. Primers 37 3.2.4. Enzymes 37 3.2.5. DNA test kits 38 3.2.6. Molecular weight standard solutions 38 3.2.7. Antibodies 38 3.2.8. Other chemicals 38 3.3. Experiments Culture Medium and Reagents 3.3.1. Culture medium 39 3.3.2. Reagents 41 3.4. Experiment Apparatus 43 3.5. Bacterial cellulose production enhancement procedures 3.5.1. Transformation of A. xylinum with pBla-VHb-122 43 3.5.2. PCR colony selection 44 3.5.3. PCR selection confirmation using agarose gel electrophoresis 45 3.5.4. VHb expression confirmation 46 3.5.5. Plasmid segregational stability 46 3.5.6. Bacterial cellulose production 47 3.5.7. Analysis of bacterial cellulose production 47 3.5.8. Scaling Up the bacterial cellulose production 49 3.6. Construction of placP-DAAO-122 Procedures 3.6.1. Polymerase Chain Reaction (PCR) primers design 49 3.6.2. Construction of placP-DAAO-122 50 3.6.3. Transformation of A. xylinum with placP-DAAO-122 71 3.6.4. PCR colony selection 71 3.6.5. PCR selection confirmation using agarose gel electrophoresis 72 3.6.6. daao expression confirmation 72 3.7. DAAO Production in Recombinant A. xylinum 3.7.1. Substrate selections 73 3.7.2. Optimum initial concentration of glycerol selection 73 3.8. Self immobilization of DAAO expressing A. xylinum 3.8.1. Culture cultivation effect 74 3.8.2. Operational Stability 76 3.9. SDS PAGE analysis 77 3.10. Immuno/Western Blot analysis 78 3.11. Colorimetric quantification of DAAO activity 79 3.12. Competent cells preparation 80 CHAPTER 4 RESULTS AND DISCUSSIONS 4.1. pBla-VHb-122 expression plasmids 82 4.2. A. xylinum transformation 83 4.3. vgb gene expression in recombinant A. xylinum 84 4.4. The pBla-VHb-122 expression plasmid segregational stability 85 4.5. Enhancementof bacterial cellulose production 4.5.1. Effect of shaking 86 4.5.2. Effect of medium composition 90 4.5.3. Effect of vgb expression 94 4.5.4. Effect of O2 tension 96 4.5.5. Scaling up the bacterial cellulose production 4.6. Construction of placP-DAAO-122 4.6.1. Construction of intermediate plasmid, placP-30 101 4.6.2. Construction of intermediate plasmid, placP-DAAO-30 103 4.6.3. Construction of placP-DAAO-122 expression plasmid 105 4.7. A. xylinum transformation with placP-DAAO-122 107 4.8. daao gene expression in recombinant A. xylinum 108 4.9. Effect of substrate selection 110 4.10. Effect of substrate initial concentration 113 4.11. Effect of culture cultivation 115 4.12. Operational stability 4.12.1. Effect of Temperature 117 4.12.2. Effect of pH 118 4.12.3. Effect of storage time 119 4.12.4. Recycle usage of the cell 120 CHAPTER 5 CONCLUSIONS 5.1. Bacterial cellulose production enhancement 122 5.2. Self immobilization of DAAO expressing cells 123 REFERENCES xiii

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