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研究生:王嘉宏
研究生(外文):Jia-Hung Wang
論文名稱:基因重組嗜熱菌海藻糖合成酶之蛋白質工程及其在轉殖水稻的應用
論文名稱(外文):Protein Engineering of Recombinant Thermus thermophilus Trehalose Synthase and Its Application in Transgenic Rice
指導教授:蕭介夫蕭介夫引用關係
學位類別:博士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
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:海藻糖嗜熱菌海藻糖合成酶重組融合蛋白質轉殖水稻生物反應器
外文關鍵詞:TrehaloseThermus thermophilustrehalose synthaseRecombinant fusion proteinTransgenic ricebioreactor
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摘 要

來自嗜熱菌(Thermus thermophilus)的海藻糖合成酶(TtTS)是一種具有催化麥芽糖轉化成海藻糖能力,及擁有高熱穩定性之酵素;相較於其他已知的海藻糖合成酶,其對於海藻糖的生成具有相對較佳的熱穩定性和較高的生成轉化率。藉由多種不同微生物來源之相關海藻糖合成酶的氨基酸序列及概要性單元組成序列比對,在嗜熱菌海藻糖合成酶中,發現其蛋白質序列含有一段在其他多數海藻糖合成酶所沒有的特殊C-端片段。為了證實此片段存在的功能,我們利用蛋白質工程技術分別執行了海藻糖合成酵素蛋白質C-端的刪除與接合,以闡明此片段在海藻糖的生成上扮演之重要角色。實驗結果發現被刪除C-端片段之TtTS其在海藻糖生成的催化反應上,失去了原有之絕佳的熱穩定性,又在反應中產生了更多不經濟的副產物;相反的,在融合酵素的實驗方面,來自抗輻射菌(Deinococcus radiodurans)的海藻糖合成酶(DrTS)於其C-端接合了此特殊片段後,與原始酵素相較,則融合酵素擁有了較佳的熱穩定性和較低的副產物生成率;由研究結果推論,TtTS的C-端片段在維持酵素的熱穩定性上,扮演了一個關鍵性角色;在高溫作用時,並可藉此調節葡萄糖從側反應生成的比例。
再者,為了獲得在高溫下可使澱粉轉化成海藻糖的耐熱性酵素,使催化反應步驟簡單化且達到高效率的目標,本研究採用蛋白質融合方式,將源自耐熱梭孢桿菌(Clostridium thermosulfurogenes)的β-澱粉酶(BA)與TtTS融合,建構成TtTSBA和BATtTS兩種重組融合蛋白質,利用E. coli表現系統大量表達,並經多重純化步驟後獲得具雙功能活性的重組融合酵素。在澱粉轉化成海藻糖的反應上,其融合酵素催化效率分別是混合個別原始酵素(BA/TtTS)的3.4及2.4倍;顯見融合酵素在連續式的催化作用上,可藉由動力學上的優勢達到更佳的催化效率。
海藻糖在生物體遭遇外在環境壓力時,具有保護生物分子抵禦逆境的獨特能力;藉由增加海藻糖在植物體中之累積量,可增進它們對環境壓力的耐受度已獲證實。研究中採用玉米ubiquitin 的啟動子(Ubi1),分別建構含DrTS或BADrTS基因的兩個轉殖質體,進行水稻轉殖試驗,由南方墨漬之雜交實驗驗證,成功獲得了分別含有此兩種基因的水稻轉殖植株;高量的海藻糖累積可分別在其穀粒與葉片中測得,且其對於鹽度、乾旱和寒冷等壓力之耐受性測試,與非轉基因水稻相較,也皆有較持續性生長的優勢。
此外,本研究更觀察到水稻轉殖植株,含有海藻糖蓄積之穀粒,具有降低本身酸敗發生速率的功能,使維持儲藏一段時間仍可保持良好之稻米品質;其葉片在生長的老化進程也有延緩現象,或許此優勢可供做生長代謝與穀粒產量關聯性的進一步研究,以期提升整體產量。總之,利用雙功能重組融合酵素轉殖水稻之策略成功的證明,促進海藻糖在水稻中的生成、累積,不但可增進植物體對抗逆境的耐受性,更可採用此一轉殖水稻表達系統做為『天然的生物反應器』,提供海藻糖大量生產之新策略。


關鍵字:海藻糖、嗜熱菌海藻糖合成酶、重組融合蛋白質、轉殖水稻、生物反應器。
ABSTRACT

Trehalose synthase (TS) from Thermus thermophilus (TtTS) is a thermostable enzyme that catalyzes the conversion of maltose into trehalose. It has a relatively higher thermostability and a better conversion ratio for trehalose production than other known TSs at present. By amino acid sequences and the schematic motif alignment of TS-related enzymes, it was discovered that TtTS contains a particular C-terminal fragment that is not found in most other TSs. To verify the function of this fragment, C-terminal deletion and enzyme fusion were respectively performed to explain the important role this fragment plays in the formation of trehalose.
It was found that the recombinant TtTSΔC enzyme had a lower thermostability and a higher byproduct than TtTS in catalyzing the conversion of maltose into trehalose. On the other hand, the recombinant DrTS-TtTSΔN enzyme had a higher thermostability and a lower byproduct than DrTS in their reactions. The above-mentioned results allowed the inference that the C terminus of TtTS plays a key role in maintaining its thermostability and hence in modulating the side reaction to reduce glucose production at a high temperature.
Moreover, a fusion gene that encoded a polypeptide of 1495 amino acids was constructed from the β-amylase (BA) gene of C. thermosulfurogenes and TtTS. It was overexpressed in E. coli, and a recombinant bifunctional fusion protein with BA at the N-terminal (BATtTS) or C-terminal (TtTSBA) of TtTS having both BA and TS activities. BATtTS or TtTSBA catalyzes the sequential reaction in which maltose is formed from starch and then is converted into trehalose. TtTSBA showed much higher sequential catalytic efficiency than the separately expressed BA/TtTS mixture. The catalytic efficiency of TtTSBA or BATtTS was 3.4 or 2.4 times higher, respectively, than that of a mixture of individual enzymes, showing the kinetic advantage of the fusion enzyme. These results apparently demonstrate that fusion enzymes catalyzing sequential reactions have kinetic advantages over a mixture of both enzymes.
Trehalose is an uncommon saccharide with unique abilities to protect biomolecules from abiotic stresses. Increasing the trehalose accumulation in plants could improve their environmental stress tolerance. We have generated transgenic rice plants expressing a novel D. radiodurans TS (DrTS) gene alone and a bifunctional fusion (BADrTS) of C. thermosulfurogenes BA (CtBA) and DrTS genes, respectively, under the control of the maize ubiquitin promoter, and investigated their responses to various stresses. Southern blot-positive lines with the correct hybridization pattern for each construct were obtained. The trehalose accumulation from DrTS and BADrTS plants was increased up to 1.86 and 2.26 mg g fresh weight-1 in leaf extracts, and up to 2.16 and 3.06 mg g fresh weight-1 in seed extracts, respectively. In stress tolerance, several independent transgenic lines displayed a sustained plant growth as compared to nontransgenic rice, more propitious for salinity, drought, and cold stress conditions.
Furthermore, we also observed the effect of time on maintained seeds flavor quality during storage and the postponed leaves senescence in transgenic rice plants. These findings clearly demonstrate that the strategies of trehalose biosynthesis in transgenic rice plants can increase tolerance with regard to rice plant improvement. Therefore, we could nevertheless confirm that it has a tremendous potential for large scale production of trehalose through this fusion enzyme using a heterologous source like starch from rice as “natural bioreactors”.


KEY WORDS:Trehalose, Thermus thermophilus trehalose synthase, Recombinant fusion protein, Transgenic rice, bioreactor.
TABLE OF CONTENTS

ABSTRACT (Chinese) i
ABSTRACT (English) iv
TABLE OF CONTENTS vii
LIST OF FIGURES xi
LIST OF TABLES xiv
ABBREVIATION xv

I. INTRODUCTION 1
1.1 A MULTIFUNCTIONAL MOLECULE “TREHALOSE” 1
1.1.1 Structure and Distribution of Trehalose 1
1.1.2 Functions of Trehalose 3
1.1.2.1 As an energy and carbon reserve 3
1.1.2.2 As a stabilizer and protectant of proteins and membranes 4
1.1.2.3 As a sensing compound and/or growth regulator 7
1.1.2.4 As a structural component of the bacterial cell wall 7
1.1.3 Manipulations of Trehalose Accumulation in Plants and Animal Cells 8
1.2 INDUSTRIAL APPLICATIONS OF TREHALOSE 10
1.3 PRODUCTION OF TREHALOSE BY ENZYME SYSTEMS
10
1.3.1 Trehalose Synthase 11
1.3.2 Thermus Thermophilus Trehalose Synthase 12
1.3.3 Clostridium Thermosulphurogenes β-Amylase 12
1.4. MECHANISMS OF PROTEIN THERMOSTABILIZATION
13
1.5 PROTEIN THERMOSTABILITY ENGINEERING 14
1.5.1 Potential for Protein Thermostabilization 15
1.5.2 Choice of Thermostabilization Strategy 16
1.5.3 Strategies for Stabilization by Site-Directed Mutagenesis 16
1.5.4 Computational Methods in the Design of Stabilizing Strategies 17
1.5.5 Directed Evolution 20
1.6 ADVANTAGES OF RECOMBINANT FUSION ENZYME 21
1.7 A NATURAL BIOREACTOR “TRANSGENIC PLANTS” 22

II. Role of the C-Terminal Domain of Thermus thermophilus
Trehalose Synthase in the Thermophilicity, Thermostability,
and Efficient Production of Trehalose 23
2.1 MATERIALS AND METHODS 23
2.1.1 Materials 23
2.1.2 Bacterial Strains and Cultivation 24
2.1.3 Construction of Recombinant Expression Plasmids 24
2.1.4 Expression of Recombinant Enzymes 26
2.1.5 Purification of Recombinant Enzymes 27
2.1.6 Protein Assay 28
2.1.7 Enzyme Characterization 28
2.1.8 Analysis of Carbohydrate 29
2.2. RESULTS 30
2.2.1 Molecular Cloning of DrTS Gene 30
2.2.2 Characterization of the Recombinant Trehalose Synthases 30
2.2.3 Effects of the C-Terminal Domain of TtTS (TtTSΔN) on
Thermophilicity and Thermostability 31
2.2.4 Effects of the C-terminal domain of TtTS (TtTSΔN) on the Glucose
Formation of TS-Catalyzed Reaction 32
2.3 DISCUSSION 33
III. Construction of a Recombinant Thermostable β-Amylase-
Trehalose Synthase Bifunctional Enzyme for Facilitating the
Conversion of Starch to Trehalose 36
3.1 MATERIALS AND METHODS 36
3.1.1 Materials 36
3.1.2 Bacterial Strains and Cultivation 36
3.1.3 Construction of Recombinant Expression Plasmids 37
3.1.4 Expression of Recombinant Enzymes 39
3.1.5 Purification of Recombinant Enzymes 40
3.1.6 Protein Assay 42
3.1.7 Enzyme Characterization 42
3.1.8 Analysis of Carbohydrate 43
3.2 RESULTS 43
3.2.1 Expression and Purification of the Bifunctional Fusion Enzymes
BATtTS and TtTSBA 43
3.2.2 Recombinant Enzyme-Catalyzed Trehalose Synthesis 44
3.2.3 Biochemical Properties of the Bifunctional Fusion Enzymes TtTSBA
and BATtTS 45
3.2.4 Kinetic Properties of the Bifunctional Fusion Enzymes BATtTS and
TtTSBA 46
3.3 DISCUSSION 47

IV. Engineering Maintained Seeds Flavor Quality during Storage
and Delayed Leaves Senescence in Transgenic Rice Plants by
Overexpression of a Bifunctional Fusion Protein of the
β-amylase and Trehalose Synthase 51
4.1 MATERIALS AND METHODS 52
4.1.1 Plasmid Construction and Rice Transformation 52
4.1.2 DNA-Blot Hybridization Analysis 53
4.1.3 Protein Extraction and Immunoblotting 54
4.1.4 Enzyme Assays 55
4.1.5 Detecting Trehalose and Soluble Carbohydrates 56
4.1.6 Estimation of Growth Condition and Stress Tolerance of Transgenic
Rice Plants 56
4.1.7 Free Fatty Acid and Total Lipid Content of Rice Grain Analyses 57
4.1.8 Chlorophyll Determination 57
4.2 RESULTS 58
4.2.1 Production of Transgenic Rice Lines with the Recombinant Gene DrTS
and Fusion Gene BADrTS 58
4.2.2 Expression of DrTS and BADrTS in Transgenic Rice Plants and
Trehalose Synthase Activity Analysis 58
4.2.3 Determination of Transgenic Plants that Produced Increased Trehalose
Content and the Effect of Trehalose Production upon Rice Growth
Phenotype 59
4.2.4 Stress Tolerance Conferred by DrTS or BADrTS Expression in
Transgenic Plants 60
4.2.5 The Seeds of Transgenic Plants Show Improved Flavor Quality by
Delaying FFA Formation during Storage with Time 62
4.2.6 Transgenic Plants have Increased Senescence-resistant Capacity 62
4.3 DISCUSSION 63

V. CONCLUSIONS 68

VI. BIBLIOGRAPHY 70

VII. FIGURES AND TABLES 90
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