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研究生:羅濟威
研究生(外文):Chi-Wei Lo
論文名稱:菌產聚羥基烷酸之分離純化策略之研究
論文名稱(外文):Strategy of Separation and Purification for Polyhydroxyalkanoates From Microorganism
指導教授:吳和生吳和生引用關係
學位類別:博士
校院名稱:元智大學
系所名稱:化學工程與材料科學學系
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:120
中文關鍵詞:聚羥基烷酸溶解度組合式化學分離純化
外文關鍵詞:polyhydroxyalkanoatesolubilityseparationpurificationcombinatorial chemistry
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聚羥基烷酸 (Polyhydroxyalkanoate, PHA),為一生物可分解性塑膠,其無毒且可利用微生物自行分解的特性,且因連接不同側鏈而有不同的性質,極具取代現有部分塑膠材料的潛力。但由於此種產品大部分藉由微生物生產製造,故在聚羥基烷酸的取得上,將通過純化提取的步驟來取得,本研究主要就針對Cupriavidus taiwanensis 184、基因改質過的大腸桿菌以及可生產中長碳鏈聚羥基烷酸的Pseudomonas putida KT2442這幾種菌株,利用溶劑萃取以及消化提取的方式取得聚羥基烷酸並討論性質之改變。
首先在聚羥基烷酸的測定上是將高分子經由硫酸水解並與甲醇反應成單體,而後利用氣相分析儀來進行定量分析,為了增加其準確性,此部份分別探討了硫酸濃度、反應時間、反應溫度、氯化鈉濃度以取得各種不同聚羥基烷酸 : (poly-3-hydroxybutyrate, P(3HB)), poly(3-hydroxybutyrate-co- 3-hydroxyvalerate) (P(3HB3HV)) 和poly(3-hydroxybutyrate-co-3-polyhydroxyhexanoate) (P(3HB3HHx))所需要前處理條件,同時測試於C. taiwanensis 184 and Burkholderia sp. PTU9這兩株菌株, 結果發現氯化鈉的加入將可抑制單體進入水相而增加分析的準確性,而不同側鏈的聚羥基烷酸將使用不同的硫酸濃度,如: 1 %的硫酸適用於聚羥基丁酸 (P(3HB)),而當共聚合物為P(3HB3HV)以及P(3HB3HHx)則硫酸濃度則需增加到5 %以及7 %。
在探討聚羥基烷酸溶解度的部份則是探討高分子聚羥基烷酸在溶劑中的熱力學相互關係以獲得聚羥基烷酸的溶解度。經由實驗值以及溶解度參數可獲得一新的模型,其重要的參數有溫度、分子量、結晶度以及聚羥基烷酸的種類,其結果利用232個實驗結果建立了P(3HB)和P(3HB3HHx)共聚合物的模組,這個模組(SHB= (141.7 + 0.454 exp(0.063 T) + 292.8 exp(-0.210 × 10-4 × Mw)) exp (-0.692 r) 和 SHHx = (20.06+100.6 exp(12.3 XHHx)+0.454 exp(0.063 T)+292.74 exp (-0.211 Mw)) exp ((-0.272 -0.422 exp(-1351 XHHx)) r))的建立將可以有效的選擇高效率、低價格以及低毒性的溶劑來進行溶劑萃取聚羥基烷酸。
對Cupriavidus taiwanensis 184所生產的聚羥基烷酸純化方面,利用十二烷基硫酸鈉 (SDS, 1%) 20毫升以及次氯酸鈉(4 %, w/v) 10毫升在50 oC下,以125 rpm轉速先後反應以十分鐘以及三分鐘,最終將得到純度99 %的聚羥基丁酸。
對基因改質大腸桿菌(E. coli XL1 bule)所生產的聚羥基烷酸進行純化方面,利用組合式化學以及高倍速分析方式,將可快速的取得最佳純化方式,最後以十二烷基硫酸鈉 (SDS, 10 %) 10毫升以及次氯酸鈉(NaOCl, 10 %) 10毫升在50 oC下,以125 rpm轉速對高壓均質法進行預處理的乾菌 (> 120 mesh)進行純化,最終將得到純度99 %的聚羥基丁酸。而此種方式將使150次實驗次數減少至16次。
在聚羥基烷酸的性質探討方面,則是對於不同提取方式對分子量的影響進行討論,對Cupriavidus taiwanensis 184提取P(3HB) (Mw: 1,600,000)而言,其結果發現,在預處理部份,高溫的乾燥過程將會使得分子量降低至880,000;與溶劑萃取法相較,消化提取方式的使用,將會使得分子量從1,600,000耗損成930,000,而不同的溶劑對Pseudomonas putida KT2442 (Mw:89,000)所產之中長碳鏈聚羥基烷酸進行溶劑萃取,當氯仿萃取法之萃取溫度從40 oC上升至60 oC時分子量由89,000降到50,000,而不同溶劑也會使分子量改變,當溶劑選用二氯乙烷時分子量為70,000,選用丁酸乙酯時分子量則為68,000。
Polyhydorxyalkanoates (PHAs) is a biodegradable plastic that can be produced or decomposed by microorganism. They have different properties when the different side chains link to polymer. Due to these reasons, they could replace the plastic popularly used nowadays for environmental protection. Most of PHAs were produced via the fermentation by microorganism. However the PHAs must be obtained with purification method from other residues. This study chooses different kinds of bacteria (i.e. Cupriavidus taiwanensis 184, recombinant Escherichia coli and Pseudomonas putida KT2442) to produce PHA via different purification method to recover PHAs. In addition, the diversity of characteristics obtained by different purification methods will be discussed.
For PHA analysis, the quantification method was usually used through acidic methanolysis and gas chromatographic (GC) analysis. The operation parameters for analyzing the quantitation of PHAs included sulfuric acid concentration, reaction time, salt (e.g. NaCl) addition, temperature and kind of PHAs (commercial products of poly-3-hydroxybutyrate (P(3HB)), poly(3-hydroxybutyrate-co- 3-hydroxyvalerate) (P(3HB3HV)) and poly(3-hydroxybutyrate-co-3-polyhydroxyhexanoate) (P(3HB3HHx)), as well as the strain of PHA-producing microorganisms (C. taiwanensis 184 and B. sp. PTU9). The results showed that the esters would preferentially accumulate in the organic phase, thereby significantly increasing the accuracy of GC analysis when NaCl was added in the two-phase system. This findings indicated that the decomposition efficiency of different types of PHAs was found to be dependent upon sulfuric acid concentration. For example, 1 % H2SO4 was favorable for P(3HB) decomposition. However 5 % and 7 % H2SO4 were more favorable to decompose P(3HB3HV) and P(3HB3HHx).
For solubility of PHAs, this study also presented and developed a thermodynamic correlation to obtain the solubility of PHAs in the organic solvent. This correlation offers a new perspective to evaluate solubility profile of the polymer and regression between experimental solubility and its parameters. Crucial parameters influencing solubility (e.g. temperature, molecular weight, crystallinity and kind of PHAs) were mentioned herein. Finally, a model with consideration of these parameters were established by using 232 experimental points for PHBHHx copolymers. These equations (SHB= (141.7 + 0.454 exp(0.063 T) + 292.8 exp(-0.210 × 10-4 × Mw)) exp (-0.692 r) and SHHx = (20.06+100.6 exp(12.3 XHHx)+0.454 exp(0.063 T)+292.74 exp (-0.211 Mw)) exp ((-0.272 -0.422 exp(-1351 XHHx)) r)) developed could be used for the selection of a solvent with high recovery efficiency, economic feasibility and low toxicity.
For the purification of PHB from C. taiwanensis 184, sodium dodecyl sulfate - sodium hypochlorite method seemed to be the best method. The P(3HB) in 99% could be obtained through SDS - sodium hypochlorite method for 0.5 g cell dry weight, 20 mL NaOH (4 %), 10 mL SDS (1%), 10 min reaction time and the agitation rate of 125 rpm.
For recombinant E. coli, the SDS-NaOH method was the best method in purification from the perspective of combinatorial chemistry. Using combinatorial chemistry, 150 sets of experimental data would be significantly decreased to 16 runs. Finally, nearly pure P(3HB) (ca. 99%) was yielded when 2 g (particle size > 120 mesh) biomass treated with 20 mL NaOH (10 %) and 10 mL SDS (10 %) for 10 min, 125 rpm.
The properties of PHAs would be discussed via different of kinds of separation methods. For molecular weight of PHB was 1,600,000 obtained from C. taiwanensis184. It would be decreased to 880,000 during pretreatment and high dried temperature. Using the organic solvent extraction as digestion method for separation would decrease average molecular weight form 1,600,000 to 930,000. For separation of P. putida KT2442, by chloroform extraction, the molecular weight of PHA was 89,000 at 40oC. It would be decreased to 50,000 when extraction temperature increased to 60oC. Using different solvent would obtained different molecular weight of resulted products. The molecular weight in 70,000 and 68,000 could be obtained using dichloroethane and butyl acetate, respectively.
Contents
Abstract I
中文摘要 III
致謝 V
Content VI
List of Tables X
List of Figure XI
Chapter 1 Introduction 1
1.1 Introduction 1
1.2 Polyhydroxyalkanoates 1
1.3 Production of PHAs by Microorganisms 3
1.4 Recovery of PHA 9
1.4-1 Solvent Extraction 13
1.4-2 Digestion Methods 14
1.4-2.1 Digestion by Surfactants 14
1.4-2.2 Digestion by Sodium Hypochlorite 15
1.4-2.3 Digestion by Sodium Hypochlorite and Chloroform 16
1.4-2.4 Surfactant-hypochlorite Treatment 16
1.4-2.5 Surfactant-chelate Digestion 17
1.4-2.6 Enzymatic Digestion 17
1.4-3 Mechanical Disruption 18
1.4-3.1 Bead mill Disruption 18
1.4-3.2 High Pressure Homogenization 18
1.4-3.3 Supercritical Fluid 19
1.4-3.4 Using Cell Fragility 19
1.5 Motivation and Objective of This Study 20
Chatper 2 Materials and method 22
2.1 Microorganisms 22
2.2 Materials 22
2.3 Equipment 23
2.4 Purification System 23
2.4-1 Pertreatment of Crude Cell 23
2.4-2 Chemical Digestion by Sodium Dodecyl Sulfate(SDS),
Sodium Hydroxide, Potassium Hydroxide and Sodium Hypochlorite 24
2.4-3 Solvent Extraction 25
2.4-4 Enzymatic Digestion 26
2.4-5 Preparation of PHB Library 26
2.4-6 Screening PHB for Purity 27
2.4-6.1 Screening Second Stage Digestion Solution from Sublibrary 27
2.4-6.2 Screening First Stage Digestion Solution from Sbulibrary 27
2.4-6.3 Screen the Optimum Pretreatment in the Library 28
2.5 Determination of Sodium Hypochlorite Titration 28
2.6 Bacteria Strain and Culture Conditions 29
2.7 Analysis of PHA by GC 30
2.7-1 Pre-treatment by Acidic Methanolysis for PHA Analysis 30
2.7-2 GC Calibration Curve 31
2.8 Molecular Weight Determination 33
2.9 Solubility Measurements of PHA Polymer 33
2.10 Determination of Crystallinity 34
2.11 Degradation Procedure of PHB 34
Chapter 3 Improved Gas Chromatography Analysis of
Poly(3-hydroxyalkanotes) by Optimizing acidic Methanolysis and Ether Partition Conditions 35
3.1 Introduction 35
3.2 Effect of Purification on the Characteristics of PHA 37
3.3 Quantitative Analysis of Standard PHB Using Braunegg’s Method 38
3.4 Effect of NaCl Concentration on Salting Out 40
3.5 Effect of H2SO4 Concentration 43
3.6 PHB Analysis from PHB-Containing Bacterial Cells 48
3.7 Conclusion 48
Chapter 4 Solubility of Polyhydroxyalkanoates by Experiment and
Thermodynamic Correlations 49
4.1 Introduction 49
4.2 Theoretical of Solubility Predictions 50
4.3 Evaluation of PHB Solubility Parameters 51
4.3-1 Group Contribution Methods 51
4.3-2 Experimental Methods 52
4.4 Comments on Solubility Results 54
4.5 Solubility and Solubility Parameters 55
4.6 Discussion on the Model 57
4.7 Effect of Molecular Weight 59
4.8 Effect of Temperature 61
4.9 Polymer Crystallinity 64
4.10 PHB Copolymers 65
4.11 Conclusion 70
Chapter 5 Effect of Separation Methods on PHAs’ Properties 71
5.1 Introduction 71
5.2 Purification of Polyhydroxybutyrate from C. taiwanensis184 72
5.3 Chloroform Extraction 77
5.3-1 Molecular Weight of PHA with Different Purification 77
5.3-2 Effect of Temperature on PHA for Solvent Extraction 80
5.3-3 Effect of Molecular Weight on Dry Temperature for C.184. 82
5.4 Effect of Sodium Hypochlorite on PHB Molecular Weight 84
5.4-1 Effect of Amount Sodium Hypochlorite for PHB Molecular Weight 84
5.4-2 Effect of Reaction Temperature 85
5.5 Effect of Alkali on PHB Properties 86
5.6 Conclusion 87
Chapter 6 High Throughput Study of Purification of
Polyhydroxybutyrate from Recombinant Escherichia coli 89
6.1 Introduction 89
6.2 Preparation of P(3HB) Library by Using Mix-split Method 90
6.3 Screen of PHB Purity Using Combinatorial Chemistry 92
6.3-1 Screening of Second Digestion Stage in P(3HB) Purity 92
6.3-2 Screening of First Digestion Stage in P(3HB) Purity 94
6.3-3 Screening of Pretreatment 94
6.4 Single Digestion Method for Purification 95
6.5 Two Stage Digestion Method 98
6.6 Conclusion 101
Chapter 7 Conclusions 102
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