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研究生:高維忱
研究生(外文):Wei-Chen Kao
論文名稱:以表現真核金屬鍵結蛋白Metallothionein之大腸桿菌開發重金屬生物吸附劑
論文名稱(外文):Development of heavy metal biosorbent using recombinant Escherichia coli expressing eucaryotic metal-binding protein - metallothionein
指導教授:張嘉修張嘉修引用關係
指導教授(外文):Jo-Shu Chang
學位類別:博士
校院名稱:國立成功大學
系所名稱:化學工程學系碩博士班
學門:工程學門
學類:化學工程學類
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:167
中文關鍵詞:融合蛋白重金屬細胞固定化填充床管柱聚乙烯醇間質表達生物吸附麥芽糖鍵結蛋白金屬硫蛋白
外文關鍵詞:cadmiumBiosorptioncell immobilizationfixed bed columnfusion proteinheavy metalsmaltose-binding proteinmetallothioneins (MTs)periplasmic expressionpolyvinyl alcohol (PVA)
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本研究利用在大腸桿菌中表達哺乳動物及魚類的金屬鍵結蛋白質metallothioneins (MTs)的策略,以提升細菌的生物吸附劑對重金屬鉛、銅、鎘及鋅的金屬吸附效率,並將MT蛋白質表現於宿主細胞的細胞質及細胞間質,以探討金屬鍵結蛋白表達位置對金屬吸附的影響。由實驗結果得知,大腸桿菌宿主細胞在表現MT蛋白質後,大幅增加了其對Cd的總生物吸附效率(ηads) (5-210 %)。表達MT在金屬生物吸附的改善上,主要是增加其生物吸附的速率(r2),其效果比增加平衡生物吸附容量(qmax)更為明顯。在細胞間質部分表達MTs,似乎比在細胞質表達時更能促進其對金屬鍵結的能力,並且MT蛋白質的來源對其金屬生物吸附能力有不同程度的影響。和源自於老鼠(MT1)以及魚類的MTs (OmMT)相比,來自人類的MT (MT1A)對大腸桿菌宿主之生物吸附促進效果較為顯著。此外,亦發現表現MT蛋白質的基因重組生物吸附劑的總生物吸附效率(ηads)是和吸附金屬之種類相關,且ηads的大小依次為Cd > Cu > Zn > Pb。
由上述實驗初步證實,在細胞間質部分表現人類金屬鍵結蛋白質MT的重組大腸桿菌種對鎘之吸附效果極佳,因此以聚乙烯醇將該基因重組生物吸附劑固定化,並以該固定化生物吸附劑來移除水中的重金屬鎘。為評估固定化生物吸附劑的吸附能力以及再使用能力,本研究探討不同的操作條件如吸附的pH值、溫度、起始的鎘濃度、菌體固定量以及吸附/脫附的循環(A/D cycles)對鎘吸附之影響。由實驗結果可知,pH值對吸附能力有很大的影響,且在pH 5.0時有較佳的吸附結果,但在溫度在範圍為20-42oC時,對吸附能力的影響較小。和Pseudo-first order model相比,pseudo-second order model對吸附的動力性質有較好的描述。另外由Langmuir isotherm的分析可知,對菌體固定量為9.71及15.4 wt.%的固定化顆粒而言,其qm及Kd值分別為8.67 mg/g、141.3 mg/l及10.58 mg/g、71.94 mg/l。上述結果和沒有固定菌體的PVA顆粒(qm及Kd分別為6.35 mg/g及229.0 mg/l)相比,皆有較佳的表現。在起始鎘濃度為10-150 mg/l時,有固定化菌體的PVA顆粒和沒有固定菌體的顆粒相比有較佳的鎘移除百分比。但是,鎘移除百分比會隨著起始鎘濃度的增加而減少,並在起始濃度10 mg/l、菌體固定量15.4 wt.%時有最高的移除百分比(82.7%)。當進一步增加菌體含量時,其對鎘的吸附能力也跟著增加。而在起始鎘濃度100 mg/l、菌體固定量為33.0 wt.%時,其吸附能力(4.29 mg/g)和菌體固定量9.71及15.4 wt.%相比,分別提高了73%及7%的吸附能力。再者,當固定化顆粒達飽和吸附後,以0.01 M Na3NTA溶液為脫附劑進行4個吸附/脫附循環(A/D cycles)時有最好的脫附效率(57-89%),且固定化顆粒再生後仍保有51-61%的原始吸附能力。
接著,本研究亦以表現人類金屬鍵結蛋白質(MT)的重組菌種之PVA固定化細胞進行連續式管柱吸附實驗,並探討不同的程序參數 (如床高、體積流速以及進料金屬濃度) 對填充床生物吸附效率的影響,所使用之吸附效率功能指標包括穿透時間,總吸附量,吸附容量,初始吸附速率等。由實驗結果得知,減少流率(從2.0到0.5 ml/min,20 mg/l進料濃度)及增加進料金屬濃度(從5到20 mg/l,0.5 ml/min)皆可使金屬吸附量增加,但初始吸附速率則與重金屬進料濃度與負載速率有正相關。在所探討的程序參數中,重金屬進料濃度是對連續填充床管柱生物吸附程序效能最重要的影響因子。而生物吸附在突破濃度時所得的曲線,可藉由Bohart-Adams model及Thomas model加以描述。而以串聯填充管柱進行之吸附/脫附程序顯示,該程序可穩定的操作,且穿透時間能有效地延長。以0.01 M Na3NTA對吸附飽和的管柱進行脫附時,鎘濃度最高可濃縮達12.1-15.5倍之多,且脫附後的管柱具有不錯的再生吸附能力。
In this study, we examined the expression of mammalian and fish metallothioneins (MTs) in E. coli as a strategy to enhance metal biosorption efficiency of bacterial biosorbents for lead (Pb), copper (Cu), cadmium (Cd), and zinc (Zn). In addition, MT proteins were expressed in either the cytoplasmic or periplasmic compartment of host cells to explore the localization effect on metal biosorption. The results showed that MT expression led to a significant increase (5-210 %) in overall biosorption efficiency (ηads), especially for biosorption of Cd. The MT-driven improvement in metal biosorption relied more on the increase in the biosorption rates (r2; a kinetic property) than on the equilibrium biosorption capacities (qmax; a thermodynamic property), despite a 10-45 % and 30-80 % increase in qmax of Cd and Zn, respectively. Periplasmic expression of MTs appeared to be more effective in facilitating the metal-binding ability than the cytoplasmic MT expression. Notably, disparity of the impacts on biosorption ability was observed for the origin of MT proteins, as human MT (MT1A) was the most effective biosorption stimulator than MTs originating from mouse (MT1) and fish (OmMT). Moreover, the overall biosorption efficiency (ηads) of the MT-expressing recombinant biosorbents was found to be adsorbate-dependent: the ηads values decreased in the order of Cd > Cu > Zn > Pb.
Due to its excellent ability on Cd biosorption, recombinant E. coli strain expressing human metal-binding protein metallothionein (MT) in the periplasmic compartment was immobilized with polyvinyl alcohol (PVA) and used as biosorbent for the removal of cadmium (Cd) in aquatic environment. Investigations were conducted to examine the effect of adsorption pH, temperature, initial Cd concentration, biomass loading, and adsorption/desorption (A/D) cycles on the adsorption ability and reusability of the immobilized biosorbent. The adsorption ability was strongly affected by pH with an optimal performance at pH 5.0, while it was less sensitive to temperature over the range of 20-42oC. The adsorption kinetics was best described by pseudo-second order model. Langmuir isotherm analysis shows a qm and Kd value of (8.67 mg/g, 141.3 mg/l) and (10.58 mg/g, 71.94 mg/l) for PVA-immobilized cells with a biomass loading of 9.71 and 15.4 wt.%, respectively. This performance is much better than biomass-free PVA beads, displaying a qm and Kd value of 6.35 mg/g and 229.0 mg/l, respectively. Over the initial Cd concentrations range of 10 to 150 mg/l, PVA-immobilized cells had better Cd removal percentage than the biomass-free PVA beads, but the removal percentage decreased with an increase in initial Cd concentration, giving the highest Cd removal percentage of 82.7% at an initial concentration of 10 mg/l and a biomass loading of 15.4 wt.%. Further increase in biomass loading resulted in better adsorption ability. With an initial Cd concentration of 100 mg/l, a biomass loading of 33.0 wt.% attained an adsorption capacity of 4.29 mg/g, which is 73 and 7% higher than that obtained from a biomass loading of 9.71 and 15.4 wt.%, respectively. An aqueous solution of 0.01 M Na3NTA was found to display the best desorption efficiency (57-89%) for four A/D cycles, while 51-61% of the original adsorption capacity was retained after regeneration.
Finally, recombinant E. coli cells expressing human metal-binding protein (metallothionein; MT) were immobilized by polyvinyl alcohol (PVA) and packed into fixed-bed columns to carry out continuous biosorption of cadmium. The effect of various process parameters, such as bed depth, flow rate, and influent metal concentration on the fixed-bed biosorption was investigated. Decrease in flow rate (from 2.0 to 0.5 ml/min, 20 mg/l) and increase in influent metal concentration (from 5 to 20 mg/l, 0.5 ml/min) led to an increase in metal uptake (bed depth = 30 cm) from 1.37 to 2.22 mg/g and 0.53 to 2.22 mg/g, respectively. Other factors such as total capacity, initial adsorption rate and breakthrough bed volume were also discussed. In addition, it is found that among the process parameters discussed (bed depth, flow rate, and influent concentration), the influent concentration is the most critical factor influencing the performance of continuous column biosorption process. The biosorption breakthrough curves were described by Bohart-Adams model and Thomas model with good agreement. Serial adsorption/desorption operations of the fixed-bed columns shows a good stability of repeated uses of the columns, and desorption of loaded Cd2+ ions with 0.01 M Na3NTA could concentrate Cd2+ ions up to 12.1-15.5 fold when compared with the feeding Cd2+ ion concentration. The regenerated column still exhibited good adsorption ability, indicating that the proposed serial column biosorption/regeneration system is feasible and potentially applicable in treating real heavy-metal-polluted effluents.
Abstract (Chinese)……………………………………………………..…………….……...I
Abstract (English)…………………………………………………………………………IV
Acknowledgements……………………………………………………………………...VIII
Contents………………………………………………………………………………….....X
List of Tables…………………………………………………………………………….XVI
List of Figures………………………………………………………………………….XVIII
Chapter 1 Introduction……………………………………………………………………1
1.1 Background of this study…………………………………………………………..1
1.2 Motivation and purpose……………………………………………………………5
1.3 Content of this dissertation………………………………………………………...6
Chapter 2 Literature review………………………………………………………………9
2.1 Heavy metals pollution………………………………………………………….…9
2.1.1 Definition of heavy metals…………………………………………………9
2.1.2 Source of heavy metals pollution…………………………………………10
2.1.3 Metals and related diseases to human body………………………………12
2.1.4 Methods used to remove heavy metal from the environment…………….15
2.2 Biosorption……………………………………………………………………….17
2.2.1 Definition of biosorption………………………………………………….18
2.2.2 Comparison of biosorption and bioaccumulation………………………...18
2.2.3 Natural and commercial biosorbents……………………………………...22
2.2.4 Development of biosorbent…………………………………………….…24
2.3 Metallothionein (MT)…………………………………………………………….25
2.3.1 Introduction to metallothioneins………………………………………….25
2.3.2 History of metallothioneins……………………………………………….27
2.4 Cell immobilization………………………………………………………………29
2.4.1 Introduction to cell immobilization……………………………………….30
2.4.2 Characteristics of some polymers used for cell immobilization………….31
2.4.3 Polyvinyl alcohol (PVA)………………………………………………….32
2.5 Processes and reactor design for heavy metals biosorption……………………...34
2.5.1 Processes for heavy metals biosorption…………………………………...34
2.5.2 Type of reactors for heavy metals biosorption……………………………34
Chapter 3 Materials and methods………………………………………………………36
3.1 Chemicals and materials………………………………………………………….36
3.2 Equipment………………………………………………………………………..38
3.3 Methods for analysis, preparation and measurement…………………………….40
3.3.1 Plasmids and host strains………………………………………………….40
3.3.2 Strain cultivation and proteins expression in E. coli……………………...41
3.3.3 Preparation of the recombinant bacterial biosorbents…………………….42
3.3.4 Measurements of heavy metals…………………………………………...42
3.3.5 Cell immobilization with polyvinyl alcohol (PVA)………………………43
3.3.6 Total protein concentration analysis………………………………………44
3.3.7 SDS-PAGE analysis………………………………………………………44
3.3.8 Scanning electron microscope (SEM) analysis…………………………...45
3.3.9 Calibration of peristaltic pump……………………………………………46
3.4 Construction of MT-bearing recombinant E. coli strains………………………...46
3.4.1 Molecular cloning of MTs of human, mouse, and Tilapia fish…………...46
3.4.2 Production of cytosolic and periplasmic MBP-MT fusion proteins in E. coli………………………………………………………………………..50
3.5 Free cells biosorption by MT-bearing recombinant E. coli strains………………51
3.5.1 Time-course of biosorption……………………………………………….51
3.5.2 Determination of adsorption isotherms…………………………………...51
3.5.3 Selection of desorption agents…………………………………………….52
3.6 PVA-immobilized cells biosorption by MT-bearing recombinant E. coli strains...53
3.6.1 Biosorption at different pH………………………………………………..53
3.6.2 Biosorption at different temperature……………………………………...53
3.6.3 Determination of adsorption isotherms…………………………………...54
3.6.4 Time-course profile of biosorption………………………………………..54
3.6.5 Biosorption using PVA-immobilized cells with different biomass loading55
3.6.6 Desorption of Cd-loaded immobilized biosorbents……………………….56
3.6.7 Repeated adsorption/desorption operations in shake flasks………………56
3.7 Fixed-bed column biosorption by MT-bearing recombinant E. coli strains……...57
3.7.1 Fixed-bed column biosorption experiments………………………………57
3.7.2 Adsorption/desorption in serial fixed-bed columns……………………….58
3.7.3 Quantitative analysis of fixed-bed column adsorption……………………60
3.7.4 Bohart-Adams model……………………………………………………..62
3.7.5 Thomas model………………………………………………………….…63
Chapter 4 Construction of MT-bearing recombinant E. coli strains………………….64
4.1 Introduction………………………………………………………………………64
4.2 Construction of expression vectors encoding MBP-MT fusion proteins………...64
4.3 Expression of cytosolic and periplasmic MBP-MT fusion proteins……………..67
4.4 Confirmation of human MT expression from X-ray results……………………...68
Chapter 5 Free cells biosorption by MT-bearing recombinant E. coli strains………..72
5.1 Introduction………………………………………………………………………72
5.2 Biosorption capacities of the biosorbents at an identical metal concentration…...73
5.3 Time-course biosorption profile and biosorption rate……………………………76
5.4 Biosorption isotherms…………………………………………………………….81
5.5 Effects of expression and localization of MT proteins on heavy metal biosorption………………………………………………………………………82
5.6 Effect of the origin of MT proteins on biosorption………………………………89
5.7 Effect of the type of metal adsorbates on biosorption……………………………91
Chapter 6 PVA-immobilized cells biosorption by MT-bearing recombinant E. coli strains………………………………………………………………………..93
6.1 Introduction………………………………………………………………………93
6.2 Effect of pH on Cd2+ uptake by PVA-immobilized cells………………………...94
6.3 Effect of temperature on Cd2+ uptake by PVA-immobilized cells……………….97
6.4 Kinetics of biosorption…………………………………………………………...99
6.5 Percentage Cd2+ removal under different initial concentrations………………..102
6.6 Biosorption equilibrium………………………………………………………...104
6.7 Effect of biomass loading on Cd2+ biosorption…………………………………107
6.8 Selection of desorption agents and repeated adsorption/desorption cycles…….109
Chapter 7 Fixed-bed column biosorption by MT-bearing recombinant E. coli strains……………………………………………………………………...114
7.1 Introduction……………………………………………………………………..114
7.2 Effect of bed depth on Cd2+ uptake by PVA-immobilized cell…………………114
7.3 Effect of flow rate on Cd2+ uptake by PVA-immobilized cell…………………..119
7.4 Effect of initial inlet concentrationon Cd2+ uptake by PVA-immobilized cell….123
7.5 Effect of loading rate on Cd2+ uptake by PVA-immobilized cell……………….130
7.6 Model analysis of column biosorption behavior using Bohart-Adams equation.133
7.7 Analysis with Thomas model…………………………………………………...136
7.8 Adsorption/desorption in serial fixed-bed columns…………………………….141
Chapter 8 Conclusions and future work………………………………………………144
8.1 Conclusions……………………………………………………………………..144
8.2 Future work……………………………………………………………………..146
References………………………………………………………………………………..148
Curriculum vitae………………………………………………………………………….165
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