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研究生:張所宜
研究生(外文):So-Yi Chang
論文名稱:Thermoanaerobacteriumsaccharolyticum之β-xylosidase基因選殖與表現及重組蛋白之生化特性分析
論文名稱(外文):Molecular cloning and expression of Thermoanaerobacterium saccharolyticumβ-xylosidase and Biochemical properties of recombinant β-xylosidase
指導教授:鍾雲琴
指導教授(外文):Yun-Chin Chung
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2008/07/
畢業學年度:96
語文別:中文
論文頁數:144
中文關鍵詞:畢赤酵母寡木糖水解酶
外文關鍵詞:β-xylosidasepichia pastoris
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  β-xylosidase不但能水解寡木糖亦可利用其保留型轉糖苷活性進行寡糖合成。本研究目的為選殖Thermoanaerobacterium saccharolyticum之β-xylosidase基因,使其在Pichia pastoris中大量表現,並探討重組酵素的生化特性。以colony polymerase chain reaction (colony PCR)大量複製T. saccharolyticum β-xylosidase之DNA,將其與表現載體(pGAPZαA、pPICZαA)形成重組DNA後再轉型於不同表現宿主細胞內(x-33、SMD1168H)。經基因定序後得知此研究所得之β-xylosidase DNA含1500 bp,雖然β-xylosidase DNA序列為His 455、Ala 456、Tyr230及Arg 380與Lee等人所發表之Gln 455、Pro 456、Thr 230不同,然β-xylosidase的活性中心在His228及Glu323上,因此推測不會對活性造成太大影響。最後篩選所得之轉殖株中,由於x-33作用表現宿主時,只得以pGAPZαA的表現質體中的轉殖株。為比較兩種重組質體(xynB-pGAPZαA、xynB-pPICZαA)之β-xylosidase在表現後其活性等是否有差異,以及考量SMD1168H不會分泌protease,可能較不易對目標蛋白質產生蛋白質水解的情況出現,因此之後的誘導表現便只針對SMD1168H分別所含之兩組重組質體(xynB-pGAPZαA、xynB-pPICZαA)進行探討。以SMD1168H為表現宿主時,可將重組於pGAPZαA之β-xylosidase表現出胞外酵素,其比活性較pPICZαA接上β-xylosidase所產生之胞內酵素高。胞外β-xylosidase分子量為61 kDa,比活性為530.8U/mg,pI值約為6.28;最適反應pH值與溫度分別為5.0與60oC,於pH5.0~8.0反應1小時其殘留活性約還有50~60%,而在pH為酸性時較頗為穩定,pH > 8時其酵素活性急速下降;在50~70oC反應半小時其活性仍有80%以上。β-xylosidase分解基質ρ-Nitrophenyl-β-D- xylopyranoside之Km為0.115mM,Kcat值為1.038 mM/min,Kcat/Km為9.03/min,Vmax為0.077mM/min/mg,活化能為11.84 Kcal /mM。本研究所得之胞外重組β-xylosidase對於基質ρ-Nitrophenyl-β-D- xylopyranoside的親和力及催化速率較差,比活性則較Lee等學者發表之重組酵素高出144倍,然是否具潛力發展為木糖苷合成酶則須作進一步的確認。
The β-xylosidase can not only hydrolyze xylooligosaccharides but also synthesize oligosaccharides with its retaining mechanism of transglycosylatic activity. The aims of this study were to clone β-xylosidase from Thermoanaerobacterium saccharolyticum and then express in Pichia patoris, and the biochemical properties of recombinant β-xylosidase were evaluated as well. Recombinant β-xylosidase gene was constructed by ligating β-xylosidase DNA obtained from colony polymerase chain reaction with either pGAPZαA or pPICZαA. Expression hosts, including P. patoris x-33 and P. patoris SMD1168H, were used to express the recombinant β-xylosidase. DNA sequencing data showed that the cloned β-xylosidase contained 1500 bp, and had the homology of 99% with the β-xylosidase gene reported by Lee et al. Four amino acids were difference between the previously reported β-xylosidase and the present cloned β-xylosidase. Fortunately, these four amino acids did not locate at the catalytic site of the enzyme (His228 and Glu323). When x-33 was used as the expression host, only the recombinant β-xylosidase -pGAPZαA was transformed successfully. To compare the expression levels for two recombinant expression vectors, xynB-pGAPZαA and xynB-pPICZαA, and since SMD1168H is free of protease and this makes it can not proteolysis expressed recombinant protein, The SMD1168H transforms with recombiniant expression vector, xynB-pGAPZαA and xynB-pPICZαA, were chosen for further study. An extracellular β-xylosidase was produced by xynB-pGAPZαA with higher specific activity than an intracellular β-xylosidase produced by xynB-pPICZαA. Molecular weight of the extracellular β-xylosidase was approximately 61 kDa, and the specific activity and pI were 530.8 U/mg and 6.28, respectively. Optimal reaction pH and temperature for the recombinant extracellular β-xylosidase were 5.0 and 60oC, repectively. The recombinant extracellular β-xylosidase was stable at pH range of 5.0-8.0 and maintained 50~60% residual activity after incubating at 30oC for 1hr. Acid condition did not affect the enzyme activity of the recombinant extracellular β-xylosidase, and the enzyme activity decreased dramatically at pH higher than 8. For thermal stability of the enzyme activity, about 80% of the original activity was left even at 50~70oC for 30 min. When ρ-Nitrophenyl-β-D- xylopyranoside was served as a substrate, the kinetic parameters Km, Kcat, Kcat/Km Vmax, and activiation energy of the recombinant extracellular β-xylosidase were 0.115mM, 1.038 mM/min, 9.03/min, 0.077mM/min/mg, and 11.84 Kcal/mM respectively. Low substrate affinity and catalytic velocity were observed in the recombinant extracellular β-xylosidase. Even the specific activity of recombinant β-xylosidase was 144 times higher than that of genetic recombinant β-xylosidase reported by Lee et al, further study is needed to confirm the potential of β-xylosidase as a xylosynthase via site-directed mutation of recombinant β-xylosidase.
目錄..............................................................................................................................I
圖目錄……………………………………………………………………………...VI
表目錄……………………………………………………………………………...IX
摘要…………………………………………………………………………………X
Abstract …………………………………………………………………………....XII
第一章 前言…………………………………………………………………………1
第二章 文獻回顧…………………………………………………………………....3
一、 糖苷水解酶…………………………………………………………………3
1. 簡介……………………………………………………………………......3
二、糖苷合成酶…………………………………………………………………5
1. 催化機制………………………………………………………………......5
2. 應用………………………………………………………………………10
三、β-xylosidase…………………………………………………………………12
1. 水解特性…………………………………………………………………14
2. 轉糖苷作用及應用例子............................................................................16
四、Thermoanarobacterium saccharolyticum β-xylosidases……………………17
1. 來源 ……………………………………...………………...…………..17
2. 催化機制....................................................................................................18
五、木糖(Xylose)...........................................................................................20
1. 簡介............................................................................................................20
2. 生理功能....................................................................................................21
3. 加工應用性................................................................................................22
六、木寡糖(xylooligosaccharides)之簡介....................................................23
1. 簡介............................................................................................................23
2. 生理活性....................................................................................................24
七、Pichia Pastoris..............................................................................................25
1. 簡介............................................................................................................25
2. 於基因工程上之應用................................................................................26
第三章 材料與方法 ……………………………………………………………….30
第一節 實驗流程..............................................................................................30
第二節 實驗材料..............................................................................................31
一、菌種與載體..................................................................................................31
二、化學藥品.......................................................................................................31
三、使用儀器........................................................................................................33
第三節 實驗方法..............................................................................................36
一、菌種活化與保存 ..........................................................................................36
二、Thermoanaerobacterium saccharolyticumi菌株之β-xylosidase基因選殖 ……………………………………………………………….............................37
1. β-xylosidase基因primers之設計..............................................................37
2. 聚合酶連鎖反應 (Polymerase Chain Reaction;PCR)............................37
3. 接合反應 (Ligation)..................................................................................38
4. 製備通透性細胞 (Competent cell)...........................................................38
5. 轉殖 (Transformation)..............................................................................38
6. 製備小量質體............................................................................................38
7. 建立限制酶圖譜 (Mapping).....................................................................39
8. 鑑定DNA序列 (DNA Sequencing)........................................................39
三、β-xylosidase基因之表現與純化.................................................................39
1. 製備大量質體............................................................................................39
2. 限制酶剪切與膠上純化............................................................................39
3. 接合作用 (Ligation)..................................................................................40
4. 轉殖 (Transformation)...............................................................................40
5. 製備小量質體............................................................................................40
6. 建立限制酶圖譜 (Mapping).....................................................................41
7. 鑑定DNA基因序列 (DNA Sequencing).................................................41
8. 製備通透性細胞 (Competent cell)...........................................................41
9. 電穿孔 (Electroporation)...........................................................................42
四、重組菌株鑑定...............................................................................................42
1. RT-PCR鑑定法...........................................................................................42
2. Colony PCR鑑定法....................................................................................44
五、重組β-xylosidase之誘導與大量表現 ........................................................45
六、重組蛋白質鑑定 ..........................................................................................46
七、β-xylosidase之純化.....................................................................................49
八、重組β-xylosidase之生化特性探討............................................................50
1. β-xylosidase活性測定................................................................................50
2. 蛋白質定量................................................................................................52
3. β-xylosidase分子量測定............................................................................53
4. 等電點測定................................................................................................56
5. 最適反應之pH值與穩定性.....................................................................57
6. 最適之反應溫度與穩定性........................................................................58
7. 酵素動力參數............................................................................................59
8. 基質特異性................................................................................................60
第四章 結果與討論............................................................................................….61
一. Thermoanaerobacterium saccharolyticum 之β-xylosidase基因選殖 ........61
1. β-xylosidase基因之選殖............................................................................61
2. β-xylosidase基因之鑑定............................................................................61
二. β-xylosidase基因之表現與純化...................................................................62
1. β-xylosidase基因之表現............................................................................62
2. β-xylosidase基因之誘導與純化................................................................64
三、SMD1168H-pGAPZαA β-xylosidase之生化特性......................................68
1. 分子量 ........................................................................................................68
2. pI值.............................................................................................................68
3. 最適反應之pH值.....................................................................................69
4. pH值穩定性...............................................................................................69
5. 最適之反應溫度........................................................................................70
6. 溫度穩定性................................................................................................70
7. 基質飽和曲線............................................................................................71
8. 反應活化能................................................................................................72
9. 基質特異性................................................................................................72
第五章 結論..............................................................................................................74
第六章 參考文獻....................................................................................................115
Appendix…………………………………………………………………………..126
胡蘿蔔汁對SD大鼠之腸道功能評估...................................................................126
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