臺灣博碩士論文加值系統

高果糖玉米糖漿的成分為55%的果糖和45%的葡萄糖，其甜度是蔗糖的1.3倍，葡萄糖的1.7倍，製造成本較低廉，且原料容易取得，因此成為現今食品工業最廣為使用的甜味添加劑。在製造高果糖玉米糖漿的過程中，其中的一個步驟是依靠葡萄糖/ 木糖異構酵素將葡萄糖轉變成為果糖，若能增加酵素耐酸耐熱的活性或是提高酵素的產量，便可以降低工業生產的成本。本論文目的是要利用強啟動子(T7啟動子)於大腸桿菌中表現嗜酸耐熱菌Alicyclobacillus acidocaldarius葡萄糖異構酵素的基因xylA，期望能產生更高量的酵素蛋白質。為了提高蛋白質正確褶疊的機率，在選殖質體pWL28時，將一段pelB訊號序列加在xylA基因的上游，表現出融合蛋白質，但結果顯示因大量製造出蛋白質，產生了包涵體。降低誘導溫度及降低誘導的IPTG濃度都不能明顯提高菌株的酵素活性，但將菌株改以Ｍ9 minimum培養基於攝氏37度培養並誘導後，葡萄糖異構酵素活性有明顯的提高。

High fructose corn syrup( HFCS ) is mixture of 55% fructose and 45% glucose. It is sweeter than sucrose by 1.3 fold and glucose by 1.7 fold. The production cost is lower than common sugars, and the raw material is easy to obtain. So it becomes the most widely used sweetener. The HFCS production from starch includes the isomerization of glucose to fructose, by glucose isomerase/ xylose isomerase. Using thermostable and acid tolerant glucose isomerase in HFCS production and improving the yield of glucose isomerase can lower the cost. We studied the expression of glucose isomerase gene, xylA, from the thermoacidophile, Alicyclobacillus acidocaldarius 49-1 isolated from the hot spring of Yangming Mountain. We cloned the xylA gene into expression vector pET-25b. The coding sequence of xylA was fused with a signal sequence, pelB and expressed under control of T7 promoter. The result appeared that the fusion protein formed the inclusion body. By decreasing the growth, it did not express active protein, but when growing in M9 minimum medium, active GI protein was produced apparently.

第一章 前言 1
第一節 葡萄糖/木糖異構酵素用於製糖的發展 1
第二節 葡萄糖/木糖異構酵素 2
第三節 高果糖玉米糖漿的優點與生產過程 3
第四節 高果糖玉米糖漿工業生產的改善 5
第五節 實驗目的與架構 6
第二章 材料與方法 10
第一節 材料 10
一、菌株 10
二、質體 10
三、藥品與器材 10
四、培養基配方 11
五、試劑配方 14
第二節 方法 22
一、大腸桿菌質體DNA之製備 22
二、聚合酵素鏈反應 24
三、DNA回收 24
四、聚合酵素鏈反應產物之選殖 25
五、限制酵素切割反應 26
六、黏接反應 26
七、勝任細胞的製備 26
八、大腸桿菌之轉型 27
九、洋菜醣膠體電泳 27
十、菌株的誘導 27
十一、菌體的酵素活性分析 28
十二、菌體乾重的測量 29
十三、菌體萃取物的製備 29
十四、蛋白質濃度的測定 30
十五、周質空間蛋白質萃取 31
十六、蛋白質SDS-聚丙烯醯胺膠體電泳 31
十七、包涵體 ( inclusion body ) 的萃取 32
十八、包涵體蛋白質之復性 34
十九、相位差顯微鏡觀察菌體 35
第三章 結果 36
第一節 pWL102 質體的建構 36
第二節 pWL102- HMS174中GI蛋白質的表現 38
第三節 pWL28 質體的建構 39
第四節 pWL28- HMS174中GI蛋白質的表現 40
第五節 49-1菌株與其他菌株GI活性的比較 44
第六節 包涵體復性實驗 44
第四章 討論 46
第一節 pET25b(+) /HMS174表現系統的選用策略 46
第二節 pWL102/HMS174菌株表現葡萄糖異構酵素 47
第三節 pWL28 / HMS174菌株表現葡萄糖異構酵素 48
第四節 49-1菌株與其它菌株GI活性的比較 51
第五節 包涵體的復性 51
第六節 未來研究的方向 53
參考文獻 54
表 58
圖 60
附錄 81

林聖展。2003。高溫嗜酸菌葡萄糖異構酵素基因選殖─於大腸桿菌及枯草桿菌之表現。東吳大學微生物學系碩士論文。

柯舜文。1996。分離生產葡萄糖異構酵素之高溫嗜酸菌及酵素純化與定性。東吳大學微生物學系碩士論文。

Bandlish, R. K., J. M. Hess, K. L. Epting, C. Vieille, and R. M. Kelly. 2002. Glucose-to-fructose conversion at high temperatures with Xylose ( Glucose ) isomerase from Streptomyces murinus and two hyperthermophilic Thermotoga species. Biotechnol. Bioeng. 80 (2): 185-194.

Bhosale, S. H., M. B. Rao, and V. V. Deshpande. 1996. Molecular and industrial aspect of Glucose Isomerase. Microbiol. Rev. 60 (2): 280-300.

Chang, Changsoo, Park Byung Chul, Lee Dae-Sil and Suh Se Won. 1999. Crystal structures of thermostable xylose isomerase from Thermus caldophilus and Thermus thermophillus: possible structural determinants of thermostability. J. Mol. Biol. 288:623-634.
Choi, J. H., Lee, S. Y. 2004. Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol. 64: 625-635.

Grossman, T. H., Kawasaki, E. S., Punreddy, S. R. and Osburne, M. S. 1998. Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability. Gene. 209:95-103.

Hu, H., H. Liu, and Y. Shi. 1997. The reaction pathway of the isomerization of D-xylose catalyzed by the enzyme D-xylose isomerase: a theoretical study. Proteins. 27: 545-555.

Joo, Gil-Jae, Shin Jae-Ho, Heo Gun-Young, Kim Toung-Mog, Rhee In-Koo. 2005. Molecular cloning and expression of a thermostable xylose (glucose) isomerase gene, xylA, from Streptomyces chibaensis J-59. J. Microbiology. 43:34-37.

Kane, James F. 1995. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr. Opin. Biotechnol. 6:494-500.

Kobayashi, Michihiko,and Sakayu Shimizu. 1999. Cobalt proteins. Eur. J. Biochem. 261:1-9.

Lee, Hak Jin, and Jin Hyung Jong. 2003. Renaturation of recombinant ErmSF and its refolding behavior. Mol. Cells. 16:187-193.

Lehmacher, A., and H. Bisswanger. 1990. Isolation and characterization of an extremely thermostable D- xylose isomerase from Thermun aquaticus HB8. J. Gen. Microbiol. 136:679- 686.

Marshall, P. O. and E. R. Kooi. 1957. Enzymatic conversion of D-glucose to D-fructose . Science. 125: 648-649.

Raykovska, V., P. Dolashka-Angelova, D. Paskaleva, S. Stoeva, J. Abashev, L. Kirkov, and W. Voelter. 2001. Isolation and characterization of a xylose-glucose isomerase from a new strain Streptomyces thermovulgaris 127, var. 7-86. Biochem. Cell Biol. 79: 195-205.



Vallejo, Luis Felipe, and Ursula Rinas. 2004. Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microbial Cell Factories. 3:11.

Wuxiang, L., and K. Jeyaseelan. 1993. High level expression of a thermostable Bacillus xylose (glucose) isomerase in Escherichia coli. Biotechnol. Lett. 15:1101-1106.