利用乳酸菌表達大腸桿菌之酸性磷酸

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曾季清(Chih-Ching Tseng) 查詢紙本館藏

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生命科學系

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利用乳酸菌表達大腸桿菌之酸性磷酸

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摘要(中)

本研究首先利用聚合?鏈鎖反應（PCR）的方式成功的篩選出大腸桿菌（Escherichia coli B strain CCRC 12567）之酸性磷酸?基因，其長度為1237 bp；接著將該基因送入大腸桿菌之蛋白質表現系統（pET system），並證明所篩選出之基因的確能轉譯成具有能夠分解抗營養因子植酸之酵素。
然後將酸性磷酸?基因與穿梭載體pGIT032重新構築後，以電穿孔（electroporation）將此重組載體送入Lactobacillus plantarum CCRC 10069、Lactobacillus casei subsp. rhamnosus GG、Lactobacillus casei Shirota等三種不同乳酸菌。結果顯示轉殖後之菌種胞內酵素活性表現以Lactobacillus casei subsp. rhamnosus GG轉殖株之植酸?表現最高，達未轉殖單一宿主菌株之62.4倍，而Lactobacillus casei Shirota之轉殖株之植酸?表現亦分別為2倍，但Lactobacillus plantarum的活性幾乎與對照菌株相同。
最後將此三種轉殖菌株進行模擬體內消化道環境酸度、膽鹽耐受性試驗。在酸度耐受性試驗方面，三種轉殖株經pH 2.0之Rogosa and Sharpe（MRS）處理、培養後均不生長；但經pH 3.0處理後，任一轉殖菌均發現當酸度處理時間愈長，其菌液於培養皿內培養後之生成菌落數愈少。此外，三種菌轉殖株均不具0.3和0.5％Oxygall膽鹽之耐受性。

摘要(英)

The acid phosphatase gene of Escherichia coli B strain was cloned using polymerase chain reaction (PCR) technique. Subsequently, the gene was transferred into pET expression system using BL21 cells as host. The recombinant Escherichia coli successfully expressed fused protein affirmatively carrying phytase activity.
The same gene was set to transform the Lactobacillus using shuttle vector pGIT032. After the recombinant vector was constructed, it was transferred to Lactobacillus plantarum, Lactobacillus casei subsp. rhamnosus GG and Lactobacillus casei Shirita using electroporation technique. The results showed the intracellular phytase activity of the transformants of Lactobacillus casei subsp. rhamnosus GG increased 62.4 times and Lactobacillus casei Shirita increased 2 times while Lactobacillus plantarum remained unchanged.
All three different transformed Lactobacillus were subjected to simulation tests for gastrointestinal tract in animals including acid tolerance and bile-salt tolerance. In pH 2.0, all three showed no tolerance. In pH 3.0, the tolerance decreased as the incubation time increased. Furthermore, no tolerance was observed in the treatments by bile salt of 0.3 and 0.5% of Oxygall.

關鍵字(中)

★ 大腸桿菌
★ 酸性磷酸
★ 電穿孔
★ 植酸酵素
★ 乳酸菌

關鍵字(英)

★ Escherichia coli
★ acid phosphatase
★ electroporation
★ phytase
★ Lactobacillus

論文目次

中文摘要…………………………………………………………...I
英文摘要…………………………………………………………...III
目錄…………………………………………………………...IV
圖目錄…………………………………………………………...VI
表目錄…………………………………………………………...VIII
第一章前言……………………………………………………...1
1.1研究緣起………………………………………………...1
1.2研究目的與內容………………………………………...3
第二章文獻回顧………………………………………………...4
2.1基因工程在乳酸菌之應用……………………………...4
2.2植酸與植酸分解?……………………………………...7
2.3大腸桿菌植酸分解?…………………………………...12
第三章實驗材料與方法………………………………………...15
3.1研究材料與儀器設備…………………………………...17
3.1.1菌種與宿主細胞………………………………………...17
3.1.2引子(primer)……………………………………………..17
3.1.3載體(vecter)……………………………………………...21
3.1.4蛋白質表現系統（expression system）………………..23
3.1.5培養基…………………………………………………...23
3.1.6化學藥品與耗材………………………………………...24
3.1.7儀器設備………………………………………………...24
3.2研究方法………………………………………………...26
3.2.1酵素基因之篩選與活性確認…………………………...26
3.2.2乳酸菌的蛋白質表現…………………………………...38
3.2.3模擬動物體內環境耐受試驗…………………………...40
第四章結果與討論……………………………………………...43
4.1植酸分解酵素酸性磷酸?基因之篩選與活性之確認...43
4.2乳酸菌的轉型作用與酵素活性表現測定……………...53
4.3模擬動物體內環境耐受試驗…………………………...60
第五章結論與建議……………………………………………...63
5.1結論……………………………………………………...63
5.2建議……………………………………………………...65
參考文獻……………………………………….…………………..66

參考文獻

參考文獻
田蔚城 (1999) 生物技術的發展與應用，九州圖書文物有限公司， p.49。
陳勁初、蔡士昌、黃進發、許文輝 (1990) ?酸生產菌電轉型條件之探討，經濟部研究報告第623號。
廖啟成 (1998) 乳酸菌之分類與應用，食品工業月刊，30(2)，p.1-10。
高野俊明 (1998) 乳酸菌保健營養效果之研究動向，生物產業，9(2)，p.118-122。
佐佐木隆(1998) 乳酸菌生物技術之進展，生物產業，9(4)，p.227-233。
黃遵錫，章克昌 (1998) 植酸酵素基理與應用概況， Food and Fermentation Industries，25(2)，p.54-58.
Adrianus, J. Engelen, F. C., van der Heeft, P., Randorp, H.G., and Ed L.C. Smit. (1993) Simple and Rapid Determination of Phytase Activity. Journal of Association of Offical Analytical Chemists International, 77(3), p.760-764.
Anderson, R. J.（1914）A contribution to the chemistry of phytin. Journal of Biological Chemistry , 17, p171-190.
Caldwell, R. A. (1992) Effect of calcium and phytic acid on the activation of trypsinogen and the stability of trypsin. Journal of Agricultural Food Chemistry, 40, p.799-800
Cawley, R. W. and Mitchell, T. A. (1968) Inhibition of wheat alpha-amylase by bran phytic acid. Journal of the Science of Food and Agriculture, 19, p.106.
Cho, J. S., Choi, Y. J., and Chung, D. K. (2000) Expression of Clostridium thermocellum Endoglucanase gene in Lactobacillus gasseri and Lactobacillus johnsonii and characterization of the genetically modified probiotic Lactobacilli. Current Microbiology, 40, p.257-263.
Dassa, E., and Boquet, P. L. (1985) Identification of the gene appA for the acid phosphatase ( pH optimum 2.5) of Escherichia coli. Molecular & General Genetics：MGG, 200, p.68-73.
Dassa, J., Marck, C., and Boquet, P. L. (1990) The complete nucleotide sequence of the Escherichia coli gene appA rerveals significant homology between pH 2.5 acid phosphatase and glucose-1-phosphatase. Journal of Bacteriology, 172, p.5497-5500.
de Rham, O. and Jost, T. (1979) Phytate-protein interactions in soybean extracts and low-phytate soy protein products. Journal of Food Science, 44, p.596-601.
Despande, S. S. and Cheryan, M. (1984) Effects of phytic acid, divalent cations, and their interactions on alpha-amylase activity. Journal of Food Science, 49, p.43-46.
Erdman, J. W. Jr. (1972) Oilseed phytates: nutritional implications. Journal of American Oil Chemists' Socity, 56, p.763-741.
Ertl, D. S. A, Young, K. A. and Robert V. (1998) Plant genetic approaches to phosphoroue management in agricultural production. Journal of Enviromental Quality, 27, p.299-304.
Gerard, A., Walsh, Ronan, F. P., and Denis, R. H. (1994) Enzymes in the animal-feed industry. Trends in Food Science & Technology, 5, p.81-87.
Gilson, D. M., and Ullah, A. H. J. (1988) Purification and characterization of phytase from cotyledons of germinating soybean seed. Archives of Biochemistry and Biophysics, 260, p.503-513.
Greene, D. A., De Jesus, P. V., and Winegrad, A. I. (1975) Effects of insulin and dietary myo-inositol on impaired peripheral motor nerve conduction velocity in acute streptozotocin diabetes.The Journal of Clinical Investigation, 55, p.1326-1336.
Greiner, R., Konietzny, U., and Jany, K. D. (1993) Purification and characterization of two phytases from Escherichia coli. Archives of Biochemistry and Biophysics, 303, p.107-113.
Howson, S. J., and Davis, R. P. (1983) Production of phytate-hydrolyzing enzyme by some fungi. Enzyme and Microbial Technology, 5, p.377-382.
Janne, K., Mardo, L., Paivi, N., Nisse, K., and Juha, A. (1998) Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis. Applied and Enveronmental Microbiology , 64(6), p.2079-2085.
Komegay, E. T., Denbow, D. M., YI, Z., and Ravindran, V. (1996) Response of broilers to graded levels of Natuphos phytase added to corn-soybean meal based diets containing three levels of nonphytate phosphorus. British Journal of Nutrition, 75, p.839-852.
Lim, D., Golovan, S., Forsberg, C. W., and Jia, Z. (2000) Crystal structures of Escherichia coli phytase and its complex with phytate. Nature structural biology, 7(2), p.108-113.
Liu, B., Rafiq, A., Tzeng, Y. M., and Rob, A. (1998) The induction and characterization of phytase and beyond. Enzyme and Microbial Technology, 22, p.415-424.
Mayo, B., Gonzalez, B., Arca, P., and Suarez, E. (1994) Cloning and expression of the plasmid encoded b-galactosidase gene from a lactobacillus plantarum strain of dairy origin. FEMS Microbiology Letters, 122, p.145-152.
McIntyre, D. A., and Harlande, S. K. (1989) Improved electroporation efficiency of intact lactococcus lactis subsp. Lactis cells grown in defined media. Applied and Enveronmental Microbiology, 55(10), 2621-2626.
Mroz, Z., Jongbloed, A. W., and Kemme, P. A. (1994). Apparent digestibility and retention of nutrients bound to phytate complexes as influenced by microbial phytase and feeding regimen in pigs. Journal of Animal Science, 72, p.126-132.
Powar, V. K. and Jagannathan, V. (1982) Purification and properties of phytase-specific phosphatase from Bacillus subtilis. Journal of Bacterial, 151, 1102-1108.
QIAGEN Product Guide (2000) p.59
Sanoja, R. R., Morlon-Guyot, J., and Guyot, J. P. (1999) Electrotransformation of lactobacillus manihotivorans LMG 18010 and LMG 18011. Journal of Applied Microbiology. 87, p.99-107.
Sebastian, S., Touchburn, S. P., and Chavez, E. R. (1998) Implications of phytic acid and supplemental microbial phytase inpoultry nutrition: a review. World's Poultry Science Association, 54, p.27-47.
Sebastian, S., Touchburn, S. P., and Chavez, E. R. (1997) Apparent Digestibility of protein and amino acids in broiler chickens fed a corn-soybean diet supplemented with microbial phytase. Poultry Science , 76, p.1760-1769.
Segueilha, L., Lambrechts, C., Boze, H., Moulin, G., and Galzy, P. (1992) Purification and properties of the phytase from Schwanniomyces castellii. Journal of Fermental Bioengineering, 74, p.7-11.
Singh, M. and Krikorain, A.D. (1982) Inhibition of trypsin activity in vitro by phytate. Journal of Agricultural Food Chemistry, 30, p.799-800.

指導教授

董啟功(Chi-Gong Tong)

審核日期

2000-7-18

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