臺灣博碩士論文加值系統

植酸酶將飼料中之植酸水解，除了降低植酸之抗營養性外，亦可提供植酸水解後所釋放出之無機磷酸鹽供禽畜攝取，是一種極具經濟價值的單胃型動物飼料添加劑。本研究探討以重組酵母菌Pichia pastoris KM 71- 61表現Escherichia coli植酸酶之生長與生產條件，及以此方法生產的植酸酶之生化特性。在三角瓶規模中，待菌體生長至穩定期後每24小時添加0.5 %甲醇誘導植酸酶合成，可以使酵素活性在誘導144小時後達150 U/ml。在誘導前以新鮮培養基置換培養液，可使酵素活性提高大約1倍。在五公升醱酵槽規模時，更可在誘導168小時後得到600 U/ml的高酵素活性。探討置換培養基時可提升植酸酶活性的因素，並非由於提供額外之養分所致，而可能是移除舊有培養基中之抑制因子之故。以單位體積不同初始細胞密度做誘導，發現密度愈低，單位細胞的酵素產量愈高。而誘導前給予一段時間的飢餓，並無法提高菌體對於甲醇之利用能力，因此酵素活性沒有明顯提高。

Phytase, a valued feed additive for monogastric animals, has been used to hydrolyze phytic acid in feeds and to reduce the antinutrient ability of phytic acid. It also helps the release of phosphorous from phytic acid to increase phosphorous digestibility. In this study, the production of Escherichia coli phytase in recombinant Pichia pastoris KM 71-61 was investigated and the biochemical characteristics of said phytase were presented. By adding 0.5% methanol every 24 h during the stationary phase in the flask cultures, the phytase activity reached up to 150 U/ml after 144 h of induction. The phytase activity increased one fold in the case of replacing culture with fresh medium before induction. With the same strategy, the phytase activity is about 600 U/ml in fermentor cultures after 168 h of induction. The increase of phytase activity is not resulted from adding extra nutrients but from removing inhibitors. The phytase activity on cellular base was higher when initiated with lower cell density. The starvation before induction has little effect on the phytase activity due to the slow utilization of methanol in Pichia pastoris KM 71- 61.

第一章 前 言 1
一、畜產業與環境污染 1
二、植酸(Phytic acid) 2
三、植酸酶(Phytase) 7
四、重組酵母菌Pichia pastoris概述 11
五、重組酵母菌之高細胞密度培養 14
六、饋料批次培養之控制原理 15
七、研究動機與目的 16
第二章 材料與方法 18
一、菌 株 18
1.1 宿主 18
1.2 質體構築 18
1.3 菌種保存 18
二、三角瓶培養及誘導表現 22
2.1 活化及種菌培養 22
2.2 Baffled三角瓶之生長培養 22
2.3 Baffled三角瓶之生產培養 24
三、醱酵槽之培養及誘導表現方法 25
3.1 醱酵槽之構造簡介 25
3.2 醱酵槽之操作程序 25
3.3 醱酵槽之批次培養 27
3.4 饋料批式醱酵之高細胞密度培養 28
3.5 高細胞密度培養之誘導表現 30
四、分析方法 31
4.1 菌體生長量之測定(OD600、生菌數、乾重) 31
4.2 甘油濃度之測定 32
4.3 植酸酶之部分純化 34
4.4 植酸酶活性分析 35
4.5 蛋白質濃度的定量 37
4.6 蛋白質電泳分析 38
五、酵素生化性質測試 42
5.1 最適反應pH之測試 42
5.2 最適反應溫度之測試 44
5.3 酵素熱穩定性測試 44
5.4 植酸酶活性染色 45
5.5 去醣基化(Deglycosylation) 46
第三章 結果與討論 47
一、分析方法的建立 47
1.1 菌體濃度(OD600)之測定條件 47
1.2 酵素活性分析 47
1.3 最適酵素反應時間 47
二、三角瓶規模之生長 53
2.1 培養基之選擇 53
2.2 討論 53
三、五公升醱酵槽規模之高細胞密度培養 56
3.1 醱酵槽規模之生長 56
3.2 高細胞密度培養模式 56
3.3 饋料液組成份及模式 57
3.4 討論 58
四、植酸酶誘導條件之決定 63
4.1 誘導時機之探討 63
4.2 誘導條件之決定 63
4.3 討論 63
五、五公升醱酵槽規模之植酸酶誘導表現 66
5.1 五公升醱酵槽之植酸酶生產 66
5.2 高細胞密度培養之植酸酶生產 66
5.3 討論 66
六、置換培養基對酵素活性之影響 72
6.1 三角瓶規模之置換培養 72
6.2 醱酵槽規模之置換培養 72
6.3 討論 72
七、飢餓對酵素活性之影響 75
7.1 不同飢餓狀態對酵素活性之影響 75
7.2 討論 75
八、添加濃縮營養液對酵素活性之影響 77
8.1 添加不同成分營養液之影響 77
8.2 討論 77
九、不同起始細胞密度對酵素活性之影響 79
9.1 提高起始細胞濃度與酵素活性之關係 79
9.2 降低起始細胞密度與酵素活性之關係 79
十、植酸酶酵素特性探討 83
10.1 植酸酶分子量 83
10.2 植酸酶最適反應溫度 83
10.3 植酸酶最適反應pH值 83
10.4 植酸酶熱穩定性 83
10.5 討論 90
第四章 結 論 91
未來工作項目 93
參考文獻 94

Berridge M. J. Inositol trisphosphate and calcium signaling. Nature 361：315-325. (1993)
Binita, R., Khetarpau, N. Probiotic fermentation: effect on antinutrients and digestibility of starch and protein of indigenously developed food mixture. Nutr. Health. 11：139-147. (1997)
Botstein, D., Falco, S. C., Stewart, S. E., Brennan, M., Scherer, S., Stinchcomb, D. T., Struhl, K., Davis, R. W. Sterile host yeasts (SHY)：A eukaryotic system of biological containment for recombinant DNA experiments. Gene：17-24. (1979)
Cheryan, M. Phytic acid interactions in food systems. Crit. Rev. Food Sci. Nutr. 13：297-335. (1980)
Clare, J. J., Rayment, F. B., Ballantine, S. P., Sreekrishna, K., Romanos, M. A. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Biotechnology：455-460. (1991)
Couderc, R., Baratti, J. Oxidation of methanol by the yeast Pichia pastoris：Purification and properties of alcohol oxidase. Agric. Biol. Chem. 44：2279-2289. (1980)
Cregg, J. M., Vedick, T. S., Raschke, W. C. Recent advances in the expression of forgein genes in Pichia pastoris. Bio/Technology 11：905-910. (1993).
Danicke, S., Simon, O., Jeroch, H., Bedford, M. Interactions between dietary fat type and xylanase supplementation when rye-based diets are fed to broiler chickens 2. Performance, nutrient digestibility and the fat-soluble vitamin status of livers. Br. Poult. Sci. 38：546-556. (1997)
de Boland, A. R., Garner, G. B., O''Dell, B. L. Identification and properties of "phytate" in cereal grains and oilseed products. J. Agric. Food Chem. 23：1186-1189. (1975)
Ene-Obong, H. N. Content of antinutrients and in vitro protein digestibility of the African yambean, pigeon and cowpea. Plant Foods Hum. Nutr. 48：225-233. (1995)
Fernandez, J. M., Hoeffler, J. P. Gene expression systems. (1999)
Gibson, D. M., and Ullah, A. H. J. Inositol metabolism in plants. (1990)
Graf, E. Formation of [3H, 32P] phytic acid in germinating wheat. Anal. Biochem. 131：351-355. (1983)
Graf, E., Empson, K. L., Eaton, J. W. Phytic acid. A natural antioxidant. J. Biol. Chem. 262：11647-11650. (1987)
Greiner, R., Konietzny, U., Jany, K. D. Purification and characterization of two phytases from Escherichia coli. Arch. Biochem. Biophys. 303：107-113. (1993)
Hitzeman, R. A., Hagie, F. E., Levine, H. L., Goeddel, D. V., Ammerer, G., Hall, B. D. Expression of a human gene for interferon in yeast. Nature. 293：717-722. (1981)
Howson, S. J., Davis, R. P. Production of phytate-hydrolysing enzyme by some fungi. Enzyme Micro. Technol. 5：377-382. (1983)
International Union of Biochemistry, Enzyme nomenclature：Recommendations of the Nomenclature Committee of the International Union of Biochemistry. 242-247. (1979)
Kerovuo, J., Lauraeus, M., Nurminen, P., Kalkkinen, N., Apajalahti, J. Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis. Appl. Environ. Microbiol. 64：2079-2085. (1998)
Kim, T. R., Goto, Y., Hirota, N., Kuwata, K., Denton, H., Wu, S. Y., Sawyer, L. High-level expression of bovine β-lactoglobulin in Pichia pastoris and characterization of its physical properties. Protein Eng. 10：1339-1345. (1997)
Knuckles, B. E., Kuzmicky, D. S., Gumbmann, M. R., and Betschart, A. A. Effect of myo-inositol hexaphosphate esters on in vitro and in vivo digestion of protein. J. Food Sci. 54：1348-1350. (1989)
Lolas, G. M., Palamidis, N., Markakis, P. Thy phytic acid-total phosphorus relation in barley, oats, soybeans and wheat. Cereal Chem. 23：13. (1976)
Lonnerdal, B., Sandberg, A. S., Sandstrom, B., Kunz, C. Inhibitory effects of phytic acid and other inositol phosphates on zinc and calcium absorption in suckling rats. J. Nutr. 119：211-214.(1989)
Mitchell, D. B., Vogel, K., Weimann, B. J., Pasamontes, L., van Loon, A. P. The phytase subfamily of histidine acid phosphatases: isolation of genes for two novel phytases from the fungi Aspergillus terreus and Myceliophthora thermophila. Microbiology. 143：245-252. (1997)
Mosha, T. C., Gaga, H. E., Pace, R. D., Laswai, H. S., Mtebe, K. Effect of blanching on the content of antinutritional factors in selected vegetables. Plant Foods Hum. Nutr. 47：361-367. (1995)
Nahm, K. H., Carlson, C. W. Effects of cellulase from Trichoderma viride on nutrient utilization by broilers. Poult. Sci. 64：1536-1540. (1985)
Nelson, T. S., Ferrara, L. W. Phytate phosphorus content of feed ingredients derived from plants. Poult. Sci. 47：1372-1374. (1968)
Newkirk, R. W., Classen, H. L. In vitro hydrolysis of phytate in canola meal with purified and crude sources of phytase. Anim. feed sci. technol 72：315-327. (1998)
Ogata, K., Nishikawa, H., Ohsugi, M. A yeast capable of utilizing methanol. Agric. Biol. Chem. 33：1519-1520. (1967)
Pasamontes, L., Haiker, M., Wyss, M., Tessier, M., van Loon, A. P. Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus. Appl. Environ. Microbiol. 63：1696-1700. (1997)
Powar, V. K., Jagannathan, V. Purification and properties of phytate- specific phosphatase from Bacillus subtilis. J. Bacteriol. 151：1102-1108. (1982)
Reddy, N. R., Salunkhe, D. K., Sathe, S. K. Biochemistry of black gram (Phaseolus mungo L.): a review. Crit. Rev. Food Sci. Nutr. 16：49-114. (1982)
Reddy, N. R., Sathe, S. K., Salunkhe, D. K. Phytates in legumes and cereals. Adv. Food Res. 28：1-92. (1982)
Sabin, R. U. S. Patent No. US4758430：Method of treatment of Alzheimer''s disease using phytic acid. (1988)
Sabin, R. U. S. Patent No. US5112814：Method of treatment of Parkinson''s disease using phytic acid. (1992)
Schingoethe, D. J, Stegeman, G. A, Treacher, R. J. Response of lactating dairy cows to a cellulase and xylanase enzyme mixture applied to forages at the time of feeding. J. Dairy Sci. 82：996-1003. (1999)
Selinger, L. B., Forsberg, C. W., Cheng, K. J. The rumen：A unique source of enzymes for enhancing livestock production. Anaerobe. 2：263-284. (1996)
Shamsuddin, A. M, Vucenik, I. Mammary tumor inhibition by IP6: a review. Anticancer Res. 19：3671-3674. (1999)
Shieh, T. R., Ware, J. H. Survey of microorganism for the production of extracellular phytase. Appl. Microbiol. 16：1348-1351. (1968)
Skowronski, T. Some properties of partially purified phytase from Aspergillus niger. Acta. Microbiol. Pol. 27：41-48. (1978)
Smith, R. A., Duncan, M. J., Moir, D. T. Heterologous protein secretion from yeast. Science 229：1219-1224 (1985).
Stratton, J., Chiruvolu, V., Meagher, M. High cell-density fermentation. Methods Mol. Biol. 103：107-120. (1998)
Tikhomirova, L. P., Ikonomova, R. N., Kuznetsova, E. N., Fodor, I. I., Bystrykh, L. V., Aminova, L. R., Trotsenko, Y. A. Transformation of methylotrophic yeast Hansenula polymorpha：cloning and expression of genes. J. Basic Microbiol. 28：343-351. (1988)
Tuntawiroon, M., Sritongkul, N., Rossander-Hulten, L., Pleehachinda, R., Suwanik, R., Brune, M., Hallberg, L. Rice and iron absorption in man. Eur. J. Clin. Nutr. 44：489-497. (1990)
van Arsdell, J. N., Kwok, S., Schweichart, B. L., Ladner, M. B., Gelfand, D. H., and Innis, M. A. Cloning, characterization, and expression in Saccharomyces cerevisiae of endoglucanase I from Trichderma reesei. Bio/Technology 5：60-64. (1987)
van Hartingsveldt, W., van Zeijl, C. M., Harteveld, G. M., Gouka, R. J., Suykerbuyk, M. E., Luiten, R. G., van Paridon, P. A., Selten, G. C., Veenstra, A. E., van Gorcom, R. F. Cloning, characterization and overexpression of the phytase-encoding gene (phyA) of Aspergillus niger. Gene. 127：87-94. (1993)
Wegner, G. H. Emerging applications of the methylotrophic yeasts. FEMS Microbiol Rev. 7：279-283. (1990)
Wodzinski, R. J. and Ullah, A. H. J. Phytase. Adv. appl. microbiol. 264-302. (1996)
Yanke, L. J., Bae, H. D., Selinger, L. B., Cheng, K. J. Phytase activity of anaerobic ruminal bacteria. Microbiology. 144：1565-1573. (1998)
Yee, L., Blanch, H. W. Recombinant protein expression in high cell density fed-batch cultures of Escherichia coli. Biotechnology. 10：1550-1556. (1992)
Zhu, A., Monahan, C., Zhang, Z., Hurst, R., Leng, L., Goldstein, J. High-level expression and purification of coffee bean alpha-galactosidase produced in the yeast Pichia pastoris. Arch Biochem Biophys. 324：65-70. (1995)
Zhu, A., Monahan, C.,Wang, Z. K., Goldstein, J. Expression, purification, and characterization of recombinant α-N-acetyl- galactosaminidase produced in the yeast Pichia pastoris. Protein Expr Purif. 8：456-462. (1996)
Zyla, K., Ledoux, D. R., Garcia, A., Veum, T. L. An in vitro procedure for studying enzymic dephosphorylation of phytate in maize-soyabean feeds for turkey poults. Br J Nutr. 74：3-17. (1995)