張鴻民臺灣大學：食品科技研究所朱信嘉Chu, Hsin-ChiaHsin-ChiaChu2007-11-272018-06-292007-11-272018-06-292004http://ntur.lib.ntu.edu.tw//handle/246246/56300黃豆(學名為Glycine max Merrill)(亦稱為大豆)，營養價值高，具有高蛋白質及高油脂的特性。其含有異黃酮、植物動情激素等一些植物性化學物質，亦被認為有助於捕捉自由基，減輕婦女更年期症狀，甚至於預防癌症。但文獻對黃豆發芽過程中，酵素的作用機制並沒有明確的探討。因此，本論文之目的在觀察黃豆發芽過程中，主要酵素的活化情況。實驗中所選用的酵素有：果膠酯酶(Pectinesterase)、脂氧合酶(Lipoxidase)、胰蛋白酶抑制劑(Trypsin inhibitor)以及過氧化酶(Peroxidase)。利用二維電泳(Two-dimensional electrophoresis)與免疫轉印技術(Western blotting)分析黃豆蛋白質的分佈，以觀察在黃豆發芽過程中酵素的變化情形，進而探討各酵素對黃豆發芽過程的影響。 結果顯示，利用果膠酯酶、脂氧合酶、胰蛋白酶抑制劑以及過氧化酶免疫白兔後，血清抗體效價(titer)最高值分別為105、105、105及106。由此可知，白兔經過免疫處理可增加抗血清抗特殊抗原的抗體量。而這些抗特殊抗原的抗體將可作為免疫轉印時的一次抗體。 迷你電泳圖中，隨著黃豆發芽天數的增加，大分子蛋白質含量減少，小分子蛋白質含量增加。由此可知，隨著發芽天數增加，蛋白酶活性增加，進而分解大分子蛋白質以產生黃豆芽生長所需的營養及能量。而在免疫轉印結果發現，在黃豆發芽過程中，果膠酯酶、過氧化酶及胰蛋白酶抑制劑的含量有下降的趨勢。而脂氧合酶含量呈現上下起伏的現象。 經二維電泳分析及CBR染色結果發現，在黃豆發芽過程中，大分子蛋白質有減少的現象，小分子蛋白質有明顯的增加，結果與迷你電泳的結果相同。在免疫轉印結果方面，胰蛋白酶抑制劑含量有下降的趨勢，結果與迷你電泳相同。而過氧化酶、果膠酯酶及脂氧合酶的含量則是逐漸增加，此結果與迷你電泳結果不盡相同。文獻指出，在黃豆發芽時，蛋白酶活性增加而產生所需的營養，胰蛋白酶抑制劑含量減少則可以避免蛋白酶活性減少。在過氧化酶、果膠酯酶及脂氧合酶這方面，我們認為其含量增加比較合理的，因為二維電泳的解析度較高，結果較準確。此外，黃豆發芽過程中，油脂會被酵素代謝產生過氧化物，這些物質通常具有毒性，過氧化酶可以代謝過氧化物減少有毒物質，對黃豆芽的生長有保護的效果。在發芽過程中，果膠酯酶的作用會造成果膠上甲基去酯化，進而導致甲醇的累積，使黃豆變質。而脂氧合酶的含量增加，可以產生一些抗微生物物質，讓黃豆在發芽時，避免遭受病原菌的傷害。Soybean (Glycine max Merrill) is highly nutritive due to its high protein and lipid contents. Some bioactive compounds such as isoflavones and phytoestrogen were reported to help scavenge free radicals and alleviate feminine menopause syndrome, respectively, and furthermore, prevent cancer. Activation of enzymes during germination is relevant to hydrolysis of nutritive components as well as to the plant growth. Proteomic researches on the activity changes of some prominent enzymes, such as lipoxidase, peroxidase, pectinesterase (PE) and trypsin inhibitor, appeared to be of great interest during germination. Therefore, two-dimensional electrophoresis (2-DE) and immunoblotting techniques were tried to investigate the quantitative changes of enzymes during germination. First, four rabbits were immunized with one of above four enzymes to prepare polyclonal antibodies. Subsequently, soybeans germinated 1/3, 1, 3, 5, and 7 days were homogenized and the obtained protein portions were applied on a SDS-PAGE and a 2-D electrophoresis. Finally, Western blotting analysis was conducted to compare the color developed. Results showed that ELISA value increase with the increasing time of boosting, and the antiserum titer against PE, lipoxidase, trypsin inhibitor, and peroxidase were determined to be 105, 105, 105, and 106, respectively, by an indirect ELISA method. For mini gel SDS-eletrophoresis, high molecular weight proteins reduced and low-molecular weight proteins increased with the increasing cultivation time of soybeans, suggesting marked protein hydrolysis occurred during germination. Immunoblotting results suggested that PE, peroxidase, and trypsin inhibitor reduced during germination, while lipoxidase remained unchanged. Changes in the trends of protein distribution assayed by 2-DE during germination were similar to those in mini gel SDS-electrophoresis. Immunoblotting stains revealed the decreasing trypsin inhibitor and increasing peroxides during germination. Decrease in trypsin inhibitor was considered to be favorable for the enhanced protease activity during germination, which was consistent with the phenomenon of sprout growth. However, increase in peroxidase activity during germination facilitated the reduction of fat hydroperoxide, which could be toxic to sprout growth. 2-DE and the subsequent immunoblotting results showed that PE, and lipoxidase both increased during germination. Increase in lipoxidase was favorable for the plant anti-microbial actions during germination to avoid the possible attack. Combination of 2-DE with immunoblotting stains provided convenient techniques for the detection of proteins in quantitative changes during germination.中文摘要……………………………………………………………… Ⅰ 英文摘要……………………………………………………………… Ⅲ 目錄…………………………………………………………………… Ⅴ 表次…………………………………………………………………… Ⅸ 圖次…………………………………………………………………… Ⅹ 第一章 文獻整理…………………………………………………….. 1 壹、黃豆………………………………………………………… 1 一、黃豆之簡介……………………………………………….. 1 二、黃豆主要營養成分及功效……………………………….. 1 1.蛋白質(Protein)……………………………………………. 1 2.多元不飽和脂肪酸(Polyunsaturated fatty acids, PUFA)…. 2 3.卵磷脂(Lecithin)…………………………………………... 2 4.異黃酮(Isoflavone)………………………………………… 4 5.維生素E(Vitamin E)………………………………………. 4 6.纖維(Fiber)………………………………………………… 4 7.胰蛋白酶抑制劑(Trypsin inhibitor)………………………. 4 貳、酵素…………………………………………………………. 5 一、脂氧合酶(Lipoxidase) ……………………………………. 5 1.亞麻油酸(Linoleic acid) …………………………………... 5 2. 次亞麻油酸(Linolenic acid) ……………………………... 5 3. 花生四烯酸(Arachidonic acid)…………………………... 7 二、過氧化酶(Peroxidase)……………………………………… 11 1.第一類(Class I)……………………………………………. 11 2.第二類(Class II)…………………………………………… 11 3.第三類(Class III)…………………………………………... 12 三、果膠酯酶(Pectinesterase)………………………………….. 13 1.果膠酯酶之作用與結構…………………………………... 13 2.基質專一性………………………………………………... 13 3.PE之分子量與等電點……………………………………. 14 參、蛋白質體學………………………………………………… 18 一、蛋白質體學(Proteomics)…………………………………. 18 二、二維電泳分析(two-dimensional gel electrophoresis, 2-D-GE)……….…………………………………………. 19 三、蛋白質體學之應用……………………………………….. 20 第二章、黃豆芽果膠酯酶之純化及抗體製備……………………… 22 壹、實驗材料……………………………………………………. 22 一、黃豆芽…………………………………………………….. 22 二、實驗動物………………………………………………….. 22 三、藥品與耗材……………………………………………….. 22 1.實驗藥品…………………………………………………... 22 2.實驗耗材…………………………………………………... 24 貳、儀器設備與裝置…………………………………………… 26 參、實驗方法…………………………………………………… 26 一、黃豆芽果膠酯酶(PE)之萃取……………………………… 26 二、果膠酯酶活性之測定…………………………………….. 26 三、蛋白質含量測定………………………………………….. 27 四、硫酸銨劃分……………………………………………….. 29 五、膠體過濾層析法………………………………………….. 29 六、CM-Sepharose CL-6B離子交換層析法…………………. 30 七、比色法…………………………………………………….. 30 八、疫苗製備及免疫處理…………………………………….. 30 九、紐西蘭大白兔之耳靜脈採血及血清分離……………….. 31 十、ELISA測定抗四種酵素(Peroxidase、Lipoxidase、Pectinesterase and trypsin inhibitor)抗體之活性及效價(titer)測定………………………………………………… 32 肆、結果與討論………………………………………………… 34 一、黃豆芽果膠酯酶萃取及純化…………………………….. 34 二、ELISA測定法中結合體(conjugate)濃度的選定………… 35 三、免疫期間白兔血清中抗四種酵素(Peroxidase、Lipoxidase、Pectinesterase and trypsin inhibitor)抗體活性及效價(titer)之變化…………………………………... 39 第三章、蛋白質體學分析黃豆芽中四種酵素的變化……………… 43 壹、實驗材料…………………………………………………… 43 一、黃豆芽…………………………………………………….. 43 二、藥品與耗材……………………………………………….. 44 1.實驗藥品…………………………………………………... 44 2.實驗耗材…………………………………………………... 45 貳、儀器設備與裝置……………………………………………. 45 參、實驗方法…………………………………………………… 47 一、黃豆芽之孵育…………………………………………….. 47 二、不同時間點之黃豆芽採收……………………………….. 47 三、樣品蛋白質萃取法……………………………………….. 47 四、樣品蛋白質溶解………………………………………….. 48 五、電泳檢定法……………………………………………….. 48 1.試劑………………………………………………………... 48 2.實驗方法…………………………………………………... 50 六、免疫轉印法……………………………………………….. 52 1.試劑………………………………………………………... 52 2.操作流程…………………………………………………... 53 七、二維電泳分析…………………………………………….. 53 1.試劑………………………………………………………... 53 2.第一維電泳………………………………………………... 54 3.第二維電泳………………………………………………... 55 4.膠片染色及免疫轉印……………………………………... 56 肆、結果與討論………………………………………………… 57 一、迷你電泳分析…………………………………………….. 57 1.Coomassie Blue 染色……………………………………... 58 2.免疫轉印法………………………………………………... 58 二、二維電泳分析…………………………………………….. 60 1.Coomassie Blue 染色……………………………………... 60 2.黃豆儲存性蛋白質(Soybean storage protein)…………….. 60 3.免疫轉印法………………………………………………... 61 第四章、參考文獻…………………………………………………… 72en-US蛋白質體學過氧化酶果膠酯酶發芽黃豆蛋白酶脂氧合酶抑制劑SoybeanPectinesteraseGerminationTrypsin inhibitorProteomicsLipoxidasePeroxidase[SDGs]SDG3thesis