臺灣博碩士論文加值系統

白胺酸N端切位酶（Leucine aminopeptidase；LAP）可切除胜肽兩端的疏水性胺基酸，去除苦味。本研究以Aspergillus oryzae (米麴菌) LAP基因轉殖番茄植株為材料，探討米麴菌LAP 基因在番茄體內的異源表現。因番茄本身有兩種異構型酵素LAP-A、LAP-N的存在，但胺基酸序列比對結果顯示，米麴菌LAP與番茄LAP，在序列上並無明顯的相關性。轉殖殖株之鑑定，首先先分析各植株之LAP 酵素活性，確定轉殖植株酵素活性後，再分別以PCR和RT-PCR進行DNA、RNA的分析工作。然於酵素活性分析時，除了發現活性比wild type高之植株外，也發現活性和wild type相當或較wild type低者。這可能是基因插入的位置不同，造成表現上的差異。但是，為何連番茄本身內在的LAP活性也受到影響，則須進一步釐清。而於酵素特性分析方面，發現溫度在50到70℃、pH在 8.5 到 10.5之間，以及16% NaCl高鹽存在之下，酵素有較佳的活性表現。特性分析結果顯示，異源表現之LAP，較原始來源之LAP更為耐熱、耐鹼以及耐鹽。此結果暗示，米麴菌LAP 酵素，經番茄異源性表現後，可能經過某種後修飾作用，造成此酵素性質發生改變。

Leucine aminopeptidase (LAP) is widely applied in food industry to reduce the bitterness of protein hydrolysate by liberating the hydrophobic residues in the N terminus of peptides. For the overproduction of the enzyme, transgenic tomato plants had been developed in our previous work. The transgene, Aspergillus oryzae LAP, as well as the NPT II marker gene were introduced into tomato via Agrobacterium-mediated transformation. After antibiotic selection, approximately 100 independent lines were obtained. Although two main LAP isoforms, LAP-A and LAP-N, are present in tomato, sequence comparison showed no apparent homology between the heterologous LAP and the endogenous LAPs. In this study, these transgenic plants were further identified and characterized. The enzyme activities were assayed firstly, and then, according to the level of activity, genomic PCR and RT-PCR were performed to confirm the presence of NPT II gene and Aspergillus LAP mRNA respectively. As expected, several transgenic plants showed higher enzyme activity than wild type, especially line 38; meanwhile, some showed no significant difference compared to wild type, such as lines 29, 34, and 67. Surprisingly, some lines were found to possess lower activity than wild type. Moreover, the characteristic analysis of the heterologous LAP showed some variations in enzymatic properties such as optimal temperature, optimal pH, and salt requirement. These results indicated that the Aspergillus LAP, expressed heterologously in tomato, differs from the original form, which is isolated from its natural source.

封面內頁
簽名頁
授權書.............................................................iii
中文摘要............................................................iv
英文摘要.............................................................v
誌謝..................................................................vi
目錄.................................................................vii
圖目錄...................................................................x

1. 前言...................................................................1
1.1 Leucine aminopeptidase與苦味胜肽的關係............................1
1.1.1 Leucine aminopeptidase........................................1
1.1.2蛋白質製品苦味之發生..............................................2
1.1.3 Leucine aminopeptidase於去除苦味之應............................3
1.2米麴菌LAP 轉基因番茄...............................................4
1.2.1米麴菌LAP 酵素之特性.............................................5
1.2.2轉基因番茄之建立.................................................5
1.3研究目的..........................................................6
2. 材料與方法........................................................7
2.1 實驗材料及藥品....................................................7
2.1.1實驗材料........................................................7
2.1.2實驗藥品........................................................7
2.1.3培養基..........................................................7
2.1.3.1番茄轉殖株之繼代...............................................8
2.1.4植物荷爾蒙.......................................................8
2.1.5其他緩衝液及試劑.................................................9
2.2 實驗方法........................................................10
2.2.1 Genomic DNA 之製備與純化.......................................10
2.2.2 NPTⅡ基因專一性引子之設計.......................................11
2.2.3聚合酶鏈鎖反應鑑定轉基因植株......................................11
2.2.4瓊脂凝膠電泳 (Agarose gel electrophoresis)
..............................................................12
2.2.5製備cDNA......................................................13
2.2.5.1製備RNase-free 溶液..........................................13
2.2.5.2番茄Total RNA 之萃取..........................................13
2.2.5.3甲醛變性瓊脂凝膠電泳...........................................14
2.2.5.4 LAP基因專一性引子之設計.......................................15
2.2.5.5逆轉錄聚合酶鏈鎖反應...........................................15
2.2.5.5.1第一股cDNA之合成............................................15
2.2.5.5.2聚合酶鏈鎖反應..............................................15
2.2.6 LAP之活性測定..................................................16
2.2.6.1 LAP之酵素粗萃取..............................................16
2.2.6.2蛋白質濃度的測定..............................................16
2.2.6.3 p-nitroaniline 的標準曲線製作................................17
2.2.6.4酵素呈色法....................................................17
3. 結果與討論........................................................18
3.1米麴菌與番茄LAP基因之比較..........................................18
3.2 番茄轉殖株之建立與鑑定............................................19
3.3 轉基因番茄LAP之酵素特性分析......................................20
3.3.1 LAP之最適反應pH值............................................21
3.3.2 LAP最適反應溫度...............................................21
3.3.3 NaCl對LAP活性的影響...........................................22
3.4未來展望........................................................23
4. 結論.............................................................24
參考文獻............................................................32
附錄................................................................35

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