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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭光成 | |
dc.contributor.author | Wen-Chun Yang | en |
dc.contributor.author | 楊雯鈞 | zh_TW |
dc.date.accessioned | 2021-06-08T01:13:27Z | - |
dc.date.copyright | 2014-08-26 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-14 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18589 | - |
dc.description.abstract | 玉米烯酮(zearalenone ; ZEN)為Fusarium產生的一種類雌激素黴菌毒素,利用酵素水解玉米烯酮毒素可有效減少毒素含量,由Clonostachys rosea中選殖到的zhd101即為一可表現具有分解玉米烯酮的内酯水解酶(lactonohydrolase)的基因。應用益生菌做為表現宿主除了其安全性高、耐酸、耐膽鹽和腸道吸附性以外,也具很高的便利性,本實驗中所使用的乳酸桿菌為由肉雞腸道中分離出的Lactobacillus reuteri Pg4,已證實具有益生菌之特性。將zhd101基因構築於乳酸菌-大腸桿菌穿梭載體pNZ3004,並利用電穿孔轉形獲得L. reuteri pNZ-zhd101轉形株,在DNA、RNA及蛋白質層次可證實重組菌株具表達Zhd101之能力;以高效能液相層析儀進行酵素活性檢測,由於乳酸菌生長代謝產生酸會使Zhd101酵素失活,因此藉由發酵槽控制pH值,結果顯示重組乳酸菌之生長相較於L. reuteri Pg4慢,但在pH 7的環境下具有降解ZEN的活性,質體及基因的存在率在發酵14小時後分別為91%及100%。本實驗成功利用重組乳酸桿菌L. reuteri pNZ-zhd101表達玉米烯酮內酯水解酶Zhd101,利用發酵槽放大培養的轉形株具有降解玉米烯酮的能力而且表現高度穩定度。 | zh_TW |
dc.description.abstract | Mycotoxins are fungal secondary metabolites and contaminate agricultural products during crops growing, harvest, transportation, processing, or storage. Zearalenone (ZEN) is a nonsteriod estrogenic mycotoxin, produced by Fusarium species, and has been frequently implicated in reproductive disorders of farm animals and occasionally in hyperoestrogenic syndromes in humans. Biological, chemical, and physical detoxification procedures are trying to figure out mycotoxin contaminations by absorption or biotransformation, and enzymatic detoxification could be an efficient method for ZEN detoxification. ZEN-detoxifying gene, zhd101, is isolated from Clonostachys rosea IFO 7063. Zhd101 converts ZEN to the nonestrogenic product. Maximal activity of Zhd101 toward ZEN was measured at pH 10.5 and 37 to 45°C. Lactobacillus reuteri Pg4 is a lactic acid bacteria isolated from the gastrointestinal tract of broilers and possesses probiotic characteristics, so it has great potential for application. This study is aimed to construct expression vector with zhd101 and electroporation into L. reuteri Pg4 to express Zhd101. The zhd101 gene was cloned into Escherichia coli-Lactobacillus shuttle vector, pNZ3004, and the plasmid pNZ-zhd101 was electoporotion into L. reuteri Pg4. Zhd101 was successfully expressed by recomninant L. reuteri pNZ-zhd101 at DNA, mRNA, and protein levels. L. reuteri pNZ-zhd101 transformants showed ZEN degradation activity at pH 7.0 and highly plasmid stability. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T01:13:27Z (GMT). No. of bitstreams: 1 ntu-103-R01641017-1.pdf: 1611117 bytes, checksum: 3eed5712e60485da8d397c322caa2201 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 謝誌 I
序言 II 中文摘要 III Abstract IV 圖目錄 VII 表目錄 VIII 第一章 、文獻探討 1 第一節 、黴菌毒素之簡介 1 (一) 玉米烯酮 (zearalenone, ZEN)簡介 1 (二) 黴菌毒素於食品及飼料之影響 2 (三) 黴菌毒素防治 3 第二節 、Zhd101 8 (一) Zhd101簡介 8 (二) Zhd101相關研究與應用 8 第三節 、益生菌之介紹與特性 10 (一) 益生菌之定義 10 (二) 益生菌之特性 10 第四節 、 乳酸菌(lactic acid bacteria) 14 (一) 乳酸菌特性及應用 14 (二) 基因改造乳酸菌的研究 14 (三) Lactobacillus reuteri Pg4 16 第二章 、 材料與方法 18 第一節 、 本實驗中使用的質體及菌株 18 第二節 、大腸桿菌系統表達內脂水解酶 19 (一) 大腸桿菌表現質體建構 19 (二) 組胺酸標幟融合蛋白純化 21 第三節 、乳酸菌系統表達內酯水解酶 23 (一) 大腸桿菌-乳酸桿菌載體建構 23 (二) 乳酸菌勝任細胞製備 26 (三) 電穿孔轉形 27 (四) 菌落聚合酶連鎖反應 28 (五) 反轉錄聚合酶連鎖反應 28 (六) 蛋白質純度之檢測-聚丙烯醯胺膠體電泳分析(sodium dodecyl sulfate-polyacryamide gel electrophoresis;SDS-PAGE) 30 (七) 西方轉漬 32 第四節 、重組乳酸菌生長及玉米赤黴毒素降解能力分析 34 (一) 生長曲線 34 (二) 以發酵槽培養重組菌株 35 (三) 重組乳酸菌在發酵槽中之質體穩定度 36 (四) 以高效能液相層析儀分析玉米赤黴毒素濃度檢測 36 第三章 、結果與討論 39 第一節 、以大腸桿菌大量表達玉米烯酮內酯水解酶及蛋白質純化 39 (一) 大腸桿菌之玉米烯酮內酯水解酶表達質體構築 39 (二) 大腸桿菌之玉米烯酮內酯水解酶表現與純化 41 (三) 以高效能液相層析儀分析Zhd101蛋白質之酵素活性 43 第二節 、乳酸桿菌表達玉米烯酮內酯水解酶 45 (一) 大腸桿菌-乳酸桿菌之玉米烯酮內酯水解酶穿梭質體構築 45 (二) 以菌落聚合酶連鎖反應確認轉形株 47 (三) 以反轉錄聚合酶連鎖反應確認zhd101基因之表達 49 第三節 、重組乳酸菌生長及降解玉米烯酮毒素之能力分析 53 (一) 重組乳酸菌之生長曲線測定 53 (二) 重組乳酸菌之玉米烯酮降解能力分析 55 (三) 發酵槽培養重組乳酸菌之質體穩定度 60 第四章 、結論與未來展望 62 參考文獻 63 | |
dc.language.iso | zh-TW | |
dc.title | 以乳酸桿菌Lactobacillus reuteri Pg4表達內酯水解酶Zhd101以降解玉米烯酮毒素 | zh_TW |
dc.title | Expression of lactonohydrolase Zhd101 for detoxifying zearalenone by Lactobacillus reuteri Pg4 | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 劉?睿 | |
dc.contributor.oralexamcommittee | 劉啟德,謝建元,彭及忠 | |
dc.subject.keyword | 玉米烯酮,內酯水解?,乳酸桿菌, | zh_TW |
dc.subject.keyword | zearalenone,lactonohydrolase,Lactobacillus, | en |
dc.relation.page | 70 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2014-08-14 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 食品科技研究所 | zh_TW |
顯示於系所單位: | 食品科技研究所 |
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