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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48696

Title: 牛樟芝酒精氧化酶之基因選殖、表現以及紫紅硬孔菌(Rigidoporus vinctus) α-酮戊二酸依賴型黃嘌呤雙加氧酶之基因選殖、表現
Cloning, expression of alcohol oxidase from Taiwanofungus camphorata and alpha-ketoglutarate-dependent xanthine dioxygenase from Rigidoporus vinctus
Authors: Cnen, Jia-Shing
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: 牛樟芝;酒精氧化酶;紫紅硬孔菌;黃嘌呤
Taiwanofungus camphorata;alcohol oxidase;Rigidoporus vinctus;xanthine
Date: 2014
Issue Date: 2018-08-22T06:30:29Z
Abstract: 本研究共包含兩部分,分別為牛樟芝酒精氧化酶以及紫紅硬孔菌α-酮戊二酸依賴型黃嘌呤雙加氧酶。 首先為牛樟芝酒精氧化酶,酒精氧化酶(alcohol oxidase)為一種常見的酒精氧化酵素,大多存在於嗜甲醇真菌(methylotrophic yeasts),主要功能為:將酒精氧化,得到相對應的乙醛以及過氧化氫。牛樟芝酒精氧化酶基因全長為1953 bp轉譯651個胺基酸,分子量為72.2 kDa。 將牛樟芝酒精氧化酶以pET-20b(+)以及pYEX-S1表現載體,分別以大腸桿菌以及酵母菌作為表現宿主。 α-酮戊二酸依賴型黃嘌呤雙加氧酶(alpha-ketoglutarate-dependent xanthine dioxygenase)是一種能將黃嘌呤氧化的酵素同時將α-酮戊二酸氧化酶琥珀酸,與黃嘌呤的代謝有關。 紫紅硬孔菌α-酮戊二酸依賴型黃嘌呤雙加氧酶基因全長為1191bp,轉譯出397個胺基酸,分子量為44.3k Da,利用pET-20b(+)以及pYLEX1表現載體,分別利用大腸桿菌以及真菌系統做為蛋白質表現宿主。 將表現之重組蛋白質,利用鎳離子親合性膠體純化,具結果所示,純化效果不如預期。
We study an enzyme which is alcohol oxidase from Taiwanofungus camphorata and alpha-ketoglutarate-dependent xanthine dioxygenase from Rigidoporus vinctus. Alcohol oxidase (AOX) from T. camphorata , is the first enzyme of the methanol utilization pathway in methylotrophic yeasts and is strongly induced by methanol as a sole carbon source. It contains an open reading frame of 1953 bp which encodes a protein of 651 amino acid residues, molecular mass is 72.2 kDa. To characterize AOX , the coding region of TcAOX was introduced into an expression vector pET-20b(+) and pYEX-S1, and transfered into Escherichia coli and Saccharomyces cerevisiae . The alpha-ketoglutarate-dependent xanthine dioxygenases use one atom of the oxygen molecule to oxidize a specific substrate (xanthine) while the second atom oxidizes a co-substrate (alpha - ketoglutarate) to succinate. The DNA sequence of RvXDO has 1191 bp which encodes a RvXDO of 397 amino acids, and molecular mass is 44.3 kDa. To characterize the RvXDO, the coding region of RvXDO was introduced into an expression vector pET-20b (+) and pYLEX1, and transform into E. coli and Yarrowia lipolytica . The recombinant His-tag RvXDO was overexpressed and purified by Ni2+-nitrilotriacetic acid agarose.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010136008.id
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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