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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/30571

Title: 噬鹼性真菌、白腐真菌或樟芝等其氧化還原相關脢之選殖、表現、特性分析和在環境解毒、生物燃料電池、解毒等之應用
Oxidoreduction Related Enzymes from Alkalinphilic Fungi, White Rod Fungi or Antrodia Camphorata: Cloning, Expression, Characterization and Application in Environmental Detoxification, Biofuel Cell, or Detoxification, Etc.
Authors: 林棋財
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立臺灣海洋大學:生物科技研究所
Date: 2011-08
Issue Date: 2012-04-13T01:06:33Z
Publisher: 行政院國家科學委員會
Abstract: 摘要:為研發氧化還原相關酶[GFD(glutathione-dependent formaldehyde dehydrogenase),PDI(protein disulphide isomerase), NR (nitroreductae), Srx (sulfiredoxin), ADD (aryl-alcohol dehydrogenase), COD (catechol 1,2 dioxygenase), DR (diacetyl reductase),2-Cys Prx2(2-Cys peroxyredoxin2), MonoGrx (monothiol glutaredoxin),laccase] 在解毒、環境解毒、生物燃料電池等之應用。以噬鹼性真菌及白腐真和採自鹿谷之新鮮樟芝子實體為材料,抽取RNA,合成cDNA,建立EST,藉由EST序列資訊設計引子,用噬鹼性真菌、白腐真菌或樟芝子實體cDNA為模板,以PCR增生氧化還原相關酶(GFD,PDI,NR,Srx,ADD,COD,DR,2-Cys Prx2 isozyme, MonoGrx,laccase) DNA並進行篩選,篩得噬鹼性真菌、白腐真菌或樟芝子實體氧化還原相關酶相關基因之片段DNA,再進行5′RACE和3′RACE,最後得到全長氧化還原相關酶之cDNA,繼之與表現型載体連接,送入 E.coli和酵母菌進行大量生產並純化及進行生化性質測試和動力學研究。另進行GFD cDNA轉染至細胞及其在細胞的表現、並探討GSNO(nitroglutathione)處理含有GFD之細胞和對照組細胞之nitrosative stress差異性及調節細胞重要的metabolic enzymes之 S-nitrososylation的程度。另將全長laccase之cDNA,送入 E.coli和酵母菌進行大量生產並純化及進行生化性質測試和動力學之研究外,進一步藉由定位突變技術進行laccase之改良,試圖找出大量生產最適於環境解毒和作為生物燃料電池陰極的laccase。Laccase固定在燃料電池陰極,以開發直接電子移動型或電子傳遞媒電子移動型生物燃料電池。
Abstract:In order to study oxidoreduction related enzymes [GFD (glutathione-dependent formaldehyde dehydrogenase), PDI (protein disulphide isomerase), NR (nitroreductae), Srx (sulfiredoxin), ADD (aryl-alcohol dehydrogenase), COD (catechol 1,2 dioxygenase), DR (diacetyl reductase), 2-Cys Prx2 (2-Cys peroxyredoxin2), MonoGrx (monothiol glutaredoxin), laccase], which shall be used in detoxification, environmental detoxification, or biofuel cell from alkalinphilic fungi, white rod fungi or Antrodia camphorata. Fresh alkalinphilic fungus, white rod fungus or Antrodia camphorata shall be used to isolate Poly(A) mRNAs , then synthesize cDNAs as templates. Based on the conserved sequence of oxidoreduction related enzymes from other organisms or EST, the degenerate primers be synyhesized. Using PCR technique, full length oxidoreduction related enzyme cDNAs will be obtained. In addition, the coding regions of oxidoreduction related enzyme cDNAs will be introduced into expression vectors then transformed to E.coli or yeast. High amount and high activity of oxidoreduction related enzymes would obtained. Furthermore, we will investigate the characters of oxidoreduction related enzymes which are probably applied in environmental detoxification or detoxification. In addition, GFD cDNA will be transfected to 293T human embryotic kidey cell or SH-SY5Y human neuronblastoma cell for expression, then using GSNO (nitroglutathione) to challenge these cells to study whether GFD affect nitrosative stress among these cells. In addition, in order to improve the recombinant laccse activity or stability, site mutation technique will be used. Hope the recombinant laccase would be more suitably used in a cathode of biofuel cell.
Relation: NSC100-2313-B019-003-MY3
URI: http://ntour.ntou.edu.tw/handle/987654321/30571
Appears in Collections:[生命科學暨生物科技學系] 研究計畫

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