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研究生:李奕彥
研究生(外文):I-YEN Lee
論文名稱:黑色素的生產與應用
論文名稱(外文):Production and Application of Melanin
指導教授:陳建源陳建源引用關係
指導教授(外文):Chien-Yuan Chen
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:農業化學研究所
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:黑色素紫外線輻射光保護醱酵
外文關鍵詞:MelaninUVRadiationPhotoprotectionFermentation
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黑色素在人體皮膚的形成作用是一種對UV照射的保護反應,而且形成的黑色素能防止UV光進一步的傷害。也有文獻指出,黑色素參與了油脂過氧化效應中的活性基反應,而扮演了一種天然的光保謢或輻射防護的角色。
為能有效利用黑色素的功能,本研究室以遺傳工程方法成功建構出4個具有鏈黴菌來源tyrosinase 基因之表現載體(pLCJ-1、pLCJ-2、pLCJ-3、pLCJ-4),利用其中黑色素表現情況最佳之表現載體pLCJ-1,以E.coli JM109為宿主系統,進行黑色素表現條件探討,設計出一適合其表現黑色素的培養基,其組成成份為sucrose 1%、NH4Cl 2%、tyrosine 1%、yeast extract 0.5%、Na2HPO4 0.05M,並進一步探討醱酵生產條件,建立了合適的醱酵生產條件:通氣量 1.5 vvm、溫度 37℃、初始 pH 7.4、轉速 200 rpm、接種量1%、種菌培養時間6小時。
生產出的黑色素在分離純化後,進行了基本性質分析,針對黑色素之輻射與UV防護能力,進行探討,發現黑色素添加濃度達0.1 mg/ml,能降低UV對哺乳動物細胞造成的傷害,當黑色素添加濃度達0.5 mg/ml,能在輻射照射下保護哺乳動物細胞。

It has long been believed that the formation of melanin in human skin is a protective response to the damaging effects of UV radiation and that melanin offers protection against futher UV-induced damage. There are indication that melanins are involved in free radical reactions of lipid peroxidation , where thy may act as natural radio- and photo- protectors.
We have construction four plasmid (pLCJ-1 pLCJ-2 pLCJ-3 pLCJ-4), which have tyrosinase gene from Streptomyces sp which can produce melanin, and design an adaptive medium and expression situation for the best strain pLCJ-1, more distant we had design an adaptive fermentation situation for the strain. The medium is composed of sucrose 1%、NH4Cl 2%、tyrosine 1%、yeast extract 0.5%、Na2HPO4 0.05M, the adaptive fermentation situation is 1.5 vvm、37℃、initial pH 7.4、agitation rats 200 rpm、inoculum size 1%、inoculum age 6 hr.
We also studied the antioxidant and anti-UV habitudes of melanin made from the gene transformants strain, and attempt to use the melanin we produced to explore it’s application in the antiradiation and anti-UV of mammalian cell.
We find an additive of 0.1mg/ml melanin can protect mammalian cell from the damage of UV radiation,and an additive of 0.5mg/ml melanin can increase the resistance of mammalian cell in radioactivity.

目錄 I
中文摘要 Ⅱ
英文摘要 Ⅳ
緒言 1
壹、研究動機 1
貳、文獻探討 3
參、總論 13
研究架構 14
材料與方法 15
第一部份、黑色素的生產
壹、建構基因轉殖之黑色素生產菌株 15
貳、於天然環境中篩選能生產黑色素之天然株 17
參、菌種的保存與製備 18
肆、黑色素的取樣與測定 19
伍、基因轉型菌株最適表現與醱酵條件之探討 20
陸、黑色素的純化 26
柒、黑色素的性質分析 28
捌、儀器與藥品 30
第二部份、黑色素的應用
壹、UV照射下黑色素對微生物細胞的保護 32
貳、黑色素對哺乳動物細胞的保護 33
參、儀器與藥品 41
結果與討論 43
壹、基因轉殖建構之黑色素生產菌株 44
貳、於天然環境中篩選出黑色素之生產菌株 44
參、基因轉型菌株最適培養基與醱酵條件之探討 44
肆、黑色素的性質分析 53
伍、UV照射下黑色素對微生物細胞的保護 54
陸、哺乳動物細胞檢量線的建立 54
柒、UV照射下黑色素對哺乳動物細胞的保護 56
捌、放射線照射下黑色素對哺乳動物細胞的保護 56
結論 58
表 59
圖 63
參考文獻 104

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