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研究生:黃雅琪
研究生(外文):Huang, Yachi
論文名稱:重組人類酪胺酸酶最適復性條件、生化特性探討及抑制劑搜尋
論文名稱(外文):Investigation Of The Optimal Renaturation Conditions And The Biochemical Properties Of Recombination Human Tyrosinase, And Screening Its Inhibitors
指導教授:江善宗陳俊宏陳俊宏引用關係
指導教授(外文):Jiang, ShanntzongChen, Genhung
口試委員:曹欽玉殷儷容江善宗陳俊宏
口試委員(外文):Tsao, ChingyuYin, LijungJiang, ShanntzongChen, Genhung
口試日期:2012-06-04
學位類別:碩士
校院名稱:靜宜大學
系所名稱:化粧品科學系
學門:民生學門
學類:美容學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:103
中文關鍵詞:重組人類酪胺酸酶復性純化特性分析抑制劑
外文關鍵詞:recombinant human tyrosinaseRenaturationPurificationBiochemical propertyTyrosinase inhibitors
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酪胺酸酶 (tyrosinase) 是一個結構複雜的含銅多酚類氧化酶。為了以人類酪胺酸酶當指標酵素來篩選其抑制劑,本研究首先將重組人類酪胺酸酶於大腸桿菌表現系統進行內涵體形式的表現,之後利用變性緩衝溶液將內涵體展開後於含銅離子的復性緩衝溶液中進行重新摺疊可大量獲得有活性的重組人類酪胺酸酶。利用陰離子交換管柱和膠體過濾進行純化,酵素純化倍率提高 9 倍,並獲得 4.2% 的回收率,純化酵素經由 SDS-PAGE 電泳分析鑑定其分子量約為 57 KDa,純化酵素的最適作用溫度為 70oC,最適作用pH值為 8.5。額外加入 Ca2+、Cd2+、Cu2+ 、Mg2+、Zn2+ 等金屬離子 (最終濃度為 10 mM)、β-ME、DTT 可略為增加 RHT 的活性,而 DMSO (最終濃度 20%) 可增加 1.5 倍的 RHT 活性,相反的加入 EDTA、Urea、SDS、Kojic acid、α-Arbutin、L-ascorbic acid 具有抑制部分的RHT 活性,但是只要添加 Co2+、Hg2+、Cr3+ (最終濃度為 5 mM) 金屬離子就可以 100% 抑制重組人類酪胺酸酶活性。本研究酪胺酸酶抑制劑來源有天物植物萃取及化學合成的化合物兩類,在天然植物方面,變葉木、鵝掌藤、矮仙丹乾燥葉均無抑制 RHT活性,而大王仙丹(葉)水萃物其抑制酪胺酸酶 (IC50:58.38 mg/ml) 能力幾乎可以媲美麴酸 (IC50:58.31 mg/ml)。在化學合成的化合物方面,本實驗利用化妝品法規美白劑 Kojic acid 為正控制與化合物 1-acetyl-5-(5,7-dichloro-8-hydroxyquinolin-2-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole 進行對照,利用酵素動力學方法了解該化合物其抑制機轉為競爭型抑制劑。未來,期盼大王仙丹(葉)水萃物與1-acetyl-5-(5,7-dichloro-8-hydroxyquinolin-2-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole化合物有未來或許可以應用於化妝品美白產品中。
Tyrosinase (EC 1.14.18.1) is a complex structure of copper-containing polyphenolic oxidase. For the purpose of using human tyrosinase as indicator to search its inhibitors, recombinant human tyrosinase was first expressed in the form of inclusion bodies from Escherichia coli expression system. After being solubilized with denaturation buffer and following dialyzed against renaturation buffers, high quantity of active form of recombinant human tyrosinase (RHT) was obtained. The RHT could be purified by DEAE Sepharose and Sephadex G-75 chromatography with purification fold of 9 and a final yield of 4.2%. The molecular weight of RHT is about 57.0 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature and pH of RHT were at 70oC and pH 8.5. Purified RHT activity could be increased by addition of Ca2+, Cd2+, Cu2+, Mg2+, Zn2+ (Final concentration of 10 mM). Dimethy sulphoxide (DMSO, Final concentration of 20%) increased 1.5-fold RHT activity. Purified RHT could be moderately inhibited by EDTA, Urea, SDS, Kojic acid, α-Arbutin and L-ascorbic acid, and inhibited completely by Co2+, Hg2+, Cr3+ (Final concentration of 5 mM). In this study, two source of tyrosinase inhibitors, natural plant extract and chemical synthesis compounds, have been tested. At natural plant extract, Codiaeum variega, Schefflera arboricola, Ixora williamsii did not exhibit RHT inhibitory activity. However, hot water extract from Ixora duffii cv exhibited seriously inhibitiory activity of RHT (IC50 of 58.38 mg/ml) equivalent to Kojic acid (IC50 of 58.31 mg/ml). At chemical synthesis compounds, the inhibition kinetics analyzed by Lineweaver-Burk plot found that 1-acetyl-5-(5,7-dichloro-8-hydroxyquinolin-2-yl)-3-(4-methoxyphenyl) -4,5-dihydro-1H-pyrazole to be a competitive inhibitor for RHT. In the future, hot water extract of Ixora duffiicv and 1-acetyl-5-(5,7-dichloro-8-hydroxyquinolin-2-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole might be used as whiting reagent in cosmetic applications.
中文摘要 I
Abstract III
謝誌 V
表目錄 IX
圖目錄 VIII
名詞縮寫 X
第一章、前言 1
第二章、文獻回顧 3
1.美白成分其發展趨勢 3
2.皮膚的構造 13
3.黑色素的形成 21
4.酪胺酸酶 (tyrosinase) 27
5.酵素動力學探討抑制機轉 36
第三章、實驗設計 36
第四章、材料與方法 42
1. 菌種 42
2. 質體 42
3. 藥品 42
4. 儀器設備 42
第五章、 實驗方法 45
1.重組人類酪胺酸酶之表現 45
2.重組人類酪胺酸之最適復性條件 48
3.重組人類酪胺酸酶之純化 53
4.重組人類酪胺酸酶之特性分析 55
5.天然物-大王仙丹(葉) 59
6.合成化合物- C21H17Cl2N3O3 之酵素動力學測定 59
第六章、結果與討論 61
1.重組人類酪胺酸酶之最適復性條件 64
2.重組人類酪胺酸酶之純化 66
3.人類酪胺酸酶之特性分析 68
4.天然物-大王仙丹(葉) 68
5.合成化合物- C21H17Cl2N3O3 75
第七章、結論 77
參考文獻 98
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