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研究生:廖敏淳
研究生(外文):Min-Chun Liao
論文名稱:名間長紅山藥塊莖幾丁質酶之純化及性質研究
論文名稱(外文):Studies on the purification andproperties of chitinase from purple yam tubers
指導教授:張珍田張珍田引用關係
指導教授(外文):Chen-Tien Chang
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養研究所
學門:醫藥衛生學門
學類:營養學類
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
論文頁數:158
中文關鍵詞:幾丁質酶山藥
外文關鍵詞:chitinaseyam
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名間長紅山藥塊莖(Discorea alata L. var. purpurea (Roxb.) M. Pouch),經添加0.46% cetylpyridinium chloride (CPC)四級銨界面活性劑處理後,可使黏質物沈澱而獲得幾丁質酶粗酵素液,粗酵素液再經Sephacryl S-100 HR膠體過濾層析,HiLoad Sepharose Fast Flow離子交換層析及Mono P HR等電焦集層析等系列步驟純化,可使酵素純度提高98倍,並獲得2.78 % 之活性回收率。以SDS-PAGE分析顯示酵素已達均質純度,其水解乙二醇幾丁質之最適pH為4,最適溫度為60℃,Km值為1.86 mg/mL,kcat為1.01/sec而kcat/Km為0.543 mL/mg/sec;水解4-methyl-umbelliferyl N, N’, N”-triacetyl-β-chitotrioside (4-MU-β-(GlcNAc)3之最適pH為4,最適溫度為50℃,Km值為20μM,kcat為8.4x10-3/seckcat/Km為0.420/sec/mM;水解p-nitrophenyl-β-N-acetyl-
glucosamine (pNp-β-GlcNAc)3)之Km值為1237μM,kcat為0.074/sec,kcat/Km為0.06/sec/mM,故其水解(pNp-β-GlcNAc)3)較水解(4-MU-β-(GlcNAc)3) 具有較高之催化速率(較高之kcat值)。以膠體過濾層析法及SDS-PAGE測得酵素分子量皆為31 kDa,故屬單元體酵素(monomeric enzyme),以等電焦集電泳測得酵素之等電點( pI )小於3.55。重金屬離子Hg2+ (0.25mM)及Ag+ (0.25mM)與化學修飾劑NBS(0.25mM)及Woodward’s reagent K (2.5mM)等,顯著抑制酵素活性。
Chitinase was extracted from purple yam tubers [Dioscorea alata L. var. purpurea (Roxb) M. Pouch ] by the application of 0.46%cetylpyridinium chloride (CPC) quaternary ammonium ion detergent for removal of viscous mucilage. Chitinase was purified from the crude extract by sequentially appling Sephacryl S-100 HR gel filtration, HiLoad S Sepharose ion-exchange chromatography and Mono P HR chromatofocusing. By these steps, the enzyme was purified 98 fold over the crude extract chitinase, and the total yield of 2.78%. The purified enzyme was homogeneous, as examined by SDS-PAGE. With respect to ethylene glycol chitin hydrolysis, optimal pH was 4 ; optimal temperature was 60℃; Km was 1.86 mg/mL; kcat was 1.01/sec and kcat/Km was 0.543 mL/mg/sec. With respect to 4-methylumbelliferyl N, N’, N”-triacetyl-β- chitotrioside (4-MU-β-(GlcNAc)3 hydrolysis, optimal pH was 4 ; optimal temperature was 50℃; Km was 20μM; kcat was 8.4x10-3/sec and kcat/Km was 0.420/sec/mM. With respect to p-nitrophenyl-β-N-acetyl-glucosamine (pNp-β-(GlcNAc)3) hydrolysis, Km was 1237μM; kcat was 0.074/sec; kcat/Km was 0.06 sec/mM. The enzyme has higher catalytic rate for (pNp-β-GlcNAc)3) hydrolysis than for (4-MU-β-(GlcNAc)3) hydrolysis. The molecular mass of the enzyme was 31 kDa, as estimated by both SDS-PAGE and gel filtration. Thus, the enzyme is a monomeric enzyme. The isoelectric point of the enzyme was < 3.55, as estimated by isoelectrofocusing electrophoresis. Heavy metal ions of Hg2+ (0.25mM) and Ag+ (0.25mM), NBS (0.25mM) and Woodward’s reagent K (2.5mM) significantly inhibited the enzyme activity.
目 錄 ---------------------------------------------------------------------- 
Ⅰ~Ⅲ
表目錄 ---------------------------------------------------------------------- 

圖目錄 ---------------------------------------------------------------------- 
Ⅴ~Ⅶ
中文摘要 ------------------------------------------------------------------- 

英文摘要 ------------------------------------------------------------------- Ⅸ
第一章  前言 ----------------------------------------------------------- 1
第二章  文獻回顧 ----------------------------------------------------- 4
第一節 山藥 ------------------------------------------------------- 4
壹、簡介 ------------------------------------------------------- 4
貳、山藥的品種與分布 ------------------------------------- 4
參、山藥之化學組成與營養成分 ------------------------- 5
肆、山藥的生理活性及保健功能 ------------------------- 6
第二節 幾丁質水解酵素 ---------------------------------------- 9
壹、簡介 ------------------------------------------------------- 9
貳、幾丁質水解酵素之分類 ------------------------------- 9
參、幾丁質水解幾丁質之作用機制 ------------------- 13
    肆、幾丁質之天然分佈 ---------------------------------- 24
伍、幾丁質活性測定方法 ------------------------------- 40
第三節 幾丁質之應用 ---------------------------------------- 43
壹、真菌原生質體製備 ------------------------------------- 43
貳、N-乙醯幾丁寡醣之製備 ------------------------------- 43
參、植物病蟲害防治上之利用 ---------------------------- 44
    肆、菌體生產之應用 ---------------------------------------- 44
  第四節 研究動機與目的 ---------------------------------------- 46
第三章 材料與方法 ---------------------------------------------------- 47
第一節 實驗材料 ------------------------------------------------- 47
壹、山藥塊莖 ------------------------------------------------- 47
貳、化學藥品 ------------------------------------------------- 48
參、重要儀器及器材 ---------------------------------------- 50
第二節 實驗方法 ------------------------------------------------- 51
壹、山藥幾丁質活性測定方法 ------------------------- 51
貳、蛋白質定量 ---------------------------------------------- 54
參、不同濃度四級銨界面活性劑處理對幾丁質活性之影響---------------------------
-------------------------- 56
肆、山藥塊莖幾丁質之抽取與純化 ------------------- 56
一、粗酵素液製備 ----------------------------------------- 56
二、Sephacryl S-100 HR管柱膠體過濾層析 --------- 56
三、HiLoad S Sepharose Fast Flow 16/10管柱離子交換層析 ------------------------
------------------------- 57
四、Mono P等電焦集層析 ------------------------------ 57
伍、酵素性質測定 ------------------------------------------- 58
一、Superdex 75 HR膠體過濾層析之原態分子量測定 --------------------------------
----------------------- 58
二、SDS-聚丙烯醯胺膠體電泳之次單元分子量測定 58
三、等電點pI值之測定 --------------------------------- 60
四、幾丁質水解螢光基質(4-MU-β-(GlcNAc)3)之動力學參數 -----------------------
-------------------- 61
五、幾丁質水解乙二醇幾丁質動力學參數測定--- 62
六、幾丁質水解對硝基酚N-乙醯幾丁寡醣(pNp-β-(GlcNAc)3)之基質飽和曲線及Km
值 ------- 63
七、不同聚合度對硝基酚N-乙醯幾丁寡醣之水解-- 63
八、金屬鹽類對酵素活性之影響 ---------------------- 63
九、化學修飾劑對酵素活性之影響 ------------------- 64
十、幾丁寡醣之薄層層析 ------------------------------- 64
第四章 結果與討論 ---------------------------------------------------- 66
第一節 CPC界面活性劑對幾丁質活性之影響----------- 66
第二節 山藥塊莖幾丁質之純化 ---------------------------- 66
第三節 山藥塊莖幾丁質之生化性質 ---------------------- 73
壹、分子量 ---------------------------------------------------- 73
貳、等電點 ---------------------------------------------------- 78
參、水解4-MU-β-(GlcNAc)3及pNp-β-(GlcNAc)3之動力學 ---------------------------
------------------------- 81
    肆、水解乙二醇幾丁質之動力學 ------------------------- 91
伍、不同聚合度對硝基酚N-乙醯幾丁寡糖之水解 --- 98
陸、金屬鹽類對酵素活性之影響 ------------------------- 103
柒、化學修飾劑對酵素活性之影響 ---------------------- 105
捌、幾丁聚醣及幾丁寡醣之薄層層析-------------------- 107
玖、N-端胺基酸序列分析 ---------------------------------- 107
第五章 結論 ------------------------------------------------------------- 109
第六章 參考文獻 ------------------------------------------------------- 110
附 錄 132
附錄一 山藥塊莖幾丁質活性測定 ------------------------- 132
附錄二 對硝基酚N-乙醯幾丁寡糖之水解動力學測定 --- 141
附錄三 化學修飾劑對酵素活性之影響測定 ---------------- 143
附錄四 SDS-PAGE聚丙烯醯胺膠體電泳 ------------------- 146
附錄五 IEF等電焦集電泳 ------------------------- 149
附錄六 幾丁寡糖及N-乙醯幾丁寡糖之薄層層析 --------- 150
附錄七 N-端胺基酸序列分析 ---------------------------------- 155
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