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研究生:王育廷
研究生(外文):Yu-ting Wang
論文名稱:愛玉子瘦果幾丁質酶純化與生化性質研究
論文名稱(外文):Studies on the purification and biochemical properties of chitinase from jelly fig (Ficus awkeotsang Makino) achenes
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:2010
畢業學年度:98
語文別:中文
論文頁數:210
中文關鍵詞:愛玉子瘦果幾丁質酶純化與生化性質研究
外文關鍵詞:chitinasepurification and biochemical propertiesjelly fig (Ficus awkeotsang Makino) achenes
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  • 下載下載:211
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愛玉子瘦果所含酵素經由硫酸銨劃分、DEAE - Sephacel離子交換層析及Sephacryl S–100 HR膠體過濾層析等步驟純化,獲得一純化之幾丁質酶,以SDS - PAGE電泳及蛋白質染色顯示純化之幾丁質酶已達均質純度,然而,以SDS - PAGE電泳及酵素活性染色則顯示純化之幾丁質酶尚含有微量之其他幾丁質酶及幾丁聚醣酶異構酶。純化之幾丁質酶可水解幾丁質及對硝基苯基N-乙醯幾丁寡醣 (pNP-β-GlcNAcn,n = 2-5) ,其水解水溶性幾丁質─羧甲基幾丁質 (CM-chitin) 之最適pH為4,最適溫度為65℃,Km值為5.07 mg/mL 及最大反應速率為13.15 μmol GlcNAc min-1mg-1,以SDS - PAGE電泳及酵素活性染色分析,估計酵素之分子量為28.9 kDa,以等電焦集電泳分析,估計酵素之等電點為3.8。純化之幾丁質酶於30至70℃保溫90分鐘頗為穩定,但高於80℃時則顯著失去活性,其pH穩定性範圍在2.5至9.5間,化學修飾劑 N-bromosuccimide (1 mM)、Woodward’s reagent K (50 mM)及chloramine T (1 mM) 顯著抑制酵素活性,由pNP-β-GlcNAc3-5水解動力學參數分析,顯示酵素對聚合度4之pNP-β-GlcNAc4具有最高活性,以pNP-β-GlcNAc4為基質,於不同pH下測酵素動力學參數,由Dixon-Webb作圖分析顯示酵素分子有一pKa約4之質子解離基團參與基質結合及產物轉換。純化之幾丁質酶水解水溶性幾丁質─乙二醇幾丁質之產物為低分子量幾丁質及N - 乙醯幾丁寡醣。純化之幾丁質酶亦可水解不同去乙醯度 (22 - 94%) 之幾丁聚醣,其中以去乙醯度~ 30%及50 - 60%幾丁聚醣聚具有最高活性,此水解活性可能因純化之幾丁質酶含有微量幾丁聚醣酶異構酶所致。
A chitinase (EC 3.2.1.4) was purified from jelly fig achenes through successive steps of ammonium sulfate fractionation, DEAE-Sephacel ion-exchange chromatography and Sephacryl S-100 HR gel filtration. The purified chitinase appeared homogeneous on SDS-polyacrylaminde gel electrophoresis (SDS-PAGE) and protein staining. However, there was contamination with trace amounts of isoforms of chitinase and chitosanase in the purified chitinase, as analyzed by SDS-PAGE and enzyme activity staining. The purified enzyme catalyzed the hydrolysis of chitin polymer and p-nitrophenyl-N-acetylchitooligosaccharides (pNP-β-GlcNAcn , n = 2~5). The optimum pH of the enzyme for carboxymethyl chitin (CM-chitin) was 4, the optimum temperature was 65℃, the Km was 5.07 mg/mL, and the Vmax was 13.15 μmol GlcNAc min-1mg-1. The molecular mass of the enzyme was 28.9 kDa, as estimated by SDS-PAGE and enzyme activity staining. The isoelectric point of the enzyme was pH 3.8, as estimated by isoelectric focusing electrophoresis. The enzyme was thermally stable after 90 min at 30 to 70℃, but it’s activity decreased significantly at temperatures greater than 80℃. The pH stability range of the enzyme was between pH 2.5 to 9.5. Chemical modification reagent N-bromosuccinimide (1 mM), Woodward’s reagent K (50 mM) and chloramine T (1 mM) significantly inhibited enzyme activity. Kinetic parameters analysis for the enzyme for hydrolysis of p-nitrophenyl-N-acetylchitooligosaccharides with chain length from 3 to 5 indicated that the most effectively hydrolyzed substrate was p-nitrophenyl tetra N-acetyl-β-chitotetraoside (pNP-β-GlcNAc4) .A prototropic group with pKa value of approximate 4 on the enzyme may be involved in substrate binding and transformation, as examined by Dixon-Webb plots for kinetic parameters at different pH using pNP-β-GlcNAc4 as a substrate. The end products of EG-chitin hydrolysis catalyzed by the purified enzyme was low molecular weight chitin and N-acetylchitooligosaccholrides. The purified chitinase also showed activity toward chitosan polymers exhibiting various degrees of deacetylation (22-94%), most effectively hydrolyzing chitosan polymers that were ~ 30% and 50-60% deacetylated. This may be due to contamination with trace amounts of chitosanase isoforms in the purified chitinase.
目錄------------------------------------------------------------------------------------ I
表目錄--------------------------------------------------------------------------------- III
圖目錄--------------------------------------------------------------------------------- IV
縮寫表--------------------------------------------------------------------------------- VI
中文摘要------------------------------------------------------------------------------ VIII

英文摘要------------------------------------------------------------------------------ X

第一章 前言
第一節 研究背景---------------------------------------------------------------- 1-7
第二節 研究目的---------------------------------------------------------------- 18
第二章 文獻回顧
第一節 幾丁質之水解酵素---------------------------------------------------- 19
第二節 愛玉子瘦果幾丁質酶------------------------------------------------- 53
第三章 材料與方法
第一節 實驗材料---------------------------------------------------------------- 57
壹、愛玉子瘦果---------------------------------------------------------------- 57
貳、化學藥品------------------------------------------------------------------- 57
參、重要儀器及器材---------------------------------------------------------- 59
第二節 實驗方法
壹、幾丁質酶活性測定
還原醣增量法------------------------------------------------------------- 60
貳、β-NAHA活性測定------------------------------------------------------ 62
参、幾丁聚醣酶活性測定---------------------------------------------------- 62
肆、蛋白質定量
一、BCA呈色法------------------------------------------------------------ 64
二、Micro BCA呈色法---------------------------------------------------- 65
伍、Ethylene glycol chitin (EG-chitin) 、carboxymethyl chitin (CM-chitin) 及膠態幾丁質 (colloidal chitin) 之製備
一、EG-chitin製備--------------------------------------------------------- 66
二、CM-chitin製備-------------------------------------------------------- 68
三、colloidal chitin製備------------------------------------------------- 68
陸、胺基乙醯幾丁聚醣 (Aminoethyl-chitosan; AE-Chitosan)之製備 69
柒、不同去乙醯度幾丁聚醣之製備---------------------------------------- 70
  捌、幾丁聚醣去乙醯度之測定---------------------------------------------- 72
玖、醣類之酚-硫酸呈色法定量--------------------------------------------- 73
拾、GlcNAc之DMAB呈色定量----------------------------------------- 73
拾壹、愛玉子瘦果幾丁質酶之純化---------------------------------------- 75
拾貳、愛玉子瘦果幾丁質酶之性質測定--------------------------------- 77
第四章 結果與討論
第一節 幾丁質酶純化與性質
壹、幾丁質酶純化---------------------------------------------------------- 94
貳、幾丁質酶性質
一. 分子量 1.膠體過濾層析------------------------------------------- 98
2. SDS-聚丙烯醯胺膠體電泳
101
二. 等電點---------------------------------------------------------------- 105
三. 最適pH及pH 穩定性-------------------------------------------- 108
四. 最適溫度及熱穩定性---------------------------------------------- 114
五. 熱失活活化能------------------------------------------------------- 118
六. 動力學參數
1. 幾丁質和幾丁聚醣基質-------------------------------------- 120
2. 人工合成基質-------------------------------------------------- 129
七. 活性影響物質------------------------------------------------------- 138
八. 不同去乙醯度幾丁聚醣對幾丁聚醣酶活性之影響--------- 141
九. 幾丁質酶水解EG – chitin產物--------------------------------- 143
十. pH對動力學參數KmH+及Vmax H+之影響----------------------- 146
第五章 結論------------------------------------------------------------------------- 149
第六章 未來展望------------------------------------------------------------------- 150
第七章 參考文獻------------------------------------------------------------------- 151
附錄
附錄一 幾丁質酶活性測定方法 (螢光法 )----------------------------- 159
附錄二 幾丁質酶活性及幾丁聚醣酶活性測定方法------------------- 163
附錄三 BCA蛋白質校正曲線---------------------------------------------- 167
附錄四 Micro BCA蛋白質校正曲線-------------------------------------- 168
附錄五 總醣之酚-硫酸呈色定量法--------------------------------------- 169
附錄六 Glucosamin (GlcN) 之MBTH呈色定量校正曲線--------- 170
附錄七 N-Acetyl-D-glucosamin (GlcNAc) 之DMAB呈色定量校 正曲線--------------------------------------------------------------
172
附錄 八 迷你SDS-聚丙烯醯胺膠體電泳-------------------------------- 173
附錄 九 VisPRO 蛋白質染色----------------------------------------------- 179
附錄 十 GE公司Gel filtration calibration kits 說明圖----------------- 180
附錄 十一 含chitosan酸性迷你聚丙烯醯胺膠片製備--------------------- 181
附錄 十二 PhastSystem等電焦集電泳---------------------------------------- 183
附錄 十三 酵素之熱失活模式------------------------------------------------- 185
附錄 十四 對硝基苯N-乙醯幾丁寡糖之水解動力學測定---------------
189
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