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研究生:吳雅瑛
研究生(外文):Ya-Ying Wu
論文名稱:綠竹筍殼幾丁三糖酶之純化與性質研究
論文名稱(外文):Studies on the purification and characterization of chitotriosidase from outer sheathes of bamboo(Bambusa oldhamii)shoots.
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:2007/11/
畢業學年度:96
語文別:中文
論文頁數:221
中文關鍵詞:幾丁三糖酶純化綠竹筍生化性質
外文關鍵詞:biochemical propertieschitotriosidasepurificationBamboo shoots
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綠竹筍採收後,以幾丁聚醣處理,置室溫下貯存48小時,可使幾丁質酶及幾丁聚醣酶等幾丁聚合物水解酵素活性顯著增加。貯存後之綠竹筍,其外殼所含幾丁三糖酶(一種可水解合成基質4-methylumbelliferyl N, N’N”-triacetyl-β-chitotrioside;4-MU-GlcNAc3之幾丁質水解酵素)經由緩衝液萃取、硫酸銨劃分、Sephacryl S-100膠體過濾層析、製備式等電焦集電泳及HiLoad Superdex 75HR膠體過濾層析等連續步驟純化,可使酵素純度提高341倍並獲得13﹪酵素活性回收率。純化之幾丁三糖酶水解合成基質4-MU-GlcNAc3之最適pH為4,最適溫度為60℃,Km為6.93μM。以膠體過濾法測得酵素分子量為35.5 kDa,等電焦集電泳測得等電點在pH 3.5~5.2間,SDS-PAGE及幾丁三糖酶活性染色顯示含有兩種異構酶,重金屬離子Ag+(0.25 mM)及Hg2+(0.25 mM)顯著抑制酵素活性,化學修飾劑N-bromosusccinimide(NBS)、diethyl-pyrocarbonate(DEPC)及2,4-dintro-1-fluorobenzene(DNFB)等修飾劑則幾乎完全抑制酵素活性。將純化之幾丁三糖酶置30至80℃水浴40分鐘,結果顯示酵素在60℃以下頗穩定,70℃下保溫40分鐘尚殘留70﹪活性,80℃下保溫30分鐘則完全失去活性。幾丁三糖酶對合成之螢光基質4-MU-GlcNAc2-3及呈色基質p-nitrophenyl di-N-acetyl-β-chitobioside(pNp-GlcNAc2)和p-nitrophenyl tri-N-acetyl-β-chitotrioside(pNp-GlcNAc3),皆具活性,其水解pNp-GlcNAc3、4-MU-GlcNAc3、pNp-GlcNAc2及4-MU-GlcNAc2之催化效率(Vmax / Km)值分別為317、156、27.9和7.69 nmole/min/mg/μM,此外幾丁三糖酶對幾丁質多醣ethylene glycol chitin及carboxymethyl chitin,亦具活性。由酵素水解合成基質及ethylene glycol chitin產物分析,顯示幾丁三糖酶水解幾丁質寡糖及多醣為內切方式。將幾丁三糖酶和pNp-GlcNAc2及4-MU-GlcNAc保溫反應產生4-MU螢光物質,顯示酵素具有轉糖基活性。
Fresh harvest bamboo shoots were treated with chitosan and stored at room temperature for 48 hours. After storage, activities of chitinolytic enzymes, such as chitinase and chitosanase in the shoots were significantly increased. A chitotriosidase, which can hydrolyze synthetic substrate 4-methylumbelliferyl N,N’,N”-triacetyl-β-chitotrioside(4-MU-GlcNAc3), was purified from outer sheaths of the chitosan treated bamboo shoots by sequential steps of buffer extraction, ammonium sulfate fractionation, Sephacryl S-100 HR gel filtration, preparative isoelectrofocusing electrophoresis in Rotofor cell and HiLoad Superdex 75 HR gel filtration. By these steps, the purity of chitotriosidase was increased by 314 fold and the recovery of enzyme activity was 13%. The optimal pH for 4-MU-GlcNAc3 hydrolysis of the enzyme was 4, the optimal temperature was 60℃ and the Km was 6.93μM. The molecular mass of the enzyme was 35.5 kDa, as estimated by gel filtration. The isoelectric point of the enzyme was between pH 3.5 and 5.2, as estimated by isoelectrofocusing electrophoresis. Two isoforms of chitotriosidase were observed, as analyzed by SDS-Polyacrylamide gel electrophoresis and enzyme activity staining. Heavy metal ions of Ag+ and Hg2+ (0.25 mM)significantly inhibited the enzyme,s activity. Chemical modification by N-bromosusccinimide(0.5 mM), diethyl-pyrocarbonate(2.5 mM)or 2,4- dintro-1-fluorobenzene(2.5 mM)almost completely inhibited the enzyme’s activity. Incubation of the chitotriosidase for 10 to 40 min at temperatures ranging from 30 to 80℃ revealed that the enzyme was fairly stable up to 60℃, retained 70% of its activity at 70℃ for 40 min and completely inactivated at 80℃ for 30 min. The chitotriosidase showed activity toward fluorogenic substrates 4-MU-GlcNAc2-3 and chromogenic substrates, p-nitrophenol di-N-acetyl-β-chitobioside(pNp-GlcNAc2)and p-nitrophenol tri-N-acetyl-β-chitotrioside(pNp-GlcNAc3). The catalytic efficiency (Vmax / Km) values of the enzyme for pNp-GlcNAc3, 4-MU-GlcNAc3, pNp-GlcNAc2 and 4-MU-GlcNAc2 were 317, 156, 27.9 and 7.69 nmole/min/mg/μM, respectively. In addition, the enzyme also showed activity toward chitin polymer, ethylene glycol chitin and carboxymethyl chitin. Based on the analysis of hydrolysis products of synthetic substrates and ethylene glycol chitin catalyzed by the enzyme, the chitotriosidase split chitin oligomer and polymer in an endo-manner. Incubation of the chitotriosidase with pNp-GlcNAc2 and 4-MU-GlcNAc results in formation of fluorescent 4-MU, indicating that the enzyme has transglycosidase activity.
頁次
目錄---------------------------------- Ⅰ
表目錄-------------------------------- Ⅳ
圖目錄---------------------------------- Ⅴ
縮寫表---------------------------------- Ⅷ
中文摘要------------------------------ Ⅸ
英文摘要-------------------------------- XI

第一章 前言
第一節 研究背景------------------------- 1~5
第二節 研究目的-------------------------5
第二章 文獻回顧
第一節 幾丁質水解酵素---------------------6
一 、簡介-----------------------------6
二 、幾丁質水解酵素之分類---------------6
三 、幾丁質酶之天然分佈-----------------9
四 、幾丁質酶水解幾丁質之作用形式--------33
五 、幾丁質酶之催化機制-----------------35
六 、幾丁質酶活性測定方法---------------45
第二節 N-乙醯幾丁寡醣與幾丁寡醣------------48
一、簡介----------------------------------48
二、寡醣之應用----------------------------48
三、N-乙醯幾丁寡醣與幾丁寡醣之製備--------------------50
四、N-乙醯幾丁寡醣與幾丁寡醣之測定--------------------51
第三節 幾丁質酶之應用------------------------------------52
一 、N-乙醯幾丁寡醣之製備-------------------------------52
二 、真菌原生質體製備-----------------------------------53
三 、菌體生產之應用-------------------------------------53
四 、植物病蟲害防治上之利用------------------------------54
第四節、綠竹筍之幾丁質水解酵素-------------------------56~57
第三章 材料與方法
第一節 實驗材料----------------------------------------58
壹、綠竹筍材料---------------------------------------58
貳、化學藥品-----------------------------------------58
參、重要儀器及器材------------------------------------60
第二節 實驗方法-----------------------------------------61
壹、幾丁三糖酶活性測定---------------------------------61
一、螢光光度法-------------------------------------61
貳、β-N-乙醯胺基六碳糖苷酶活性測定----------------------63
參、幾丁聚醣酶活性測定---------------------------------64
肆、幾丁質酶活性測定-----------------------------------65
伍、蛋白定量------------------------------------------66
一、BCA呈色法--------------------------------------66
二、Micro BCA呈色法--------------------------------67
陸、綠竹筍外殼幾丁三糖酶之純化--------------------------69
一、粗酵素液製備-----------------------------------69
二、硫酸銨劃分-------------------------------------69
三、Sephacyl S-100 HR 管柱膠體過濾層析--------------70
四、製備式等電焦集電泳第一次劃分---------------------71
五、製備式等電焦集電泳再劃分------------------------71
六、HiLoad Superdex 75管柱快速蛋白質液相層析-------- 72
柒、幾丁三糖酶之性質測定-------------------------------73
一、原態分子量測定---------------------------------73
二、SDS-聚丙烯醯胺膠體電泳及酵素活性染色------------ 74
三、等電點(pI)及β-N-乙醯胺基六碳糖苷酶活性染色------75
四、pH值對酵素活性之影響----------------------------77
五、溫度對酵素活性之影響----------------------------78
六、基質飽和曲線、Km值及最大反應速率Vmax值------------79
七、活化能測定-------------------------------------80
八、基質特異性-------------------------------------80
九、金屬離子及鹽類對酵素活性之影響-------------------81
十、化學修飾劑對酵素活性之影響----------------------81
十一、幾丁三糖酶水解不同聚合度合成基質pNp-GlcNAcn 之動力
學測定-----------------------------------82
十二、幾丁三糖酶水解合成基質4-MU-GlcNAc3及4-MU-GlcNAc2之
動力學測定-------------------------------82
十三、幾丁三糖酶水解不同聚合度合成基質pNp-GlcNAcn之產物分
析---------------------------------------83
十四、幾丁三糖水解不同聚合度幾丁寡糖和水溶性幾丁質之產物分析-
-----------------------------------------85
十五、幾丁三糖酶水解合成基質pNp-GlcNAc2及pNp-GlcNAc3於不
同反應時間下之pNp產物分析------------------86

十六、轉糖基作用---------------------------------87


第四章、結果與討論------------------------------------88
第一節、幾丁三糖酶及β-N-乙醯胺基六碳糖苷酶之純化-------- 88
第二節、β-N-乙醯胺基六碳糖苷酶之分子量及等電點------------ 98
第三節、幾丁三糖酶之生化性質----------------------------103
一、分子量-----------------------------------------103
二、pI值-------------------------------------------109
三、pH值對酵素之影響--------------------------------113
四、溫度對酵素之影----------------------------------119
五、Km值、最大反應速率Vmax及催化效率Vmax / Km--------- 123
六、活化能------------------------------------------130
七、金屬離子對酵素活性之影響---------------------------132
八、化學修飾劑對酵素活性之影響-------------------------134
九、基質專一性---------------------------------------136
十、人工合成基質之水解動力學---------------------------139
十一、合成基質pNp-GlcNAcn之水解產物分析----------------142
十二、幾丁三糖酶水解不同聚合度N-乙醯幾丁寡糖產物與乙二醇幾丁質
產物之HPLC分析---------------------------148
十三、幾丁三糖酶水解pNp-GlcNAc2及 pNp-GlcNAc3不同時間下之
pNp產物分析------------------------------------156
十四、轉糖基作用-------------------------------------158
第五章 結論----------------------------------------------161
第六章 未來展望-------------------------------------------162
第七章 參考文獻-------------------------------------------163
附錄
附錄一 幾丁三糖酶活性測定方法----------------------------185
附錄二 還原榶增量法-------------------------------------189
附錄三 BCA蛋白質校正曲線-------------------------------192
附錄四 Micro BCA蛋白質校正曲線-------------------------193
附錄五 迷你SDS-聚丙烯醯胺膠體電泳-----------------------194
附錄六 PhastSystem等電焦集電泳-------------------------201
附錄七 β-N-乙醯胺基六碳糖苷酶之活性染色------------------204
附錄八 對硝基苯N-乙醯幾丁寡糖之水解動力學測定-------------208
附錄九 酵素之熱失活模式---------------------------------211
附錄十 幾丁三糖酶水解對硝基苯N-乙醯幾丁二糖(pNp-GlcNAc2)及對硝基苯N-乙醯幾丁三糖(pNp-GlcNAc3)於不同反應時間下之pNp產物分析-----215
附錄十一人類原態幾丁三糖酶之立體結構分析數據---------------- 221
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