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研究生:葉貞妤
研究生(外文):Chen-Yu Yeh
論文名稱:綠竹筍(Bambusaoldhamii)外殼幾丁聚醣酶之純化及性質研究
論文名稱(外文):Studies on the purification and properties of chitosanase from outer hulls of bamboo shoots (Bambusa oldhamii)
指導教授:張珍田張珍田引用關係
指導教授(外文):Chen-Tein 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:2003
畢業學年度:91
語文別:中文
論文頁數:158
中文關鍵詞:綠竹筍幾丁聚醣幾丁聚醣酶純化
外文關鍵詞:bamboo shootschitosanchitosanasepurification
相關次數:
  • 被引用被引用:6
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幾丁聚醣可抑制微生物生長,並改善採收後蔬果之保鮮。本研究將新鮮採收綠竹筍浸泡包覆幾丁聚醣後置室溫(24 ± 2℃)貯存18至66小時,試驗結果發現可降低貯存期間綠竹筍之濕重下降速率,並增加幾丁聚醣酶活性。綠竹筍外殼及可食部基部均含有幾丁聚醣酶活性,其中以外殼含量較高,而可食部上端部則活性頗低。外殼粗酵素萃取液所含幾丁聚醣酶經40~80 % 飽和度硫酸銨劃分、膠體過濾層析及Mono P HR 管柱等電焦集層析等連續步驟純化,可使其純度提高447倍並獲得7.5 %活性收率。純化之幾丁聚醣酶經SDS-PAGE分析顯示已達均質純度,其水解幾丁聚醣之最適pH為5.0,最適溫度為40℃,由動力學分析顯示酵素水解幾丁聚醣並不遵守Michaelis-Menten動力學方程式,以不同去乙醯度幾丁聚醣為基質顯示酵素對30至90 %去乙醯度幾丁聚醣均具水解能力,其中以對50 %去乙醯度幾丁聚醣具有最高活性,然酵素對幾丁質、幾丁三糖衍生物4-MU-β-(GlcNAc)3及纖維素則不具水解活性。由熱穩定性分析顯示酵素於60℃貯存1小時顯著失去活性,其熱失活屬二級失活模式,化學修飾劑2,4-dinitro-1-fluorobenzene(5 mM)完全抑制酵素活性,離胺酸(Lys)可能為構成活性中心所必須或位於其附近。以SDS-PAGE測得分子量為16.6 kDa,以膠體過濾層析管柱測得酵素分子量亦接近此值,故為單元體酵素,等電焦集電泳測得等電點為7.1,由幾丁聚醣水解產物分析顯示酵素以內切方式水解幾丁聚醣,首先幾丁聚醣水解為低分子量幾丁聚醣,再進一步水解為幾丁寡糖,幾丁寡糖組成則有待進一步探討與確認。
Chitosan can inhibit the growth of a wide range of microorganisms. It can also improve the store ability and decay of postharvest fruits and vegetables. In this study, fresh harvest bamboo shoots were coated with chitosan and stored at room temperature (24 ± 2℃) for 18 to 66 hours. The chitosan treated bamboo shoots showed less decrease than the control group in fresh weight during storage. This treatment retarded the decrease of chitosanase activity in the outer hulls of bamboo shoots and enhanced the increase of chitosanase activity in the edible base section of bamboo shoots during storage. The outer hulls had higher chitosanase activity then the edible base section. Only small amount of chitosanase activity was detected in the edible upper section of bamboo shoots. Chitosanase was purified from the crude extract of hulls of bamboo shoots by successive steps of 40 to 80 % saturation of ammonium sulfate fractionation, gel filtration and Mono P HR column chromatofocusing. By these steps, the enzyme was purified 447-fold, and the total yield was 7.5 %. The purified enzyme was homogeneous, as analyzed by SDS-PAGE. For chitosan hydrolysis, the enzyme had an optimal pH of 5.0, optimal temperature of 40℃. From kinetic analysis, the enzyme didn’t obey Michaelis-Menten equation for chitosan hydrolysis. The chitosan polymers with 30 to 90 % deacetylation were susceptible to the enzyme action. The most susceptible to hydrolysis was 50 % deacetylation. However, the enzyme had no activity toward chitin, 4-MU-β-(GlcNAc)3 and CM-cellulose. From thermo- stability analysis, the enzyme was significantly lost activity above 60 ℃. The rate of thermal inactivation of the enzyme was not a first-order rate denaturation. Chemical modification agent 2,4-dinitro-1-fluorobenzene (5 mM) completely inhibited the enzyme activity. Lysine is probably located at or near the active site of the enzyme. The molecular mass of the enzyme was 16.6 kDa, as estimated by SDS-PAGE. This value was close to that estimated by gel filtration, indicating the enzyme to be monomer. The isoelectric point (pI) of the enzyme was 7.1, as estimated by isoelectric focusing electrophoresis. From the products composition of chitosan hydrolysis for different time by the enzyme indicate the enzyme split chitosan in an endo-manner. The enzyme first hydrolyzed chitosan to yield low molecular weight chitosan and then further hydrolyzed into chito-oligosaccharides. The composition of the end product chito-oligosaccharides remains to be further confirmed.
目錄--------------------------------------------------------------------------------Ⅰ~Ⅳ
表目錄-----------------------------------------------------------------------------Ⅴ
圖目錄-----------------------------------------------------------------------------Ⅵ
中文摘要--------------------------------------------------------------------------Ⅸ
英文摘要--------------------------------------------------------------------------Ⅹ
第一章 前言--------------------------------------------------------------1
第二章 文獻回顧--------------------------------------------------------3
第一節 綠竹筍-----------------------------------------------------------3
一、 簡介-------------------------------------------------------------3
二、 竹筍之品種與分布-------------------------------------------3
三、 綠竹筍採收後之生理與保鮮-------------------------------9
第二節 幾丁質與幾丁聚醣之結構---------------------------------11
一、 幾丁質---------------------------------------------------------11
二、 幾丁聚醣------------------------------------------------------12
第三節 幾丁質與幾丁聚醣之理化學性質------------------------13
第四節 幾丁質、幾丁聚醣與幾丁寡醣之生理功能------------16
一、 降低血脂------------------------------------------------------16
二、 免疫及抗腫瘤活性------------------------------------------18
三、 抗菌活性------------------------------------------------------18
四、 植物防禦性酵素之誘發------------------------------------20
第五節 植物幾丁質酶和幾丁聚醣酶------------------------------22
第六節 本研究之動機與目的---------------------------------------34
第三章 材料與方法---------------------------------------------------35
第一節 實驗材料、試劑與設備------------------------------------35
一、 實驗材料-------------------------------------------------------35
二、 化學藥品-------------------------------------------------------36
三、 重要器材及儀器----------------------------------------------37
第二節 實驗方法-------------------------------------------------------38
一、 綠竹筍採收後之幾丁聚醣處理----------------------------38
二、 水分測定-------------------------------------------------------39
三、 蛋白質定量----------------------------------------------------40
四、 幾丁三糖活性測定----------------------------------------42
五、 幾丁質活性測定-------------------------------------------44
六、 幾丁聚醣活性測定----------------------------------------46
七、 過氧化活性測定-------------------------------------------49
八、 胺基葡萄糖之呈色定量-------------------------------------51
九、 總醣之酚-硫酸呈色定量------------------------------------52
十、幾丁聚醣酶純化------------------------------------------------53
1. 粗酵素液製備-----------------------------------------------53
2. 硫酸銨劃分--------------------------------------------------53
3. HiLoad Superdex 75管柱快速蛋白質液相層析------55
4. Mono P等電焦集層析-------------------------------------55
5. Superose 12 HR膠體過濾層析---------------------------56
十一、 幾丁聚醣酶性質測定----------------------------------------57
1. 最適pH---------------------------------------------------------57
2. 最適溫度-------------------------------------------------------57
3. SDS-聚丙烯醯胺膠體電泳----------------------------------58
4. 等電點( pI )----------------------------------------------------62
5. Superose 12 HR 10/30膠體過濾層析之原態
分子量測定-----------------------------------------------------65
6. 幾丁聚醣去乙醯度對幾丁聚醣酶活性影響-------------67
7. 基質飽和曲線-------------------------------------------------67
8. 金屬鹽類對酵素活性之影響-------------------------------68
9. 化學修飾劑對酵素活性之影響----------------------------68
10. 幾丁聚醣水解產物分析------------------------------------72
11. Dextran之Superose 12 HR管柱膠體過濾層析校正
曲線製作------------------------------------------------------74
12. Chitosan oligomer之Superdex peptide PE 7.5/300
管柱膠體過濾層析校正曲線製作------------------------74
十二、 乙二醇幾丁質之合成----------------------------------------76
十三、 不同去乙醯度幾丁聚醣之製備----------------------------77
十四、 N-端胺基酸序列分析----------------------------------------78
十五、 統計分析-------------------------------------------------------79
第四章 結果與討論-----------------------------------------------------80
第一節 幾丁聚醣處理對綠竹筍室溫貯存濕重及數種防禦
性酵素活性之影響-------------------------------------------80
一、 濕重之影響----------------------------------------------------80
二、 幾丁聚醣酶活性之影響-------------------------------------80
三、 幾丁質酶活性之影響----------------------------------------85
四、 過氧化酶活性之影響----------------------------------------86
第二節 幾丁聚醣酶純化----------------------------------------------92
第三節 幾丁聚醣酶生化學性質-------------------------------------99
一、 最適溫度-------------------------------------------------------99
二、 最適pH---------------------------------------------------------100
三、 熱穩定性-------------------------------------------------------101
四、 基質飽和曲線-------------------------------------------------102
五、 不同去乙醯度幾丁聚醣對酵素活性之影響-------------104
六、 分子量----------------------------------------------------------105
七、 活性影響物質-------------------------------------------------108
八、 等電點----------------------------------------------------------112
九、 基質專一性----------------------------------------------------114
十、 水解產物分析-------------------------------------------------115
十一、 N-端胺基酸序列分析---------------------------------------121
第四節 不同品種竹筍外殼幾丁聚醣酶----------------------------121
第五節 討論-------------------------------------------------------------124
第五章 結論與展望-----------------------------------------------------126
第六章 參考文獻--------------------------------------------------------127
附 錄
附錄一 幾丁質去乙醯酶活性測定----------------------------------143
附錄二 溫度對酵素穩定性影響之測定----------------------------145
附錄三 蛋白質測定標準曲線----------------------------------------148
附錄四 4-MU螢光校正曲線-----------------------------------------149
附錄五 N-乙醯胺基葡萄糖及胺基葡萄糖定量校正曲線-------150
附錄六 N-端胺基酸序列分析數據----------------------------------151
表 目 錄

表一 台灣主要食用竹筍種類--------------------------------------------------5
表二 植物來源幾丁質酶之特性-----------------------------------------------25
表三 葉、種子或果實抽出液幾丁聚醣酶電泳分析之相對移動度
(Rm)和相對活性強度(Ri)-------------------------------------------30
表四 幾丁聚醣酶之來源與性質-----------------------------------------------33
表五 各種化學修飾劑之溶劑及反應緩衝液--------------------------------71
表六 綠竹筍殼幾丁聚醣酶之純化結果--------------------------------------93
表七 各種金屬離子及鹽類對幾丁聚醣酶活性之影響-------------------109
表八 各種化學修飾劑對幾丁聚醣酶之影響-------------------------------110
表九 不同濃度DNFB和2-mercaptoethanol對幾丁聚醣活性之
影響-------------------------------------------------------------------------111
表十 幾丁聚醣酶之基質專一性----------------------------------------------114

圖 目 錄

圖一 綠竹筍與烏腳綠竹筍之外觀圖---------------------------------------------6
圖二 麻竹筍植株與筍和桂竹筍之外觀圖---------------------------------------7
圖三 箭竹筍與孟宗竹筍之外觀圖------------------------------------------------8
圖四 竹筍中含有peroxidase- H2O2- tyrosine system可產生螢光物質---10
圖五 幾丁質(A)與纖維素 (B)之化學結構式-----------------------------------11
圖六 幾丁質(A)與幾丁聚醣 (B)之化學結構式--------------------------------12
圖七 a型幾丁質的結晶構造------------------------------------------------------14
圖八 b型幾丁質的結晶構造------------------------------------------------------15
圖九 膽酸結合清除劑和膽固醇生合成抑制劑治療高膽固醇之
效應--------------------------------------------------------------------------17
圖十 幾丁聚醣對腐皮鐮孢子菌(Fusarium solani)和豌豆間交互作用
之基因作用模式假說圖-------------------------------------------------21
圖十一 參與幾丁質代謝之幾丁質相關酵素----------------------------------23
圖十二 不同類型幾丁質酶結構差異圖解-------------------------------------26
圖十三 幾丁質酶研究流程圖----------------------------------------------------28
圖十四 綠竹筍外觀圖-------------------------------------------------------------35
圖十五 綠竹筍室溫貯藏過程之濕重變化-------------------------------------81
圖十六 幾丁聚醣處理後置室溫貯存十天之綠竹筍縱切面比較----------82
圖十七 綠竹筍室溫貯存過程之native-PAGE幾丁聚醣酶活性圖譜-----83
圖十八 綠竹筍室溫貯藏過程中之幾丁聚醣酶活性變化-------------------84
圖十九 綠竹筍貯藏過程中之幾丁質酶活性變化----------------------------87
圖二十 綠竹筍貯藏過程中幾丁三醣酶活性變化----------------------------88
圖二十一 pH對過氧化酶活性之影響-----------------------------------------89
圖二十二 溫度對過氧化酶活性之影響---------------------------------------90
圖二十三 綠竹筍貯藏過程中之過氧化酶活性變化------------------------91
圖二十四 幾丁聚醣酶之硫酸銨劃分------------------------------------------94
圖二十五 幾丁聚醣酶之HiLoad Superdex 75 管柱膠體過濾層析------95
圖二十六 幾丁聚醣酶之Mono P HR 5/20管柱等電焦集層析-----------96
圖二十七 幾丁聚醣酶與Polybuffer 74之Superose 12 HR管柱膠
體過濾層析------------------------------------------------------------97
圖二十八 幾丁聚醣酶之SDS-PAGE及水解乙二醇幾丁聚醣之
活性染色---------------------------------------------------------------98
圖二十九 溫度對幾丁聚醣酶活性之影響------------------------------------99
圖三十 pH對幾丁聚醣酶活性之影響--------------------------------------100
圖三十一 幾丁聚醣酶於不同溫度下之殘留活性百分率對數值對
保溫時間作圖---------------------------------------------------------101
圖三十二 幾丁聚醣濃度對幾丁聚醣酶活性影響---------------------------102
圖三十三 乙二醇幾丁聚醣濃度對幾丁聚醣酶活性影響------------------103
圖三十四 不同去乙醯度幾丁聚醣對酵素活性之影響---------------------104
圖三十五 幾丁聚醣酶之Superose 12 HR管柱膠體過濾層析------------105
圖三十六 幾丁聚醣酶之Superose 12 HR管柱膠體過濾層析之分
子量測定---------------------------------------------------------------106
圖三十七 幾丁聚醣酶之SDS-PAGE分子量測定--------------------------107
圖三十八 幾丁聚醣酶之等電焦集電泳---------------------------------------112
圖三十九 幾丁聚醣酶之等電焦集電泳等電點(pI)測定-------------------113
圖四十 幾丁聚醣產物之Superose 12 HR管柱膠體過濾層析--------116
圖四十一 幾丁聚醣水解產物之Superose 12 HR管柱膠體過濾
層析分子量測定-----------------------------------------------------117
圖四十二 幾丁聚醣不同時間水解產物之Superose 12 HR管柱
膠體過濾層析--------------------------------------------------------118
圖四十三 幾丁聚醣水解產物之Superdex peptide PE 7.5/300
管柱膠體過濾層析--------------------------------------------------119
圖四十四 幾丁聚醣水解產物於Superdex peptide PE 7.5/300管柱
之分子量測定--------------------------------------------------------120
圖四十五 幾丁聚醣酶之Superdex peptide 7.5/300管柱膠體過濾-----122
層析
圖四十六 不同品種竹筍外殼之幾丁聚醣酶活性和SDS-PAGE
活性圖譜-------------------------------------------------------------123
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