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研究生:陳雅惠
論文名稱:龍鬚菜果膠酯酶所催化轉醯基反應之探討
論文名稱(外文):The study on the transacylation reaction catalyzed by pectinesterase from tendril shoots of chayote
指導教授:吳明昌張鴻民張鴻民引用關係江啟銘江啟銘引用關係
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
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:169
中文關鍵詞:龍鬚菜果膠酯酶轉醯基反應
外文關鍵詞:tendril shoots of chayote (Sechium edule (Jacq.) Swartz)pectinesterasetransacylation
相關次數:
  • 被引用被引用:9
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中文摘要
學號:M8936019
論文名稱:龍鬚菜果膠酯酶所催化轉醯基反應之探討 總頁數169
學校名稱:國立屏東科技大學食品科學系碩士班
畢業時間及摘要別:九十學年度第二學期碩士學位論文摘要
研究生:陳雅惠 指導教授:吳明昌 博士
張鴻民 博士
江啟銘 博士
論文摘要內容:
本研究以龍鬚菜為材料來探討PE之轉醯基反應,為了更確切瞭解龍鬚菜PE所催化的轉醯基反應,因此將龍鬚菜PE加以萃取及純化,以瞭解龍鬚菜PE是否能催化轉醯基反應及最適反應條件。此外,並利用不同的方法來證明轉醯基反應,所得結果如下:
龍鬚菜PE活性以葉部最高,以葉部為材料,先以蒸餾水均質,再以pH 8.0的磷酸緩衝液反覆水洗後,再用1 M 氯化鈉(NaCl)萃取PE,此步驟可提高比活性約2.1倍。經硫酸銨沉澱劃分收集40~70%飽和度,進行CM-Sepharose CL-6B 離子交換層析後再以Sephacryl S-100膠濾層析純化,即可得到純化的PE酵素。以SDS-PAGE鑑定呈單一蛋白質帶,表示酵素純度己達均質,利用膠濾層析法求得分子量為50 kDa為一單元體。
以Fractogel TSK HW-65(S)膠濾層析的方法來探討龍鬚菜PE催化轉醯基反應的最適條件。結果發現當龍鬚菜PE在pH 6.5、0.2 M NaCl濃度及60℃下反應30 min時,具有最高轉醯基反應效果。此外,所產生的新酯鍵(非甲基酯鍵)不會被PE所水解。
本研究開發幾種新的檢測技術來偵測PE轉醯基反應,例如黏度、硬度及濁度。結果發現隨著PE酵素活性或反應時間的增加,反應前後在黏度、膠體硬度及濁度皆有顯著改變。顯示以上數種新開發檢測技術,應可利用在PE轉醯化活性之檢測上。
關鍵字:龍鬚菜、果膠酯酶、轉醯基反應
Abstract
In this study, the transacylation catalyzed by pectinesterase(PE) from tendril shoots of chayote(TSC) was investigated, from PE TSC was extracted and partially purified to find the optimal conditions for transacylation reactions, and the transacylation was proved by differnet methods. The results obtained were as follows:
It was found that PE activity was higher in the leaves of TSC, therefore, the leaves were firstly homogenized with water, then repeatedly washed with in pH 8.0 phosphate buffer, finally, the crude PE was extracted with 1 M NaCl solution to increase the specific activity to 2.1 fold. The crude PE extract was fractionated at 40~70% saturation of ammonium sulfate separated by CM-sepharose CL-6B cation exchange chromatography, and further separated with Sephacryl S-100 gel permeation chromatography. SDS-PAGE appraisal single band of protein was used as the pure PE, the molecular weight of PE was 50,000 dalton proved by gel filtration.
Fractogel TSK HW-65(S) gel permeation chromatography was used to study PE transacylation from TSC. It was found that the optimum condition for transacylation in TSC was at 60℃, pH 6.5
for 30 min. Besides, the new ester linkages (non-methoxy ester linkages) would not hydrolyzed by PE.
In this study, new methods were built to measure the transacylation reaction from PE, such as viscosity, hardness and turbidity. It was found when the PE activity or the reaction time increased, the viscosity, gel hardness and turbidity of the reaction solution were changed remarkably, so the above techniques could be used to detect the transacylation reaction catalyzed by PE.
Key words:tendril shoots of chayote (Sechium edule (Jacq.) Swartz), pectinesterase, transacylation
目錄
頁次
中文摘要--------------------------------------------------- Ⅰ
英文摘要--------------------------------------------------- Ⅲ
誌謝------------------------------------------------------- Ⅴ
目錄------------------------------------------------------- Ⅵ
圖索引------------------------------------------------------ⅩⅠ
表索引----------------------------------------------------ⅩⅠⅤ
第一章、前言------------------------------------------------ 1
第二章、文獻整理---------------------------------------------3
2.1果膠酯酶--------------------------------------------------3
2.1.1果膠酯酶及其作用機制--------------------------- ----3
2.1.2 PE作用方式及基質專一性----------------------- -------5
2.2 PE的理化性質---------------------------------------------9
2.2.1分子量和等電點------------------------------------------9
2.2.2 PE之化學組成---------------------------------------- --9
2.3 PE活性測定及影響PE活性之因素----------------------------10
2.3.1 PE活性的測定方式--------------------------------------10
2.3.2影響PE活性的因子探討-----------------------------------12
2.4 PE在蔬果發育過程中所扮演的角色及生理意義------------- 18
2.4.1 PE在蔬果發育過程中所扮演的角色---------------------- 18
2.4.2 PE及其同功酶在蔬果組織中之分布與消長------------- 20
2.4.3 PE同功酶之生理意義------------------------------------22
2.5 PE在蔬果之加工上之意義----------------------------------27
2.5.1中溫預煮時PE與蔬菜加工質地之關係------------------ 27
2.5.2 PE對果實加工品之影響---------------------------------30
2.6果膠(物)質---------------------------------------------- 32
2.6.1果膠的理化特性-----------------------------------------32
2.6.2果膠質之化學組成及分子結構-----------------------------34
2.6.3果膠質分子之化學變化-----------------------------------39
2.6.4果膠之物理性質-----------------------------------------43
2.6.5果膠的成膠機制-----------------------------------------46
2.6.6果膠在食品上之應用----------------------------------48
2.7有關梨瓜與龍鬚菜之介紹---------------------------------- 49
第三章、材料與方法------------------------------------------51
3.1 實驗材料------------------------------------------------51
3.2實驗藥品------------------------------------ ------------51
3.2.1酵素分析試藥------------------------------------------51
3.2.2電泳試藥----------------------------------------------52
3.3裝置與儀器-----------------------------------------------53
3.3.1酵素分析儀器-------------------------------------- ----53
3.3.2物理測定儀器-----------------------------------------54
3.4實驗方法-------------------------------------------------54
3.4.1實驗設計-----------------------------------------------54
3.5 PE之萃取及純化------------------------------------------54
3.5.1梨瓜及龍鬚菜PE之萃取---------------------------------54
3.5.2硫酸銨劃分-------------------------------------------56
3.5.3 離子交換管柱層析------------------------------------59
3.5.4 膠濾層析--------------------------------------------59
3.5.5蛋白質電泳分析---------------------------------------60
3.6 PE活性測定方法------------------------------------------67
3.7蛋白質含量的測定-----------------------------------------69
3.8影響PE活性因子之探討----------------------------------- 70
3.8.1 pH之影響--------------------------------------------70
3.8.2溫度之影響-------------------------------------------70
3.8.3氯化鈉之影響------------------------------ ----------71
3.8.4 PE儲存安定性--------------------------------------- 71
3.9 PE之轉醯基反應試驗------------------------- ------------71
3.9.1果膠之前處理-----------------------------------------71
3.9.2轉醯基反應條件之探討---------------------------------72
3.9.3轉醯基反應之判定-------------------------------------72
3.9.4果膠含量之測定---------------------------------------73
3.10物理測定------------------------------------------------73
3.10.1膠體硬度測定------------------------------------------73
3.10.2黏度的測定--------------------------------------------74
3.10.3濁度測定法------------------------------------------74
第四章、結果與討論 ------------------------------- ---------76
4.1龍鬚菜PE之萃取及純化-------------------------------------76
4.1.1龍鬚菜PE之萃取----------------------------------------76
4.2龍鬚菜葉部之硫酸銨劃分-----------------------------------86
4.2.1 龍鬚菜葉部之硫酸銨劃分(細劃分) --------------------86
4.2.2 龍鬚菜葉部之硫酸銨劃分(主劃分)---------------------86
4.3 CM-Sepharose CL-6B 離子交換層析之純化結果------------ 89
4.4膠濾層析之純化結果---------------------------------------91
4.5龍鬚菜葉部PE之電泳分析-----------------------------------95
4.5.1 SDS-聚丙烯醯胺膠體電泳------------------------------- 95
4.5.2酸性電泳和PE活性染色-----------------------------------98
4.6龍鬚菜葉部PE性質之探討----------------------------------100
4.6.1不同pH對龍鬚菜葉部PE活性之影響------------------ 100
4.6.2不同濃度之NaCl對龍鬚菜葉部PE活性之影響------- 100
4.6.3不同溫度對龍鬚菜葉部PE活性之影響----------------- 102
4.6.4龍鬚菜葉部PE之儲存安定性---------------------------- 104
4.7龍鬚菜PE催化轉醯基反應最適條件之探討------------------ 107
4.7.1 pH對龍鬚菜PE轉醯基反應之影響--------------------- 108
4.7.2不同NaCl濃度對龍鬚菜PE催化轉醯基反應之影響- 112
4.7.3溫度對龍鬚菜PE轉醯化活性之影響-------------------- 113
4.7.4不同果膠濃度對龍鬚菜PE轉醯基反應的影響-------- 115
4.7.5反應時間對龍鬚菜PE所催化轉醯基反應之影響----- 117
4.8 物理測定-----------------------------------------------119
4.8.1利用膠體硬度測定法檢測龍鬚菜PE轉醯化活性----- 119
4.8.2利用粘度測定法檢測龍鬚菜PE轉醯化活性----------- 122
4.8.3利用濁度測定法檢測龍鬚菜PE轉醯化活性----------- 123
4.9 PE轉醯基活性不同測定方法之比較------------------------ 137
第五章、結論-----------------------------------------------142
第六章、參考文獻-------------------------------------------144
符號索引---------------------------------------------------168
作者簡介---------------------------------------------------169
圖索引
頁次
圖一、PE作用方式-------------------------------------------------------7
圖二、PE催化水解及轉醯基反應的可能機制----------------------29
圖三、熟成期間果膠性狀變化與酵素作用情形-------------------35
圖四、果膠分子構造-----------------------------------------------------38
圖五、果膠醣苷鍵之化學變化(a)β-脫去反應(b)水解反應--------44
圖六、果膠之凝膠機制(a)高甲氧果膠 (b)低甲氧果膠------------50
圖七、本研究實驗內容簡要--------------------------------------------55
圖八、龍鬚菜PE之抽取與純化程序流程圖-------------------------57
圖九、龍鬚菜粗PE酵素溶液經硫酸銨沉澱劃分之流程圖-------58
圖十、不同蔬果的PE活性之比較-----------------------------------78
圖十一、不同成熟度梨瓜PE之活性變化----------------------------79
圖十二、抽取溶液之NaCl濃度及抽取液放置時間對龍鬚菜PE抽取率的影響-------------------------------------------------82
圖十三、龍鬚菜不同部位間PE活性含量之差異-------------------83
圖十四、龍鬚菜葉部PE粗抽物之硫酸銨沉澱劃分(細劃分)----87
圖十五、龍鬚菜葉部PE粗抽物之硫酸銨沉澱劃分(主劃分)-----88
圖十六、經40~70%飽和度硫酸銨沉澱回收之龍鬚菜葉部PE之
CM-Sepharose CL-6B離子交換層析圖-------------------90
圖十七、龍鬚菜葉部PE之Sephacryl S-100膠濾層析圖----------92
圖十八、龍鬚菜葉部PE分子量測定之Sephacryl S-100 膠濾層析圖------------------------------------------------------------------94
圖十九、純化後龍鬚菜葉部PE之SDS-PAGE----------------------96
圖二十、以SDS-PAGE決定龍鬚菜葉部PE分子量----------------97
圖二十一、龍鬚菜葉部PE的酸性電泳(a)和活性染色(b)圖------99
圖二十二、龍鬚菜葉部PE活性之pH效應-------------------------101
圖二十三、龍鬚菜葉部PE活性之鹽濃度效應--------------------103
圖二十四、龍鬚菜葉部PE活性之溫度效應-----------------------105
圖二十五、經膠濾層析後龍鬚菜葉部PE在4℃下的儲存安定性--------------------------------------------------------------106
圖二十六、以膠濾層析製作不同分子量多醣類標準曲線-------110
圖二十七、龍鬚菜PE於不同pH轉醯基反應之膠濾層析圖----111
圖二十八、龍鬚菜PE於不同NaCl濃度下轉醯基反應之膠濾層析圖--------------------------------------------------------114
圖二十九、龍鬚菜PE於不同溫度(25, 50, 60, 85℃)PE轉醯基反應之膠濾層析圖--------------------------------------116
圖三十、龍鬚菜PE於不同果膠濃度(0.1~1%)下轉醯基反應之膠
濾層析圖----------------------------------------------------118
圖三十一、商品果膠溶液經龍鬚菜PE於pH6.5,30℃下反應2hr (A),4hr (B)後之膠濾層析圖,及由管柱回收的果膠在經0.1M 冰冷的Na2CO3於4℃處理24小時(A”)、(B”)後之膠濾層析圖-------------------------------------------121
圖三十二、不同酵素活性對膠體硬度之影響----------------------124
圖三十三、不同的PE酵素活性及反應時間對黏度之影響-----126
圖三十四、(a)果膠溶液添加PE及經熱不活化PE反應後之吸收光譜圖-----------------------------------------------------128
(b)果膠溶液添加PE及經熱不活化PE反應後之吸收光譜圖的差值--------------------------------------------129
圖三十五、不同酵素活性對PE轉醯基活性之影響-------------131
圖三十六、轉醯基反應於不同時間之吸光值--------------------132
圖三十七、不同溫度下對於PE轉醯基反應活性之影響--------135
圖三十八、不同鹽濃度下對於PE轉醯基活性之影響-----------136
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