臺灣博碩士論文加值系統

Polyphenol oxidase（PPO）又稱Tyrosinase（E.C. 1.14.18.1），為一種內含銅離子之酵素，使用氧分子催化兩種不同的反應，首先將monophenols經脫氫氧化作用形成o-diphenols ，再於含氧的環境下氧化o-diphenols 形成o-quinones。繼續進行非酵素性聚合反應產生非水溶性之褐色物質。此酵素廣泛的存在於微生物、動物、以及植物中，並且會造成動物形成黑色素(melanian) 以及蔬菜水果褐變反應(browning reaction)。我們研發出新的方式”PAGE-blot assay”可以快速鑒別及分析PPO之存在及分子量，而此技術更提供了一個初步的研究方法來找出潛在於蔬果中的天然抑制劑，並利用此方法研究分析荔枝皮PPO。
本實驗利用HPLC及SDS-PAGE萃取，成功的純化荔枝皮PPO，其分子量分別為86及66 kDa，使用catechol當 PPO的substrate定義出86 kDa的 PPO，其Km及Vmax分別為66 mM及382 �嵱/min；而66 kDa PPO之值分別為102 mM及290 �嵱/min。有趣的是86 kDa PPO可使用，3,3’-diaminobenzidine 及 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)當為substrate，但是它們通常只會被雙氧水氧化產生的自由基所氧化反應，故荔枝PPO除了氧化酵素的活性外，同時具有超氧化酵素之活性，即可解釋荔枝之快速褐變的現象。此外，我們證明荔枝內含揮發性之PPO抑制劑，且藉由掃瞄式電子顯微鏡觀察荔枝皮表面結構，其結構隨時間的增長由平滑緊密漸漸地變為粗糙孔洞變大，促使PPO抑制劑揮發愈快速，降低對PPO之制劑效果，加速了褐變現象。這些新發現解釋了荔枝採收後果皮快速褐變現象之反應機制。此外，PPO於人類中又稱為Tyrosinase會造成皮膚形成黑斑，發現此揮發性的PPO抑制劑，可以用於發展新的化妝、保養品。

Polyphenol oxidase (PPO) is a copper-containing enzyme that catalyzes the chain-oxidation from monophenol or polyphenols to o-diphenols and subsequent o-quinones. The enzyme reflects the browning reaction in fruits. In the present study, we investigated the oxidation activity of PPO in litchi pericarp and the mechanism by which PPO instantly makes pericarp browning.
PPO of litchi pericarp was initially extracted and isolated through gel filtration chromatography and then eluted directly from SDS-PAGE. Two molecular forms of litchi PPO were identified as 86 and 66 kDa, and thereafter named as PPO-86 and PPO-66, respectively. The Km and Vmax were determined as 66 mM and 382 mM/min for PPO-86 and 102 mM and 290 mM/min for PPO-66, respectively. Most importantly, PPO-86 could trigger the 3,3’-diaminobenzidine and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation, which was not found in other plant PPO. Thus, the unique PPO activity of litchi might account for its superior rapid-browning reaction. In addition, we show the presence of a potent volatile inhibitor(s) for PPO in litchi pericarp. The surface of post-harvesting litchi pericarp revealed an opening ultra structure under the scanning electron macroscopic examination, therefore allowing an instant evaporation of PPO inhibitor. As such, the PPO oxidation was proceeded. The novel finding clarifies the mechanism involved in the rapid browning phenomenon of post-harvesting litchi pericarp. Since the PPO is also know existed in human as tyrosinase responsible for the formation of “darkening spots” on skin, the finding of evaporation of potent PPO inhibitor may be potentially used as a strategy in developing a novel cosmetic product.

中文摘要---------------------------------------------------------------------------------i
英文摘要--------------------------------------------------------------------------------iii
目錄--------------------------------------------------------------------------------------iv
表目錄-----------------------------------------------------------------------------------ix
圖目錄-----------------------------------------------------------------------------------x

第一章、緒論---------------------------------------------------------------------------1

1.1荔枝之簡介-------------------------------------------------------------------------1
1.2褐變反應（browning reaction）概論------------------------------------------2
1.3 Polyphenol oxidase（PPO）之歷史及特性---------------------------------- 3
1.3.1 PPO之歷史------------------------------------------------------------------ 3
1.3.2 PPO之特性------------------------------------------------------------------ 3
1.3.3 PPO之substrate------------------------------------------------------------4
1.4 PPO於植物中所扮演之角色---------------------------------------------------4
1.5 廣泛用於預防褐化反應之方法-------------------------------------------------5
1.5.1利用PPO抑制劑降低PPO活性----------------------------------------- 5
1.5.2二氧化硫燻蒸與浸酸處理------------------------------------------------- 6
1.5.3 加熱及浸酸處理------------------------------------------------------------ 6
1.5.4 甲殼素處理------------------------------------------------------------------7
1.6研究目的----------------------------------------------------------------------------7

第二章、材料與方法-----------------------------------------------------------------9

2.1 開發快速檢測PPO活性SDS-polyacrylamide gel electrophoresis（PAGE-blot assay）之方法---------------------------------------------------9
2.1.1 catechol paper之研發------------------------------------------------------ 9
2.1.2固定於3MM層析紙上的catechol最適濃度之測試-----------------9
2.1.3 PAGE-blot assay方法之建立（Bio-Rad系統）---------------------10
2.1.4利用市售洋菇PPO 做PAGE-blot assay靈敏度之測試----------- 12
2.2 PAGE-blot assay之應用------------------------------------------------------12
2.2.1蔬菜水果之取得-----------------------------------------------------------12
2.2.2蔬菜水果粗萃取物之準備-----------------------------------------------12
2.2.3測定不同蔬果粗萃取物之PPO酵素活性----------------------------12
2.2.4不同蔬菜水果PPO活性與PAGE-blot assay之比較---------------13
2.2.5洋菇PPO之純化----------------------------------------------------------13
2.3 測試植物內生性抑制劑及化學成分之抑制劑對洋菇PPO活性抑制
效果-------------------------------------------------------------------------------14
2.3.1蕃茄與芭樂酒精萃取液之準備-----------------------------------------14
2.3.2 化學成分之抑制劑對洋菇PPO活性抑制效果之測試-------------15
2.3.3 利用catechol paper測試蕃茄與芭樂酒精萃取液對於洋菇PPO
活性之抑制效果-----------------------------------------------------------15
2.3.4 蕃茄與芭樂酒精萃取液對於洋菇PPO活性抑制效果之酵素動力
學-----------------------------------------------------------------------------15
2.4 荔枝皮PPO之研究-------------------------------------------------------------16
2.4.1荔枝皮PPO之純化--------------------------------------------------------16
2.4.2 10 % SDS-PAGE、Native- PAGE及PAGE-blot assay分析荔枝皮PPO純化前後之差異-----------------------------------------------------------17
2.4.3分析荔枝皮PPO純化各階段之產率及回收率-----------------------18
2.4.4利用Lineweaver-Burk plots 定義不同分子量荔枝皮PPO（求Km及Vmax）----------------------------------------------------------------------19
2.4.5 高分子量荔枝皮PPO具催化特殊substrate能力之測定----------19
2.5 分析荔枝內生性之PPO 抑制劑----------------------------------------------20
2.5.1 揮發性PPO抑制劑之發現-----------------------------------------------20
2.5.2荔枝皮揮發性PPO 抑制劑之準備--------------------------------------20
2.5.3分析自荔枝皮中萃取之揮發性PPO抑制劑對PPO之抑制效果--20
2.5.4荔枝皮粗萃取液揮發不同時間後之PPO活性之測定---------------21
2.6 掃瞄式電子顯微鏡(Scanning Electron Microscope；SEM)之觀察------21
2.6.1 荔枝皮之前處理------------------------------------------------------------21
2.6.2 臨界點乾燥(Critical Point Dryer；CPD) --------------------------- 22
2.6.3 掃瞄式電子顯微鏡之觀察---------------------------------------------22
2.7 免疫染色(immunolocalization)定位荔枝皮PPO存在之區域---------22
2.7.1荔枝皮PPO多株抗體之製備------------------------------------------22
2.7.2 荔枝皮冷凍切片---------------------------------------------------------23
2.7.3 荔枝皮組織免疫染色---------------------------------------------------23

第三章、結果與討論--------------------------------------------------------------25

3.1 開發快速檢測PPO活性（PAGE-blot assay）之方法------------------25
3.1.1 使用catechol當為substrate作用於PPO之酵素反應------------25
3.1.2 PAGE-blot assay之基本原理-------------------------------------------26
3.1.3 PAGE-blot assay方法之建立-------------------------------------------26
3.1.4固定於3MM層析紙上的catechol最適濃度之測試---------------27
3.1.5利用市售洋菇PPO 做PAGE-blot assay靈敏度之測試-----------27
3.2 PAGE-blot assay之應用------------------------------------------------------27
3.2.1不同蔬菜水果PPO活性與PAGE-blot assay之比較---------------27
3.2.2鑑定植物是否具有內生性的抑制劑-----------------------------------28
3.2.3洋菇PPO之純化----------------------------------------------------------29
3.3化學成分及植物內生性抑制劑對洋菇PPO活性抑制效果-------------30
3.3.1 化學成分之抑制劑對洋菇PPO活性抑制效果之測試-------------30
3.3.2鑑定PPO與內生性抑制劑的關連--------------------------------------30
3.4 PAGE-blot assay其他的應用---------------------------------------------------31
3.5 荔枝皮PPO之研究-------------------------------------------------------------31
3.5.1荔枝皮PPO之純化--------------------------------------------------------31
3.5.2 荔枝皮PPO之酵素特性分析-------------------------------------------32
3.6 分析荔枝內生性之PPO 抑制劑----------------------------------------------32
3.6.1 揮發性PPO抑制劑之發現----------------------------------------------32
3.6.2證明揮發性抑制劑之存在------------------------------------------------33
3.6.3分析荔枝皮揮發性PPO 抑制劑對PPO之抑制效果---------------33
3.6.4將PPO抑制劑揮發後分析荔枝皮PPO活性-------------------------34
3.7 荔枝皮表面結構之影響--------------------------------------------------------34
3.8 利用免疫染色（immunolocalization）定位PPO存在之區域----------34

第四章、結論-------------------------------------------------------------------------35

第五章、參考文獻-------------------------------------------------------------------37

附錄一、Manuscript

表目錄
表一：不同蔬菜水果PPO活性與PAGE-blot assay之比較------------------47
表二：荔枝皮PPO純化各階段之產率及回收率分析表----------------------48
表三：高分子量荔枝皮PPO具有特殊催化substrate之能力----------------49


圖目錄
圖一：固定於3MM層析紙上的catechol最適濃度之測試-------------------50
圖二：利用10% SDS-PAGE與catechol-paper測試PPO活性---------------51
圖三：PAGE-blot assay靈敏度之測試---------------------------------------------52
圖四：21種不同蔬果PPO酵素活性之酵素動力學-----------------------------53
圖五：利用PAGE-blot assay回復具代表性的PPO活性----------------------54
圖六：蕃茄與芭樂酒精萃取液對於洋菇PPO活性之抑制效果--------------55
圖七：蕃茄與芭樂酒精萃取液對於洋菇PPO活性抑制效果之酵素動力學-56
圖八：利用HPLC Sepharose G-200純化洋菇PPO-----------------------------57
圖九：化學成分之抑制劑對洋菇PPO活性抑制效果之測試-----------------58
圖十：利用HPLC Sepharose G-200純化荔枝皮PPO--------------------------60
圖十一：10%SDS-PAGE及PAGE-blot assay分析荔枝皮PPO純化前後之差異------------------------------------------------------------------------------61
圖十二：利用Lineweaver-Burk plots 定義不同分子量荔枝皮PPO---------62
圖十三：荔枝皮高分子量PPO具有特殊催化substrate之能力。-----------63
圖十四：荔枝的褐變反應------------------------------------------------------------65
圖十五：分析自荔枝皮中萃取之揮發性PPO抑制劑對PPO之抑制效果-66
圖十六：將PPO抑制劑揮發後分析其PPO活性-------------------------------67
圖十七：荔枝皮表面結構之Scanning Electron Microscope (SEM)圖------68
圖十八：荔枝皮PPO免疫染色（immunolocalization）圖--------------------69

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