臺灣博碩士論文加值系統

中文摘要
蠟蚧輪枝菌 (Verticillium lecanii) 過去被人發現為一種昆蟲病原真菌，當與病原菌細胞或是類似幾丁質、幾丁聚醣的物質接觸時，會誘導促成幾丁質相關酵素的合成，這是蠟蚧輪枝菌對於含幾丁質的植物病源性微生物的防禦機能的一種現象。

在培養蠟蚧輪枝菌生產幾丁質□過程中，我們利用一種實驗設計方法－回應曲面法(Response surface methodology, RSM )，此方法是利用電腦軟體作統計運算，回歸出一階和二階模式，從模式中觀察培養基各成分對酵素產值的影響，找到我們最適培養基。由回應曲面法回歸之最大值點換算出各成對應的濃度為glucose 3.4 %、yeast extract 1.3 %、SCSP 0.3 %及蛋白質精0.5 %。其酵素產量之理論計算值為2.79 U/mL；而近一步以此培養基搖瓶培養所得酵素產量為 2.74U/mL；由此顯示回應曲面模式之可靠性。以實驗結果所設計最適培養基所生產之幾丁質□產量，比較交叉測試實驗中所得酵素活性相比，提高 1~2 倍。另外，將回歸出得最適培養基擴大到 5-L 攪拌式發酵槽，利用發酵槽培養蠟蚧輪枝菌生產幾丁質□。控制菌生長時的條件，如溫度、通氣量、轉速等，觀察酸鹼值、酵素活性、殘醣、溶氧量等變化及之間的關係，最後決定酵素最適量的培養條件。目前實驗結果發現，培養條件在通氣量為0.6 vvm、溫控在 2.4 ℃、酸鹼質為 8 及轉速為 150 rpm 時，酵素產量最好，而且可以培養時間由 6 天縮短至 3 天，酵素活性即可達到最高點。
在過去對幾丁質酶的用途報導中，除了本研究強調的生物防治上的功能以外，也可添加在食物之中作腐延長保存期限，以及此酵素反應後產物－六個單元體的幾丁寡NACOS (N-acetlchitooligo saccharide)，在醫學上具抗腫瘤活性。針對蠟蚧輪枝菌生產幾丁質□酶的研究中指出，此類胞外酵素是屬於醣蛋白的一種，分子量為 42 kDa 左右、等電點約在 4 左右，根據上述酵素的特牲，我們將 Verticillium lecanii 發酵液經透析濃縮，然後把濃縮之發酵液經硫酸銨鹽析，陰離子交換樹脂、膠體過濾層析管柱等分離步驟，再由活性分析及 SDS-PAGE 鑑定，始將幾丁質酶自發酵液分離。根據實驗結，純化倍率和原發酵液相比，提高六十多倍，單位總蛋白質的酵素活性為 163U/mg 。期望對於分離之 Chitinase 對於未來後續的研究，如酵素特性、應用、立體結構之活性位置等有幫助。

Abstract
Verticillium lecanii is regarded as the insect-infectious fungi. When contact with the pathogen cells or the materials composed of something like chitin, chitosan etc., the chitin related enzymes would be synthesized. This is the defensive phenomenon of Verticillium lecanii to chitin-containing pathogens.

In the process of Verticillium lecanii chitinase production, the experiment design method, RSM (Response Surface Methodology) is utilized. The method is doing statistical calculation to reverse the first and second class modes. The optimal medium could be found via the effects of the constitutions to enzyme production. Via the calculation of RSM, the correspondent concentrations, glucose 3.4%, yeast extract 1.3%, SCSP 0.3% and peptone 0.5%, are obtained. The theoretical concentration of enzyme production is 2.79 U/mL, and the concentration of enzyme production via flask incubation is 2.74 U/mL. It reveals the reliability of RSM mode. The chitinase concentration produced from the designed optimal medium is elevated to 1-2 fold, comparing with the cross-test experiments. Extending the conditions of optimal medium to 5-L stirring fermentator, and controlling the conditions of bacteria growth, i.e. temperature, air concentration, and rotating rate, make us retrieve the optimal incubating condition for enzyme production. The condition of 0.6vvm, temperature 2.4℃, pH 8, rotating rate 150rpm, makes maximal enzyme production; the incubation time shorten from 6 days to 3 days.

From the reports of chitinase applications, beside the application on biological control emphasized in this research, chitinase could be used to extend the food reservation time by adding, and the anti-tumor function of NACOS (N-acetlchitooligosaccharide), the product after reaction of chitinase. According to the reports of Verticillium lecanii chitinase production, the extra-cellular enzyme is belonged to glycoprotein. The molecular weight is about 42 kDa, and the iso-electric point is about 4. After the dialysis, the concentrated Verticillium lecanii fermentation solution is processed via the separation processes of ammonia sulphate salting out, anion ion-exchanged resin, and chromatography column. The chitinase is separated from the fermentation solution. The concentration of purified chitinase is elevated more than 60s fold comparing with the fermentating extract, and the chitinase activity per unit protein is 163 U/mg. It is convinced that the purified chitinase in this research would be beneficial to the following studies, i.e. chitinase properties, applications, active site of 3-D structure etc.

目錄
封面內頁
授權書
中文摘要…………………………...…………………...……………………………..ii
英文摘要…………………………...…………………………………………...…….iv
目錄………………………………...…………………………………………………vi
圖目錄……………………….………………………………………………………viii
表目錄……………………...………………………………………………………….x
第一章緒論
1-1 前言………………………………………………………………………………1
1-2 文獻回顧…………………………………………………………………………2
1-2-1 蠟蚧輪枝菌的簡介……………………………………………………………2
1-2-2 幾丁質簡介與應用…………………………………………………………....8
1-2-3 幾丁質的天然分佈…………………………………………………………...12
1-2-4 幾丁質酶的分類……………………………………………………………...14
1-2-5 幾丁質受酵素分解可能的途徑……………………………………………...18
1-2-6 幾丁質□活性之測定………………………………………………………...20

第二章Verticillium Lecanii 液態發酵 Chitinase 最適培養基及活性分析

2-1 前言……………………………………………………………………………...26
2-2 材料與方法…………………………………………………………………...…27
2-3 幾丁質□活性分析……………………………………………………………...28
2-4 回應曲面法設計培養基………………………………………………………...32
2-5 結果與討論………………………………………………………………...……37

第三章Verticillium Lecanii 之 Chitinase 的分離純化

3-1 前言……………………………………………………………………………...55
3-2 材料與方法……………………………………………………………………...56
3-3 結果與討論……………………………………………………………………...63

第四章利用5-L 發酵槽生產幾丁質□

4-1 前言……………………………………………………………………………...68
4-2 材料與方法……………………………………………………………………...69
4-3 結果與討論……………………………………………………………………...77

參考文獻………………………………………………………..……………………80
附錄……………………………………………………………..……………………83



圖目錄
圖 1-1 蠟蚧輪枝菌枝的型態………………………………………………………..4
圖 1-2 蠟蚧輪枝菌圖………………………………………………………………..7
圖 1-3 幾丁質、幾丁聚醣之分子結構……………………………………………..11
圖 1-4 自 Hevea brasliensis 分泌幾丁質酶之3D蛋白質結構圖………………...16
圖 1-5 Serratia marcescens 及大豆中之chitinase 3D蛋白質結構圖……………..17
圖 1-6 幾丁質酶之作用機制……………………………………………………….19
圖 1-7 乙二醇幾丁質之構造……………………………………………………….23
圖 2-1 CM-chitin-RBV在不同pH值的吸收值變化……………………………….30
圖 2-2 發酵活性對OD550吸光值的變化…………………………………………..31
圖 2-3 RSM實驗設計之概念圖…………………………………………………….41
圖 2-4 RSM陡升路徑之概念圖…………………………………………………….42
圖 2-5 發酵時間對產生酵素活性變化之走勢圖………………………………….43
圖 2-6 蛋白質精和SYSP對chitinase之回應曲面圖……………………………..51
圖 2-7 蛋白質精和Yeast extract對chitinase之回應曲面圖………………………51
圖 2-8 蛋白質精和Glucose對chitinase之回應曲面圖…………………………..52
圖 2-9 SCSP和Yeast extract對chitinase之回應曲面圖…………………………..52
圖 2-10 SCSP和Glucose對chitinase之回應曲面圖……………………………...53
圖 2-11 Yeast extract和Glucose對chitinase之回應曲面圖………………………53
圖 2-12 Yesst extract的濃度對酵素活性之趨劫圖………………………...………54
圖 3-1 分離純化之流程圖………………………………………………………….62
圖 3-2 離子交換層析圖…………………………………………………………….65
圖 3-3 不同純化階段之蛋白質電泳分析圖……………………………………….66
圖 3-4 SDS-PAGE的軟體分析圖…………………………………………………..67
圖 4-1 不同轉速下Glucose的濃度及酸鹼值的變化……………………………..7
圖 4-2 在150 rpm時，酵素活性、Glucose濃度及總蛋白的變化………………76
圖 4-3 在150 rpm時，chitinase的活性及和酸鹼值之間的關係………………...78
圖 4-4 不同酸鹼值對活性的變化………………………………………………….79






表目錄
表1-1 不同幾丁質成分分析………………………………………………………..11
表1-2 幾丁質□活性測定之方法…………………………………………………..22
表1-3 不同菌株所得幾丁質□產量比較…………………………………………..23
表1-4 植物中幾丁質□的分佈……………………………………………………..25
表2-1 比較不同第一氮、碳源組合對幾丁質□的活性變化……………………..44
表2-2 比較不同第二氮、碳源組合對幾丁質□的活性變化……………………...44
表2-3 RSM一階實驗設計圖表……………………………………………………..45
表2-4 RSM一階實驗設計之結果…………………………………………………..46
表2-5 RSM陡升路徑之實驗設計圖表……………………………………………..47
表2-6 RSM中心混成(二階)實驗設計圖表………………………………………...48
表2-7 RSM中心混成(二階)實驗設計之結果……………………………………...49
表2-8 四種因子中心混成實驗之迴歸分析表……………………………………..50
表2-9 由二階模式去除因子相關之變異係數分析………………………………..54
表3-1 依蛋白質之性質所衍生出分離的方法……………………………………..56
表3-2 電泳(SDS-PAGE)分析試劑之配製………………………………………….61
表4-1 不同攪拌速度所測得各種參數值…………………………………………..75

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