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研究生:李京樺
研究生(外文):Jing-Hua Li
論文名稱:角蛋白酶之生產菌篩選、純化及其特性
論文名稱(外文):Screening of the Keratinase-Producing Bacteria, Purification and Characterization of Keratinase
指導教授:江善宗殷儷容殷儷容引用關係
指導教授(外文):Shann-Tzong JiangLi-Jung Yin
學位類別:碩士
校院名稱:國立臺灣海洋大學
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:2006
畢業學年度:94
語文別:中文
論文頁數:77
中文關鍵詞:角蛋白角蛋白酶篩選純化蛋白酶
外文關鍵詞:KeratinKeratinaseScreenPurificationprotease
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中文摘要

由土壤中篩選可生產keratinase降解羽毛之Pseudomonas aeruginosa之菌種。樣品在羽毛培養基中於37oC、150rpm下振盪培養三天後,再取其培養液轉入另一羽毛培養基,重複三次後取其培養液在羽毛Agar上進行畫線分離,挑出可在羽毛Agar上產生透明環之菌落,進行培養及活性測定。再挑選出活性最高的Pseudomonas aeruginosa菌株放入羽毛培養基中於37oC、150rpm下振盪培養54小時,收集胞外酵素液,經0.45 µm膜過濾後,測其蛋白酶活性約為141 unit。將粗酵素液經5 kDa超過濾濃縮後,進行sephacryl s-100 HR管柱層析可得到具有活性之角蛋白酶,其比活性為 4068 units/mg,回收率為10.6%,純化倍率為6.79倍。經SDS-PAGE電泳分析確定純化之角蛋白酶分子量為39 kDa。其最適pH為 9,而在pH 5.5-9.0之間有較好的安定性。而最適溫度為60oC,在10oC-50oC之間有較好的溫度安定性。該角蛋白酶被金屬離子Hg+、Cu2+、Fe3+和Ni2+及蛋白酶抑制劑pCMB、IAA與NEM會抑制其活性,但在還原劑β-mercaptoethanol、dithiothreitol和glutathione影響下,能促進其活性,故推測此角蛋白為硫醇類蛋白質且其活化能為53.54 kcal/mole。
Abstract

The feather-degrading strain of Pseudomonas aeruginosa was isolated from soil under aerobic conditions. In the primary screening of feather-degrading bacteria, strains obtained from feather broth were spread on feather powder agar plates and incubated at 37oC for 72 h. Single colony appeared in the transparent ring on the plates were selected and scrubed. It was inoculated into 100 mL of feather broth and then incubated at 37oC for 54 h. After removing the cells by passing through a 0.45 µm membrane, the crude enzyme was concentrated by Amicon ultrafiltration. The keratinase was purified to homogenity after Sephacryl S-100 HR chromatograph. The purification was up to 6.79 fold with 10.6 % recovery. It's apparent molecular mass was estimated to be 39 kDa based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature for the enzyme activity were 9 and 60oC, respectively. It was stable at pH 5.5-9.0 and from 10oC to 50oC. The activity was inhibited by Hg+, Cu2+, Fe3+ and Ni2+. The protease was considered to be a cysteine protease according to its sensitivity to protease inhibitor pCMB, IAA and NEM, and strong activation by mercaptoethanol, dithiothreitol and glutathione. The activation energy of purified keratinase was 53.54 kcal/mole。
目 錄
頁數
名詞縮寫--------------------------------------------------------------------- 1
中文摘要--------------------------------------------------------------------- 2
英文摘要--------------------------------------------------------------------- 3
壹、前言--------------------------------------------------------------------- 4
貳、文獻整理--------------------------------------------------------------- 5
一、蛋白酶之簡介------------------------------------------------------ 5
(1)蛋白酶的命名------------------------------------------------------ 5
(2)蛋白酶的特性-------------------------------------- 5
(3)蛋白酶的分類------------------------------------- 6
(4)鹼性蛋白酶在工業上之應用----------------------- 8
二、角蛋白之簡介------------------------------------------------------- 9
(1)角蛋白之來源----------------------------------------------------- 9
(2)角蛋白之種類---------------------------------------------------- 10
(3)角蛋白之化學水解---------------------------------- 10
(4)角蛋白之酵素水解---------------------------------- 11
(5)角蛋白酶之微生物來源----------------------------- 12
(6)環境因子對角蛋白酶生產之影響------------------------------ 13
(7)角蛋白之工業用途------------------------------------------------ 14
三、角蛋白酶之生化特性--------------------------------------------- 14
(1)pH與溫度之影響-------------------------------------------------- 14
(2)分子量--------------------------------------------------------------- 15
(3)金屬離子影響------------------------------------------------------ 16
(4)抑制劑與還原劑之影響------------------------------------------ 16
參、實驗材料與方法------------------------------------------------------- 17
一、實驗材料------------------------------------------------------------ 17
(1)藥品------------------------------------------------------------------ 17
(2)儀器------------------------------------------------------------------ 17
二、實驗方法------------------------------------------------------------ 19
(1)試驗菌株篩選------------------------------------------------------ 19
A.樣本來源------------------------------------------------- 19
B.分離菌株----------------------------------------- 19
C.酵素活性測定------------------------------------- 19
(2)角蛋白酶的純化---------------------------------- 19
A.粗酵素液的製備---------------------------------- 19
B.超過濾濃縮--------------------------------------- 20
C.Sephacryl S-100 High Resolution管柱層析------- 20
D.SDS-PAGE聚丙烯胺膠體電泳---------------- 20
E.蛋白質分子量測定------------------------------ 21
F.蛋白質濃度測定--------------------------------------------- 21
(3)角蛋白酶的生化特性試驗--------------------------------------- 21
A.最適反應溫度------------------------------------------------- 21
B.最適反應pH--------------------------------------------------- 22
C.溫度安定性---------------------------------------------------- 22
D.pH安定性----------------------------------------------------- 22
E.抑制劑對角蛋白酶的影響---------------------------------- 22
F.還原劑對角蛋白酶的影響--------------------------------- 23
G.金屬離子對角蛋白酶的影響------------------------------- 23
H.角蛋白酶的活化能------------------------------------------- 23
肆、結果與討論------------------------------------------------------------- 25
一、角蛋白酶生產菌之篩選及其生長情形------------------------ 25
二、角蛋白酶之純化--------------------------------------------------- 26
(1)粗酵素液的製備--------------------------------------------------- 26
(2)Sephacryl S-100 High Resolution管柱層析------------------ 26
(3)電泳分析------------------------------------------ 27
三、蛋白酶的生化特性試驗------------------------------------------ 27
(1)最適酸鹼度--------------------------------------------- 27
(2)酸鹼度安定性--------------------------------------------------- 28
(3)最適溫度------------------------------------------------------ 28
(4)溫度安定性--------------------------------------------------------- 29
(5)活化能--------------------------------------------------------------- 29
(6)金屬鹽類的影響--------------------------------------------------- 30
(7)抑制劑的影響------------------------------------------------------ 30
(8)還原劑的影響------------------------------------------------------ 31
伍、結論---------------------------------------------------------------------- 32
陸、參考文獻---------------------------------------------------------------- 33










表目錄

表一、鹼性蛋白酶的工業利用------------------------------------------ 39
表二、Pseudomonas aeruginosa之微生物脂肪酸鑑定系統分析
結果------------------------------------------------------------------ 41
表三、Pseudomonas aeruginosa 角蛋白酶之純化表。--------------- 42
表四、金屬離子對Pseudomonas aeruginosa 角蛋白酶活性之
影響。-------------------------------------------------------- 43
表五、抑制劑對Pseudomonas aeruginosa角蛋白酶活性之影
響。------------------------------------------------------------------ 44
表六、還原劑對Pseudomonas aeruginosa 角蛋白酶活性之影
響。------------------------------------------------------------------ 45














圖目錄


圖一、胰凝乳蛋白酶作用機制------------------------------------------ 46
圖二、Thermolysin之作用機制------------------------------------------- 47
圖三、豬胃蛋白酶之作用機制------------------------------------------ 48
圖四、木瓜蛋白酶之作用機制------------------------------------------ 49
圖五、角蛋白中雙硫鍵之結構------------------------------------------ 50
圖六、角蛋白之α-螺旋結構----------------------------------------------- 51
圖七、角蛋白之β-褶板結構----------------------------------------------- 52
圖八、Pseudomonas aeruginosa之 16S rDNA部分序列------------- 53
圖九、Pseudomonas aeruginosa之電子顯微鏡照片----------------- 54
圖十、Pseudomonas aeruginosa之微生物脂肪酸鑑定系統分析
結果------------------------------------------------------------------ 55
圖十一、Pseudomonas 與 Stenotrophomonas 角蛋白酶活性變化
情形--------------------------------------------------------------- 56
圖十二、Pseudomonas aeruginosa生長曲線與酵素活性之變化--- 57
圖十三、Pseudomonas aeruginosa keratinase 在第一次
Sephacryl S-100 HR管柱層析-------------------------------- 58
圖十四、Pseudomonas aeruginosa keratinase 在第二次
Sephacryl S-100 HR 管注層析------------------------- 59
圖十五、純化之Pseudomonas aeruginosa keratinase 電泳
圖---------------------------------------------------------- 60
圖十六、SDS-PAGE測定純化之角蛋白分子量之檢量曲線-------- 61
圖十七、純化之角蛋白酶溫度安定性----------------------------------- 62
圖十八、純化之角蛋白酶酸鹼安定性---------------------------------- 63
圖十九、純化之角蛋白酶最適反應溫度------------------------------- 64
圖二十、純化之角蛋白酶溫度安定性---------------------------------- 65
圖二十一、Pseudomonas aeruginosa ketatinase之熱失活曲線圖- 66
圖二十二、Pseudomonas aeruginosa keratinase之
Arrhenius plot圖---------------------------- 67
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