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研究生:劉佳玲
論文名稱:橘青黴脂肪純化、特性及基質特異性之研究
論文名稱(外文):Studies on Purification , Characterization and the Substrate Specificity of a Lipase from Penicillium citrinum
指導教授:吳清熊
學位類別:碩士
校院名稱:國立海洋大學
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:2004
畢業學年度:90
語文別:中文
論文頁數:94
中文關鍵詞:橘青黴脂肪
外文關鍵詞:Penicillium citrinumLipase
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  • 被引用被引用:1
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本研究利用橘青黴Penicillium citrinum產生之脂肪,在培養溫度 30oC,振盪速率 120 rpm 最適培養條件下,得到最佳活性 10.07 U/mL。經離心、超過濾後製成粗酵素液,回收率為 88.66% ,在經由 40-60% 飽和硫酸胺鹽析,Sephacyl S-200膠體過濾管柱,DEAE-Sephdex A-50離子交換樹酯等過程後順利將此酵素純化出,最終純化倍率為 27.08 倍,總產率4.31%。
此酵素之分子量為 74.78 kDa 左右。酵素最適反應溫度為50 oC,最適反應pH值為8.0左右,在pH 6.0-8.0,30 oC下維持四小時酵素仍相當穩定。本酵素受EDTA強烈抑制,顯示為金屬依存性酵素,受Cu2+、Fe2+及Cd2+離子之抑制,而Mn2+、Mg2+、K+ 等金屬離子對活性表現有促進作用。本脂肪活性能被非離子性界面活性劑如 Triton X-100 所促進。在多種有機溶劑環境中脂肪仍可維持水解活性,甚至有些情況下還比原活性高,其中以Cyclohexane對脂肪活性最有助益。
在本脂肪水解油脂後之水解產物中發現明顯產生單酸甘油酯,同時 游離脂肪酸含量也明顯增加,推測脂肪對三酸甘油酯分子的某兩個位置具有特異性,但不能水解單酸甘油酯,所以水解作用會蓄積單酸甘油酯,但從其水解產物之脂肪酸組成來看,似乎沒有顯示脂肪酸種類的特異性。
The role of culture conditions on a lipase production by Penicillium citrinum (P) , the optimal culture conditions from the shaked culture at 30 oC and 120 rpm, lipase activity reached a maximum of 10.07 U/mL. By employing cenrifugal and ultrafitration to remove cells and water, respectively, the concentrated broth to obtain the crude enzyme with 88.66% recovery. The lipase was purified to homogeneity from the crude extract of whean bran culture ammonium sulfate precipition, Sephacyl S-200 gel filtration chromatography, DEAE-Sephdex A-50 ion exchange chromatography. A 27.08-fold purification was obtained, with a recovery of 4.31%.
The molecular weight of this lipase was determined to be 74.78 kDa. The optimal pH and temperature for the hydrolysis was pH 8.0 and 50 oC, respectively. It was found that the enzyme had stable over a pH range of 6 to 8. A complete inhibition by EDTA, suggesting the lipase was a metal dependent enzyme. Partial inhibition were shown by either Cu2+, Fe2+ or Cd2+. However, it is activated Mn2+, Mg2+, K+ . The enzyme activity persisted in detergents, and was stimulated by non-ionic surfactants triton X-100. It remains hydrolytic activity at various solvent conditions, even higher in some situation than the control. It was most benefitical for P. citrinum lipase to incubate with cyclohexane.
By using olive oil, safflower oil or fish oil as a substrate for determining the specicity of P. citrinum lipase. It is shown that TG are the favored substrates for this lipase, where MG is poor ones.Therefore, after hydrolysis, the TG was decreased and MG and FFA are accumulated, and by determining the fatty acid composition of FFA in the hydrolyzate, suggesting that this lipase does not shown fatty acid specificity.
中文摘要 I
英文摘要 II
目錄 IV
表目錄 IX
圖目錄 X
一、研究背景及目的 1
I. 研究背景 1
II. 研究目的 2
二、文獻回顧 3
I. 脂肪的種類與來源 3
I-1. 動物脂肪 3
I-2. 植物脂肪 3
I-3. 微生物脂肪 4
II. 脂解的特性 4
II-1. 脂肪的催化反應 4
II-2. 脂肪的特異性 5
II-2-1. 受質特異性脂肪 5
II-2-2. 區域位置特異性脂肪 6
II-2-3. 非特異性脂肪 7
II-2-4. 脂肪酸特異性脂肪 7
II-2-5. 立體位置特異性脂肪 7
III. 以薄層層析、氣相層析法測定脂肪之特異性 8
III-1. 對三酸甘油酯作用位置特異性 8
III-2. 對脂肪酸作用特異性 9
IV. 影響脂肪活性表現之因子 10
IV-1. 有機溶劑對酵素活性之影響 10
IV-2. 水分對酵素活性之影響 10
IV-3. 基質種類之影響 11
IV-4. 溫度對酵素活性之影響 12
IV-5. 金屬離子對酵素活性之影響 12
IV-6. 界面活性劑對酵素活性之影響 13
V. 增加酵素穩定性之研究 13
V-1. 溫度穩定性 14
V-2. 固定化 14
V-3. 添加安定劑 14
VI. 脂肪的應用 15
VI-1. 濃縮魚油之 w3多元不飽和脂肪酸 15
VI-2. 將 w3多元不飽和脂肪酸結合至食用油及甘油酯 15
VI-3. 乳化劑方面 16
VI-4. 醫藥品方面 16
VI-5. 食品方面 17
VI-6. 清潔劑方面 18
VI-7. 污水處理方面 18
VII. 關於橘青黴 19
三、實驗材料與方法 20
I. 實驗材料 20
I-1. 菌株 20
I-2. 化學試藥 20
I-3. SDS-聚丙醯胺膠體溶液調配法 22
I-3-1. SDS-聚丙醯胺膠體溶液 22
I-3-2. 電泳膠體染色 23
I-4. 儀器設備 23
II. 實驗流程 25
III. 實驗方法 26
III-1. 黴菌菌株之保存與培養 26
III-1-1. 菌種保存 26
III-1-2. 孢子懸浮液製作 26
III-1-3. 菌株培養條件 26
III-2. 粗酵素液之製備 26
III-3. 酵素純化 27
III-3-1. 超過濾膜濃縮 27
III-3-2. 硫酸銨沉澱 27
III-3-3. 透析 27
III-3-4. 膠體過濾層析分析 28
III-3-5. 離子交換管柱層析 28
III-4. 脂肪特性分析 29
III-4-1. 不同受質濃度對酵素活性表現之影響 29
III-4-2. 反應 pH 值對酵素活性之影響 29
III-4-3. pH 安定性 29
III-4-4. 反應溫度對酵素活性之影響 29
III-4-5. 熱安定性 30
III-4-6. 界面活性劑種類對酵素活性之影響 30
III-4-7. 不同金屬離子對酵素活性之影響 30
III-4-8. 有機溶劑對酵素活性之影響 31
IV. 酵素之基質特異性 31
IV-1. 對天然油脂之水解特異性 31
IV-1-1. 脂肪酸量 31
IV-1-2. 脂肪酸分佈情形 32
IV-2. 對三酸甘油酯之位置特異性 32
V. 主要分析方法 33
V-1. 脂肪活性測定 33
V-2. 蛋白質定量分析 33
V-3. SDS-聚丙烯醯胺膠體電泳分析 33
VI. 薄層層析-火焰離子檢測儀 34
VI-1. 分析方法 34
VI-2. 薄層火焰離子分析儀器分析條件 35
VII. 氣相層析分析法 35
VII-1. 氣相層析儀操作條件 35
VII-2. 脂肪酸組成. 35
四、結果與討論 36
I. 酵素純化 36
I-1. 橘青黴所產脂肪生產情形 36
I-2. 以 p-nitrophenyl stearate 為受質之活性分析 36
I-3. 粗酵素液濃縮 37
I-4. 酵素蛋白質沉澱劃分 37
I-5. Sephacryl S-200 之膠體過濾層析 38
I-6. DEAE Sephadex A-50 離子交換管柱層析 38
I-7. 純化總表 39
I-8. SDS-PAGE 39
II. 脂肪之生化特性 39
II-1. 最適反應 pH 值 39
II-3. pH 安定性 40
II-4. 最適反應溫度 41
II-5. 熱安定性 41
II-6. 界面活性劑 41
II-7. 金屬離子 42
II-8. 有機溶劑 43
III. 脂肪之基質特異性 44
III-1. 脂肪對天然油脂作用之特異性 44
III-2. 脂肪對三酸甘油酯作用之特異性 47
五、結論 49
六、參考文獻 50
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