由台灣北部土壤篩選出一株能以黃豆為生產脂肪分解酶之主要碳氮源的新品種菌株TKU009。此菌株在pH10、25℃培養4天，可得較高脂肪分解酶活性。經培養條件探討發現，所得發酵上清液在pH8，60℃對基質p-NPP反應可得最高活性(1.81U/mL)。在酵素安定性方面，發酵上清液之脂肪分解酶活性在50℃以及pH6-8之間均能保持安定；在25℃儲存5天仍殘存60％活性；在4℃下儲存18天則仍可維持67％的活性。在酵素動力學部分，發酵上清液之脂肪分解酶之Km、Vmax及Ea分別為6.25 mM、3.25U/mL和6.58 kcal。
    最適培養條件所得發酵上清液經硫酸銨沉澱及DEAE-Sepharose CL-6B離子交換層析，可回收兩個酵素活性區F1及F2。其中F1經純化之後呈現乳化狀態；而F2於後續Sephacryl-100膠體層析之後，可純化出分子量(以SDS-PAGE測得)為43.7 kDa之脂肪分解酶。F1和F2酵素在pH6，40℃以及pH7，40℃分別對於基質p-NPP有最佳活性為1.58U/mL以及4.29U/mL。在安定性方面，F1在pH6-7以及50℃內均能保持安定；F2則在pH 8-11以及50℃內能保持安定。F1和F2在25℃下分別儲存4天和6天之後仍殘存50%和54%活性；在4℃下分別儲存6天仍能維持59%以及56%活性。在酵素動力學部分，F1酵素液之Km、Vmax及Ea分別為3.70 mM、1.58U/mL和2.64kcal；F2酵素液之Km、Vmax及Ea則分別為13.2mM、 4.29U/mL和6.31 kcal。
    在金屬離子對酵素活性的影響方面，Cu2+、Na+、Zn2+的添加對於發酵上清液的酵素活性具有增強效果，Cu2+對F1具增強效果；Ca2+、Na+、Zn2+的添加則對F2具增強效果。界面活性劑Triton X-100和Tween40的添加，對於發酵上清液、F1和F2的酵素活性均有增強效果；Tween20則對發酵上清液和F2具有抑制效果，但對F1則具增強的效果。
    在有機溶劑對酵素活性的影響方面，正丁醇的添加對於發酵上清液的酵素活性具有促進作用，正己烷對F1和F2均有增強效果，甲苯的添加則對發酵上清液、F1和F2皆具促進效果，乙醇則可提高F2的活性。
    在基質特異性方面，對於短鏈的基質幾乎沒有水解能力。發酵上清液和F1對於中鏈p-NP caprylate(C8)的水解能力最佳。F2則對中短鏈基質的水解能力皆欠佳，而對p-NP myristate(C14)則有最佳的反應活性。綜合言之，TKU009所生產脂肪分解酶對於越短鏈的基質，其分解能力越差，對於鏈長(C12-C14)的基質則有最佳的水解能力，對於鏈長C16-C18的基質則次之。

In this study, we have isolated a new strain (TKU009), from soil in north of Taiwan, which can use the soybean as the main nutrition source. The bacterium could produce the best activity when the cultivation condition was pH10 and 25℃ accompanied with shaking for 4 days. 
In enzymatic study, the optimal activity of crude lipases was 1.81U/mL at pH8 and 60℃ if the p-NPP was used as substrate. The enzyme was stable in the pH range of 6 to 8 at 50℃. High storage stability with 67% of the relative activity remaining was observed when the crude enzyme was stored at 4℃ for 18 days. However, 60% of activity remained when the crude enzyme was stored at 25℃ for 5 days. In enzymatic kinetic study, the kinetic parameters, Michaelis-Menten constant (Km), maximum velocity (Vmax) and activation energy, were 6.25mM, 3.25U/mL and 6.58 Kcal, respectively.
  In purification study, the crude lipase was separated into two protein fractions, F1 and F2, by using the ion exchange chromatography of DEAE- Sepharose CL-6B. The F1 protein was in emulsion phase after purification.  The molecular weight of F2 was 43.70 kDa if electrophoresis of SDS-PAGE was used. The F1 and F2 enzyme had a optimal activity of 1.58U/mL and 4.29U/mL if the optimum pH value were respectively 6 and 7 at 40℃. The F1 and F2 had a worse stability than crude enzyme. In enzymatic kinetic study for F1 and F2 we found that F1 had lower Km, Vmax and Ea than crude enzyme, but the F2 enzyme had a higher Vmax than crude enzyme. 
  Enhancement of crude and F1 lipase activity by the presence of Cu2+ was observed.On the other hand, all the enzymes (crude, F1 and F2) could be enhanced by adding the detergents Triton X-100 and Tween 40. But the addition of Tween 20 inhibited the activity of crude and F2 enzyme.
  We also found that the addition of toluene could enhance the activity of all types of enzymes. For the individual enzymes, addition of n-butanol could enhance the crude enzyme activity, n-hexane could enhance both the F1 and F2 enzyme activity, and ethanol could enhance the F2 enzyme activity. 
  In substrate specificity, lipase had almost no hydrolytic ability for short chain (C4) substrates. F1 and fermentation broth had better hydrolysis ability for p-NP caprylate (C8) than F2, but F2 had a good ability of hydrolysis of p-NP myristate (C14). In brief, the lipase produced from TKU009 showed better hydrolytic ability for substrates of long C-length than those of short C-length.

中文摘要………………………………I
英文摘要………………………………II
謝誌……………………………………IV
目錄……………………………………V
圖目錄…………………………………IX
表目錄…………………………………XI
第一章 緒論
1.1脂肪分解酶………………………………1
1.2脂肪分解酶的應用………………………11
1.3微生物之營養需求………………………17
1.4脂肪分解酶的純化分離…………………21
1.5實驗設計流程……………………………26
第二章 材料與方法
2.1菌株………………………………………27
2.2實驗材料…………………………………27
2.3實驗方法…………………………………30
第三章 結果與討論
3.1脂肪分解酶之較適生產條件探討………40
3.2脂肪分解酶之分離純化…………………46
3.3酵素反應之最適溫度與熱安定性………56
3.4酵素反應之最適pH與pH安定性…………59
3.5酵素之儲存安定性………………………62
3.6微量元素對酵素活性之影響……………65 
3.7界面活性劑對酵素活性之影響…………67
3.8有機溶劑對酵素活性之影響……………69 
3.9酵素之基質特異性………………………71
3.10活化能研究…………………………….73
3.11脂肪分解酶的酵素動力學反應……….77
3.12脂肪分解酶對油催化水解的能力…….80
第四章 結論與未來展望……………………85
參考文獻…………………………………….87
附錄………………………………………….103
圖目錄
圖1-1 脂肪分解酶水解三酸甘油酯產生甘油和脂肪酸…….……………..8
圖1-2 脂肪分解酶催化之反應…………………………………………….10
圖3-1黃豆粉添加濃度對脂肪分解酶活性之影響……………..………….41
圖3-2培養體積對脂肪分解酶生產之影響………………..………...……..42
圖3-3培養溫度對脂肪分解酶生產之影響….……………………………..43
圖3-4培養基酸鹼值對脂肪分解酶生產之影響…………………………...44
圖3-5發酵生產脂肪分解酶之時間趨勢圖………………………….……..45
圖3-6 DEAE-Sepharose CL-6B之離子交換層析圖………………….……48
圖3-7 F2 Sephacryl S-100之膠體過濾層析圖……………………….……49
圖3-8 F1 之蛋白質電泳分子量測定圖……..……………………….…..…53
圖3-9 F1蛋白質區的HPLC分析圖譜……………………………...……...54
圖3-10 F2 之蛋白質電泳分子量測定圖………………………....………...55
圖3-11溫度對酵素活性的影響……………………………….……………57
圖3-12酵素的熱安定性…………………………………………….………58
圖3-13酸鹼值對酵素活性的影響…………………………………...……..60
圖3-14酵素的pH安定性…………………………………………….…..…61
圖3-15酵素於4℃之儲存安定性……………………………………….….63
圖3-16酵素於25℃之儲存安定性…………………………………….…...64
圖3-17 Arrhenius方程式發酵上清液的lnk對1/T作圖…………………...74
圖3-18 Arrhenius方程式F1的lnk對1/T作圖…………………………..…75
圖3-19 Arrhenius方程式F2的lnk對1/T作圖…………………………….76
圖3-20不同p-NPP基質濃度對各階段脂肪分解酶水解活性的影響…….78
圖3-21 酵素液Lineweaver-Burk作圖……………………………………..79
圖3-22 發酵上清液之HPLC 層析圖……………………………………...82
圖3-23 F1之HPLC層析圖…………………...……………………...……..83
圖3-24 F2之HPLC層析圖…………………...……………………...……..84
表目錄
表1-1以真菌為脂肪分解酶生產菌的種類………………………………... 3
表1-2以細菌為脂肪分解酶生產菌的種類………………………………....5
表1-3以酵母菌為脂肪分解酶生產菌的種類………………………………7
表1-4微生物來源的脂肪分解酶在工業上之應用………..………….……16
表1-5氮源對於脂肪分解酶生產菌之酵素產量影響………………..…….19
表1-6微量元素對於脂肪分解酶生產菌之酵素產量影響………….…….20
表2-1a電泳膠體之配方….............................................................................37
表2-1b 電泳膠體之配方. ..............................................................................37
表2-2電泳膠體溶液之配方……………………………………………...…38
表3-1脂肪分解酶之蛋白質純化結果……………………………..….……50
表3-2微量元素對TKU-009脂肪分解酶酵素活性之影響……………..…66
表3-3界面活性劑對TKU-009脂肪分解酶酵素活性之影響……….…….68
表3-4有機溶劑對TKU-009脂肪分解酶酵素活性之影響…….….………70
表3-5 TKU-009脂肪分解酶之基質特異性……….………….……………72

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