臺灣博碩士論文加值系統

本研究取麵包蟲(Tenebrio molitor)中腸道段中的共生菌，以甘油三丁酸酯作為單一碳源培養，篩選出可能帶有脂肪酶之菌株，將所得之菌種用16s核醣體去氧核醣核酸(16s rDNA)作為鑑定標準，比對資料庫並使用比對分析後知其為Pseudomonas nitroreducens，再從物種Pseudomonas nitroreducens的蛋白質資料庫中尋找可能為脂肪酶之α/β水解酶，以帶有限制酶酵素的切位和組胺酸標記的候選引子進行聚合酶連鎖反應，經限制酵素、接合酵素反應，建構以pET21a質體為載體的重組質體，以轉型作用送入大腸桿菌BL21(DE3)中用異丙基-β-D-硫代辦乳糖苷(IPTG)誘導，續用親和性管柱進行純化，發現洗脫後重組蛋白質可在甘油三丁酸(tributyrin)洋菜膠盤上產生降解圈。將純化過後的重組蛋白質進行酵素活性測試，以具有不同碳鍊長度的對─硝基苯酚酯類 (p-nitrophenyl ester)作為受質，偵測波長405 nm的吸光值來計算受質的分解狀況，藉此推估酵素活性。經實驗數據，重組蛋白質在溫度40℃、pH 8 時具有最高活性，且對於丁酸對─硝基苯酚酯 (p-nitrophenyl butyrate)分解效率最佳，在熱穩定性實驗中，當處理溫度大於50℃時，其活性下降劇烈。離子對活性影響分析中發現MgCl2和NH4Cl可提高酵素活性，MnSO4、NiSO4、CaCl2、ZnSO4、CoCl2、CuSO4、FeSO4、FeCl3 則會使活性降低，NaCl 則對於活性沒有顯著性的影響。有機溶劑和界面活性劑的添加方面，發現DMSO (Dimethyl sulfoxide)可幫助提升酵素活性，正己烷 (Hexane)、甲醇 (Methanol)、乙醇 (Ethanol)、丙酮 (Acetone)、異丙醇 (Isopropanol)、三氯甲烷 (Chloroform)、乙酸乙酯 (Ethyl acetate)會降低酵素活性，而甘油 (Glycerol)則不會對酵素有顯著的影響，界面活性劑方面，Triton x-100、Tween 80、 Tween 20、Brij35等界面活性劑皆會使酵素活性減少。經Lineweaver-Burk雙倒數作圖法，可得出之Km 值為0.488 (mM)，Vmax為0.0644 (mM/min)， kcat為3.01 (s-1)，kcat / Km為6.17 (mM-1/s-1)。之後，經脂肪酶親緣性分析，LipD為第四型脂肪酶，為此蛋白質的分類依據，並探討其所具有之獨特的特性。

In this study, a symbiotic bacteria from yellow mealworm’s (Tenebrio molitor) mid-gut was isolated with characteristics of growth on minimal-tributyrin medium. After a PCR-amplification of its 16s rDNA, the resultant nucleotide sequences were then analyzed by schemes of the phylogeny trees. Accordingly, it was designated as Pseudomonas nitroreducens D-01. Next, by searching the lipolytic enzymes in its protein data bank, one of those potential lipolytic α/β hydrolases was identified, again using PCR-amplification and nucleotide-sequencing methods. To construct an expression of this lipolytic gene in plasmids, the target-gene primers were then designed, carrying the C-terminal his-tag sequences. Using the vector pET21a, a recombinant lipolytic hydrolase D gene with his-tag nucleotides was successfully cloned into it, of which the lipolytic D gene is under a control of the T7 promoter. After transformation of the resultant plasmids into Eescherichia coli BL21 (DE3), an IPTG inducer was used for the induction of the recombinant proteins. The protein products were then purified by metal-ion affinity column and the purified proteins were found capable of forming a clear zone on tributyrin agar plate. Shortly, its enzyme activities were determined by degradation of p-nitrophenyl ester(s) and the substantial yellow end-product, p-nitrophenol, was measured at O.D.405 nm. Specifically, this lipolytic enzyme efficiently targets p-nitrophenyl butyrate. As well, it shows the most reactive activities at 40℃, pH 8 in potassium phosphate buffer. In thermal stability assays, the activities of this enzyme dramatically drop when the temperature is above 50℃. In metal ion assays, MgCl2 and NH4Cl induce the enzyme activities while MnSO4, NiSO4, CaCl2, ZnSO4, CoCl2, CuSO4, FeSO4, and FeCl3 reduce its activities. Besides, NaCl has no effects on its enzyme activities. Most organic solvents decrease the activities of this enzyme such as hexane, methanol, ethanol, acetone, isopropanol, chloroform and ethyl acetate. However, its enzyme activities increase when DMSO exists. All the surfactants like Triton X-100, Tween 80, Tween 20, and Brij35 decrease its lipolytic activities. Using Lineweaver-Burk double reciprocal methods, the function of the enzyme kinetics were determined such as Km = 0.488 (mM), Vmax = 0.0644 (mM/min), and kcat = 3.01 (s-1), as well the total efficiency of kcat / Km is 6.17 (mM-1/s-1). Afterwards, based on the phylogenetic analyses, this lipolytic protein is classified to type IV lipase by its homologous conserved region in this lipase family.

中文摘要……………………………………………………………………...……...i
Abstract………………………………………………………………………….…..iii

壹、緒論………………………………………………………………………….......1
貳、材料及方法…………………………………………………………………….7
一、 實驗材料…………………………………………………………..………..7
1.實驗菌株及質體………………………………………………..…………7
2.藥品、酵素及引子…………………………………………..……………7
3.套組…………………………………………………………..……………7
4.實驗設備……………………………………………………..……………7
5.分析軟體…………………………………………………………..………8
二、 實驗方法…………………………………………………………..………..8
1.蟲體處理和篩菌………………………………………………..…………8
2.菌種鑑定…………………………………………………………………..9
3.基因處理…………………………………………………………………11
4.重組質體建立……………………………………………………………12
5.重組質體放大……………………………………………………………14
6.蛋白質表現及純化………………………………………………………15
7.酵素活性及特性測試……………………………………………………17
8.重組蛋白質親緣性分析…………………..…………………………..…19
參、結果……………………………..…………………………………..…………20
1.菌種篩選及其鑑定………………………………………………………20
2.篩選目標基因與選殖於pET21a載體……………………..……………20
3.重組蛋白質表現、純化及其生理活性測試……………………………21
4.測定酵素基質特異性……………………………………………………21
5.測定酵素最適pH值……………………………………….……………21
6.測試酵素最適反應溫度及熱穩定性…………………….……………...21
7.含陽離子下酵素反應活性測試…………………….…………………...22
8.含有機溶劑下酵素反應活性測試…………………….………………...22
9.含界面活性劑下反應活性測試…………………….…………………...22
10.酵素動力學測試及分析…………………….………………………….22
11.重組蛋白質親緣性分析…………………….………………………….23
12. 重組蛋白質結構預測及區塊活性分析…………………...……….…23
肆、討論……………………………………………………………………………24
伍、結論……………………………………………………………………………29
陸、參考文獻…………………………………..…………………………………30
柒、表…………………………………………………………………….…………34
捌、圖……………………………………………………….………………………37
玖、附錄……………………………………………………….…….……………..52

表次
表一、本篇使用之菌株…………………………………………………..……..........34
表二、本篇使用之質體…………………………………………………..……..........34
表三、本篇所使用之引子………………………………………….……..…...…......35
表四、Braford檢量線………………………………………………….……............36
表五、活性測試檢量線…………………………………………………….…..........36

圖次
圖一、16s rDNA親緣性分析………………………………………………….…….37
圖二、目標基因篩選結果……………………………………………….……....…..38
圖三、重組蛋白質表現及純化………………………………………….……….….39
圖四、酵素基質特異性測試………………………………………………………...41
圖五、最適反應pH值測試…………………………………………………………42
圖六、最適反應溫度測試…………………………………………………………...43
圖七、熱穩定性測試…………………………………………………………………44
圖八、含陽離子條件下反應活性測試………………………………………………45
圖九、含有機溶劑條件下反應活性測試……………………………………………46
圖十、含界面活性劑條件下反應活性測試…………………………………………47
圖十一、酵素動力學測試及分析……………………………………………………48
圖十二、脂肪酶親緣性分析…………………………………………………..…….49
圖十三、重組蛋白質結構預測………………………………………………………50
圖十四、重組蛋白質和其他bHSL保守序列比較………………………………..51

附錄次
附錄一、基質特異性測試……………………………………………………....……52
附錄二、最佳pH值測試…………………………………………….………………53
附錄三、最適反應溫度測試…………………………………..………….….....……54
附錄四、熱穩定性測試………………………………………………………………55
附錄五、含陽離子下活性測試………………………………………………………56
附錄六、含有機溶劑下活性測試……………………………………………………58
附錄七、含界面活性劑下活性測試…………………………………………………60
附錄八、本篇之候選蛋白質…………………………………………………………61
附錄九、LipD之核酸及胺基酸序列………………………………..………………62

附圖次
附圖一、Pseudomonas nitroreducens電顯圖………………………………....………2
附圖二、脂肪酶二級結構圖……………………………………….………………….3
附圖三、脂肪酶蓋子及界面示意圖…………………………..………….….....…..…3
附圖四、小鼠HSL (Hormone sensitive lipase)………………………………………..5
附圖五、人類HSL和四種bHSL (bacterial HSL)的保守區域比較………………….5
附圖六、本研究所分離之重組蛋白LipD二級結構示意圖………………………..27
附圖七、人類胰脂酶二級結構示意圖………………………………………………28
附圖八、Geotrichum candidum脂肪酶二級結構示意圖……………………………28

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