麵包蟲中腸分離菌株Pseudomonas nitroreducens之酯酶特性分析

Characterization of an esterase of Pseudomonas nitroreducens from Tenebrio molitor mid-gut

關榮恩(Jung-En Kuan)

指導教授 ： 吳蕙芬

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202004048

摘要

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。

關鍵字

假單孢菌屬 ； 硝酸根還原菌 ； 酯酶 ； 脂肪酶 ； 第四型脂肪酶

並列摘要

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.

並列關鍵字

Pseudomonas nitroreducens ； esterase ； type IV lipase ； lipase

參考文獻

1. Anzai, Y., Kim, H., Park, J.Y., Wakabayashi, H., and Oyaizu, H. (2000). Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence. Int J Syst Evol Microbiol 50 Pt 4, 1563-1589.

2. Arpigny, J.L., and Jaeger, K.E. (1999). Bacterial lipolytic enzymes: classification and properties. Biochem J 343 Pt 1, 177-183.

3. Beer, H.D., Wohlfahrt, G., McCarthy, J.E., Schomburg, D., and Schmid, R.D. (1996). Analysis of the catalytic mechanism of a fungal lipase using computer-aided design and structural mutants. Protein Eng 9, 507-517.

4. Bergmann, F., and Rimon, S. (1958). The effect of pH variations on the activities of esterase. Biochem J 70, 339-344.

5. Boratyn GM, Thierry-Mieg J, Thierry-Mieg D, Busby B, Madden T.L. (2019) "Magic-BLAST, an accurate RNA-seq aligner for long and short reads." BMC Bioinformatics. 25, 20:405.