產鹼假單胞菌A25之LipA純化與特性分析

Abstract

本實驗室先前研究已經發現，Pseudomonas alcaligenes A25具有降解磷苯二甲酸二（2-乙基己基）酯 [di(2-ethylhexyl)phthalate, DEHP] 能力，此菌株能夠分泌胞外脂肪酶，在三丁酸甘油脂平板培養基上產生透化圈。透過選殖得到脂肪酶LipA，經過胺基酸序列比對建立親緣關係樹，確定LipA屬於Family1.I脂肪酶，需要Lif (lipase- specific foldase)幫助摺疊成具有活性的構型，單獨異源表現lipA會因為摺疊不完全導致蛋白質彼此聚集以不可溶形式存在包涵體中。因此利用異源共表達系統同時表現LipA以及LifB，在經過誘導以及純化後，得到可溶且具活性的LipA，由LipA基因序列轉譯成蛋白質序列，預測LipA蛋白質大小為35 kDa，序列比對得到活化位為Ser111，Asp257，His279其中Serine位在保守五胜肽序GHSHG上，和另外兩胺基酸殘基構成催化三元。由LifB基因序列轉譯成蛋白質序列，預測LifB蛋白質大小為38 kDa。針對LipA進行其他酵素特性分析，受質特異性測試分析結果顯示，相較於長碳鏈酯類，LipA偏好分解短碳鏈酯類，尤其以pNPC2(p-nitrophenyl acetate) 為受質時水解能力最佳，因此判斷其為酯解酵素 (3.1.1.1)。LipA反應溫度非常廣泛，溫度範圍在40~70℃下進行反應都具有良好活性，在溫度50℃有最大相對活性。其最適反應溫度為50℃，最適反應pH值為8。穩定性測試方面，在20℃以及pH 8條件下能維持良好活性。對於大部分金屬離子耐受性佳，重金屬Zn2+對酵素活性抑制較為顯著，非離子型介面活性劑Brij 35對酵素活性有提升效果，離子型介面活性劑Sodium Dodecyl Sulfate (SDS) 則會抑制酵素活性。多數有機溶劑對酵素活性有不同程度抑制，僅有ethanol對酵素活性影響較小，其中以chloroform以及isopropanol對活性抑制最為顯著。

Pseudomonas alcaligenes A25 was firstly selected for its capable of degrading DEHP. This bacterial strain also could secret extracellular lipases, grown on tributyrin agar plate with surrounded transparent zone. In this study, Pseudomonas alcaligenes A25 lipA+ gene was cloned and its encoded protein was referred to the phylogenetic tree analysis. As results, LipA belongs to lipase family 1.I, which needs a lipase specific foldase to fold itself into an active structure. LipA was folded incompletely when it was expressed alone. Therefore, it was aggregated and formed insoluble inclusion body. However, soluble and active LipA was obtained by simutaneously expressing LipA and LifB (lipase specific foldase) in bacterial cells. LipA was about 35 kDa and LifB was about 38 kDa. By amino-acid sequence alignment, an active site, Ser111, Asp257 and His279 were revealed in LipA. Ser 111 was in the conserved site, GHSHG; the disulfide bond which formed by Cys 213 and Cys 219, stabilizes its structure. In degradation assay, LipA prefered to degrade short chain p-nitrophenyl esters. Especially, LipA degrades pNPC2 (p-nitrophenyl acetate) efficiently. Therefore, LipA belongs to the esterase family (3.1.1.1). The optimal temperature for LipA activity is wide; it matains a higher activity beTween 40℃ and 70℃. Specifically, it had the highest activity at 50℃. An optimal pH for LipA activity was pH 8. LipA retains its most stability at pH 8 and 20℃. In addition, LipA could tolerate most of the metal ions used in the assays. However, its enzymatic activity was inhibited by a heavy metal ion, Zn2+. Furthermore, its enzymatic activity was elevated by nonionic-surfactants, Brij 35, but it was inhibited by ionic-surfantant, SDS. Similarly, the enzymatic activity of LipA was inhibited by most organic solvents.