由環境中分離的Pseudomonas alcaligenes菌株能夠分泌胞外脂肪酶,可在三丁酸甘油酯平板培養基上產生透化圈。透過生物資訊的篩選,得到脂肪酶LipA胺基酸序列。經過胺基酸序列比對建立親緣關係樹圖,確定LipA屬於Family 1脂肪酶,需要LifB(lipase- specific foldase)伴隨蛋白質,幫助摺疊成具有活性的蛋白質。因此,利用具有異源共表達系統的質體(pACYCDuet-1),可同時表現LipA及LifB。在經過IPTG誘導,蛋白質純化及西方墨點法偵測後,得到具可溶活性的LipA。經由胺基酸序列比對,得到活化位為Ser111、Asp257、His279,其中Serine位在具有保守性的五胜肽序列GHSHG上,和另外兩個胺基酸殘基構成催化三組合(catalytic triad)。針對LipA進行酵素特性分析,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,因此被歸類為酯解酵素(3.1.1.1)。最適合LipA酵素活性溫度非常廣泛,在溫度範圍40℃~70℃下進行測試,皆具有良好活性,50℃為其最適反應溫度。當pH值為8,酵素活性較佳。針對酵素穩定性測試,在20℃下保存,能維持良好活性。此酵素對於大多數金屬離子的耐受性佳,Zn^(2+)離子對酵素活性抑制性較為顯著。非離子型介面活性劑Brij 35對酵素活性具有提升效果;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的抑制,其中以異丙醇對LipA酵素活性抑制性最為顯著。
Pseudomonas alcaligenes, which was isolated from the environments, could secret extracellular lipases and grown on tributyrin agar plate with surrounded transparent zone. By the bioinformatics analysis, the amino-acid sequences of its LipA were referred to the phylogenetic tree analysis. The LipA belongs to lipase family 1, which needs a lipase-specific foldase (LifB) to assist an active folding of itself. In this study, using the dual active promoter plasmid, lipA^+-ha and lifB^+ genes were in series cloned into pACYC-duet-1. Through a coincident expression of LipA and LifB by IPTG induction, the soluble LipA protein could be purified and detected by Western blot analysis. By amino-acid sequence alignment, an active site, Ser111, Asp257 and His279, were revealed in LipA. Ser 111 was in a conserved position, GHSHG and a catalytic triad site was formed with the other two amino acids. In the degradation assay, LipA preferred to degrade short chain p-nitrophenyl esters. Especially, LipA degraded pNPC2 (p-nitrophenyl acetate) efficiently. Therefore, LipA belongs to the esterase family (3.1.1.1). The optimal temperatures for LipA activity are wide-range; it maintains a higher activity between 30℃ and 70℃. Specifically, it had the highest activity between 40oC~ 50℃. At pH 8, the enzyme activity was also optimal. LipA retains its 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 Zn^(2+). In contrast, its enzymatic activity was elevated by nonionic-surfactants, Brij 35, but it was inhibited by ionic-surfactant, SDS. Similarly, the enzymatic activity of LipA was inhibited by most organic solvents, such as isopropanol.
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