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Pseudomonas alcaligenes脂肪酶(LipA)純化與特性分析

Purification and Characterization of Lipase A from Pseudomonas alcaligenes

摘要


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。

關鍵字

假單孢菌屬 脂肪酶 酯酶 摺疊酶

並列摘要


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.

並列關鍵字

Pseudomonas alcaligenes Lipase Esterase Foldase

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