篇名 | Pseudomonas alcaligenes脂肪酶(LipA)純化與特性分析 |
---|---|
卷期 | 57:4 |
並列篇名 | Purification and Characterization of Lipase A from Pseudomonas alcaligenes |
作者 | 林佳慧 、 吳蕙芬 |
頁次 | 165-172 |
關鍵字 | 假單孢菌屬 、 脂肪酶 、 酯酶 、 摺疊酶 、 Pseudomonas alcaligenes 、 Lipase 、 Esterase 、 Foldase 、 Scopus 、 TSCI |
出刊日期 | 201908 |
DOI | 10.6578/TJACFS.201908_57(4).0001 |
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Lif B (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,Zn2+ 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 30oC and 70oC. Specifically, it had the highest activity between 40oC~ 50oC. At pH 8, the enzyme activity was also optimal. LipA retains its stability at pH 8 and 20oC. In addition, LipA could tolerate most of the metal ions used in the assays. However, its enzymatic activity was inhibited by Zn2+. 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.