Acinetobacter junii C-01胞外脂肪酶之分離純化及特性

Purification and Characterization of Extracellular Lipase A from Acinetobacter junii C01

陳筱蓉(Hsiao-Jung Chen) ； 吳蕙芬(Whei-Fen Wu)

《臺灣農業化學與食品科學》 57卷5&6期 (2019/12) Pp. 201-209

https://doi.org/10.6578/TJACFS.201912_57(5_6).0001

摘要

從日月潭湖水中所分離出的Acinetobacter junii菌株，其所分泌的胞外酵素在三丁酸甘油酯平板培養基上產生透化圈。收集細菌生長之胞外上清液，經由分離純化將所得的單一蛋白質，亦可在上述平板培養基上，產生透化圈。利用ESI-MS-MS（electrospray ionization-tandem mass spectrometry）進行分析，確認其蛋白質序列為lipase A，分子量約為36 kDa。經由胺基酸序列比對，得到活化位為Ser124、Asp252、His294，其中Serine位在具有保守性的五胜肽序列GHSQG上，和另外兩個胺基酸殘基構成催化三聯體（catalytic triad）。針對胞外脂肪酶LipA進行酵素特性分析，發現相對於短鏈脂質，其更偏好長鏈脂質，其中當受質為p-nitrophenyl palmitate（p-NPC16），其水解能力為最佳。最適作用溫度為42℃、最適作用酸鹼值為pH 7；此脂肪酶在16-65℃、pH 9-10穩定性皆良好，且酵素活性於pH 4-10皆具有活性。在鋰、鈉、鎂離子，1 mM濃度下，皆會增進酵素活性；但是，1 mM的銨離子會抑制其活性。相對的，非離子型介面活性劑Tween 80、Triton X-100及Tween 20皆能增進酵素活性，但離子型介面活性劑sodium dodecyl sulfate（SDS）則會抑制其活性。添加有機溶劑，二甲基亞碸dimethyl sulfoxide（DMSO）亦會造成酵素活性的降低。

關鍵字

不動桿菌屬 ； 胞外脂肪酶 ； 電灑法質譜儀分析 ； 酵素活性分析

並列摘要

The bacterium, Acinetobacter junii, which was isolated from the water of Sun Moon Lake, could secret extracellular lipases and grown on tributyrin agar plate with encircled transparent zone. After purification, an extracellular lipase protein alone also showed an encircled transparent zone on tributyrin agar plate. By ESIMS- MS (electrospray ionization-tandem mass spectrometry), the lipase A, with MW 36 kDa, was identified. By phylogenetic tree analysis, this LipA belongs to lipase family 1. By amino-acid sequence alignment, an active site, Ser124, Asp252 and His294, were revealed in LipA. The Ser 124 was in the conserved sequences, GHSQG; a catalytic triad site was formed with the above two amino acids. It has preferred long-chain esters over short-chain esters, since an optimal activity was revealed while using p-nitrophenyl palmitate (p-NPC16) as substrates. The maximum activity was revealed at pH 7.0 under 42oC. It exhibits lipase activity over a broad range of temperatures and good stability. Although, the LipA only stable at pH 9 and 10, it maintains an activity over a broad range of pHs from 4.0 to 10.0. Also, the K^+, Na^+ and Mg^(2+) ions, at 1 mM, enhance the LipA activity; but partially inhibited by NH_4^+. As shown, Tween 80 and Triton X-100 and Tween 20 surfactants increase the LipA activity. But an ionic-surfactant, sodium dodecyl sulfate (SDS), inhibits its activity. The other organic solvent, such as dimethyl sulfoxide (DMSO), also decreases its activity.

並列關鍵字

Acinetobacter spp. ； Extracellular lipase ； ESI-MS-MS ； Enzyme activity test