Acinetobacter junii C-01 胞外脂肪酶之分離純化及特性

Purification and characterization of extracellular lipase from Acinetobacter junii C-01

陳筱蓉(Hsiao-Jung Chen)

指導教授 ： 吳蕙芬

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201900930

摘要

微生物所分泌的脂肪酶最常被應用於工業，因其容易取得，且分離純化上比 較容易，因此脂肪酶在研究之中依然佔有一席之地。而其中對於 Acinetobacter spp.的研究相較於其他菌株所知甚少，但從目前的文獻可以發現 Acinetobacter spp. 的耐熱及耐鹼的特性經常被應用在工業及製藥等等方面。
本篇從日月潭湖水中所分離出的菌株經過 16S rDNA 定序確認其基因序列與 Acinetobacter junii strain WCHAJ59、Acinetobacter junii strain VITSSJ、 Acinetobacter junii strain KNDSW28、Acinetobacter junii strain CAM121、 Acinetobacter junii strain IZXH15 皆有 98%的相似性，暫時命名為 Acinetobacter junii C-01 且其所分泌的胞外酵素能在 Trybutyrin agar 上產生透明的降解圈且菌株 能在 Tween20 單一營養源平板培養基上生長，經過分離純化將所得的蛋白質利用 ESI-MS-MS(electrospray ionization-tandem mass spectrometry)進行分析，確認其 蛋白質序列與 Acinetobacter junii triacylglycerol lipase 有 99%的相似性，分子量約 為 36kDa。並利用蛋白質序列建立親緣性樹圖發現其與來自 family 1 的 Pseudomonos aeruginosa PAO1 蛋白質序列親緣關係最為相近。所分離出的胞外脂 肪酶其最適作用溫度為 42°C、最適作用酸鹼值為 pH7 ; 此脂肪酶在 16°C~65°C、 pH9~10 穩定性皆良好。相對於短鏈酯質更偏好長鏈酯質，其中當受質為 p- nitrophenyl palmitate(p-NPC16)時，其水解能力為最佳。在 5mM 濃度下五種金屬離子皆會造成明顯的抑制情形。在 0.5%及 5%濃度下，Tween 80 和 Triton X-100 以及 Tween20 皆顯著增加活性，但 SDS 則會明顯抑制活性。在 20%濃度下所有 的有機溶劑皆會造成活性降低。

關鍵字

胞外脂肪酶 ； 不動桿菌屬 ； 酵素活性測試 ； 16S rDNA 定序 ； 電灑法質譜儀分析

並列摘要

The lipase secreted by microorganisms is most commonly used in industry. Because it is easy to obtain and easy to separate and purify, lipase still very important in research. However, the lipase in Acinetobacter spp. is poorly known while compared with that in other strains. Since the lipase is heat-resistant and alkali-resistant properties of Acinetobacter spp., it is often used in industry and pharmaceutics.
The bacterium isolated from the water of Sun Moon Lake was PCR-amplified the 16S rDNA and its resulting nucleotides were shown 98% similar to Acinetobacter junii strain WCHAJ59、Acinetobacter junii strain VITSSJ、Acinetobacter junii strain KNDSW28、Acinetobacter junii strain CAM121、Acinetobacter junii strain IZXH15. It was therefore named Acinetobacter junii C-01 and its secreted extracellular enzyme was able to generate a degradation zone on Trybutyrin agar and the strain can grow on Tween20 single nutrient carbon source plate medium. After separation and purification, the obtained single protein was analyzed by ESI-MS-MS (electrospray ionization- tandem mass spectrometry) to identify the protein ID and it was found that this lipase protein has 99% similarity to the Acinetobacter junii triacylglycerol lipase with a molecular weight of approximately 36 kDa. Also, a phylogenetic tree of lipase proteins was constructed, the A. junii C-01 lipase is mostly related to the family 1 Pseudomonos aeruginosa PAO1 lipase protein. The isolated extracellular lipase has an optimal activity at temperature 42°C and pH 7; also it has stabilities from 16°C to 65°C and pH 9 to 10. Also, it has preferred long-chain esters over short-chain esters, where its hydrolysis activity has an optimal function when the substrate is p-nitrophenyl palmitate (p- NPC16). However, five metal ions at 5 mM concentration caused a significant inhibition of its lipase activity. In contrast, Tween 80 and Triton X-100 and Tween20significantly increased the lipase A activity. But SDS considerably inhibited its activity. Also, the other organic solvents cause a decrease in its activity.

並列關鍵字

Acinetobacter spp. ； Extracellular lipase ； Enzyme activity test ； 16S rDNA sequencing ； ESI-MS-MS

參考文獻

Ahmed, E. H., T. Raghavendra and D. Madamwar (2010). An alkaline lipase from organic solvent tolerant Acinetobacter sp. EH28: Application for ethyl caprylate synthesis. Bioresour Technol 101: 3628-3634.

Christopher, L. P., V. P. Zambare, A. Zambare, H. Kumar and L. Malek (2015). A thermo-alkaline lipase from a new thermophile Geobacillus thermodenitrificans AV-5 with potential application in biodiesel production. Journal of Chemical Technology and Biotechnology 90: 2007-2016.

Esteban-Torres, M., J. M. Mancheño, B. de las Rivas and R. Muñoz (2015). Characterization of a halotolerant lipase from the lactic acid bacteria Lactobacillus plantarum useful in food fermentations. LWT - Food Science and Technology 60: 246-252.

Guncheva, M. and D. Zhiryakova (2011). Catalytic properties and potential applications of Bacillus lipases. Journal of Molecular Catalysis B-Enzymatic 68: 1-21.

Gupta, R., N. Gupta and P. Rathi (2004). Bacterial lipases: an overview of production, purification and biochemical properties. Appl Microbiol Biotechnol 64: 763- 781.