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標題: 耐冷菌Pseudomonas sp. P116產生之胞外脂質分解的基因選殖,純化與特性分析
Lipolytic enzyme from the Psychrotrophic Bacterium Pseudomonas sp. P116: Gene Cloning and enzyme Purification and Characterization
作者: 李慧貞
Lee, Hui-Chen
關鍵字: cold-active enzymes;低溫活化酵素;Pseudomonas sp. P116;lipolytic enzyme;protease;Pseudomonas sp. P116;脂質分解;蛋白質分解
出版社: 植物學系
在自然與人為的環境中不乏低溫的所在,在這些地方棲息著許多能適應寒冷的微生物(cold-adapted microorganism),它們不但能在寒冷下生存,有些甚至還能在接近0℃的低溫下生長。適低溫性微生物,包括嗜冷微生物(psychrophiles)和耐冷微生物(psychrotrophs),會產生在低溫狀態下具有高活性的酵素,這些酵素稱為低溫活化酵素(cold-active enzymes)。
目前在國內,似乎未見有人對適冷酵素進行研究。近來本實驗室由台灣中部合歡山山區長年低溫的生育地,分離出一株具有脂質分解與蛋白質分解活性的耐冷細菌,經初步鑑定暫時命名為Pseudomonas sp. P116。選殖此菌染色體DNA上帶有脂質分解基因的片段並加以定序,結果顯示有一個包含933 個核酸的open reading frame,可轉譯出310個胺基酸。進一步分析此氨基酸序列,發現在其活化位置的絲氨酸(serine)周圍的胺基酸序列上具有一個高度保留的pentapeptide sequence:GXSXG(“G”表Glycine ,“S”表Serine,“X”表任意的胺基酸),而組成其活化位置的催化三聯體( catalytic triad )則分別為Serine-154、Aspartate-252及Histidine-281。
將Pseudomonas sp. P116的脂質分解與蛋白質分解純化出來,並分別分析其生化特性。結果顯示脂質分解的分子量約為33 kDa,最適作用溫度為35℃,在 pH 7 - 9時具有最高的酵素活性,其活性表現會受抑制劑EDTA及EGTA的影響;而蛋白質分解的分子量約為50 kDa,最適作用溫度為37 ℃,最佳反應的酸鹼度為 pH 5 - 7,抑制劑EDTA及EGTA對蛋白質分解活性的表現影響不大。

In this study, Pseudomonas sp. P116, a gram-negative psychrotrophic bacterium displays extracellular lipolytic and proteolytic activities,was isolated from Ho-Huan Mountain of middle Taiwan. Following shotgun cloning, a DNA fragment containg lipolytic enzyme gene was obtained and sequenced. Analysis of the complete nucleotide sequence of this fragment revealed an open reading frame of 930 nucleotids, which may encode a sequence of 310 amino acids. Alignment comparison showed this amino sequence was homologous to the lipase/esterase of Pseudomonas aeruginosas PAO1 and Bacillus halodurans.
Both extracellular lipase and protease have been purified from the culture supernatant of P116 with DEAE-cellulose column. SDS-PAGE revealed their molecular weight were 33 kDa and 50 kDa , respectively. Results of enzymatic activity analysis indicated the optimal temperature and pH for lipase are 35℃and pH 7.0, and for Protease are 37℃and pH 7.0, respectively.
Appears in Collections:生命科學系所

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