醋酸鈣不動桿菌RAG-1脂解酶之生物化學特性

The biochemical properties of the lipase produced by Acinetobacter calcoaceticus RAG-1

吳昶志(Chang-Jr Wu)

指導教授 ： 陳文章

國立臺北科技大學/工程學院/生物科技研究所/碩士(2006年)

摘要

生物乳化素是由脂解酶和多醣組成的複合物，能穩定油/水乳化物，能應用在污染防治、生物復育、農業、清潔劑和化妝品工業。本研究利用不動桿菌屬醋酸鈣種(Acinetobacter calcoaceticus RAG-1)生產生物乳化素，以酒精為碳源培養基做為對照組，植物油及礦物油為碳源培養基做為實驗組，所得培養液離心去除菌體後以膜過濾系統濃縮純化胞外脂解酶，接著以Bradford染劑分析蛋白質濃度法、蛋白質電泳法、陰離子交換樹脂純化法鑑定胞外脂解酶，再以辛酸4-硝苯酯(4-Nitrophenyl caprylate)為受質測量胞外脂解酶的酵素催化反應條件：pH值、反應溫度及酵素動力。以酒精及植物油為碳源培養基所生產的胞外脂解酶過程中，酵素釋放至胞外的時間、途徑相同，pH反應範圍8~10相同，溫度反應範圍30~50℃相同；以植物油為碳源培養基所生產的胞外脂解酶的催化動力學參數Km=1.35 mM，以酒精為碳源培養基所生產的胞外脂解酶的Km=11.16 mM，植物油為碳源培養胞外脂解酶的酵素活性中心對受質辛酸4-硝苯酯有較強的結合力，其反應生成物對硝基酚(p-nitrophenol)之反應速率較慢，以酒精為碳源培養基所生產的胞外脂解酶之反應速率較快。

關鍵字

胞外脂解酶不動桿菌屬 醋酸鈣種 ； 酵素動力

並列摘要

Bioemulsans are extracellular lipase-complexed polysaccharides that stabilize oil-in-water emulsions and have applications in industrial areas of pollution control, bioremediation, agriculture, detergent, and cosmetics, etc. The Acinetobacter calcoaceticus RAG-1 grew in batch cultivations on the respective ethanol, vegetable oil and mineral oil medium as the sole carbon source to produce bioemulsans. The cell-free, lipase-containing culture supernatants were concentrated by using centrifugation and membrane dialysis for the separation of impurities. The protein contents of extracellular enzymes were characterized by Bradford essay method, SDS-PAGE, and the fast-flow gel filtration. The enzymatic, kinetic activities of extracellular lipases were investigated how to affect the catalytical rate of hydrolysis of 4-nitrophenyl caprylate (pNC8) as a function of pH and temperature. The experimental results showed that the nutrients of either ethanol or vegetable oil have similar effects on the microbial lipase production such as extracellular enzyme-release in the stationary phase, protein characteristics, and the pH-, and temperature-dependent lipolytical activities etc. The enzymatically kinetic analyses also showed that the Michaelis-Menten constant, Km of extracellular lipase by the addition of ethanol nutrient was 11.16 mM, eight times higher than Km = 1.35 mM of that by addition of vegetable oil nutrient. It was therefore inferred that the substrate pNC8-enzyme complexes of the latter active lipase have slower rates of 4-nitrophenol formation than that of the former lipase.

並列關鍵字

extracellular lipase ； Acinetobacter calcoaceticus ； enzyme kinetic

參考文獻

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