- 學位論文
Serratia plymuthica 的分離及其蛋白質分解酵素的研究
Characterization of extracellular protease from the isolation Serratia plymuthica
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摘要
在自然環境中存在著許多可以適應寒冷環境的微生物，通稱為「適冷菌」，包括嗜冷菌和耐冷菌，有些可以在低溫狀態下產生具有高活性的酵素，稱之為「低溫活化酵素」。本實驗從家庭儲存食物的冰箱中取一些水樣或腐敗食物，選殖出一株在低溫環境下具有高產量的蛋白質分解酵素，以10％的SDS-gelatin-PAGE測定蛋白質分子量，估計約為48 kDa。根據菌種的生理特性鑑定結果，可得此菌種學名為Serratia plymuthica。 將此菌株培養在不同營養成份的培養液中，測量此菌種的生長情形及蛋白質分解酵素活性的變化。發現此菌株的生長會受到環境中L型纈胺酸、L型半胱胺酸、L型酥胺酸的抑制；DL型丙胺酸、L型絲胺酸、L型麩胺酸可促進此菌生長；而硫酸銨不影響細菌生長。 以上的氮源對酵素活性大小影響如下：在礦物培養基中，L型絲胺酸、L型麩胺酸的添加會抑制酵素活性；在含有1％明膠的礦物培養基中，L型半胱胺酸、硫酸銨的添加也會抑制酵素活性。但DL型丙胺酸除了可促進細菌的生長外，亦會隨著濃度的增加而促使酵素活性增大，在0.25％DL型丙胺酸存在時，可達最高值，但超過此濃度，則開始抑制使得酵素活性降低，故DL型丙胺酸的調控可能具有胞外分解酵素演化上的意義：當環境含量少時會酵素產生促進，但環境中含量多時則抑制酵素的產生，而直接從環境取得。
並列摘要
Serratia plymuthica, a psychrotropic bacterium isolated from a refrigerating environment was found to produce an extracellular protease functioning on a wide range of temperature from a low 4℃ to a moderate 37℃. Crude extracts of this enzyme was characterized regarding to its properties, its production and regulation of its enzyme activity. Particularly, effects on the growth of Serratia plymuthica and its extracellular protease production by varying amino acid end-products were extensively investigated. Physically, a proteolytic enzyme activity separated on a sodium dodecyl sulfate-gelatin-polyacrylamide gel electrophoresis was located on a spot corresponding to 48 kDa. Addition of L-valine, L-cystein monochloride and L-threonine as the carbon and energy sources to a mineral medium, exhibited an inhibitory effect on the growth of this bacterium. On the contrary, addition of DL-alanine, L-serine and L-glutamic acid as the carbon and energy sources to a mineral medium, an accelerating promotion of growth was observed. Whereas, using (NH4)2SO4 as a replaced nitrogen source in the same basal medium for culturing, no any effects were appeared. Regulatory effects by various amino acids on the synthesis of extracellular protease by Serratia plymuthica were also evaluated. Its proteolytic activity accumulated in the growth medium was feedback inhibited by many different amino acids including L-serine, L-glutamic acid, or L-cystein monochloride and (NH4)2SO4 plus 1% gelatin as a protein source. However, the most striking characteristic pattern of changing protease yields was exhibited by using DL-alanine as an interesting regulator. When increasing concentrations of DL-alanine (0 to 0.25%) were added to the culture media, yields of protease were also gradually increased until reaching a maximum level corresponding to 0.25% of DL-alanine. Beyond that, any concentrations of DL-alanine present in the medium exceeding 0.25% resulted in lowering yields of this extracellular enzyme. Apparently, Serratia plymuthica has evolved a very simple regulatory mechanism by using alanine both as a protease inducer and an inhibitor. When this bacterium senses a lower concentration of alanine in environment, it responses by producing more protease in order to harvest more amino acids substrates; otherwise, stops protease production.
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