台灣鳥來溫泉嗜熱細菌Bacillus thermocatenulatus WL 65之篩選

Abstract

本實驗由台灣北部溫泉和其他地熱環境分離得到80株嗜熱細菌。將這些菌株純化後，先做初步外泌酵素（蛋白?、澱粉水解?、脂質水解?）測試，選取其中9株做形態及生理特性之分析，並依據165 rDNA基因序列加以鑑定。其中7株會產生內抱子的被鑑定為 Bacillus，另有兩株不產生內孢子的則是Thermus。一株自烏來溫泉中篩選出的好氧嗜熱菌，Bacillus thermocatenulatu, WL65，被桃選來進行本實驗。將此菌株在添加typtone和yeast extract於Thermus基本培養基，於55℃下培養，會產生外泌性蛋白?。此株嗜熱菌具有周圍鞭毛和革蘭氏染色呈現Gram variable的現象。將此菌大量培養去除菌體後，將粗酵素液以硫酸銨沉澱、疏水性管柱層析、陰離子交換樹脂管柱層析進行純化。得到此蛋白?的比活性為25.83U/mg蛋白質，其分子量約65kDa；酵素最適當作用的酸鹼值在pH9,最適作用溫度在37℃；即使將溫度提高到50℃時，酵素仍保有95%以上的活性。此蛋白?活性會被EDTA所抑制，添加1 mM Cu(上標 2+)會使酵素活性增加。由這些實驗結果得知，此酵素為熱穩定型之鹼性蛋白?，銅的添加可以促進酵素活性。

Eighty strains of thermophilic bacteria were isolated from hot springs and geothermal environment of Northern Taiwan. Nine strains of these isolates were selected by preliminary analysis of extracellular enzymes (protease, lipase, and amylase) productions. Conventional morphological and physiological characteristics as well as analysis based on 16S rDNA gene sequences were examined for the identification of these microorganisms. Seven endospore producing strains were identified as Bacillus. The other two non-spore forming isolates were identified as Thermus. Bacillus thermocatenulatus WL 65, isolated from a hot spring in Wu-lai, was selected for this study. This strain produced protease into extracellular medium when grown on Thermus medium containing tryptone and yeast extract at 55℃. This aerobic thermophile had peritrichous flagella and stained Gram-variable. Protease was purified from the culture supernatant by using ammonium sulfate precipitation, hydrophobic interaction chromatography, and anion exchange adsorption. The specific activity of the purified protease was 25.83 U/mg protein. The molecular weight determinted by 10% SDS-PAGE was about 65 kDa. The enzyme had maximum activity at pH 9.0 and 37℃, and more than 95% enzymatic activity was retained when temperature was raised to 50℃. The proteolytic activity was inhibited by EDTA. The activity was stimulated in the presence of 1 mM Cu(superscript 2+). These experimental results indicated that the enzyme was a thermostable alkaline protease, the addition of copper will stimulate enzyme activity.