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Isolation and Characterization of Bacteria which Tolerate High pH ( ＞ 10) and Produce Protease

Isolation and Characterization of Bacteria which Tolerate High pH ( ＞ 10) and Produce Protease. 報告學生： 林沛穎、 譚皓仁 報告日期： 2003. 12.29. 前言. Alkaliphile ： 能生長在 pH 值＞ 9 ，但無法生長於 pH 值＜ 6.5 的微生物 包括： (1) Bacillus species , Micrococcus species,

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Isolation and Characterization of Bacteria which Tolerate High pH ( ＞ 10) and Produce Protease

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  1. Isolation and Characterization of Bacteria which Tolerate High pH (＞10) and Produce Protease 報告學生： 林沛穎、 譚皓仁 報告日期：2003.12.29

  2. 前言 • Alkaliphile ：能生長在pH值＞9，但無法生長於pH值＜ 6.5的微生物 • 包括： (1)Bacillus species , Micrococcus species, Pseudomonas species, Streptomyces species (2) 酵母菌 (3)真菌

  3. Alkaliphile較容易從鹼性環境中分離出來，但也可從一般 土壤中找到

  4. Alkaliphiles的應用： • 大多用於應用工業 • 其所產生的 alkaline cellulase 或 alkaline protease 可作為生物清潔劑

  5. 細菌的 alkaline protease 的特質為： (1) 在高pH值（e.g,10）具有高度活性 (2) 廣泛的受質專一性 (3) 最佳的作用溫度為60℃

  6. 實驗目的 從環境中分離出耐鹼且會產生 protease 的微生物

  7. 實 驗 步 驟 • 分離耐鹼菌 以鹼性培養基作分離 • 檢測是否會產 protease 以 skim milk agar 培養 觀察菌落周圍有無透明環 • 將 skim milk agar 上的菌落再接到鹼性培養基作確認

  8. Alkaline medium的成分(g / L)： • Glucose / Soluble starch………………………….10g • Yeast extract……………………………………….5g • Peptone……………………………………………..5g • K2HPO4…………………………………………….1g • MgSO4 · 7H2O…………………………………...0.2g • Na2CO3 ……………………………………………10g • Agar………………………………………………..20g

  9. Skim milk agar的成分(g / L)： • Skim milk powder………………………………..10g • Yeast extract…………………………….…………1g • Agar……………………………………………….15g

  10. 結果

  11. 分離純化耐鹼菌

  12. 檢測是否產生 protease 菌株(1)、(3)菌落周圍有透明環

  13. 菌株(K)菌落周圍有透明環

  14. 真菌菌落周圍有透明環

  15. 革蘭氏染色 菌株(1)

  16. 菌株(3)

  17. 菌株(K)

  18. 革蘭氏染色結果(1)(3)(K)菌皆為G(+)球菌 推測其皆為Micrococcus spp.

  19. 討論 • 要篩耐鹼的細菌並非一定要在很鹼的環境中 • 細菌在鹼性的環境下生長較慢 • 有些菌產生的protease量可能較少，如K菌

  20. 參考文獻 • Koki Horikoshi, Alkaliphiles: Some Applications of Their Products for Biotechnology, Microbiology and Molecular Biology Reviews, 1999, 63: 735-750 • Mala B. Rao, Aparna M. Tanksale, Mohini S. Ghatge, and Vasanti V. Deshpande, Molecular and Biotechnological Aspects of Microbial Proteases, Microbiology and Molecular Biology Reviews, 1998, 62: 597-635 • Nathalie Wery, Ursula Gerike, Ajay Sharman, Julian B. Chaudhuri, David W. Hough, and Michael J. Danson, Use of a Packed-Column Bioreactor for Isolation of Diverse Protease-Producing Bacteria from Antarctic Soil, Applied and Environmental Microbiology, 2003, 69:1457-1464

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