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Isolation and characterization of organism producing thermo-stable lipase

Isolation and characterization of organism producing thermo-stable lipase. 報告學生 : 莫曉嬌 宋志潔 報告日期 :2003/12/29. 前言. Lipase ( 脂肪分解酵素 ) 可用作分解脂肪 環境中有許多微生物可產生 lipases ，催化 triglycerol 分解成 glycerol 與 fatty acid 微生物所產生的 lipase 多為胞外酵素 Lipase 多為適中溫酵素.

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Isolation and characterization of organism producing thermo-stable lipase

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  1. Isolation and characterization of organism producing thermo-stable lipase 報告學生:莫曉嬌 宋志潔 報告日期:2003/12/29

  2. 前言 • Lipase (脂肪分解酵素)可用作分解脂肪 • 環境中有許多微生物可產生lipases，催化triglycerol分解成glycerol與fatty acid • 微生物所產生的lipase多為胞外酵素 • Lipase多為適中溫酵素

  3. Lipase有許多用途： 1.食品工業：起士 2.清潔劑工業 3.油脂工業：甘油 4.廢水處裡 5.醫療：吸收不良病患 • Thermostable lipase： 1.嗜高溫微生物 2.一般溫度生長之微生物

  4. 目的 從環境中採集樣本，希望能找出並分離﹑ 鑑定能產生thermostable lipase的微生物。

  5. 實驗架構 堆肥：篩菌 Tributyrin agar plate：分離 不同受質培養基+Rhodamin B 生化測試：鑑定

  6. 材料 • 培養基 1.Tributyrin agar plate (per L)： Peptone 5.0g Beef extract 3.0g Agar 25.0g Tributyrin 15.0g 2.不同受質培養基： Peptone 5.0g Beef extract 3.0g Agar 25.0g Olive oil﹑Corn oil﹑Pig oil﹑Tween20 10.0g/L Rhodamin B 10.0ml/L

  7. 3.生化測試培養基： NA+NaCl﹑Nitrate broth﹑SIM ﹑MR-VP broth ﹑Phenol red+sugar broth ﹑MSA ﹑ Citrate ﹑Urea broth • 樣本： 木柵動物園之堆肥 • 0.85% NaCl無菌生理實驗水

  8. 實驗流程 • 培養和分離 1g樣本+9mL 0.85% NaCl ↓ 取0.1ml之101﹑102 ﹑103 ﹑104 ﹑105稀釋度塗抹平板，在Tributyrin agar plate ↓ 50℃培養1-3天 ↓ 觀察有無生長與透明環產生 ↓

  9. 選擇有透明環之菌落，在Tributyrin agar plate上作純培養，50℃培養1-3天 ↓ 在Tributyrin agar plate上作繼代培養， 50℃與60 ℃培養1-3天 ↓ 培養於Tributyrin plate﹑Olive oil plate﹑ Corn oil plate ﹑Tween20 plate ﹑ Pig oil plate，50℃與55 ℃培養1-3天 ↓ 觀察有無生長與透明環產生 ↓

  10. 培養於Tributyrin plate﹑含有Rhodamin B的Olive oil plate﹑Corn oil plate ﹑ Tween20 plate ﹑Pig oil plate， 50℃與55 ℃培養1-3天 ↓ 觀察有無生長與透明環及螢光產生 ↓ 鑑定

  11. 鑑定 Gram stain：G(+) rods(圖二) Spore stain(圖四, 圖五) + - Catalase Catalase + - - + Clostridium sp. Desulfotomaculum sp. Glucose fermentation Motile Lactobacillus sp. Erysipelothrix sp. - + - + Listeria sp. Kurthia sp. 2D (Brochothrix) 1B, 1C,1D, 1E, 1F, 2A (Bacillus sp.) Sporosacina sp.

  12. 60 ℃生長 + - 1B, 1C,1D, 1E, 1F, 2A(Bacillus sp.) 1C, 1E (B. stearothermophilus) 10%NaCl 生長 + - 1D (B. pantothenticus) 1B, 1F, 2A (B. megaterium)

  13. Gram stain: G(+) cocci Catalase • 鑑定(續) + - Oxidase Streptococcus sp. + - Motile Micrococcus sp. + - 2E (Planococcus sp.) Staphylococcus sp. Stomatococcus sp.

  14. 結果

  15. 討論 • 鑑定的結果之正確性，原因是: 1.有很多生化測試是d或ND 2.所作的生化測試並不完全 3.測得的結果很類似 4.部分菌株特性Bergey’s manual中没列 • 改善上述缺點的方法: 1.操作技術純熟 2.用Biolog分析

  16. 生化測試之糖類發酵: 糖類改為過濾滅菌應可改善結果之判讀 • 培養條件: 1.培養基受質選擇: (1)受質結構的不同 (2)受質濃度用量 2.培養溫度之選擇

  17. 參考文獻 Rohit Sharma, and Yusuf Chisti. 2001. Production, purification, characterization, and applications of lipase. Biotechnol Advances.19:627-662. Yougxiang Wang. 1995. Thermostable alkaline lipase from a newly isolated thermophilic Bacillus Strain A30-1 (ATCC 53841). J Fermen Bioeng. 79:433-438. 謝汎擎. 2001 .耐熱脂肪分解酵素分離與特性測定. Bergey’s manual. Vol. 2.

  18. 圖一､木柵動物園堆肥樣本

  19. 圖二､革蘭氏陽性桿菌(1B)

  20. 圖三､革蘭氏陽性球菌(2E)

  21. 圖四､孢子染色結果(1D)

  22. 圖五､孢子染色結果(1F)

  23. 圖六. 2D菌在Tributyrin plate產生的透明環

  24. 圖七､2E菌在olive oil plate上產生的透明環

  25. 圖八､2E菌在Pig oil plate上產生螢光

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