Burkholderia cepacia TKU025係以烏賊軟骨粉為唯一碳/氮源，篩選自台灣北部土壤之一株酪胺酸酶生產菌，經鑑定為Burkholderia cepacia，是一非葡萄糖發酵之革蘭氏陰性菌。B. cepacia TKU025生產酪胺酸酶抑制劑之較適培養條件為含有1.0 % 烏賊軟骨、0.1 % K2HPO4及0.05 % MgSO4．7H2O（pH7）之25 mL液態培養基於30 °C振盪培養4天。
發酵上清液經乙酸乙酯去除脂溶成分、後續使用水層部分進行Sephacryl S-100以及薄膜層析法(thin layer chromatography)等步驟，進行抑制劑之分離純化。此抑制劑有非常良好之熱安定性及pH安定性，於100 °C及pH 2-12反應下，其活性均不受影響。
    此外，經由Sephacryl S-100層析，所得初步分離之抑制劑對E. coli 及B. cereus TKU006之生長具有顯著抑制活性，兩者抑制效果最高都達到95 %以上。

The tyrosinase inhibitor producing strain, Burkholderia cepacia TKU025 was isolated from the soil in the northern Taiwan by using squid pen powder (SPP) as the sole carbon/nitrogen source and identified as the Burkholderia cepacia. The strain was a non-lactose-fermenting and Gram-negative bacteria. The optimal conditions for tyrosinase inhibitor production were cells used to inoculate in 25 mL of medium containing 1.0% SPP, 0.1% K2HPO4 and 0.05% MgSO4．7H2O（pH7）and shaken at 30 °C for 4 days.
The tyrosinase inhibitor was purified by ethyl acetate, chromatography of Sepharose S-100 and thin-layer chromatography (tlc). The tyrosinase inhibitor had broad thermal stability and pH stability. It maintained it’s initial activity at 100 °C and pH 2-12, respectively.
Besides, the purified inhibitor by Sepharose S-100 chromatography had antimicrobial activity against E. coli and B. cereusTKU006. Both the  best inhibition were more than 95 %.

目錄
                                                       頁次
                                                    
封面內頁
授權書
簽名頁
中文摘要.................................................Ⅰ
英文摘要	................................................Ⅱ
目錄.....................................................Ⅲ
圖目錄...................................................Ⅶ
表目錄...................................................X

第一章 緒論..............................................1
第二章 文獻回顧..........................................2
      2.1 Burkholderia cepacia TKU025之簡介..............2
      2.2黑色素簡介......................................2
      2.3黑色素形成過程..................................3
      2.4黑色素與酪胺酸酶作用機制........................4
      2.5酪胺酸酶之活性..................................6
第三章 材料與方法.......................................10
      3.1實驗菌株.......................................10
      3.2 實驗材料......................................10
      3.3實驗儀器.......................................11
      3.4 藥品配置......................................11
      3.5 菌種鑑定......................................12
      3.6 酪胺酸酶抑制率測定............................12
         3.6.1總吸光質測定..............................12
         3.6.2總空白吸光值測定..........................13
         3.6.3酪胺酸酶空白吸光值測定....................13
         3.6.4樣品液空白吸光值測定......................13
      3.7 較適培養條件探討..............................13
         3.7.1碳/氮源種類之選擇.........................13
         3.7.2培養溫度..................................14
         3.7.3碳/氮濃度之影響...........................14
         3.7.4培養體積..................................14
         3.7.5培養基酸鹼值..............................14
         3.7.6較適培養時間..............................14
      3.8酚類化合物之定量分析...........................15
      3.9分離純化.......................................15
         3.9.1樣品之製備................................15
         3.9.2膠體過濾層析..............................15
         3.9.3成分分離..................................16
      3.10抑制劑之特性分析..............................16
         3.10.1熱安定性.................................16
         3.10.2 pH安定性................................17
      3.11抗菌測定......................................17
第四章 結果與討論.......................................18
      4.1酪胺酸酶抑制劑生產菌之篩選.....................18
      4.2酪胺酸酶抑制劑較適生長條件探討.................21
         4.2.1碳氮源種類之選擇..........................21
         4.2.2培養溫度..................................21
         4.2.3碳氮源濃度之影響..........................21
         4.2.4培養體積..................................22
         4.2.5培養基酸鹼值..............................22
         4.2.6較適培養時間..............................22
         4.2.7較適培養條件..............................23
      4.3 B. cepacia TKU025與麴酸及維生素C之抑制率比較..23
      4.4酪胺酸酶抑制活性之成分分離.....................36
         4.4.1粗萃取液之初步分離........................36
         4.4.2利用有機萃取分離純化......................37
      4.5抑制劑之特性分析...............................45
      4.6 B. cepacia TKU025生產之抑制劑對試驗菌株抑菌活性之
         探討...........................................46
         4.6.1抑制Escherichia coli生長分析..............46
         4.6.2抑制Bacillus cereus TKU006生長分析........46
         4.6.3抑制真菌生長分析..........................47
         4.6.4綜合結果..................................47
第五章 結論.............................................52
參考文獻................................................53

圖目錄
                                                       頁次
圖2.1黑色素生成的反應機制圖..............................5
圖2.2對苯二酚、熊果素及麴酸之化學結構式..................8
圖4.116S rDNA 部分鹼基序列分析..........................19
圖4.2經由API鑑定B. cepacia TKU025之結果.................20
圖4.3不同碳源對於B. cepacia TKU025生產酪胺酸酶抑制劑活性之影響......................................................25
圖4.4不同碳源對於B. cepacia TKU025生產酪胺酸酶抑制劑活性之影響......................................................26
圖4.5培養溫度對於B. cepacia TKU025生產酪胺酸酶抑制劑活性之影響......................................................27
圖4.6 SPP添加濃度對於B. cepacia TKU025生產酪胺酸酶抑制劑活性之影響..................................................28
圖4.7培養體積對於B. cepacia TKU025生產酪胺酸酶抑制劑活性之影響......................................................29
圖4.8培養pH對於B. cepacia TKU025生產酪胺酸酶抑制劑活性之影響......................................................30
圖4.9 Burkholderia cepacia TKU025生產酪胺酸酶抑制劑活性、總
酚含量、pH值變化及其生長曲線圖..........................31
圖4.10以L-DOPA為基質時探討B. cepacia TKU025發酵上清液和麴酸對於酪胺酸酶活性之抑制影響..............................33
圖4.11 Sephacryl S-100分離純化流程圖....................38
圖4.12酪胺酸酶抑制劑之Sephacryl S-100層析圖譜...........38
圖4.13溶於甲醇層-酪胺酸酶抑制劑之Sephacryl S-100層析圖譜......................................................39
圖4.14不溶於甲醇層-酪胺酸酶抑制劑之Sephacryl S-100層析圖譜......................................................39
圖4.15分離純化流程圖....................................40
圖4.16 TKU025-1之1H-NMR圖譜.............................41
圖4.17 TKU025-1之13C-NMR圖譜............................41
圖4.18 TKU025-1之HMQC圖譜...............................42
圖4.19 TKU025-1之HMBC圖譜...............................42
圖4.20 TKU025-1之COSY圖譜...............................43
圖4.21 TKU025-1之NOESY圖譜..............................43
圖4.22 TKU025-1之IR圖譜.................................44
圖4.23酪胺酸酶抑制劑之熱安定性與pH安定性................45
圖4.24 B. cepacia TKU025發酵上清液對於E. coli生長之影響.48
圖4.25初步純化之酪胺酸酶抑制劑對於E. coli生長之影響.....48
圖4.26 B. cepacia TKU025發酵上清液對於B. cereus TKU006生長之影響....................................................49
圖4.27初步純化之酪胺酸酶抑制劑對於B. cereus TKU006生長之影響......................................................49
圖4.28 B. cepacia TKU025在不同濃度下之抗菌活性探討，濃度由左到右分別為0、1.66、3.33和5 mg/mL........................50
圖4.29真菌在PDA培養基上生長情形，濃度由左到右分別為0、1、和3 mg/mL.................................................51

表目錄
                                                       頁次
表2.1抑制黑色素生成之成分................................9
表4.1初篩之菌株結果.....................................18
表4.2 B. cepacia TKU025生產酪胺酸酶抑制劑之較適培養條件.32
表4.3酪胺酸酶抑制劑之半抑制率比較.......................34
表4.4發酵上清液對於真菌生長之影響.......................51

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