臺灣博碩士論文加值系統

熊果素為植物中萃取的一種美白成分。由於萃取的產量極低，故以醣化進行結構修飾將對苯二酚轉換為熊果素為較經濟可行之方法。Kurosu等人於2002年成功的利用微生物Xanthomonas campestris WU-9701做為生物觸媒，並以麥芽糖作為醣化反應之醣基來源進行對苯二酚之醣化反應，生產熊果素達轉化率達93%。然而在具醣化催活性之菌種篩選上，至今仍尚無簡單快速之篩選方法。因此，本研究目的在於建立一簡單、高效率之篩菌策略，以期經由突變及菌種篩選獲得具有醣化對苯二酚催化活性之高活性菌株。本實驗所建立的篩菌策略是利用對苯二酚相較於熊果素其光敏感性較高之特性（對苯二酚溶液照光後較容易變質而使溶液呈現深褐色），於96孔盤反應後進行光照，以顏色深淺作為指標進行初篩。本實驗經過五次的突變、多次的菌種篩選以及反應條件之最適化探討後，已獲得轉化率約85%的高活性菌株Xanthomonas campestris 5H10。

Arbutin is a whitening agent, which is extracted from plants and the yield is quite low. A more economical and environmental friendly way is to synthesize arbutin by structural modification of hydroquinone via glycosylation(biotransformation). Presently, Kurosu and coworkers(2002) have successfully synthesized arbutin by glycosylation of hydroquinone with maltose as a glucosyl donor using lyophilized cells of Xanthomonas campestris WU- 9701 as the biocatalyst. However, a fast and simple screening method for microorganisms which have catalytic activity of glycosylation has not been established up till now. So, the objective of our study was to set up a easy and efficient screening method for obtaining mutated strains that have high hydroquinone-glycosylation activity. Our screening method was based on the fact that hydroquinone solution was more light-sensitive than arbutin. We selected the mutated strains from 96-well plates after reaction and light exposure and used the color difference as the screening index. After five rounds of mutation, repeated screening and optimization of reaction conditions, we obtained a high activity strain, Xanthomonas campestris 5H10, which has a conversion of about 85%.

目錄
摘要 Ⅰ
Abstract Ⅱ
目錄 Ⅲ
圖目錄 Ⅵ
表目錄 Ⅷ
第一章 緒論 1
第二章 文獻回顧 3
2-1 生物觸媒簡介 3
2-2 醣化反應簡介 7
2-3 微生物之菌種改良 10
2-3-1人工變異(Artificial mutation) 11
2-3-2菌種篩選（Screening） 13
2-4 與黑色素相關之資料 14
2-4-1黑色素細胞 14
2-4-2酪胺酸脢簡介 15
2-4-3黑色素生成路徑 15
2-4-4黑色素生成的抑制作用 17
2-5美白產品簡介 18
2-5-1熊果素簡介 20
2-6 Xanthomonas campestris簡介 22
第三章 實驗部分 23
3-1 實驗藥品 23
3-2 實驗儀器及設備 24
3-3 實驗原始菌種 25
3-4菌種活化與保存 25
3-5 實驗及分析方法 26
3-5-1突變 26
3-5-2 Screening 28
3-5-3細胞培養與處理 29
3-5-4反應步驟及條件 30
3-5-5細胞通透處理 30
第四章 實驗結果與討論 33
4-1 對苯二酚與熊果素之液相層析儀分析 33
4-2 建立熊果素之濃度檢量線 33
4-3篩選具醣化對苯二酚活性之X. campestris突變株 37
4-4 細胞通透處理 45
4-5 不同醣基donor對醣化反應之影響 49
4-6 反應條件對菌株5H10的醣化反應活性之影響 49
4-6-1溫度 49
4-6-2 pH 49
4-6-3生物觸媒使用量 53
4-6-4基質濃度 53
4-6-5反應時間 53
第五章 結論與未來展望 58
5-1結論 58
5-2未來展望 58

參考文獻 60

附錄一 65

圖目錄

圖2-1 Production of cis-2-substituted indanols by biocatalyst 4
圖2-2 Production of D-Sorbose by biocatalyst 4
圖2-3 利用胺樹細胞醣化麴酸之反應式 9
圖2-4 利用胺樹細胞醣化檜木精之反應式 10
圖2-5 以X. campetris冷凍乾燥細胞醣化薄荷腦之反應式 10
圖2-6 以X. campetris冷凍乾燥細胞醣化對苯二酚之反應式 10
圖2-7 黑色素的生成路徑 16
圖2-8 Hydroquinone（對苯二酚）之結構圖 19
圖2-9 熊果素（Arbutin）結構圖 21
圖2-10 X. campestris之Transmission Electron Micrograph 22
圖3-1 突變劑NTG之結構式 28
圖4-1 對苯二酚與熊果素之液相層析分析圖譜 34
圖4-2 熊果素濃度之檢量線 35
圖4-3 對苯二酚濃度之檢量線 36
圖4-4 96孔盤反應及照光後之圖片 38
圖4-5 反應樣品與熊果素標準品之液相層析圖譜比對 39
圖4-6 菌液OD值VS.菌體冷凍乾燥之乾菌重量之關係圖 46
圖4-7 以介面活性劑處理全細胞對反應活性之影響 47
圖4-8 以有機溶劑處理全細胞對反應活性之影響 48
圖4-9 溫度對菌株5H10反應活性之影響 51
圖4-10 pH值對菌株5H10的醣化反應之影響 52
圖4-11 菌量對反應之影響 54
圖4-12 Maltose濃度對菌株5H10反應的影響 55
圖4-13 Hydroquinone濃度對菌株5H10反應的影響 56
圖4-14菌株5H10之time course 57

表目錄

表2-1 生物觸媒之應用 5
表2-2 全細胞與酵素之比較 7
表4-1 第一次突變篩選之菌種與其熊果素產量與轉化率 40
表4-2 第二次突變篩選之菌種與其熊果素產量與轉化率 41
表4-3 第三次突變篩選之菌種與其熊果素產量與轉化率 42
表4-4 第四次突變篩選之菌種與其熊果素產量與轉化率 43
表4-5 第五次突變篩選之菌種與其熊果素產量與轉化率 44
表4-6 不同醣基donor對醣化反應之影響 50

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