α-葡萄糖苷酶抑制劑由於其在治療糖尿病中的重要用途而受到重視。雖然一些合成的 α-葡萄糖苷酶抑制劑已經可以使用很長時間，但是它們經常會引起各種的副作用。因此，本研究旨在找到一種安全，天然的 α-葡萄糖苷酶抑制劑來源。本實驗利用篩選自淡江大學校園之菌株Staphylococcus sp. TKU 043，發酵蝦頭殼粉生產α-葡萄糖苷酶抑制劑。於含1%蝦頭殼粉培養基培養 3 天，可得較佳α-葡萄糖苷酶抑制活性（1457U / mL）。所得發酵上清液經硫酸銨鹽沉澱、DEAE-Sepharose陰離子交換管柱層析、Sepharcryl-S200膠體管柱層析，進行α-葡萄糖苷酶抑制劑之部份純化。此抑制劑於50℃處理30 分鐘後活性僅存20％，在pH 3-7反應皆具100 %之抑制活性。
　　依據研究結果顯示，TKU 043所生產之α-葡萄糖苷酶抑制劑為大分子化合物，推測此抑制劑可能為蛋白質類或是胜肽類化合物；利用酵素抑制動力學鑑定出此抑制劑為混合型(非競爭)抑制劑。

Alpha-glucosidase inhibitors have received much attention due to their important use in treating diabetes mellitus. Although some synthetic α-glucosidase inhibitors have been available for a long time, they often cause various unexpected side effects. Thus, the present study was aimed at finding a safe, natural source of α-glucosidase inhibitors. Staphylococcus sp. TKU043, a bacterium strain isolated from the soils of Tamkang University Campus, produced alpha-glucosidase inhibitors (1457 U/mL) in a medium with 1% (w/v) shrimp head powder as the sole carbon/nitrogen source. The culture supernatant obtained by culturing under optimized culture condition was purified by ammonium sulfate precipitation, DEAE-Sepharose column chromatography, and Sephacryl S-200 gel chromatography. The partially purified inhibitors remained 20 % original activity after  treatment at 50℃ for 30 min. Activity of the alpha-glucosidase inhibitors remained 100 % after treatment at pH 3-7.
　　According to the results of this study, the α-glucosidase inhibitor produced by TKU043 is a proteinous material. The inhibitor is identified as a mixed type (non-competitive) inhibitor according to the results of enzyme inhibition kinetics.

目錄
中文摘要	I
英文摘要	II
目錄	III
圖目錄	VII
表目錄	IX
第一章　緒論	1
第二章　文獻回顧	3
2.1 Staphylococcus sp. 之簡介	3
2.2 糖尿病	3
2.2.1 第一型糖尿病	3
2.2.2 第二型糖尿病	4
2.3 α-葡萄糖苷酶(alpha-glucosidase)	4
2.4 α-葡萄糖苷酶抑制劑(alpha-glucosidase inhibitors)	4
2.4.1 α-葡萄糖苷酶抑制劑之機制	7
2.5微生物發酵蝦蟹殼廢棄物	7
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生產菌株之篩選與鑑定	12
3.4.1菌株篩選	12
3.4.2 菌株鑑定	12
3.5 α-葡萄糖苷酶抑制劑抑制率活性之測定	13
3.6.1 碳/氮源對TKU043生產α-葡萄糖苷酶抑制劑活性之影響	14
3.6.2 蝦頭殼粉濃度對TKU043生產α-葡萄糖苷酶抑制劑活性之影響	14
3.6.3 培養液體積對TKU043生產α-葡萄糖苷酶抑制劑活性之影響	14
3.6.4培養液體溫度對TKU043生產α-葡萄糖苷酶抑制劑活性之影響	15
3.7 蛋白質含量測定	15
3.8 酒精沉澱	15
3.9 酚/氯仿萃取	15
3.10 TKU043生產α-葡萄糖苷酶抑制劑之分離純化	16
3.10.1 粗發酵液之製備	16
3.10.2陰離子交換樹脂	16
3.10.3 膠體過濾層析法	17
3.11 熱安定性測試	17
3.12 酸鹼安定性測試	17
3.12.1 酸鹼安定性測試	17
3.12.2 酸鹼最適反應測試	17
3.13 抑制劑動力學	18
3.13.1 決定最適抑制劑濃度	18
3.13.2 酵素抑制動力學	18
3.13.3 抑制劑種類鑑定	19
3.14 抑制劑特異性測試	19
3.14.1 不同來源α-葡萄糖苷酶抑制效果測試	19
第四章　結果與討論	20
4.1菌種鑑定	20
4.2 TKU043生產α-葡萄糖苷酶抑制劑較適培養條件探討	24
4.2.1 TKU043生產α-葡萄糖苷酶抑制劑碳/氮源之探討	24
4.2.2 TKU043生產α-葡萄糖苷酶抑制劑濃度之探討	26
4.2.3 TKU043生產α-葡萄糖苷酶抑制劑培養體積之探討	28
4.2.4 TKU043生產α-葡萄糖苷酶抑制劑培養溫度之探討	30
4.2.5 TKU043生產α-葡萄糖苷酶抑制劑較適培養條件探討結果	31
4.3 透析結果	32
4.3.1 不同粗發酵液處理方式比較	32
4.3.2酚/氯仿萃取	33
4.4 TKU043生產α-葡萄糖苷酶抑制劑之分離純化	33
4.4.1 粗發酵液之製備	33
4.4.2 陰離子交換樹脂	34
4.4.3 膠體過濾層析	34
4.5 TKU043生產α-葡萄糖苷酶抑制劑之特性分析	38
4.5.1 TKU043生產α-葡萄糖苷酶抑制劑之熱安定性	38
4.5.2 TKU043生產α-葡萄糖苷酶抑制劑最適反應pH及pH安定性	38
4.6抑制劑型態鑑定	42
4.7 不同來源α-葡萄糖苷酶抑制效果測試結果	44
4.8 不同菌株α-葡萄糖苷酶抑制劑比較	44
第五章 結論	45
參考文獻	46
 
圖目錄
圖2. 1 阿卡波糖結構圖	6
圖2. 2米格列醇結構圖	6
圖2. 3井岡黴醇胺結構圖	6
圖2. 4 幾丁質結構圖	8
圖4. 1 TKU043 革蘭氏染色圖	20
圖4. 2 API分析鑑定結果	22
圖4. 3碳/氮源對TKU043生產α-葡萄糖苷酶抑制劑之影響	25
圖4. 4蝦頭殼粉濃度及發酵時間對TKU043生產α-葡萄糖苷酶抑制劑之影響	27
圖4. 5培養體積對TKU043生產α-葡萄糖苷酶抑制劑之影響	29
圖4. 6培養溫度對TKU043生產α-葡萄糖苷酶抑制劑之影響	30
圖4. 7 Staphylococcus sp. TKU043 α-葡萄糖苷酶抑制劑之DEAE-Sepharose CL-6B層析圖譜	35
圖4. 8 Staphylococcus sp. TKU043 α-葡萄糖苷酶抑制劑之Sephacryl S-200層析法圖譜	36
圖4. 9 Staphylococcus sp. TKU043 α-葡萄糖苷酶抑制劑在不同溫度處理後之結果	39
圖4. 10 Staphylococcus sp. TKU043 α-葡萄糖苷酶抑制劑在不同pH處理後之結果圖	40
圖4. 11 Staphylococcus sp. TKU043 α-葡萄糖苷酶抑制劑在不同pH最適反應	41
圖4. 12α-葡萄糖苷酶抑制劑之雙倒數圖	43

 
表目錄
表2. 1幾丁質差異性	8
表2. 2微生物發酵水產廢棄物相關文獻	9
表4. 1 TKU043之16S rDNA 部分基因序列	21
表4. 2 TKU043之16S rDNA NCBI比較	23
表4. 3比較培養條件前後的差別	31
表4. 4上清液透析前後之抑制劑活性分布	32
表4. 5不同粗發酵液	32
表4. 6酚/氯仿萃取	33
表4. 7α-葡萄糖苷酶抑制劑之純化總表	37
表4. 8 α-葡萄糖苷酶抑制劑之抑制型態與Km及Vmax值	43
表4. 9不同來源α-葡萄糖苷酶測試	44
表4. 10不同菌株α-葡萄糖苷酶抑制劑比較	44

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