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系統識別號 U0002-1907201915415900
中文論文名稱 Paenibacillus sp.TKU047生產α-葡萄糖苷酶抑制劑及蛋白酶之條件及定性
英文論文名稱 Production and characterization of α-glucosidase inhibitors and proteases from Paenibacillus sp.TKU047
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 107
學期 2
出版年 108
研究生中文姓名 温義弘
研究生英文姓名 I-Hung Wen
學號 606160397
學位類別 碩士
語文別 中文
口試日期 2019-06-04
論文頁數 41頁
口試委員 指導教授-王三郎
共同指導教授-阮文邦
委員-糜福龍
委員-王全祿
中文關鍵字 類芽孢桿菌.  α-葡萄糖苷酶抑制劑  蛋白酶 
英文關鍵字 Paenibacillus sp.  α-glucosidase inhibitor  protease 
學科別分類 學科別自然科學化學
中文摘要 近年來水產幾丁質材料因應用廣泛而逐漸受到重視。回顧相關文獻,四種漁業加工副產品-魷魚筆粉(SPP)、蝦頭粉(SHP)、去礦物質蝦殼粉(deSSP)和去礦物質蟹殼粉(deCSP)都被用來生產α-葡萄糖苷酶抑製劑(aGI)和蛋白酶。本研究使用的菌株Paenibacillus sp.TKU047係從淡江大學土壤中篩選出來,具備能同時生產aGI和蛋白酶的特性,依據實驗結果,TKU047菌株利用蝦頭粉作為生產aGI和蛋白酶的最適合的碳/氮(C / N)源。產生aGI和蛋白酶的最佳條件為0.5%的蝦頭粉、搖瓶震盪速度150rpm、在恆溫37℃中培養3天,可得到最佳aGI活性(711 U / mL)和蛋白酶活性(3.27U / mL)。本研究也探討TKU047產生的aGI和蛋白酶特性並分離出TKU047蛋白酶,分子量為32kDa,並具有一些具發展潛力的特性,如熱穩定性、介面清潔劑穩定性和鹼穩定性,另外 TKU047 aGI也在不同反應環境下表現穩定(pH 5-7、100℃以內,可保留約90%的抑制活性),屬於非競爭混合型抑制劑(mixed non-competitive inhibitor)的抑制劑。總體而言,TKU047菌株提供了有效且環保的轉化SHP的方法,未來可發展於醫療或洗衣洗滌劑。
英文摘要 Marine chitinous materials are of interest due to their wide range of applications. In the current study, four kinds of fishery processing byproducts including squid pens powder (SPP), shrimp heads powder (SHP), demineralized shrimp shells powder (deSSP), and demineralized crab shells powder (deCSP) was used to produce aGI and protease by Paenibacillus sp. TKU047, a bacterium isolated from the soils of Tamkang University campus. Among them, SHP expressed as the most suitable carbon/nitrogen (C/N) source for α-glucosidase inhibitor (aGI) and protease productions via Paenibacillus conversion. The conditions for producing aGI and protease by TKU047 strain were found at 0.5% of SHP concentration, a shaking speed of 150 rpm and an incubation temperature of 37°C(711 U/mL of aGI activity and 3.27 U/mL of protease activity, respectively).
The character of TKU047 protease and aGI were also investigated. TKU047 protease was isolated with a molecular weight of 32 kDa and expressed various valuable properties such as thermo-, detergent-, and alkaline-stable, whereas TKU047 aGI was stable up to 100°C in a pH range of 5-7 with 90% of retained activity and its inhibitory model was mixed non-competitive inhibitor. Overall, the findings provide strong support for the potential candidacy of SHP conversion by TKU047 as an effective and eco-friendly method to produce bioactivities compounds, which could be used in the medical or laundry detergent industries.
論文目次 中文摘要 I
英文摘要 II
誌謝 III
目錄 IV
圖目錄 VIII
表目錄 X
第一章 緒論 1
第二章 文獻回顧 2
2.1 糖尿病 2
2.1.1 第一型糖尿病 2
2.1.2 第二型糖尿病 2
2.2 α-葡萄糖苷酶 3
2.2.1 α-葡萄糖苷酶抑制劑 3
2.3 蛋白酶 3
2.4 幾丁質 3
2.5 Paenibacillus sp. 4

第三章 材料與方法 5
3.1 實驗菌株 5
3.2 實驗材料 5
3.3 實驗儀器 5
3.4 菌株篩選 6
3.5 α-葡萄糖苷酶抑制劑抑制率活性測定 7
3.6 蛋白酶活性測定 7
3.7 α-葡萄糖苷酶抑制劑和蛋白酶較適產生條件實驗 7
3.7.1 碳/氮源的影響 8
3.7.2 培養溫度的影響 8
3.7.3 培養基濃度的影響 8
3.8 最適反應環境與安定性測試 9
3.8.1 最適反應溫度與熱安定性 9
3.8.2 最適反應pH值與酸鹼安定性 9
3.9 α-葡萄糖苷酶抑制劑酵素動力學 10
3.10 特異性測試 10
3.10.1 α-葡萄糖苷酶抑制劑特異性 10
3.10.2 蛋白酶特異性 11
3.11 蛋白酶之純化分離 11
3.12 蛋白質電泳分析 12
3-13 蛋白酶活性抑制 12
第四章 結果與討論 13
4.1 實驗菌株 13
4.2 菌株篩選與鑑定 13
4.3 α-葡萄糖苷酶抑制劑和蛋白酶較適產生條件實驗 17
4.3.1 碳/氮源的影響 17
4.3.2 培養溫度的影響 17
4.3.3 培養基濃度的影響 20
4.4 最適反應環境與安定性 21
4.4.1 最適反應溫度與熱安定性 21
4.4.2 最適反應pH值與酸鹼安定性 23
4.5 α-葡萄糖苷酶抑制劑酵素動力學 25
4.6 特異性測試 26
4.6.1 α-葡萄糖苷酶抑制劑特異性 26
4.6.2 蛋白酶特異性 26
4.7 蛋白酶之純化分離 27
4.8 蛋白酶電泳分析 28
4.9 蛋白酶活性抑制 32
4.10菌種蛋白酶活性比較 34
第五章 結論 35
參考文獻 36


圖目錄
圖4.1 TKU047菌株取樣地點 14
圖4.2 TKU047菌株核酸定序結果 15
圖4.3 碳/氮源對aGI活性的影響 18
圖4.4 碳/氮源對蛋白酶活性的影響 18
圖4.5 發酵溫度對aGI活性的影響 19
圖4.6 發酵溫度對蛋白酶活性的影響 19
圖4.7 SHP濃度對aGI活性的影響 20
圖4.8 SHP濃度對蛋白酶活性的影響 21
圖4.9 蛋白酶最適反應溫度與熱安定性 22
圖4.10 aGI不同溫度穩定性 22
圖4.11 蛋白酶最適反應pH與酸鹼穩定性 24
圖4.12 aGI在不同pH環境的穩定性 24
圖4.13 抑制劑酵素動力學 25
圖4.14 TKU047蛋白酶特異性 27
圖4.15 蛋白質酶在Macro-prep High S管柱的沖提曲線 28
圖4.16 蛋白酶在KW802.5管柱上的沖提曲線 29
圖4.17 TKU047蛋白酶 SDS-PAGE 31
圖4.18 金屬離子、抑製劑和表面活性劑的影響 32
圖4.19 洗衣精對蛋白酶的抑制效果 33


表目錄

表 4-1 TKU047核酸序列比對結果 16
表 4-2 TKU047 aGI特異性比較表 26
表 4-3 TKU047蛋白酶純化表 30
表 4-4不同菌種生產蛋白酶活性比較表 34
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