臺灣博碩士論文加值系統

本研究之菌種篩選自台南縣下營鄉田間土壤，經食品工業發展研究所鑑定為Aeromonas caviae，並命名為Aeromonas caviae NCHU1；以幾丁質粉末當作其主要生長源，為一株具有生產幾丁酵素之菌株。所得發酵上清液經硫酸銨沉澱、透析去除鹽類、陰離子交換層析(DEAE-Sepharose)及膠體過濾層析(Gel filitration Sephadex G-75)之純化步驟。獲得單一之酵素經SDS-PAGE測得分子量為26.2kDa，其最適反應溫度、最適反應pH值、熱穩定性及pH穩定性分別為60°C、pH6、10-40°C及 pH5-10。在酵素水解產物之幾丁寡醣分析中，由HPLC圖譜得知，反應後產物為N-乙醯幾丁單醣、二醣、五醣及六醣，因此可以推測Aeromonas caviae NCHU1具有內切型幾丁酵素。
第二階段之研究內容是以Aeromonas caviae NCHU1發酵修飾片狀幾丁質為載體，利用幾丁質對幾丁酵素之親和性來進行純化，即幾丁質親和性層析。以SEM圖觀察幾丁質經發酵修飾後之表面，發現幾丁質由平滑的表面變成多孔狀的結構，推測為幾丁質的直鏈結構，因此推知，經發酵修飾之幾丁質在吸附幾丁酵素時減少了立體障礙，進而提高對酵素的吸附量。文中探討幾丁酵素吸附之最適化條件，溫度、 pH值分別為10°C及pH7。於蛋白質脫附實驗中發現，以0.05M、pH10之Glycine緩衝液可脫附幾丁酵素，純化倍率達3.97倍；接下來探討修飾後幾丁質之再生與再利用性，以0.2M、pH10之Glycine緩衝液清洗載體3次，可脫附殘留於載體上之大部分蛋白質；而經過再生之載體也具備再次吸附酵素能力。

In this investigation, a bacterial strain with chitinase activity was isolated from soil in Tainan and identified as Aeromonas caviae NCHU1. This strain can product chitinases when it is grown in a medium containing chitin power of marine waste. A chitinase was purified from culture broth of Aeromonas caviae NCHU1 by a series of purification steps, i.e. ammonium sulfate precipitation, DEAE-Sepharose, and gel filitration with Sephadex G-75. The molecular weight of NCHU1 chitinase is 26.2kDa by sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) analysis. The optimum temperature and pH, thermal and pH stability ranges of NCHU1 chitinase are 60°C, pH6, 10-40°C, and pH5-10, respectively. To determine the oligosaccharide contents of NCHU1 chitin hydrolysates, it was found that (GlcNAc), (GlcNAc)2, (GlcNAc)5, and (GlcNAc)6 existed. This implies that Aeromonas caviae NCHU1 may produce endo-chitinase.
Furthermore, the affinity chitin chromatography with fermentation- modified chitin was employed for purification of chitinase. By observing the structural changes of chitin and fermentation-modified chitin by SEM, it was found that chitin surface became porous and branching structure, which might reveal more active sites and less the steric hinderance to the adsorption of chitinase. For chitinase adsorption capacity, factors such as loading temperature, pH and elution solutions were studied. The optimal loading conditions were found at 10℃, pH7. The elution solution was 0.05M glycine buffer. Under these conditions, a purification fold of 3.97 was obtained. Then, the modified chitin can be regeneration and reused.

目錄
摘要 I
Abstract II
目錄 III
表目錄 VI
圖目錄 VII
第一章 緒論 1
(一) 前言 1
(二) 研究動機 1
第二章 文獻回顧 3
(一) 幾丁類多聚物 3
1. 幾丁質(chitin)與幾丁聚醣(chitosan)之介紹 3
2. 幾丁質與幾丁聚醣之應用 6
3. 幾丁質與幾丁聚醣之製備 7
(二) 幾丁類寡醣 11
1. N-乙醯幾丁寡醣與幾丁寡醣 11
2. N-乙醯幾丁寡醣與幾丁寡醣之應用 11
3. N-乙醯幾丁寡醣與幾丁寡醣之製備 13
4. N-乙醯幾丁寡醣與幾丁寡醣之測定 15
(三) 幾丁酵素 16
1. 幾丁酵素的分類 16
2. 幾丁質酵素的作用機制 19
3. 幾丁酵素之應用 26
4. 幾丁質酵素的純化 28
第三章 材料與方法 32
(一) 菌種 32
(二) 實驗藥品 32
(三) 實驗儀器 32
(四) 培養基 33
1. 膠態幾丁質製備(colloidal chitin) 33
2. 膠態幾丁質的定量 33
3. 培養基組成 35
(五) 發酵流程 36
1. 菌種活化 36
2. 液態培養 36
(六) 幾丁酵素之純化與特性分析 37
1. 粗酵素液的製備 37
2. 硫酸銨劃分 37
3. 陰離子交換層析(DEAE-Sephacel chromatography) 38
4. 膠體過濾層析(Gel filitration chromatogrphy) 38
5. 幾丁酵素之生化特性分析 39
(七) 幾丁質親和性純化流程 40
1. 幾丁質的製備 40
2. 酵素液的製備 41
3. 批次實驗 41
4. 再生與再利用性 41
(八) 分析方法 41
1. 蛋白質濃度的量測 41
2. 幾丁質酵素活性分析 44
3. 還原醣分析 45
4. SDS-PAGE電泳分析 47
第四章 結果與討論 50
(一) 幾丁酵素之分離純化 50
1. 硫酸銨劃分 50
2. DEAE Sephacel 陰離子交換層析 53
3. 膠體過濾層析(Sephadex G-75) 54
4. 純化結果 55
(二) 幾丁酵素之生化特性 58
1. 最適溫度、pH值對幾丁酵素活性之影響 58
2. 溫度、pH值對幾丁酵素活性之穩定性 58
3. 幾丁酵素之水解產物分析 61
(三) 幾丁質親和性純化 64
1. 發酵修飾的幾丁質之SEM觀察 64
2. 最適吸附條件 66
3. 脫附實驗 70
第五章 結論與未來展望 79
(一) 結論 79
(二) 未來展望 80
第六章 參考文獻 81
附錄-菌種報告鑑定書 89

表目錄
表2-1幾丁質與幾丁聚醣之應用範圍 9
表2-2文獻上利用幾丁質的親和性純化 29
表3-1實驗使用之培養基配方 35
表3-2 DNS試劑之組成 46
表3-3下層膠的比例 47
表3-4上層膠的比例 48
表3-5染色劑的成分 49
表3-6脫色劑的成分 49
表3-7 loading buffer配方 49
表4-1幾丁酵素之純化步驟總表 56
表4-2 Glycine緩衝液濃度對脫附之影響 73
表4-3修飾後幾丁質載體再利用之吸附量 75
表4-4傳統純化步驟表 77
表4-5親和性純化步驟表 78

圖目錄
圖2-1幾丁質三種型態 4
圖2-2幾丁質、幾丁聚醣以及纖維素之化學結構 5
圖2-3幾丁質製備過程 10
圖2-4幾丁質分解酵素水解途徑 18
圖2-5幾丁質分解酵素之作用機制 22
圖2-6醣類水解酵素之水解機制 23
圖2-7幾丁質水解酵素過程之變旋異構物形成 24
圖2-8幾丁酵素結合幾丁質基質假說圖 25
圖2-9幾丁質藉由幾丁酵素進行生物轉化為單細胞蛋白之程序 27
圖3-1膠態幾丁質之濕重與乾重關係圖 34
圖3-2蛋白質濃度之校正曲線(0.2∼0.9 mg/ml) 42
圖3-3蛋白質濃度之校正曲線(10∼100μg/ml) 43
圖3-4幾丁質酵素活性之校正曲線(2∼7mU/ml) 45
圖3-5 DNS與還原醣之反應式 46
圖3-6 N-乙醯葡萄糖胺標準品濃度對吸光值之校正曲線 46
圖4-1硫酸銨劃分之活性、蛋白質分佈圖 51
圖4-2硫酸銨劃分之比活性 51
圖4-3硫酸銨劃分電泳圖 52
圖4-4幾丁酵素之陰離子交換層析管柱純化 53
圖4-5幾丁酵素之膠體過濾層析管柱純化 54
圖4-6幾丁酵素之傳統純化結果 55
圖4-7幾丁酵素之分子量測定 57
圖4-8 Aeromonas caviae NCHU1幾丁酵素之最適反應溫度 59
圖4-9 Aeromonas caviae NCHU1幾丁酵素之最適反應pH值 59
圖4-10 Aeromonas caviae NCHU1幾丁酵素之熱穩定性 60
圖4-11 Aeromonas caviae NCHU1幾丁酵素之pH值穩定性 61
圖4-12標準品之HPLC圖譜 62
圖4-13 N-乙醯幾丁單醣(GluNAc)標準品之HPLC圖譜 63
圖4-14 Aeromonas caviae NCHU1幾丁酵素在37℃下水解產物之HPLC圖 63
圖4-15幾丁質SEM圖 65
圖4-16經發酵處理之幾丁質SEM圖 65
圖4-17不同溫度下的活性吸附量 66
圖4-18不同溫度下的蛋白質吸附量 67
圖4-19不同溫度下產生的還原醣量 67
圖4-20不同pH下之活性吸附量 68
圖4-21不同pH下之蛋白質吸附量 69
圖4-22不同pH下產生的還原醣量 69
圖4-23緩衝液對幾丁酵素活性之影響 70
圖4-24各種緩衝液之活性脫附量 72
圖4-25各種緩衝液之蛋白質脫附量 72
圖4-26載體清洗與再生性分析 74
圖4-27幾丁酵素之傳統純化-DEAE Sephacel 76
圖4-28幾丁酵素之傳統純化-Sephadex G-75 gel filitration 76
圖4-29幾丁酵素之傳統純化電泳分析 77
圖4-30幾丁酵素之親和性純化電泳分析 78

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