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研究生:李政衛
研究生(外文):Cheng-Wei Lee
論文名稱:比較兩株法夫酵母胞外葡萄糖苷轉移酶的生產
論文名稱(外文):COMPARISON IN PRODUCTION OF XTRACELLULAR GLUCOSYLTRANSFERASES FROM TWO STRAINS OF XANTHOPHYLLOMYCES DENDRORHOUS
指導教授:許垤棋
指導教授(外文):Dey-Chyi Sheu
口試委員:許垤棋
口試委員(外文):Dey-Chyi Sheu
口試日期:2013-07-29
學位類別:碩士
校院名稱:大同大學
系所名稱:生物工程學系(所)
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:84
中文關鍵詞:醱酵異麥芽寡糖法夫酵母α-葡萄糖苷酶酵母菌
外文關鍵詞:yeastα-glucosidasefermentationisomalto-oligosaccharidesXanthophyllomyces dendrorhous
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異麥芽寡糖是一種益生源,可以促進腸道中的比菲德氏菌的生長,增進人體健康。在諸多有類似功能的寡糖之中,異麥芽寡糖具有耐酸性,可添加於酸性飲料,因此異麥芽寡糖在市場上占有極大的優勢。本實驗所使用的法夫酵母(Xanthophyllomyces dendrorhous),其胞外α-葡萄糖苷酶同時具有轉移和水解活性。以麥芽糖為受質,經酵素催化作用,產生葡萄糖、異麥芽糖、潘糖(panose)、四糖、五糖等。不止產生 α (1-6) 鍵結,也產生 α (1-2) 鍵結,這和一般商業用的葡萄糖苷轉移酶只產生α (1-6) 鍵結,有所不同。二株菌使用發酵槽培養48小時,以麥芽糖漿為受質,添加氮源,控制溫度、通氣量、攪拌速度以及酸鹼值,在過程中測定發酵液的OD660吸光值、並以HPLC分析其糖類及酵素活性,酵素活性主要測定潘糖。將發酵液離心處理,上清液收集後以分子量100 kDa限外過濾、再以等電點電泳等步驟部分純化。接著比較二個珠菌胞外酵素的活性及某些特性,胞外酵素的活性會受到溫度的影響,在50到60 °C時有較顯著的轉移活性,SDS-PAGE電泳測定分子量估計分別為145.2與142 kDa。利用此酵母菌分泌 α-葡萄糖苷酶以及代謝葡萄糖的特性,生產高純度異麥芽寡糖。在最適pH值和溫度時,胞外酵素α-葡萄糖苷酶能催化出的異麥芽寡糖濃度最高可達到每升97克。本實驗可以了解到純化後的胞外酵素-葡萄糖苷酶能催化麥芽糖生產高純度的異麥芽寡糖,對於生產異麥芽寡糖的研究有很大的潛力。
Isomaltooligosaccharides (IMO) can stimulate the proliferation of bifidobacteria intestine and thus enhance human health. Among various functional oligosaccharides, IMO is acid resistant and can be added to acidic drinks. Generally, starch undergoes catalytic reactions of α- and β-amylases, yielding maltose syrup. In this project, we use two kinds of yeast Xanthophyllomyces dendrorhous to synthesize the enzyme which catalysis to produce glucose, panose, isomaltotetraose and isomaltopent- ose. More than generate α (1-6) bond, also produced the binding of α(1-2), and different from general commercial use only produce α-glucosidase transferase α(1-6) bond. Two yeast strains were incubated in the ferment- or for 48 hours. We use maltose as a substrate, controlling nitrogen sources adding, temperature, the aeration rate and agitation speed limiting, measuring pH and absorbance value of the broth, then analysis the saccharides and enzyme activity by HPLC. Centrifuged the fermentation broth after 48hours fermentation, the supernatants were collected after a molecular weight of 100 kDa hollowfiber module was filtered and then partially purified by isoelectric focusing electrophoresis step. After the process of purification we compare two extracellular enzyme activity and some characteristics of extracellular enzyme activity which affected by temperature and pH value. We know that 50 to 60 °C is more significant for transfer activity; SDS-PAGE electrophoresis determination of molecular weight is estimated to be 145.2 and 142 kDa. We can use the characteristics of the yeast secretion of α-glucosidase and use maltose to produce high purity isomalto-oligosaccharide. In the optimal pH value and temperature, the product of highest concentration of isomalto-oligosaccharide catalyzed by extracellular enzyme α-glucosidase can be achieved to 97 g/l. This experiment can understand purified extracellular enzyme α-glucosidase can catalyze maltose to produce high purity of isomalto-oligosaccharide, there is great potential for the production of isomalto-oligosaccharides.
1前言 1
1.1研究目的 1
1.2寡糖的定義與其特點 7
1.3異麥芽寡糖的生理功能 7
1.4異麥芽寡糖(isomalto-oligosaccharides) 8
2材料與方法 11
2.1 菌種 11
2.2 藥品 11
2.3 設備 11
2.3.2 高效能層析儀(HPLC: high performance liquid chromatography) 11
2.3.1 冷凍離心機 15
2.3.3 IEF-等焦電泳(isoelectric focusing,IEF) 17
3 實驗方法 17
3.1 平板培養基配置 18
3.2 菌種保存 18
3.3 酵母菌搖瓶培養 20
3.4 酵母菌的兩公升發酵槽放大培養 22
3.4.1 測量菌液的OD660吸光值 25
3.4.2 分析發酵時菌液醣類的變化 25
3.4.3 菌液胞外酵素轉移活性的測定 28
3.5 冷凍離心 28
3.6 用Hollow fiber濃縮存化 30
3.7. 製備級等電點焦集 33
3.7.1. 純化酵素活性與pH值的測定 35
3.7.2 測定胞外酵素α-Glucosidase活性 35
3.7.3 純化酵素蛋白量的測定與比活性 36
3.7.4 SDS-PAGE電泳測定分子量 37
3.8. 測試不同因子對酵素活性的影響 38
3.8.1 測試不同pH值對酵素活性的影響 38
3.8.2 測定不同溫度對酵素活性的影響 38
3.8.3 測定酵素的熱穩定性 39
3.9 酵素反應生產異麥芽寡糖 39
4 結果與討論 40
4.1.1 兩株酵母菌之生長曲線與pH值 40
4.1.2 兩株酵母在發酵槽中醣類的變化 42
4.1.3 發酵槽中兩株菌不同時間下胞外酵素轉移活性的變化 45
4.2 Hollowfiber純化酵素濃縮液與穿透液轉移活性的比較 47
4.3.1 IEF 等焦電泳純化酵素轉移活性及pH值的測定 48
4.3.2 100 k濃縮液及IEF等焦電泳純化酵素水解PNPG活性的測定 51
4.3.3 IEF等焦電泳純化酵素蛋白量的測定和比活性 54
4.3.4 IEF等焦電泳純化酵素分子量的測定 55
4.4.1 pH值對酵素活性的影響 58
4.4.2 溫度對酵素活性的影響 60
4.4.3 酵素熱穩定性 62
4.5 異麥芽寡糖的純化 64
5結論 67
6參考文獻 69

圖目錄
圖 1. 多種異麥芽寡糖的結構 10
圖 2. 離心機轉軸基座 16
圖 3. 法夫酵母的平板培養 19
圖 4. 錐形瓶初步培養 21
圖 5. 發酵槽培養 23
圖 6. HPLC裝置示意圖 27
圖 7. 冷凍離心機 29
圖 8. Hollow fiber module MWCO 100 kDa 31
圖 9. 製備級等電點焦集設備(BioRad Preparative IEF Rotofor Cell) 34
圖 10. 比較兩株酵母X. dendrorhous在不同時間時的OD660值 41
圖 11 - a X. dendrorhous BCRC 22365在不同發酵時間時的醣類變化 43
圖 11 - b X. dendrorhous BCRC 21346在不同發酵時間時的醣類變化 44
圖 12 X. dendrorhous BCRC 22365與21346在不同發酵時間的轉移活性 46
圖 13 - a X. dendrorhous BCRC 22365 酵素經IEF純化後的轉移活性與pH值 49
圖 13 - b X. dendrorhous BCRC 21346 酵素經IEF純化後的轉移活性與pH值 50
圖 14 - a X. dendrorhous BCRC 22365 酵素經IEF純化後之水解PNPG的活性 52
圖 14 - b X. dendrorhous BCRC 21346 酵素經IEF純化後之水解PNPG的活性 53
圖 15 - a SDS-PAGE測量X. dendrorhous BCRC 22365經IEF純化之酵素的分子量 56
圖 15 - b SDS-PAGE測量X. dendrorhous BCRC 21346經IEF純化之酵的分子量 57
圖 16. pH值對BCRC 22365與21346生產之酵素酵素活性的影響 59
圖 17. 溫度對BCRC 22365與21346生產之酵素活性的影響 61
圖 18. BCRC 22365與21346生產之酵素穩定性 63
圖 19 - a BCRC 22365產生的胞外酵素生產異麥芽寡糖 65
圖 19 - b BCRC 21346產生的胞外酵素生產異麥芽寡糖 66

表目錄
表 1. 異麥芽寡糖的反應機制 5
表 2. 濃縮液與穿透液轉移活性的比較 47
表 3. BSA標準線製作 54
表 4. 蛋白量與比活性的測定 54
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