臺灣博碩士論文加值系統

N-乙醯幾丁寡醣具有抗癌、增強免疫力及抑菌等生理功效，應用價值相當高，卻受限於製備方法，使得生產成本過高，目前多種的N-乙醯幾丁寡醣製備方法各有其優缺點，而本研究採微生物發酵生產N-乙醯幾丁寡醣，考量其低污染與作用專一性。
自苗栗水尾溪篩選出幾丁質分解菌株，初步命名為DYU-Too 11，經新竹食品工業發展研究所生物資源保存及研究中心鑑定為Aeromonas hydrophila，故命名為Aeromonas hydrophila DYU-Too 11。分別以幾丁質與膠態幾丁質為碳源，比較N-乙醯幾丁寡醣與幾丁質酶之差異，結果顯示，幾丁質碳源培養可生成N-乙醯葡萄糖胺與聚合度2 ~ 5的N-乙醯幾丁寡醣，最高產量分別為0.79、0.94、0.4、0.16及0.26 g/L，而幾丁質酶活性最高為370 U/L，膠態幾丁質碳源培養可生成N-乙醯葡萄糖胺與聚合度3和4的N-乙醯幾丁寡醣，最高產量分別為15.8、0.37及0.06 g/L，而幾丁質酶活性最高為310.4 U/L，因此生產N-乙醯幾丁寡醣之碳源以幾丁質為最佳。
Aeromonas hydrophila DYU-Too 11菌株以不同濃度幾丁質培養，結果1%幾丁質可生成N-乙醯葡萄糖胺與聚合度2、4、5的N-乙醯幾丁寡醣，2%幾丁質濃度，可生成N-乙醯葡萄糖胺與聚合度2 ~ 6的N-乙醯幾丁寡醣，而3%、4%及5%幾丁質所生成的水解產物，其聚合度偏高，因此可依目標產物選擇適合的幾丁質濃度。
發酵培養Aeromonas hydrophila DYU-Too 11，並取其上清液以硫酸銨沉澱蛋白質，收集硫酸銨飽和百分比0 ~ 80的沉澱區間，將沉澱物復溶於50 mM Tris-HCl buffer (pH 7.8)中，經DEAE-Sepharose CL-6B與Sephacryl S-100膠體管柱純化後，可獲得單一幾丁質酶活性波峰，將此具活性樣品收集並進行活性電泳染色分析，發現具幾丁質酶活性之分子量分別為60與43 kDa，此酵素之最適反應溫度與pH值分別為40℃與pH 8.0，而溫度穩定性則於10 ~ 40℃時，具有70 %的殘留活性，於50℃下僅剩13.8 %的酵素活性；pH值穩定性則於pH 6 ~ 9具有50%以上的殘留活性。金屬離子對幾丁質酶活性影響之測定，Ca2+、Zn2+、Mn2+及Hg2+離子對幾丁質酶活性有抑制作用，以Zn2+離子的抑制效果最大，殘留活性僅6.5%。

N-acetylchitooligosaccharides have antitumor, immunostimulating and antibacterial functions, and therefore, are widely used in many fields. However, due to the limitation of preparation methods, the cost of production still remains high. Present methods for producing N-acetylchitooligosaccharides have advantages and drawbacks. In this study, microbial fermentation to produce N-acetylchitooligosaccharides was investigated due to its low pollution and specific action of enzyme.
A microbe, isolated from Shui-Wei Creek in the Miaoli County and named as DYU-Too 11, was used in this study to degrade chitin. The microbe had been identified to be Aeromonas hydrophila by the Food Industry Research Development Institute in Hsin-Chu, Taiwan, and was named as Aeromonas hydrophila DYU-Too 11. Two carbon sources, chitin and colloidal chitin, were used to explore the difference of N-acetylchitooligosaccharides and chitinase produced by this microbe. For the case of using chitin as a carbon source, N-acetyl-
glucosamine and N-acetylchitooligosaccharides with DP (degree of polymerization) 2 ~ 5 were produced, and their highest yields were 0.79, 0.94, 0.40, 0.16 and 0.26 g/L, respectively. The activity of crude chitinases was 370 U/L. For the colloidal chitin case, N-acetylglucosamine and N-acetylchitooligosaccharides with DP 3 ~ 4 were produced, and their highest yields were 15.8, 0.37 and 0.06 g/L, respectively. The activity of crude chitinases was 310.4 U/L. Therefore, chitin seemed to be a better carbon source for producing N-acetylchitooligosaccharides.
To investigate the effect of chitin concentration on the product, Aeromonas hydrophila DYU-Too 11 was cultivated in media with various chitin concentrations. N-acetylglucosamine and N-acetyl-
chitooligosaccharides with DP 2, 4 and 5 were produced in the culture of 1% chitin, meanwhile, N-acetylglucosamine and N-acetylchitooligosaccharides with DP 2 ~ 6 were produced in the 2% chitin medium. For higher chitin contents, e.g., 3%, 4% and 5%, N-acetylchitooligosaccharides with higher DP would be obtained. Therefore, selection of chitin concentration should depend on what target hydrolysates wanted.
In order to isolate chitinases secreted by Aeromonas hydrophila DYU-Too 11, the protein in the culture supernatant was precipitated with ammonium sulfate of 0 ~ 80% saturation. The precipitate was re-dissolved in a 50 mM Tris-HCl buffer (pH 7.8), and then purified through DEAE-Sepharose CL-6B and Sephacryl S-100. A peak showing chitinase activity was observed, and fractions of this peak were collected for further analysis. Molecular weights of the chitinases were identified to be 60 and 43 kDa by Zymogram analysis. The optimal reacting temperature of the chitinases was 40℃, and the optimal reacting pH was 8.0. The chitinases were stable in the range of 10 ~ 40℃ and still retained 70% of the original activity, while at 50℃ only 13.8% activity left. The chitinases were stable in the range of pH 6 ~ 9 and retained 50% activity. The effect of metal ions on the chitinase activity was also investigated. Results showed that Ca2+、Zn2+、Mn2+ and Hg2+ions have an inhibitive effect on the chitinases, especially the inhibition of Zn2+ was very substantial, only 6.5% activity left.

封面內頁
簽名頁
授權書…………………………………………………………………………………………iii
中文摘要...........................……............................iv
英文摘要...........................................................vi
誌謝...............................................................ix
目錄................................................................x
圖目錄............................................................xiv
表目錄 ..........................................................xvii

第一章 緒言..........................................................1
第二章 文獻回顧.......................................................3
2.1 幾丁質....................................................3
2.1.1幾丁質類之分子結構與性質...............................3
2.1.2幾丁質之功能與應用....................................6
2.1.3 幾丁質之製備.........................................8
2.2 幾丁質酶..................................................8
2.2.1 幾丁質水解酵素之分類.................................10
2.2.2 幾丁質酶的作用型式與機制.............................11
2.2.3幾丁質酶的應用.......................................15
2.2.4幾丁質酶的活性分析...................................16
2.3 N-乙醯幾丁寡醣............................................18
2.3.1 N-乙醯幾丁寡醣之功能與應用...........................18
2.3.2 N-乙醯幾丁寡醣製備..................................19
2.4 幾丁質酶純化..............................................21
2.4.1 硫酸銨分劃法........................................23
2.4.2 離子交換層析........................................23
2.4.3 膠體過濾層析........................................23
2.4.4 SDS-PAGE法.........................................24
2.5 Aeromonas hydrophila之簡介...............................24
第三章 材料與方法....................................................25
3.1 實驗架構.................................................25
3.2 實驗材料.................................................25
3.2.1 藥品...............................................25
3.2.2 儀器...............................................27
3.3 實驗方法.................................................28
3.3.1 菌株篩選、保存及鑑定.................................28
3.3.2 培養基組成..........................................28
3.3.3膠態幾丁質製備.......................................29
3.3.4 生長曲線測定........................................29
3.3.5 酵素活性分析........................................29
3.3.6 還原醣測定..........................................32
3.3.7 蛋白質濃度測定......................................32
3.3.8 幾丁質水解產物分析...................................32
3.3.9碳源和氮源對寡醣與粗酵素產量之探討...........33
3.3.10 幾丁質酶之分離純化..................................33
3.3.11聚丙烯醯胺膠體電泳分析...............................34
3.3.12純化酵素之特性分析..................................36
第四章 結果與討論....................................................38
4.1幾丁質分解菌之篩選.........................................38
4.1.1幾丁質水解產物分析....................................38
4.1.2菌株透明環測定.......................................38
4.1.3幾丁質酶之活性分析....................................40
4.1.4菌株DYU-Too 11之生長型態.............................40
4.1.5菌株DYU-Too 11之生長曲線.............................45
4.1.6菌株DYU-Too 11之鑑定…….............................45
4.2以CCB培養基培養DYU-Too 11菌株之探討.........................48
4.3不同幾丁質濃度培養DYU-Too 11菌株之探討.......................52
4.4不同氮源培養DYU-Too 11菌株之探討............................62
4.5幾丁質酶之分離純化..........................................68
4.5.1硫酸銨沉澱...........................................68
4.5.2離子交換層析.........................................71
4.5.3膠體過濾層析.........................................71
4.6 SDS-PAGE電泳分析.........................................71
4.7純化酵素之特性分析..........................................77
4.7.1最適反應溫度之測定....................................80
4.7.2最適反應pH值之測定...................................80
4.7.3 pH值穩定性之測定....................................80
4.7.4溫度穩定性之測定.....................................84
4.7.5最適基質濃度.........................................84
4.7.6金屬離子對酵素活性之影響..............................84
第五章 結論..........................................................90
參考文獻............................................................92



圖 目 錄

圖2.1纖維素、幾丁質及幾丁聚醣之結構………………………….5
圖2.2幾丁質之製備……………..…………………………………....9
圖2.3幾丁質酵素的水解路徑………………………………………12
圖2.4醣類水解酵素之水解機制可分為保留與反轉兩類……...…14
圖3.1實驗架構流程圖…..……….………………………………….26
圖4.1以CB培養基培養菌株J1 ~ J5之幾丁質酶活性的變化…..42
圖4.2以CB培養基培養菌株J1~J5之還原醣量的變化………....43
圖4.3菌株DYU-Too 11培養於CCP培養基之生長形態……...44
圖4.4菌株DYU-Too 11於LB培養基中之生長曲線………….....46
圖4.5 Aeromonas hydrophila DYU-Too 11之革蘭氏染色顯微照片......47
圖4.6以CCB培養基培養菌株DYU-Too 11之N-乙醯幾丁
寡醣的種類與含量……….………........................……........49
圖4.7以CCB培養基培養菌株DYU-Too 11之幾丁質酶、
還原醣及pH值的變化…….……….....................……........50
圖4.8以CB培養基培養菌株DYU-Too 11之幾丁質酶、還
原醣及pH值的變化….…..……......................……....…….51
圖4.9以1% ~ 5%幾丁質濃度培養DYU-Too 11菌株之幾丁質
酶活性的變化…...…..…….....................…….....……..........53
圖4.10以1% ~ 5%幾丁質濃度培養DYU-Too 11菌株之還原
醣量的變化……….…………....….....................……........54
圖4.11以1%幾丁質濃度培養DYU-Too 11菌株之水解產物
的種類與含量線…………………...................……........56
圖4.12以2%幾丁質濃度培養DYU-Too 11菌株之水解產物
的種類與含量………………...……....................……........57
圖4.13以3%幾丁質濃度培養DYU-Too 11菌株之水解產物
的種類與含量….………………….....................……........58
圖4.14以4%幾丁質濃度培養DYU-Too 11菌株之水解產物
的種類與含量……………...……………...........................59
圖4.15以5%幾丁質濃度培養DYU-Too 11菌株之水解產物
的種類與含量……………...........………….......……........60
圖4.16以不同氮源培養DYU-Too 11菌株之幾丁質酶活性
的變化……...........………….....…..……...……………….63
圖4.17以不同氮源培養DYU-Too 11菌株之還原糖量的變化.…..64
圖4.18以CB培養基培養菌株DYU-Too 11之N-乙醯幾丁
寡醣的種類與含量…….....…..……...…………………….65
圖4.19以yeast extract氮源培養菌株DYU-Too 11之N-乙
醯幾丁寡醣的種類與含量…..……...………………..……66
圖4.20以peptone氮源培養菌株DYU-Too 11之N-乙醯
幾丁寡醣的種類與含量……...…………………..………..67
圖4.21以NH4Cl氮源培養菌株DYU-Too 11之N-乙醯幾
丁寡醣的種類與含量…...……………………………….…69
圖4.22菌株DYU-Too 11發酵培養之粗酵素液經DEAE-
Sepharose CL-6B管柱層析圖…..............……...………...72
圖4.23經DEAE-Sepharose CL-6B管柱層析的幾丁質酶
以Sephacryl S-100管柱純化之膠體過濾層析圖……..…..73
圖4.24經DEAE-Sepharose CL-6B純化幾丁質酶之SDS-
PAGE分析。58’ (未加熱)、58、54 ~ 59及61為
DEAE-Sepharose CL-6B收集樣品….………….….........…75
圖4.25經DEAE-Sepharose CL-6B純化幾丁質酶之活性
染色分析。58’ (未加熱)、58、54 ~ 59、61及78
為DEAE-Sepharose CL-6B收集樣品…………………......76
圖4.26經Sephacryl S-100膠體過濾層析純化幾丁質酶
之SDS-PAGE分析。17 ~ 20為Sephacryl S-100
膠體過濾層析收集樣品…….....…....…....…....…..…….....78
圖4.27經Sephacryl S-100膠體過濾層析純化幾丁質酶
之活性染色分析。18’ (未加熱)、18、17 ~ 20為
Sephacryl S-100膠體過濾層析收集樣品…………….........79
圖4.28溫度對幾丁質酶反應活性之影響…………....………........81
圖4.29 pH值對幾丁質酶反應活性之影響…….............…..….......82
圖4.30幾丁質酶之pH穩定性………….....................……..….....83
圖4.31幾丁質酶之溫度穩定性…………....................……..….....85
圖4.32不同基質濃度對幾丁質酶活性之影響………….….………86
圖4.33幾丁質酶Lineweaver-Burk雙倒數作圖之Km與Vmax….…87


表 目 錄

表2.1幾丁類物質的來源及其含量........................................4
表2.2幾丁質與幾丁聚醣的應用..........................................7
表2.3由不同受質來測定幾丁質水解酵素之活性….......................17
表2.4各種微生物幾丁質酶之分離純化............……………...........22
表3.1培養基組成................................................30
表3.2 McIlvaine緩衝溶液.........…………………………………....31
表3.3分離膠組成…………….…………..…………………………35
表3.4堆積膠組成......................…………………………………….35
表4.1幾丁質分解菌株之來源與代號......................…………….…39
表4.2以CB培養基培養菌株J1~J5之N-乙醯幾丁寡醣的
種類與含量..……………………………………..................39
表4.3菌株J1 ~ J5於CCP培養基生成之菌落、透明環及
其比值.…………………………………………......................41
表4.4以1% ~ 5%幾丁質濃度培養DYU-Too 11之N-乙醯
幾丁寡醣的種類與含量.....……...........………………….......61
表4.5以不同氮源培養DYU-Too 11之N-乙醯幾丁寡醣的
種類與含量...........………………………………………......70
表4.6菌株DYU-Too 11之幾丁質酶純化總表.......…….................74
表4.7金屬離子對幾丁質酶活性之影響.......................89

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