臺灣博碩士論文加值系統

本研究目的為篩選土壤中具有分解幾丁質能力之菌株，利用HPLC分析幾丁質水解產物，藉由發酵槽大量培養，探討不同碳源濃度下，生質量、還原醣量、幾丁質分解酵素活性及N-乙醯葡萄糖胺之產量，以DEAE-Sepharose CL-6B離子交換層析管柱與Sephadex G-100膠體過濾層析管柱純化不同培養時間之幾丁質分解酵素，並利用SDS-PAGE分析幾丁質分解酵素之蛋白質的分子量與酵素之特性。
從雲林縣濁水溪河畔之土壤，篩選出具有分解幾丁質能力之
菌株，此菌株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 caviae，故命名為Aeromonas caviae DYU- BT4。此菌株於蝦殼粉、烏賊軟骨、幾丁質粉末及膠態幾丁質為碳源之培養基中，其幾丁質水解產物是以N-乙醯葡萄糖胺為主，其中以膠態幾丁質為碳源時所得之N-乙醯葡萄糖胺產量最高，約為39% (w/w)。以發酵槽批次培養菌株BT4可以縮短N-乙醯葡萄糖胺之生產時間，提高培養基碳源濃度則可增加N-乙醯葡萄糖胺產量。
Aeromonas caviae DYU-BT4之發酵液經純化後，以SDS-PAGE分析，在分離膠體中添加0.1%乙二醇幾丁質，可直接從膠片上得知具有幾丁質分解酵素活性之蛋白質分子量為75 kDa。此酵素之最適反應溫度為40℃；最適反應pH為7；由酵素動力反應中，Km值為1.87 g/L，Vmax值為213 U/L；Co2+可提高酵素活性，而Ag+、Ba2+、Hg2+、K+及Zn2+等金屬離子會對酵素活性有抑制作用。

The purpose of this study is to isolate an indigenous microorga- nism to degrade chitin and to produce hydrolysates of chitin. Hydro- lysates of chitin were analyzed by the method of HPLC. The effect of carbon concentration on biomass, reducing sugar, chitinase activity and N-acetylglucosamine in a batch fermenter was studied. Purification of chitinases produced at different culture times was carried out by an ion exchange chromatography column with DEAE-sepharose CL-6B gel and by a gel filtration chromatography column with Sephadex G- 100 gel. The molecular weight and the characteristics of each of chitinase proteins was identified with an SDS-PAGE.
A microbe, DYU-BT4, isolated from the soil of Zwou-Shau-Shi in the Yunlin county was used to degrade chitin. The microbe had been identified to be Aeromonas caviae by the Food Industry Research Development Institute at Hsin-Chu of Taiwan and was named as Aeromonas caviae DYU-BT4. The microbe was cultivated in media with different carbon-sources, such as shrimp shell powder, squid pens, chitin powder and colloidal chitin. The major chitin hydrolysate from the cultivation of strain DYU-BT4 was N-acetylglusamine. The highest yield of N-acetylglucosamine (about 39% (w/w)) was obtained by using the colloidal chitin as a carbon source. Experimental results have shown that the time required to produce N-acetylglucosamine can be shortened and the yield of N-acetylglucosamine can be raised as the carbon concentration in the medium was elevated during a cultivation of strain DTU-BT4 in a batch fermemter.
After the supernatant of the culture broth of Aeromonas caviae DYU-BT4 was purified, the portion of the protein with chitinase activity was examined by the SDS-PAGE containing 1% glycol chitin, and its molecular weight was identified to be 75 kDa. The optimum reacting temperature of the 75-kDa enzyme was 40℃, and the optimal pH was 7.0. The enzymatic reaction value, Km, for the 75-kDa enzyme was measured to be 1.87 g/L and the value of Vmax was 213 U/L. Metal ion Co2+ can promote the chitinase activity, and other metal ions such as Ag+, Ba2+, Hg2+, K+ and Zn2+ will inhibit the chitinase activity.

目 錄
封面內頁
簽名頁
授權書..................................................iii
中文摘要................................................iv
英文摘要................................................v
誌謝...................................................vii
目錄...................................................viii
圖目錄.................................................xiii
表目錄.................................................xvi
第一章 緒論.............................................1
第二章 文獻回顧.........................................3
2.1幾丁質.............................................3
2.1.1幾丁質之結晶結構................................3
2.1.2幾丁質之製備....................................5
2.2幾丁質分解酵素.....................................5
2.2.1幾丁質分解酵素的分佈............................5
2.2.2幾丁質分解酵素之分類............................6
2.2.3幾丁質分解酵素的活性測定........................8
2.3 N-乙醯幾丁寡醣....................................10
2.3.1 N-乙醯幾丁寡醣之製備..........................10
2.3.2 N-乙醯幾丁寡醣之分離..........................11
2.3.3 N-乙醯幾丁寡醣之應用..........................12
2.4酵素分離與純化....................................12
2.4.1硫酸銨沈澱.....................................13
2.4.2離子交換層析法.................................13
2.4.3膠體過濾層析法.................................13
2.5 SDS-PAGE之原理..................................16
2.5.1聚丙烯醯胺凝膠的形成和結構.....................16
2.5.2起始劑、增速劑和聚合...........................17
2.5.3聚丙烯醯胺凝膠之分離機制.......................17
2.5.4聚丙烯醯胺凝膠染色.............................20
2.6微生物Aeromonas caviae之簡介.......................22
2.6.1微生物Aeromonas caviae之特性...................22
2.6.2微生物Aeromonas caviae之相關研究...............22
第三章 材料與方法......................................25
3.1藥品..............................................25
3.2儀器設備..........................................26
3.3培養基與試劑的配製................................27
3.3.1培養基之組成...................................27
3.3.2膠態幾丁質之製備...............................28
3.3.3蝦殼粉之製備...................................29
3.3.4烏賊軟骨之製備.................................29
3.3.5 McIlvaine buffer之配製...........................29
3.3.6呈色劑之配製...................................29
3.3.7樣品緩衝液之配製...............................29
3.3.8 CBR染液之配製................................29
3.3.9脫色液之配製...................................30
3.3.10電泳緩衝液之配製..............................30
3.4幾丁質分解菌之篩選................................32
3.4.1採樣地點.......................................32
3.4.2篩選菌株.......................................32
3.4.3菌種保存.......................................32
3.5批次發酵槽之發酵試驗..............................33
3.5.1批次發酵培養條件...............................33
3.5.2操作步驟.......................................33
3.6分析方法..........................................34
3.6.1幾丁質分解酵素活性測定.........................34
3.6.2還原醣含量.....................................34
3.6.3生質量與幾丁質殘餘量之測定.....................35
3.7幾丁質分解酵素之特性分析..........................35
3.7.1粗酵素液之最適基質濃度.........................35
3.7.2粗酵素液之最適反應pH值........................35
3.7.3粗酵素液之最適反應溫度.........................36
3.8 N-乙醯幾丁寡醣之測定..............................36
3.8.1水解產物前處理.................................36
3.8.2高效能液相層析儀分析...........................37
3.9 N-乙醯幾丁質分解酵素純化..........................37
3.9.1 硫酸銨沉澱....................................37
3.9.2 離子交換層析..................................37
3.9.3 膠體過濾層析..................................37
3.10 SDS-聚丙醯胺膠體電泳分析.........................39
3.10.1樣品前處理....................................39
3.10.2製備SDS-聚丙醯胺膠片.........................39
3.10.3分子量之分離..................................39
3.10.4膠片染色與脫色................................41
3.10.5在電泳膠片上分析幾丁質分解酵素活性............41
3.11純化酵素之特性分析...............................41
3.11.1酵素液之最適反應pH值與pH值穩定性...........41
3.11.2酵素液之最適反應溫度與溫度穩定性..............42
3.11.3酵素液之最適基質濃度..........................42
3.11.4金屬離子對酵素活性之影響......................43
第四章 結果與討論......................................44
4.1 幾丁質分解菌之篩選...............................44
4.1.1各菌株之生長曲線..............................44
4.1.2各菌株之幾丁質分解酵素活性比較................44
4.1.3篩選菌株之水解產物分析.........................47
4.1.4菌株BT4之鑑定.................................47
4.2幾丁質粗酵素液之特性分析..........................51
4.2.1最適反應基質濃度..............................51
4.2.2最適反應pH值.................................51
4.2.3最適反應溫度..................................54
4.3不同碳源之水解產物分布............................56
4.4批次發酵培養.....................................61
4.4.1生質量.........................................61
4.4.2還原醣量.......................................61
4.4.3幾丁質分解酵素活性.............................62
4.4.4 N-乙醯葡萄糖胺產量............................62
4.5 N-乙醯幾丁質分解酵素純化..........................68
4.5.1於1%幾丁質培養基之批次發酵培養................68
4.5.2於2%幾丁質為碳源之批次發酵培養................71
4.6 SDS-聚丙醯胺膠體電泳分析..........................81
4.7純化酵素之特性分析................................91
4.7.1最適反應pH值與pH值穩定性.....................91
4.7.2最適反應溫度與溫度穩定性.......................91
4.7.3最適基質濃度...................................94
4.7.4金屬離子對酵素活性之影響.......................99
第五章 結論...........................................101
參考文獻..............................................102

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