臺灣博碩士論文加值系統

N-乙醯葡萄糖胺具有治療關節炎、小兒腸道發炎及促進皮膚保濕等生理功效，應用價值相當高，在食品加工與醫學方面有廣泛研究與應用。目前多種的N-乙醯葡萄糖胺製備方法各有其優缺點。本研究以兩階段方式發酵培養菌株Chitinibacter tainanensis生產。第一階段：改變培養基中葡萄糖濃度，以提高菌體生質量；第二階段：調控發酵培養基之pH值與幾丁質濃度，以增加N-乙醯葡萄糖胺產量。
Chitinibacter tainanensis菌株以不同濃度葡萄糖於搖瓶培養，其中以含0.3%葡萄糖之培養基的生質量最高。此菌株培養於含有0.3%葡萄糖BH培養基，培養8 h，轉換至含有2% 幾丁質濃度之BH培養基，於培養96 h時具有最高還原醣量，為14.7 g/L；幾丁質酶活性於培養至72 h時最高，為725.3 U/L；N-乙醯葡萄糖胺產量於培養至144 h達最高，為15.8 g/L，幾丁質轉化率為79%。
以不同葡萄糖濃度於發酵槽培養菌株Chitinibacter tainanensis，結果以含2.5%葡萄糖之菌體生質量最高，因此在第一階段之培養，均以2.5 % 葡萄糖為其碳源。
於發酵槽中，以兩階段調控pH，培養菌株Chitinibacter tainanensis，第一階段pH為 7，以BH-1培養基含2.5%葡萄糖培養Chitinibacter tainanensis，菌體生質量於24 h時為2.42 g/L。培養至24 h，轉換至含有2%幾丁質的培養基，進行第二階段發酵培養，還原醣於培養至96 h時達最高，為16.1 g/L，菌株生質量於轉換培養基後，培養至96 h時，生質量達最高，為1.99 g/L。幾丁質酶活性於培養至96 h時達最高，為800.3 U/L。Chitinibacter tainanensis分解幾丁質，於72 h時可生成N-乙醯葡萄糖胺15.6 g/L，幾丁質轉化率為77.8%。
菌株Chitinibacter tainanensis以兩階段培養，於第二階段不調控pH值，於培養24 h後，轉換為第二階段培養(不調控pH值)，轉換之後，培養基之pH培養至48 h時，降至5.31，幾丁質酶活性於培養72 h時達最高，為710 U/L，還原醣於96 h時最高，為16.7 g/L。第二階段發酵培養生成的N-乙醯葡萄糖胺，於培養120 h時達最高，為16.1 g/L，幾丁質轉化率為80.1%。
提高幾丁質濃度為4%，以兩階段於發酵槽中培養Chitinibacter tainanensis菌株，於第二階段(不調控pH)培養，其還原醣於120 h時達最高，為33.7 g/L，菌株生質量於轉換培養基後，於120 h生質量達最高，為2.47 g/L。幾丁質酶活性於培養72 h時達最高，為650.0 U/L。第二階段培養之N-乙醯葡萄糖胺生成量，於96 h時達最高，為31.2 g/L，幾丁質轉化率為78.0%。

N-acetylglucosamine has been used as a treatment for osteoarthritis, inflammatory bowel diseases and as an enhancer of skin moisture. N-acetylglucosamine has also become a popular research topic in the area of food processing and medical applications. Current methods for producing N-acetylglucosamine have advantages and drawbacks. In this study, N-acetyl-glucosamine was produced in a two-stage culture of Chitinibacter tainanensis. In the first stage, the medium glucose concentration was increased to create a high cell density. In the second stage, the medium pH was controlled to increase the productivity of N-acetylglucosamine.
To investigate the effect of glucose concentration on the biomass, Chitinibacter tainanensis was cultivated in media with various glucose concentrations. The highest biomass production was obtained when Chitinibacter tainanensis was cultivated in a 0.3% glucose medium. In this case, the microbe was first cultivated in the 0.3% glucose medium, and 8 h later was transferred to a Bushnell-Hass broth containing 2% chitin, the reducing sugars (mainly N-acetylglucosamine) had a maximum productivity of 14.7 g/L after 96 h of incubation. The highest chitinase activity was 725.3 U/L after 72 h of incubation; the N-acetyl-glucosamine production was the highest (15.8 g/L) at 144 h, and the conversion rate from chitin into N-acetyl-glucosamine was 79.0%.
To investigate the effect of glucose concentration on the biomass, Chitinibacter tainanensis was cultivated in media containing various glucose concentrations. The result showed that the medium containing 2.5% glucose yielded the highest biomass, and therefore, the first stage culture contained 2.5% glucose as a carbon source.
When Chitinibacter tainanensis was cultivated in a two-stage process in a fermenter, minor modifications were made, such as a set pH of 7 and use of a modified BH-1 medium containing 2.5% glucose. With this process, the maximum biomass (2.42 g/L) was attained at 24 h. In the second stage, the culture was transferred to a modified medium containing 2% chitin (designated as Medium BH-2). At 96 h, the biomass, reducing sugars and chitinase activity reached their maximums　of 1.99 g/L, 16.1 g/L, and 800.3 U/L, respectively. During cultivation of Chitinibacter tainanensis, chitin was hydrolyzed into N-acetylglucosamine with a maximum production of 15.6 g/L at 72 h, and a chitin conversion rate of 77.8%.
When Chitinibacter tainanensis was cultivated in a two-stage culture, the first stage culture was transferred into the second stage with a modified BH-2 medium after 24 h. Tthe pH in the culture was not regulated at this stage and fell rapidly from 7.0 to 5.31 by the 48 h. Maximum chitinase activity (710 U/L) was reached at 72 h, and the reducing sugars reached maximum concentration (16.7 g/L) at 96 h. The production of N-acetylglucosamine in the second stage was highest (16.1 g/L) at 120 h, and the chitin conversion rate was 80.1%.
When the chitin concentration was raised to 4%, Chitinibacter tainansnsis was cultivated using a two-stage fermentation with no pH control in the second stage. The maximum concentrations of reducing sugars (33.7 g/L) and biomass (2.47 g/L) were both achieved at 120 h. The chitinase activity reached the highest (650.0 U/L) at 72 h. N-acetyl-glucosamine production was highest (31.2 g/L) at 96 h, when the chitin conversion rate was 78.0%.

授權書iii
中文摘要iv
英文摘要vi
致謝viii
目錄ix
圖目錄xii
表目錄 xiv

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 4
2.1.3 幾丁質之製備 7
2.2 幾丁質酶 7
2.2.1幾丁質水解酵素之分類 7
2.2.2幾丁質酶的作用形機制 8
2.2.3 幾丁質酶的天然分佈 11
2.2.4 幾丁質酶的應用13
2.2.5 幾丁質酶活性分析 14
2.3 N-乙醯葡萄糖胺 15
2.3.1 N-乙醯葡萄糖胺的功能與應用 16
2.4 N-乙醯葡萄糖胺之製造方法 17
2.4.1 化學法 17
2.4.2 酵素水解法 17
第三章 材料與方法 20
3.1 實驗架構20
3.2 實驗儀器20
3.3 實驗藥品22
3.4 培養基 23
3.4.1 基礎培養基 23
3.4.2 發酵培養基23
3.5 實驗菌株 25
3.5.1 Chitinibacter tainanensis 25
3.5.2 菌種保存 25
3.6菌體顯微鏡觀察 25
3.6.1 光學顯微鏡之觀察 25
3.6.2 位相差顯微鏡之觀察 25
3.7 膠態幾丁質之製備 27
3.8 批次發酵槽培養 27
3.8.1 批次發酵培養條件 27
3.8.2 操作步驟 29
3.9 分析方法 30
3.9.1 生長曲線 30
3.9.2 N-乙醯葡萄糖胺酶活性 30
3.9.3 還原醣 31
3.9.4 生質量 31
3.9.5 殘餘葡萄糖 31
3.9.6 水解產物 32
第四章 結果與討論 33
4.1菌株基本特性 33
4.2 菌株Chitinibacter tainanensis 之生長型態 33
4.3 菌株Chitinibacter tainanensis 之生長曲線 33
4.4 葡萄糖濃度的效應(搖瓶培養) 37
4.5 葡萄糖濃度的效應(發酵槽培養)44
4.6 Chitinibacter tainanensis之兩階段培養 44
4.6.1 pH調控 47
4.6.2 第二階段培養pH不調控 49
4.6.3 以4%幾丁質於發酵槽之培養 51
第五章 結論 55
參考文獻 57
附錄 64

圖目錄

圖2.1 纖維素、幾丁質及幾丁聚糖之結構 5
圖2.2 幾丁質之製備 9
圖2.3 幾丁質酵素的水解路徑 10
圖2.4 以化學法製造N-乙醯葡萄糖胺 18
圖3.1 實驗架構流程圖 21
圖4.1於位相差顯微鏡下(1,000x)觀察之Chitinibacter tainanensis 34
圖4.2 Chitinibacter tainanensis培養於膠態幾丁質平板培養基 35
圖4.3 Chitinibacter tainanensis於BH培養基中之生長曲線 36
圖4.4不同葡萄糖濃度培養Chitinibacter tainanensis之生質量的變化 38
圖4.5不同葡萄糖濃度培養Chitinibacter tainanensis之pH的變化 39
圖4.6以2%幾丁質粉末之BH養基培養Chitinibacter tainanens之N-乙醯葡萄糖胺酶活性、還原糖及pH的變化 40
圖4.7 Chitinibacter tainanensis生產之N-乙醯葡萄糖胺的高效能液相層析圖 42
圖4.8以2%幾丁質粉末BH培養基培養Chitinibacter tainanensis 生產N-acetylglucosamine 43
圖4.9不同葡萄糖濃度培養於Chitinibacter tainanensis之生質量變化 45
圖4.10不同葡萄糖濃度於Chitinibacter tainanensis培養過程之葡萄糖的變化 46
圖4.11 Chitinibacter tainanensis於發酵培養基中N-乙醯葡萄糖胺之變化 48
圖4.12 Chitinibacter tainanensis於2.5%葡萄糖生長培養基中生質量、葡萄糖及pH值之變化 50
圖4.13 Chitinibacter tainanensis於發酵培養基中幾丁質酶活性、還原糖及生質量之變化 52

表目錄

表3.1 基礎培養基 24
表3.2 發酵培養基 24
表3.3微量金屬溶液組成 26
表3.4 Chitinibacter tainanensis 之型態特性分析 28
表3.5 Chitinibacter tainanensis 之生理特性分析 28
表 4.1以不同培養條件培養菌株Chitinibacter tainanensis之活性、還原醣、生質量、N-乙醯醯葡萄糖胺產量及轉化率之變化 54

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