臺灣博碩士論文加值系統

Chitinbacter tainanensis已被證實能夠將幾丁質分解為N-乙醯葡萄胺糖(NAG)。本研究目的為製備不同類型的幾丁質並做為碳源，再以C. tainanensis降解這些幾丁質，探討幾丁質分子鏈結構和空間結構對其分解效率的影響。α-和β-幾丁質分別於水分含量75%、50%和35%下，以壓力9 kg/cm2進行膨發，之後藉由FT-IR、XRD、色差儀等分析膨發作用對幾丁質物化特性之影響。結果顯示，無論是α-或β-幾丁質之去乙醯程度 (DD) 會隨著水分含量的增加而上升。在其顏色會隨著水份含量的降低，變化更加明顯。同時測定其總體密度和粒徑的分佈，低水份含量的幾丁質經膨發作用，其總體密度會變小，表示其表面積有變大的現象。未膨發幾丁質的粒徑為60-80 mesh，膨發後幾丁質的粒徑主要是在10-60 mesh，少數比例為大於80 mesh，其分佈在10-60 mesh 的比例會隨著膨發水份含量的增加而增加，而分佈在大於80 mesh的比例會隨著水份膨發水份含量的增加而減少。進一步以SEM觀察其表面構造，在低水份含量膨發的組別，其孔洞有明顯的增加且孔洞較大。結晶度方面，膨發作用對α-幾丁質之結晶構造破壞較β-幾丁質明顯，且膨發的水份含量越低，對結晶構造的破壞越大。
無論是α-或β-幾丁質於不同水份含量下膨發後，相較未膨發的幾丁質皆能夠有效的被C. tainanensis所利用。以水份含量75%膨發後的α-幾丁質作為碳源培養，其NAG轉化率相較控制組有明顯增加。此外，以膨發後的幾丁質作為碳源，培養至72小時，其酵素活性和菌體生質量皆高於未膨發的幾丁質。然而使用從菌體殘骸分離出的酵素進行水解幾丁質，結果顯示以膨發作用的幾丁質之水解效率較高，當中以35%的組別之水解作用最佳。

It has been confirmed that Chitinbacter tainanensis could hydrolysed chitin to perform N-acetyl-glucosamine (NAG) production. In this study, different type of chitin were prepared and were used as substrate for C. tainanensis to study the influence of molecular chain and spatial structure of chitin on degradation. α-Chitin and β-chitin were tempered to 75%, 50% and 35% moisture content and equilibrated at 25℃ then being puffed in a puffing gun. The effect of puffing to chitin were analyzed by FT-IR, XRD and so on. The results showed that the degree of deacetylation were raised with increasing moisture content both in α-chitin and β-chitin. The color values of chitin were significantly changed with the decreased of puffed moisture content. The bulk density were also significantly decreased with the decreased of puffed moisture content. The particle size of unpuffed chitin was 60-80 mesh. The particle size of puffed chitin was major in 10-60 mesh, minor below 80 mesh. The proportion of 10-60 mesh fraction was raised with increasing the moisture content. However, the proportion of below 80 mesh fraction was decreased with increasing the moisture content. The particle size of puffed chitin with lower moisture content were mostly distributed below 80 mesh and the puffed β-chitin had a significantly changed. The hole on surface of chitin were became bigger and increase with the decreased of puffed moisture content were observed by scanning electron microscopy. The result of XRD showed that the crystal structure of α-chitin were significantly destroyed than β-chitin after being puffed. The crystal structure of puffed chitin was strongly destroyed with a lower moisture content.
Moreover, this study suggested that either puffed α-chitin or β-chitin with different moisture content was more easily utilized by C. tainanensis than the unpuffed chitin. Meanwhile, there was a significantly increased of conversion rate of NAG was produced by cultivated with puffed α-chitin with 75% moisture content after 72 hours. Although -chitin were not easily utilized, it can be improved by treatment of puffing. Besides, the activity of enzyme and biomass were increased by cultivated with puffed chitin. Meanwhile, it can be observed that a high production of NAG from puffed chitin with 35% moisture content by the isolated enzyme.

目次

摘要 I
Abstract II
目次 III
圖目次 VI
表目次 VIII
附錄 IX
1. 緒論 1
1.1. 前言 1
1.2. 研究目的 1
2. 文獻回顧 2
2.1. 幾丁質的簡介 2
2.2. 幾丁質的結構 2
2.3. 幾丁質的製備 2
2.4. 幾丁質的降解方法 3
2.4.1. 化學法 3
2.4.2. 酵素法 3
2.4.3. 物理法 3
2.4.4. 製備NAG之方法 4
2.5. 常見的水解酵素 4
2.5.1. 專一性酵素 4
2.5.2. 非專一性酵素 4
2.5.3. 幾丁質水解酵素 4
2.6. 幾丁質水解酵素的純化分析 5
2.7. 幾丁質水解酵素的活性分析 6
2.7.1. 還原糖法 6
2.7.2. 螢光法 6
2.7.3. 紫色幾丁質法 6
2.7.4. 電泳膠片檢測法 7
2.8. 幾丁質結晶度的分析 7
2.9. 幾丁質和其水解產物的功能與應用 7
2.9.1. 提高免疫力 7
2.9.2. 促進凝血作用和傷口癒合 8
2.9.3. 抗氧化性 8
2.9.4. 抗腫瘤活性 8
2.9.5. N-乙醯葡萄胺糖(NAG)的應用 8
2.9.6. 幾丁寡糖的應用 9
2.10. 膨發 9
2.10.1. 影響膨發的因子 10
2.10.2. 膨發應用 10
2.10.2.1. 膨發乾燥 10
2.10.2.2. 爆米香和穀物 10
2.10.2.3. 人蔘膨發產品 10
2.10.2.4. 大豆膨發產品 11
3. 實驗架構 12
4. 材料與方法 13
4.1. 實驗材料 13
4.2. 藥品 13
4.3. 儀器設備 13
4.4. 實驗方法 15
4.4.1. 幾丁質的製備 15
4.4.1.1. α-幾丁質的製備方法 15
4.4.1.2. β-幾丁質的製備方法 15
4.4.1.3. 膠狀幾丁質 (colloid-chitin) 的製備方法 15
4.4.2. 幾丁質的膨發 15
4.4.3. 幾丁質去乙醯化程度的測定 16
4.4.4. 幾丁質色差檢測 16
4.4.5. 幾丁質總體密度和膨發比例測定 17
4.4.6. 幾丁質敲擊密度測定 17
4.4.7. 幾丁質粒徑分佈測定 17
4.4.8. 幾丁質表面微細構造之觀察 17
4.4.9. 幾丁質結晶度測定 17
4.4.10. 實驗菌株 18
4.4.10.1 菌種 18
4.4.10.2. 菌種保存 18
4.4.10.3. 菌種活化 18
4.4.11. 以搖瓶法培養菌種分解幾丁質： 18
4.4.12. 幾丁質水解酵素活性分析 19
測量NAGase活性 19
4.4.13. NAG產率測定 19
4.4.14. 菌體生質量之測定 20
4.4.15. 幾丁質水解產物分析 20
4.4.16. 幾丁質水解酵素之分離純化 21
4.4.17. 蛋白質濃度測定 21
4.4.18. 分離之酵素水解幾丁質 21
4.4.19. 統計分析 21
5. 結果與討論 22
5.1. 不同類型幾丁質之物化性質 22
5.1.1. 膨發作用對幾丁質官能基團之影響 22
5.1.2. 膨發作用對幾丁質去乙醯程度之影響 22
5.1.3. 膨發作用對幾丁質顏色之影響 23
5.1.4. 膨發作用對幾丁質顆粒密度和粒徑分佈之影響 23
5.1.5. 膨發作用對幾丁質結構型態之影響 24
5.1.6. 膨發作用對幾丁質結晶程度之影響 24
5.2. 不同類型幾丁質對Chitinbacter tainanensis水解幾丁質效率之影響 25
5.2.1. 不同類型幾丁質對NAGase活性之變化 25
5.2.2. 不同類型幾丁質對糖度之變化 25
5.2.3. 不同類型幾丁質對NAG轉化率之變化 26
5.2.4. 不同類型幾丁質對菌體生長之影響 26
5.3. 不同類型幾丁質對菌體之分離酵素水解幾丁質效率之影響 27
5.3.1. 菌體分離之酵素特性 27
5.3.2. 分離酵素之NAG水解率 27
6. 結論 29
7. 參考文獻 30
8. 圖 36
9. 表 63
10. 附錄 69

圖目次

圖一、不同水分含量膨發後a-幾丁質之紅外光圖譜 36
圖二、不同水分含量膨發後b-幾丁質之紅外光圖譜 37
圖三、不同水份含量 (a) control (b) 75% (c) 50% (d) 35%膨發作用後的a-幾丁質 38
圖四、不同水份含量 (a) control (b) 75% (c) 50% (d) 35%膨發作用後的b-幾丁質 39
圖五、不同水份含量 (a) control (b) 75% (c) 50% (d) 35%膨發後的a-幾丁質之掃描式電子顯微圖 (5000倍) 40
圖六、不同水份含量 (a) control (b) 75% (c) 50% (d) 35%膨發後的a-幾丁質之掃描式電子顯微圖 (10000倍) 41
圖七、不同水份含量 (a) control (b) 75% (c) 50% (d) 35%膨發後的a-幾丁質之掃描式電子顯微圖 (20000倍) 42
圖八、不同水份含量 (a) control (b) 75% (c) 50% (d) 35%膨發後的a-幾丁質之X-射線繞射圖譜 43
圖九、不同水份含量 (a) control (b) 75% (c) 50% (d) 35%膨發後b-幾丁質之X-射線繞射圖譜 44
圖十、N-醯葡萄胺糖酶之標準檢量線 45
圖十一、以不同水份含量膨發後a-幾丁質之BH培養基培養Chitinbacter tainanensis 之N-乙醯葡萄胺糖酶活性變化 46
圖十二、以不同水份含量膨發後b-幾丁質之BH培養基培養Chitinbacter tainanensis 之N-乙醯葡萄胺糖酶活性的變化 47
圖十三、以不同水份含量膨發後a-幾丁質粉末BH培養基培養Chitinbacter tainanensis之糖度變化 48
圖十四、以不同水份含量膨發後b-幾丁質粉末BH培養基培養Chitinbacter tainanensis之糖度變化 49
圖十五、Chitinbacter tainanensis培養72小時後之幾丁質水解產物的高效液相層析圖 (a) N-乙醯葡萄胺糖標準品、(b)a-幾丁質水解產物 (c)b-幾丁質水解產物 50
圖十六、未膨發a-幾丁質在MALDI-TOF MS下的圖譜 51
圖十七、Chitinbacter tainanensis培養72小時後之a-幾丁質水解產物在MALDI-TOF MS下的圖譜 52
圖十八、未膨發b-幾丁質在MALDI-TOF MS下的圖譜 53
圖十九、Chitinbacter tainanensis培養72小時後之b-幾丁質水解產物在MALDI-TOF MS下的圖譜 54
圖二十、不同水份含量膨發後a-幾丁質粉末BH培養基培養Chitinbacter tainanensis 72 小時之NAG轉化率 55
圖二十一、以不同水份含量膨發後b-幾丁質粉末BH培養基培養Chitinbacter tainanensis 72小時之NAG轉化率 56
圖二十二、以不同水份含量膨發後a-幾丁質粉末BH培養基培養Chitinbacter tainanensis之菌體生質量變化 57
圖二十三、以不同水份含量膨發後b-幾丁質粉末BH培養基培養Chitinbacter tainanensis之菌體生質量變化 58
圖二十四、蛋白質含量之標準檢量線 59
圖二十五、N-乙醯葡萄胺糖標準品之標準檢量線 60
圖二十六、以Chitinbacter tainanensis菌體殘骸中震盪出的酵素水解不同水份含量膨發後的a-幾丁質之NAG轉化率 61
圖二十七、以Chitinbacter tainanensis菌體殘骸中震盪出的酵素水解不同水份含量膨發後的b-幾丁質之NAG轉化率 62

表目次

表一、不同水份含量膨發後對幾丁質之去乙醯程度的影響 63
表二、不同水份含量對膨發後幾丁質顏色變化之影響 64
表三、不同水份含量下膨發後幾丁質的密度和膨發比例 65
表四、不同水份含量膨發對幾丁質粒徑分佈之影響 66
表五、不同水份含量膨發後幾丁質之結晶程度 67
表六、自菌體殘骸分離NAGase之特性 68

附錄

附錄 一、N-乙醯葡萄胺糖的結構圖 69
附錄 二、Chitinbacter tainanensis BCRC 17254的基本性質 70
附錄 三、Chitinbacter tainanensis培養基配方 71
附錄 四、幾丁質的主要官能基 72

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