臺灣博碩士論文加值系統

本研究以自行純化之Bacillus licheniformis內切聚木糖酶，分別進行纖維酵素協同性、降解產物分析及木寡醣益生特性之探討。首先將聚木糖酶 (xylanase)、纖維素酶 (cellulase) 及木糖苷酶 (xylosidase) 作複合酵素組合，並以樺木聚木醣 (birchwood xylan)、麥木聚木醣 (oat-spelts xylan)、玉米穗軸、未預處理之蔗渣、不同濃度硫酸處理之玉米穗軸及蔗渣粗半纖維素為受質，測定複合酵素間之協同性。另外以麥木聚木醣及樺木聚木醣為基質，於不同水解時間下進行木寡醣之製備，並以高效液相層析儀進行產物分析，且選擇Bifidobacterium bifidum、Lactobacillus acidophilus、Clostridium perfringens為對象，評估聚木糖酶水解產物對腸道益生菌及有害菌生長之影響。實驗結果顯示，1.5% 硫酸處理之玉米穗軸及未預處理之蔗渣，以cellulase/xylanase降解，其加乘效果最好，DSE值分別可達1.67及2.17，而樺木聚木醣、麥木聚木醣、蔗渣粗半纖維素則以xylanase/xylosidase協同性最佳，DSE值分別可得1.59、1.94及1.89。在降解產物分佈方面，麥木聚木醣以聚木糖酶反應48小時，其二醣至六醣所佔比例分別為18、25、7、18及13%，而樺木聚木醣之寡醣分佈則以二、三醣為主，兩者約可佔總寡醣含量8成以上。另外益菌作用結果指出，木寡醣於低濃度下，其促進有益菌增殖之效果並不顯著，然而提高二、三、四醣濃度總和至30,000 ppm及延長木寡醣與菌體培養時間達72小時，則麥木聚木醣降解產物可抑制C. perfringens及促進B. bifidum之增殖。

The objective of the present study is to conduct the hydrolysate profiles of xylan, prebiotic effect of xylooligosaccharides and synergistic effect by a purified endoxylanase derived from Bacillus licheniformis. First all, a synergistic effect on birchwood xylan, oat-spelts xylan, corn cob, untreated sugar cane bagasse, H2SO4 treated corn cob and hemicellulose prepared from sugar cane bagasse by single xylanase, cellulase and xylosidase or combined enzymes among them were systematically established. Xylooligosaccharides prepared from birchwood and oat-spelts xylan was colleted for prebiotic study using Bifidobacterium bifidum, Lactobacillus acidophilus, Clostridium perfringens as subjects. The results indicated that cellulase/xylanase exhibited the optimal synergistic effect on 1.5% H2SO4 treated corn cob and untreated sugar cane bagasse, where were DSE of 1.67 and 2.17, respectively. Furthermore, xylanase/xylosidase displayed the most efficient hydrolysis in Birchwood xylan, oat-spelts xylan and hemicellulose (from sugar cane bagasse), where were DSE of 1.59, 1.94 and 1.89, respectively. Hydrolysate profiles of oat-spelts xylan showed that the relative ratio of xylobiose to
xylohexaose is 18, 25, 7, 18 and 13% respectively, but the major hydrolysates of birchwood xylan are xylobiose and xylotriose which are composed of 80% of total oligomers. Prebiotic study illustrated that little acceleration on microbial growth was observed if low concentration of xylooligomers was employed. However, a moderate effect was found in C. perfringens inhibition and B. bifidum stimulation when increase the total xylobiose, xylotriose and xylotetraose concentration to 30,000 ppm and extend the incubation period up to 72 hours.

目錄

中文摘要……………………………………………………………………....I
英文摘要…………………………………………………….……………….II
誌謝………………………………………………………………………….IV
目錄…………………………………………………………………………..V
圖表目錄………………………………………………………...…………..IX
第1章 前言…………………………………………………………………..1
第2章 文獻回顧…………………………………………………………..... 3
2.1 植物纖維質之組成…………………………………………………… 3
2.1.1 細胞壁之多醣……………………………..……………………...3
2.1.2 半纖維素…………………………………………………………. 6
2.1.2.1 半纖維素之組成……………………………………………..6
2.1.3 聚木醣之構造…………………………………………………….8
2.2 本省農產廢棄物處理現況…………………………………………..11
2.2.1 玉米穗軸………………………………………………………...13
2.2.2 甘蔗渣…………………………………………………………...14
2.2.3 農業纖維質降解………………………………………………...14
2.3 嗜高溫菌……………………………………………………………..15
2.3.1 Bacillus licheniformis…………………………………………….15
2.4 聚木醣降解酵素系統………………………………………………..16
2.4.1 作用於聚木醣主鏈之酵素……………………………………...16
2.4.1.1 Endo-β-1,4-xylanase (EC 3.2.1.8)…………………………...16
2.4.1.2 β-1,4-xylosidase (EC 3.2.1.37)………………………………17
2.4.2 作用於聚木醣側鏈之酵素……………………………………...17
2.4.2.1 Arabinofuranosidase (EC 3.2.1.55)………………………….17
2.4.2.2 α-Glucuronidase (EC 3.2.1.1)……………………………….17
2.4.2.3 Acetylxylan esterase (EC 3.1.1.6)…………………………...17
2.4.2.4 Phenolic acid esterases………………………………………18
2.4.3 xylanase之種類與反應機制....................................................18
2.4.3.1 催化機制……………………………………………………18
2.4.3.2 xylanase之催化能力………………………………………..18
2.4.3.3 xylanase之結構……………………………………………...20
2.5 酵素的加乘作用……………………………………………………..22
2.5.1 協同作用之公式………………………………………………...24
2.5.2 影響酵素協同性之因子………………………………………...24
2.5.3 高聚木醣受質降解之酵素協同性……………………………...24
2.6 聚木醣降解產物……………………………………………………..25
2.6.1 木糖之應用……………………………………………………...25
2.6.2 木寡醣之應用…………………………………………………...25
2.7 膳食纖維之生理功能………………………………………………..28
2.8 木寡醣之益生特性…………………………………………………..28
第3章 材料與方法………………………………………………………....31
3.1 實驗設計…………………………………………………..…………31
3.2 實驗藥品……………………………………………………………..32
3.2.1 一般化學藥品…………………………………………………...32
3.2.2 酵素純化材料…………………………………………………...32
3.2.3 內切聚木糖酶 (endo-β-1,4-xylanase) 生產菌…………………33
3.2.4 協同作用 (Synergistic effect)…………………………………..33
3.2.4.1 受質種類……………………………………………………33
3.2.4.2 酵素種類……………………………………………………33
3.2.5 HPLC產物分析 (Product profile)……………………………34
3.2.5.1 木寡醣來源…………………………………………………34
3.2.5.2 HPLC標準品…………………………………………….....34
3.2.6 益生效果探討 (Probiotic effect)………………………………..34
3.2.6.1 菌株來源……………………………………………………34
3.2.6.2 市售寡醣產品………………………………………………34
3.3 儀器設備……………………………………………………………..35
3.3.1 一般儀器…………………………………………………...……35
3.3.2 酵素純化器材…………………………………………………...35
3.3.3 高效液相層析設備……………………………………………...35
3.3.4 微生物試驗設備………………………………………………...36
3.4 實驗方法……………………………………………………………..36
3.4.1 Xylanase之生產…………………………………….............36
3.4.1.1 Bacillus licheniformis液態培養基組成………………….36
3.4.1.2 粗酵素液之製備……………………………………………36
3.4.1.3 高濃度酵素液之製備………………………………………37
3.4.2 Xylanase之純化……………………………………................37
3.4.2.1 DEAE-Sepharose離子交換層析……………………………37
3.4.2.2 Sephacryl S-200 HR膠體過濾層析………………………...37
3.4.3 聚木醣水解酵素活性之測定…………………………………...38
3.4.3.1 磷酸緩衝液及DNS試劑之配製.………....38
3.4.3.2 木糖檢量線之建立……………………………………...….38
3.4.3.3 活性測定……………………………………………………38
3.4.4 蛋白質定量……………………………………………………...39
3.4.4.1 蛋白質檢量線之建立………………………………………39
3.4.4.2 Xylanase蛋白質濃度測定……………………..................39
3.4.5 協同作用………………………………………………………...39
3.4.5.1 受質預處理…………………………………………………39
3.4.5.2 各種受質 (2%, w/v) 溶液之製備…………………………40
3.4.5.3 經純化之xylanase其β-xylosidase、cellulase活性測定....41
3.4.5.4 葡萄糖檢量線之建立……………………………………....41
3.4.5.5 加乘作用後之還原糖測定…………………………………41
3.4.5.6 酵素加乘反應………………………………………………42
3.4.6 聚木醣水解產物分析…………………………………………...43
3.4.6.1 木寡醣製備…………………………………………………43
3.4.6.2 水解產物分析 (product profile analysis)……………...…..43
3.4.7 益生菌生長試驗………………………………………………...44
3.4.7.1 菌種選購與保存……………………………………………44
3.4.7.2 MRS Broth配製…….......……………………...…………44
3.4.7.3 AC Broth配製…………………………………………….45
3.4.7.4 保存菌株活化………………………………………………45
3.4.7.5 體外試驗……………………………………………………45
第4章 結果與討論………………………………………………………....47
4.1 聚木糖酶之分離與純化……………………………………………..47
4.2 協同性試驗…………………………………………………………..47
4.2.1 單一酵素及複合酵素作用不同受質…………………………...47
4.2.2 協同性最佳之酵素組合………………………………………...49
4.2.3 改變酵素活性比對加乘效果之影響…………………………...52
4.2.3.1 改變cellulase/xylanase活性比對協同性之影響…………..52
4.2.3.2 改變xylanase/xylosidase活性比對協同性之影響………...54
4.2.4 時間對複合酵素作用之影響…………....……………………...55
4.2.4.1 延長cellulase/xylanase作用時間…………………………..55
4.2.4.2 延長xylanase/xylosidase作用時間………………………...56
4.3 水解產物分析………………………………………………………..56
4.3.1 高效液相層析…………………………………………………...56
4.3.2 水解產物積分面積及產物分佈之探討………………………...57
4.4 益生作用……………………………………………………………..59
第5章 結論………………………………………………………………....96
參考文獻…………………………………………………………………….98
作者簡介…………………………………………………………………...110

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