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系統識別號 U0002-2607200521154600
中文論文名稱 幾丁聚醣之鳳梨酵素水解產物於抗腫瘤及抗菌之效果探討
英文論文名稱 The inhibitory effects of bromelin hydrolyzed chitosan on tumor cells and microorganisms
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 93
學期 2
出版年 94
研究生中文姓名 曾惠婷
研究生英文姓名 Hui-Ting Tzeng
學號 692290504
學位類別 碩士
語文別 中文
口試日期 2005-06-15
論文頁數 90頁
口試委員 指導教授-王三郎
委員-陳曜鴻
委員-陳裕仁
中文關鍵字 幾丁質  幾丁聚醣  幾丁寡醣  幾丁寡醣分離法  腫瘤細胞存活率試驗  鳳梨酵素 
英文關鍵字 chitinc  hitosan  chitooligosaccharides  chitooligosaccharide separating method  tumor cell viability test  bromelin 
學科別分類 學科別醫學與生命科學生物學
中文摘要 本研究主要探討幾丁質、幾丁聚醣物質,經由鳳梨酵素水解,所生成幾丁寡醣對於細菌、黴菌、腫瘤細胞之抑制效果。
將2%烏賊軟骨粉末於pH5、37℃,經0.1%鳳梨酵素水解兩天,所得水解物具有最高產量,對於Fusarium oxysporum具有1.6U的抑制活性。將此水解物利用幾丁寡醣分離法回收得到幾丁寡醣,進行腫瘤細胞存活率試驗,結果顯示對於人類白血病細胞U937剩下69%的存活率。
將0.1%水溶性幾丁聚醣在pH5、37℃,經0.1%鳳梨酵素水解一天,所得水解物具有最高產量。其對Bacillus subtilis具有抑制效果,亦對Aspergillus fumigatus具有0.8U的抑制活性。將此水解物利用幾丁寡醣分離法回收得到幾丁寡醣,進行腫瘤細胞存活率試驗,結果顯示對於小鼠大腸癌細胞CT26剩下57%的存活率。
英文摘要 The major purpose of this research was to investigate the inhibitory effects of chitosan on bacteria, fungus, and tumor cells.
An aqueous solution of 2% squid pen powder was hydrolyzed, at pH5 and 37℃, with 0.1﹪bromelain for two days to get the highest yield of hydrolysis product. Chitooligosaccharides were then recovered from this hydrolysis product by chitooligosaccharide separating method. The recovered chitooligosaccharides were used do assay its viability effect on the tumor cells tested. The result showed a 69% viability effect on leukemia cell U937. The chitooligosaccharides also showed a 1.6U inhibitory effect on Fasarium oxysporum.
An aqueous solution of 0.1% chitosan was hydrolyzed, at pH5 and 37℃, with 0.1﹪bromelain for one day to get the highest yield of hydrolysis product. The resultant hydrolysis product showed inhibitory effect on Bacillus subtilis, 0.8U inhibitory activitiy to Aspergillus fumigatus, and 57﹪viability effect on colon cell CT26.
論文目次 目 錄

封面內頁
簽名頁
授權書
中文摘要……………………………………………………………i
英文摘要……………………………………………………………ii
誌謝………………………………………………………………iv
目錄…………………………………………………………………v
圖目錄……………………………………………………………ix
表目錄………………………………………………………………xi

第一章 緒言..............................................1
第二章 文獻回顧..........................................2
2.1幾丁質、幾丁聚醣、幾丁寡醣........................2
2.1.1幾丁質之分子結構..............................2
2.1.2幾丁聚醣......................................3
2.1.3 N-乙醯幾丁寡醣及幾丁寡醣.....................6
2.2 N-乙醯幾丁寡醣及幾丁寡醣之製備...................6
2.2.1化學法........................................6
2.2.2生化法........................................7
2.3植物酵素之簡介....................................7
2.3.1鳳梨酵素......................................7
2.3.2木瓜酵素......................................8
2.4幾丁質及幾丁聚醣之應用............................8
2.5 N-乙醯幾丁寡醣及幾丁寡醣之抑菌效果..............11
2.6 N-乙醯幾丁寡醣及幾丁寡醣之抑黴效果..............11
2.7 N-乙醯幾丁寡醣及幾丁寡醣之抗癌效果..............12
2.7.1研究人類白血病及大腸癌之重要性...............13
2.7.2 U937細胞株之簡介............................14
2.7.3 CT26細胞株之簡介............................14
第三章 材料與方法.......................................16
3.1研究設備..............................................16
3.2研究材料.........................................17
3.2.1生化材料.....................................17
3.2.2微生物材料...................................18
3.3研究方法.........................................19
3.3.1研究架構.....................................19
3.3.2懸浮態幾丁質之製備...........................20
3.3.3幾丁質酶活性測定方法.........................20
3.3.4蛋白酶活性測定方法...........................21
3.3.5蛋白質定量分析...............................21
3.4酵素水解幾丁類物質以生產幾丁寡醣條件探討.........21
3.4.1酵素濃度對還原醣、胺基酸產量之影響...........22
3.4.2基質種類對還原醣、胺基酸產量之影響...........22
3.4.3基質濃度對還原醣、胺基酸產量之影響...........22
3.4.4水解時間對還原醣、胺基酸產量之影響...........23
3.4.5幾丁寡醣之製備..........................23
3.5細菌抑制試驗.....................................25
3.5.1測試之菌株...................................25
3.5.2細菌增值抑制作用.............................25
3.6 黴菌抑制試驗....................................25
3.6.1測試之菌株...................................25
3.6.2生物分析法...................................25
3.7腫瘤細胞抑制試驗.................................26
3.7.1測試之細胞株.................................26
3.7.2細胞培養........................26
3.7.3細胞計數法...................................26
3.7.4細胞存活率之分析.............................27
3.7.5細胞形態學之分析.............................27
3.7.6 NBT還原試驗. ...............................27
3.8鳳梨酵素之純化與分離.............................28
3.8.1 DEAE Sepharose CL-6B 陰離子交換層析法.......28
3.8.2 Sephacry S-200 膠體過濾層析法...............29
3.9酵素生化性質之探討...............................29
3.9.1酵素之最適pH反應測定.........................29
3.9.2酵素之pH安定性測定...........................30
3.9.3酵素之最適反應溫度測定.......................30
3.9.4酵素之熱安定性測定...........................31
3.9.5蛋白質電泳測定分子量.........................31
第四章 結果與討.論......................................33
4.1酵素水解幾丁類物質以生產幾丁寡醣條件探討.........33
4.1.1酵素濃度對還原醣、胺基酸產量之影響...........33
4.1.2基質種類對還原醣、胺基酸產量之影響...........35
4.1.3基質濃度對還原醣、胺基酸產量之影響............36
4.1.4水解時間對還原醣、胺基酸產量之影響.............37
4.2細菌抑制試驗.......................................50
4.3黴菌抑制試驗.....................................51
4.4腫瘤細胞抑制試驗.................................54
4.4.1細胞存活率之分析...............................54
4.4.2細胞形態學之分析...............................55
4.4.3 NBT還原試驗...................................56
4.5鳳梨酵素之純化與分離.............................69
4.5.1 DEAE Sepharose CL-6B 陰離子交換層析法.......69
4.5.2 Sephacry S-200 膠體過濾層析法...............69
4.5.3酵素之最適反應pH、pH安定性...................69
4.5.4酵素最適反應溫度、熱安定性...................70
4.5.5酵素之分子量判定.............................71
第五章 結論.............................................80
參考文獻.................................................83



圖目錄

圖 2.1幾丁質與幾丁聚醣之化學結構..........................4
圖 2.2幾丁質的三種排列型態................................5
圖 2.3幾丁質、幾丁聚醣之關聯圖............................9
圖 2.4幾丁聚醣對植物病原菌之作用.........................15
圖 3.1研究架構...........................................19
圖 3.2 N-幾丁乙醯寡醣製備流程圖..........................24
圖 4.1酵素濃度對還原醣產量之影響.........................40
圖 4.2酵素濃度對胺基酸產量之影響.........................41
圖 4.3基質種類對還原醣產量之影響.........................42
圖 4.4基質種類對胺基酸產量之影響.........................43
圖 4.5水溶性幾丁聚醣對還原醣產量之影響...................44
圖 4.6水溶性幾丁聚醣對胺基酸產量之影響...................45
圖 4.7烏賊軟骨粉末對還原醣產量之影響.....................46
圖 4.8烏賊軟骨粉末對胺基酸產量之影響.....................47
圖 4.9水解時間對還原醣產量之影響.........................48
圖 4.10水解時間對胺基酸產量之影響........................49
圖 4.11細菌增值抑制試驗..................................60
圖4.12水溶性幾丁聚醣之黴菌抑制試驗.......................61
圖4.13烏賊軟骨粉末之黴菌抑制試驗.........................62
圖4.14黴菌抑制試驗之F.O..................................63
圖4.15黴菌抑制試驗之A.F..................................64
圖4.16 CT26細胞存活率試.驗.......................................................65
圖4.17 U937細胞存活率試驗................................66
圖4.18 U937細胞形態之分析................................67
圖4.19 U937之NBT還原試驗 ................................68
圖4.20 DEAE-Sepharose CL-6B層析圖........................72
圖4.21蛋白酶S-200過濾層析圖..............................73
圖4.22幾丁質酶 S-200過濾層析圖...........................74
圖4.23蛋白酶之pH安定性與最適反應pH ......................75
圖4.24幾丁質酶之pH安定性與最適反應pH ....................76
圖4.25蛋白酶之熱安定性與最適反應溫度.....................77
圖4.26幾丁酶之熱安定性與最適反應溫度.....................78
圖4.27蛋白酶之SDS-PAGE...................................79
表目錄

表2.1幾丁類物質之應用....................................10
表3.1製備SDS-PAGE膠體溶液配方............................32
表4.1 U937之NBT陽性率....................................58
表4.2初步細菌抑制結果....................................59

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