陳俊任臺灣大學:生物產業機電工程學研究所林芫如Lin, Yuan-JuYuan-JuLin2010-05-052018-07-102010-05-052018-07-102008U0001-1507200814075600http://ntur.lib.ntu.edu.tw//handle/246246/180187目前已知N-乙醯幾丁寡糖 (聚合度4-7)具有特殊生理活性如能提高免疫力及抑制腫瘤生長，故本研究目的擬由富含幾丁質環境中篩選出可以分解幾丁質產生N-乙醯幾丁寡醣之微生物。在初期篩選中，以粗酵素水解膠態幾丁質並以高效能液相層析 (HPLC)確認其產物，挑選出一株產物中有較長鏈N-乙醯幾丁寡糖之微生物，經鑑定命名為Serratia marcescens NTU-17。以反應區面法 (response surface methodology, RSM)之中心混成設計 (central composite design, CCD)得到最佳酵素生產的培養基條件為0.4 g/l colloidal chitin、1.6 g/l casein於30。C及pH 7.5下培養；以此條件下震盪培養18小時後可生產出最高的酵素活性。粗酵素液經35-70%硫酸銨沉澱後，以Sephacryl 200進行膠體過濾，接著以DEAE-Sephacel離子交換層析進行純化，最後分別得到二種純化之幾丁質酶，經SDS-PAGE分析其分子量，分別為53 kDa (chitinase 1)及39 kDa (chitinase 2)，並以活性染色方式確認此2種蛋白質皆具有幾丁質酶活性，純化之幾丁質酶活性大約為0.3 U/ml且比活性增加2.5倍，而酵素回收率則約為12%。在酵素特性部分，chitinase 1在pH 3反應溫度為50℃時有最佳的活性，而chitinase 2在pH 3-12的範圍中皆具有幾丁質酶活性。Abstract-acetylchitooligosaccharides (degree of polymerization 4-6) have specific biological activities such as antitumor activity and immuno-enhancing effects. In this study, we aimed to isolate environmental microorganisms which could produce enzymes to hydrolyze chitin into N-acetylchitooligosaccharides. At the initial stage, we hydrolyzed colloidal chitin with crude microbial enzymes and analyzed the products by HPLC. From this screening, we found that the crude enzyme from one bacterial isolate could hydrolyze chitin and produce N-acetylchitooligosaccharides. The bacterial strain was identified by 16S rRNA sequencing and phylogenetic analysis to belong Serratia marcescens and was named S. marcescens NTU-17. We used central composite design (CCD) of response surface methodology (RSM) to obtain the optimal culture condition for chitinase production: 0.4 g/l colloidal chitin, 1.6 g/l casein, 30。C and pH 7.5; the highest chitinase activity was produced at 18 hours after inoculation. The crude enzyme from culture broth of S. marcescens NTU-17 was subjected to successive steps of purification. After ammonium sulfate fractionation (35-70%), gel filtration-Sephacryl 200 chromatography, and DEAE-Sephacel column chromatography, two species of chitinase were purified and the molecular weights were determined by SDS-PAGE to be 53 kDa (chitinase 1) and 39 kDa (chitinase 2). The chitinase activity of chitinase 1 and 2 were also verified by an in-gel chitinase activity assay. After purification, the specific activity of chitinase was increased by 2.5 fold and the yield was 12%. Chitinase 1 exhibited the optimal activity at pH 3 and 50℃, and chitinase 2 showed the optimal activity at 30℃ and similar activities at pH 3-12.目錄錄 i目錄 iii目錄 v謝 vi文摘要 1bstract 2言 4一章、文獻探討 51)幾丁質 5.1幾丁質之結構 5.2幾丁質之製備 5.3幾丁質之應用 62)幾丁聚醣 6.1幾丁聚醣之結構 6.2幾丁聚醣之應用 7.3幾丁聚醣之製備 73) N-乙醯幾丁寡醣 (N-acetyl chitooligosaccharides, (GlcNAc)n)與幾丁寡醣 (chito-oligosaccharides) 8.1 N-乙醯幾丁寡醣與幾丁寡醣之結構 8.2 N-乙醯幾丁寡醣與幾丁寡醣之製備 8.3 N-乙醯幾丁寡醣與幾丁寡醣之應用 94)不同的幾丁質水解酵素 9.1幾丁質酶 (chitinase；EC 3.2.1.14) 95)反應曲面法 (Response surface methodology,RSM) 11.1反應區面法實際應用之優點 12.2常用實驗設計類型及實施步驟 136)微生物菌種鑑定 15.1 核醣體核醣核酸 (ribosomal RNA, rRNA) 15二章、材料與方法 161)實驗材料 16.1菌株 16.2化學材料 16.4儀器 172)實驗方法 17.5菌種保存及菌株培養 17.6酵素活性測定方法 21.7酵素最適培養條件探討 22.8幾丁質酶之純化 23.9幾丁質酶酵素之特性分析 26.10產物分析 27三章、結果與討論 30一)菌種篩選 30二) Serratia marcescens NTU-17生產幾丁質酶之最佳培養條件探討 35三)酵素純化 45四)產物確認 57四章、結論 59五章、參考文獻 60目錄1- 1反應曲面法Response surface methodology 122- 1 HPLC樣品製備流程圖 272- 2實驗流程圖 293-1初步篩選18株菌株幾丁質酶活性 323-2以HPLC檢測No.17粗酵素水解colloidal chitin之產物 323-3 Serratia marcescens NTU-17其生長曲線及酵素曲線。 333-4以HPLC分析甲醇和丙酮分離不同聚合度之N-乙醯幾丁寡醣 333-5 16S rDNA phylogenetic tree 343-6 1%不同氮源對幾丁質酶的影響 373-7 ANOVA 二階層軟體設計方程式 383-8利用CCD設計最適培養基之RSM不同條件之活性曲線圖 423-9利用CCD設計最適培養基之RSM不同條件之活性曲線圖 423-10利用CCD設計最適培養基之RSM不同條件之活性曲線圖 433-11利用CCD設計最適培養基之RSM不同條件之活性曲線圖 433-12利用CCD設計最適培養基之RSM不同條件之活性曲線圖 443-13利用CCD設計最適培養基之RSM不同條件之活性曲線圖 443-14以不同濃度硫酸銨劃分幾丁質酶活性及幾丁二醣酶活性 483-15不同濃度硫酸銨初步劃分之幾丁質酶水解產物分析 483-16不同濃度硫酸銨初步劃分之幾丁質酶水解產物分析 493-17 35％-70%之硫酸銨劃分蛋白質以sephacryl 200膠體過濾層析 493-18膠體過濾後收集不同fractions以12.5% SDS-PAGE分析 503-19膠體過濾後幾丁質酶之活性染色 503-20以HPLC檢測膠過濾後幾丁質酶水解colloidal chitin產物 513-21 20 mM pH7 phosphate buffer進行DEAE離子交換層析 513-22 20 mM pH8.6 Tris-HCl buffer進行DEAE離子交換層析 523-23經兩次DEAE離子交換層析純化所得幾丁質酶之SDS-PAGE分析 523-24以Sephacryl 100二次膠體過濾膠體過濾分析 533-25取圖3-24膠體過濾分析後收集不同fractions以12.5% SDS-PAGE分析 533-26 Chitinase 1及Chitinase 2其活性之最適反應溫度 543-27 Chitinase 1 (53 kDa)活性之熱穩定度 543-28 Chitinase 2 (39 kDa)活性之熱穩定性 553-29 Chitinase 1及Chitinase 2其活性之最適反應pH 值 553-30 Chitinase 1及Chitinase 2其活性之最適pH穩定性 563-31以HPLC分析純化後chitinase 1水解colloidal chitin產物 583-32以HPLC分析純化後chitinase 2水解colloidal chitin產物 58目錄2. 1 PCR流程表 203.1 two level實驗設計表 373.2 ANOVA 二階層軟體設計表 383.3 RSM軟體5 level中心混成設計 393.4 以RSM軟體 5 levels、4 factors CCD實驗設計 403.5 ANOVA 五階層軟體設計表 413.6 酵素純化表 56application/pdf1225618 bytesapplication/pdfen-US幾丁質chitinthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/180187/1/ntu-97-R95631003-1.pdf