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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳俊任 | |
| dc.contributor.author | Yuan-Ju Lin | en |
| dc.contributor.author | 林芫如 | zh_TW |
| dc.date.accessioned | 2021-06-13T15:27:22Z | - |
| dc.date.available | 2013-07-23 | |
| dc.date.copyright | 2008-07-23 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-07-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37415 | - |
| dc.description.abstract | 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。 | zh_TW |
| dc.description.abstract | Abstract
N-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. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T15:27:22Z (GMT). No. of bitstreams: 1 ntu-97-R95631003-1.pdf: 1225618 bytes, checksum: 5dec15edef9da6be9de3d6f28ca390d8 (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | 目錄
目錄 i 圖目錄 iii 表目錄 v 誌謝 vi 中文摘要 1 Abstract 2 前言 4 第一章、文獻探討 5 (1)幾丁質 5 1.1幾丁質之結構 5 1.2幾丁質之製備 5 1.3幾丁質之應用 6 (2)幾丁聚醣 6 2.1幾丁聚醣之結構 6 2.2幾丁聚醣之應用 7 2.3幾丁聚醣之製備 7 (3) N-乙醯幾丁寡醣 (N-acetyl chitooligosaccharides, (GlcNAc)n)與幾丁寡醣 (chito-oligosaccharides) 8 3.1 N-乙醯幾丁寡醣與幾丁寡醣之結構 8 3.2 N-乙醯幾丁寡醣與幾丁寡醣之製備 8 3.3 N-乙醯幾丁寡醣與幾丁寡醣之應用 9 (4)不同的幾丁質水解酵素 9 4.1幾丁質酶 (chitinase；EC 3.2.1.14) 9 (5)反應曲面法 (Response surface methodology,RSM) 11 5.1反應區面法實際應用之優點 12 5.2常用實驗設計類型及實施步驟 13 (6)微生物菌種鑑定 15 6.1 核醣體核醣核酸 (ribosomal RNA, rRNA) 15 第二章、材料與方法 16 (1)實驗材料 16 2.1菌株 16 2.2化學材料 16 2.4儀器 17 (2)實驗方法 17 2.5菌種保存及菌株培養 17 2.6酵素活性測定方法 21 2.7酵素最適培養條件探討 22 2.8幾丁質酶之純化 23 2.9幾丁質酶酵素之特性分析 26 2.10產物分析 27 第三章、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 第四章、結論 59 第五章、參考文獻 60 圖目錄 圖1- 1反應曲面法Response surface methodology 12 圖2- 1 HPLC樣品製備流程圖 27 圖2- 2實驗流程圖 29 圖3-1初步篩選18株菌株幾丁質酶活性 32 圖3-2以HPLC檢測No.17粗酵素水解colloidal chitin之產物 32 圖3-3 Serratia marcescens NTU-17其生長曲線及酵素曲線。 33 圖3-4以HPLC分析甲醇和丙酮分離不同聚合度之N-乙醯幾丁寡醣 33 圖3-5 16S rDNA phylogenetic tree 34 圖3-6 1%不同氮源對幾丁質酶的影響 37 圖3-7 ANOVA 二階層軟體設計方程式 38 圖3-8利用CCD設計最適培養基之RSM不同條件之活性曲線圖 42 圖3-9利用CCD設計最適培養基之RSM不同條件之活性曲線圖 42 圖3-10利用CCD設計最適培養基之RSM不同條件之活性曲線圖 43 圖3-11利用CCD設計最適培養基之RSM不同條件之活性曲線圖 43 圖3-12利用CCD設計最適培養基之RSM不同條件之活性曲線圖 44 圖3-13利用CCD設計最適培養基之RSM不同條件之活性曲線圖 44 圖3-14以不同濃度硫酸銨劃分幾丁質酶活性及幾丁二醣酶活性 48 圖3-15不同濃度硫酸銨初步劃分之幾丁質酶水解產物分析 48 圖3-16不同濃度硫酸銨初步劃分之幾丁質酶水解產物分析 49 圖3-17 35％-70%之硫酸銨劃分蛋白質以sephacryl 200膠體過濾層析 49 圖3-18膠體過濾後收集不同fractions以12.5% SDS-PAGE分析 50 圖3-19膠體過濾後幾丁質酶之活性染色 50 圖3-20以HPLC檢測膠過濾後幾丁質酶水解colloidal chitin產物 51 圖3-21 20 mM pH7 phosphate buffer進行DEAE離子交換層析 51 圖3-22 20 mM pH8.6 Tris-HCl buffer進行DEAE離子交換層析 52 圖3-23經兩次DEAE離子交換層析純化所得幾丁質酶之SDS-PAGE分析 52 圖3-24以Sephacryl 100二次膠體過濾膠體過濾分析 53 圖3-25取圖3-24膠體過濾分析後收集不同fractions以12.5% SDS-PAGE分析 53 圖3-26 Chitinase 1及Chitinase 2其活性之最適反應溫度 54 圖3-27 Chitinase 1 (53 kDa)活性之熱穩定度 54 圖3-28 Chitinase 2 (39 kDa)活性之熱穩定性 55 圖3-29 Chitinase 1及Chitinase 2其活性之最適反應pH 值 55 圖3-30 Chitinase 1及Chitinase 2其活性之最適pH穩定性 56 圖3-31以HPLC分析純化後chitinase 1水解colloidal chitin產物 58 圖3-32以HPLC分析純化後chitinase 2水解colloidal chitin產物 58 表目錄 表2. 1 PCR流程表 20 表3.1 two level實驗設計表 37 表3.2 ANOVA 二階層軟體設計表 38 表3.3 RSM軟體5 level中心混成設計 39 表3.4 以RSM軟體 5 levels、4 factors CCD實驗設計 40 表3.5 ANOVA 五階層軟體設計表 41 表3.6 酵素純化表 56 | |
| dc.language.iso | zh-TW | |
| dc.subject | 幾丁質 | zh_TW |
| dc.subject | chitin | en |
| dc.title | Serratia marcescens NTU-17所產生幾丁質分解酵素
之研究 | zh_TW |
| dc.title | Study on Chitinolytic Enzymes from Serratia marcescens NTU-17 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 盧福明 | |
| dc.contributor.oralexamcommittee | 李允中,楊景雍 | |
| dc.subject.keyword | 幾丁質, | zh_TW |
| dc.subject.keyword | chitin, | en |
| dc.relation.page | 68 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-07-17 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 生物產業機電工程學研究所 | zh_TW |
| 顯示於系所單位： | 生物機電工程學系 | |
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