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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 柯淳涵 | |
| dc.contributor.author | Po-Heng Lin | en |
| dc.contributor.author | 林柏亨 | zh_TW |
| dc.date.accessioned | 2021-06-15T06:47:33Z | - |
| dc.date.available | 2016-07-25 | |
| dc.date.copyright | 2011-07-25 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-05-31 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48156 | - |
| dc.description.abstract | 由Paenibacillus campinasensis BL11 之基因中選殖出的基因片段(CelBL11)具有對對羧甲基纖維素（CMC）有活性的內切型葡聚醣酶於大腸桿菌中表達。CelBL11 由1,465 個核苷酸所組成，而此片段的末端並沒有停止密碼子。CelBL11經轉譯後形成約 55 kDa 的內切葡聚醣酶的蛋白質。其N 端包含一個經由預測的29 個氨基酸長度的訊息肽。催化中心(catalytic domain)是被歸類在family 5（GH5）之中。在纖維素酶的C 端有碳水化合物結合區是被歸類在在family III（CBM3）之中。重組蛋白在纖維素酶的N 端上有His-tag 的片段是用來作NTA-Agarose 親合性管柱曾析純化蛋白質所用。在SDS-PAGE 顯示分別在52 kD 和38 kDa 的位置上有內切型纖維素酶之活性。由核甘酸所推測出的長度大概與38 kDa 相差14kDa 左右。有可能是因為後修飾作用或是蛋白質酶水解所導致的結果。38 kDa纖維素酶對avicel, acid swollen avicel, CMC, 1,3B-glucan 和xylan 可作為分解的基質。經由純化後的38 kDa 纖維素酶其最適酸鹼條件為pH 7 最適溫度為60 oC。Hg2+和 N-bromosuccinimide 對纖維素酶具有劇烈的抑制；而Mn2+對纖維素酶有促進的效果。當環境pH 6 和7 時，在60 oC 的環境下八個小時後活性只減少的百分之二十以下；pH 5 和8 其相對活性分別為31％和15％。纖維素酶酵素動力學常數在基質為羧甲基纖維素時Km 和Vmax 分別為11.25 mg/ml 與1250mol/min/mg。當環境cellobiose 的濃度在5 mg/ml 以下時，基質與cellobiose 為競爭型抑制。 | zh_TW |
| dc.description.abstract | From Paenibacillus campinasensis BL11, the gene CelBL11 encoding an
endoglucanase with an activity towards carboxymethyl cellulose (CMC) was cloned and expressed in E. coli. CelBL11 is composed of 1,465 bp nucleotides without stop coden in C-terminal. Cel-BL11 encodes an endoglucanase of 55 kDa. The N-terminal of the cellulase contains a deduced signal peptide of 29 amino acids in length and a glycosyl hydrolase domain (catalytic domain) in family 5 (GH5); and a carbohydrate binding module in family III (CBM3) at C-terminal of this cellulase. The Cel-BL11 was fused with a His-tag at its N-terminal and the recombinant Cel-BL11 was purified by Ni-NTA affinity chromatography. Zymographic analysis of the recombinant Cel-BL11 exhibited two cellulase activities at 52 kDa and 38 kDa. Apparent ca. 14 kDa difference between the recombinant 38 kDa cellulase and its precursor form minus the signal peptide (ca. 3 kDa) could be due to proteolysis or post-translational modifications. The 38 kDa cellulase hydrolyzed avicel, acid swollen avicel, CMC, 1,3B-glucan and xylan. Optimum temperature and pH for the 38 kDa cellulase activity of the purified cellulase were found to be 60 °C and pH 7.0, respectively. The 38 kDa cellulase activity was strongly inhibited by Hg2+ and N-bromosuccinimide, andstrongly promoted by Mn2+. More than 80% residual activities at 60 oC for 8 h were demonstrated at pH 6 and 7; while 31 and 15% residual activities found for pH 5 and 8. The 38 kDa cellulase has a Km of 11.25 mg/ml and a Vmax of 1250 mol/min/mg with carboxymethyl-cellulose (CMC). Competitive inhibition of cellobiose below 5 mg/ml on CMC hydrolysis by Cel-BL11 was found. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T06:47:33Z (GMT). No. of bitstreams: 1 ntu-100-R97625009-1.pdf: 581600 bytes, checksum: 962796790aae88b9036200095e125c5e (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | Index……………………………………………………………………… III
Table index……………………………………………………………….. V Figure index………………………...………………………..................... V Ⅰ Introduction…………………….………………………………………... 1 Ⅱ Literature review…………….…………………………………………. 4 1. Characterization and structure of cellulose…………………………….. 4 2. Cellulolytic enzymes…………………………………………………... 5 3. The mechanisms of cellulolytic enzymes……………………………… 7 4. Classification of cellulases…………………………………………….. 9 4.1 Classification of catalytic domains………………………………….. 9 4.2 Classification of carbohydrate-binding modules…………………… 11 5. Cellulase producing microorganisms………………………………….. 12 5.1 Bacterium………...……………………………………...................... 12 5.2 Fungi……………………………………..………………………….. 17 6. Applicationsof cellulase………………………………………………... 18 Ⅲ Objective…………………………………………………………………. 21 Ⅳ Materials and methods………………………………………………….. 22 1. Materials............…………………………………………..................... 22 2. Bacterial strains and plasmids…………………………………………. 22 3. DNA isolation, genomic library construction and screening…............... 22 4. DNA sequencing and sequence analysis………………………………. 23 5. Construction of BL11 cellulase expression system……………………. 24 6. Protein analysis………………………………………………………… 24 7. Expression and purification of the recombinant cellulase……………... 24 8. SDS-PAGE and zymogram……………………………………………. 25 9. Effects of pH and temperature on CMCase activity and stability……... 26 10. Effect of additives on cellulase activity………………………………. 26 11. Substrate specificity…………………………………………………... 27 12. Kinetic parameters and hydrolysis inhibition by cellobiose………….. 27 Ⅴ Results and Discussion…………………….......... …………………….. 29 1. Cloning of the cellulase gene Cel BL11……………………………….. 29 2. DNA sequence analysis of the cellulase (Cel BL11) gene…………….. 30 3. Overexpression of cloned cellulase……………………………………. 32 3.1 Primer set design of cellulase gene and optimal PCR program…….. 32 3.2 TA cloning, restriction enzyme digestion, and fragment recovery…. 33 3.3 Restriction enzyme digestion and fragment recovery of pET15b…... 34 3.4 Ligation, electroporation, and transformation………………………. 35 4. Purification of cloned BL11 cellulase…………………………………. 37 4.1 Condition test of induction………………………………………….. 37 4.2 Pretest of purification condition…………………………………….. 38 4.3 Purification of Cel-BL11 endoglucanase…………………………… 39 5. Production and characterization of the recombinant cellulase………… 41 5.1 DNSA assay of crude Cel BL11 cellulase activity………………….. 41 5.2 DNSA assay of purify induced Cel BL11 activity…………………... 42 6. Effect of pH and temperature on CMCase stability……………………. 44 7. Effect of various additives on cellulase activity……………………….. 47 8. Substrate specificity……………………………………………………. 48 9. Kinetic parameters and hydrolysis inhibition by cellobiose…………… 50 VI Conclusion……………………………………………………………….. 52 VII References………………………………………………….…………….. 54 | |
| dc.language.iso | en | |
| dc.title | Paenibacillus campinasensis BL11 纖維素酶之大量表現、純化及基本性質 | zh_TW |
| dc.title | Overexpression, purification and characteristics of the cellulase gene from Paenibacillus campinasensis BL11 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張上鎮,常玉強,劉佳振 | |
| dc.subject.keyword | 羧甲基纖維素（CMC）,纖維素酶,clone,Paenibacillus campinasensis BL11, | zh_TW |
| dc.subject.keyword | carboxylmethylcellulose (CMC),cellulase,clone,Paenibacillus campinasensis BL11., | en |
| dc.relation.page | 63 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-06-07 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 森林環境暨資源學研究所 | zh_TW |
| 顯示於系所單位： | 森林環境暨資源學系 | |
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