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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 柯淳涵 | |
dc.contributor.author | Guan-Yu Ke | en |
dc.contributor.author | 柯冠宇 | zh_TW |
dc.date.accessioned | 2021-06-17T00:53:55Z | - |
dc.date.available | 2017-01-17 | |
dc.date.copyright | 2012-01-17 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-10-05 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66728 | - |
dc.description.abstract | 在先前的研究中,從製漿漂前黑液(58oC,pH 9.83)中分離出可耐高溫高鹼的Paenibacillus sp. BL11。從該菌株分泌出的endo-1,4-xylanase 稱為xylanase X。兩株變異株(分別是胺基酸取代株xylanase H2(T44A)及後段缺失株xylanase L2(胺基酸序列270之後鹼基缺失)與xylanase X比較。Xylanase X的最佳反應條件為60oC、pH 7。在其最佳反應條件下,xylanase X具有2392 U/mg的酵素活性。Xylanase H2的最佳反應條件為55oC、pH 7。在其最佳反應條件下,xylanase H2具有9725 U/mg的酵素活性。Xylanase L2的最佳反應條件為50oC、pH 8。在其最佳反應條件下,xylanase L2具有4710 U/mg的酵素活性。將這些xylanase 做比較,在最佳反應條件下,xylanase H2 有效地增加其酵素活性。利用不同濃度的聚乙二醇(polyethylene glycol (PEG))(最終濃度0 - 1.6%)保護xylanase X,在60oC、pH7條件下反應顯示最佳反應濃度為0.8%。Xylanase X, H2 以及L2 在50oC、PEG濃度為0.5%條件下反應160分鐘,酵素活性可以持續增加。而在55以及60oC反應條件下,xylanase X, H2 以及L2的酵素活性可以持續增加至反應80分鐘,但在反應160分鐘後,xylanase H2以及L2的酵素活性則降低。 | zh_TW |
dc.description.abstract | In previous research, a thermo-alkaline Paenibacillus sp. BL11 was isolated from black liquor (58oC, pH 9.83). The endo-1,4-xylanase secreted from this bacterium was designated xylanase X. Two mutants xylanase H2 (amino acid substitution mutant, T44A) and L2 (sequence deletion mutant after aa270) were compared with xylanase X. Optimum temperature and pH for purified xylanase X activity were 60oC and pH7. The xylanase X activity was 2392 U/mg under optimal condition. Those for purified xylanase H2 activity were 55oC and pH7. The xylanase H2 activity was 9725 U/mg under optimal condition and the activity was 8495 U/mg under 60oC and pH7. Those for purified xylanase L2 activity were 50oC and pH 8. The activity of xylanase L2 could arrive about 4710 U/mg at this condition. Comparing these xylanases, the activity of xylanase H2 was the only enzyme greatly improved by changing its assay condition. Using polyethylene glycol (PEG) to protected xylanase X in different concentration (0 - 1.6% of final volume) at 60oC and pH7 showed the optimal concentration of PEG was 0.8%. Protection of PEG 6000 at a final concentration of 0.5%, the activity of xylanase X, H2 and L2 could keep increase in 160 min at 50oC. At 55 and 60oC, the activity of xylanase X, H2 and L2 could keep increase in 80 min. But however the activity of xylanase H2 and L2 decreased after 80 min under this condition. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T00:53:55Z (GMT). No. of bitstreams: 1 ntu-100-R96625043-1.pdf: 630506 bytes, checksum: 22d742e4f256b42c211c477984ca69d7 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 摘要…………………………………………………………………………..
ii Abstract........................................................................................................... iii Index………………………………………………………………………… iv Table index………………………………………………………………….. vi Figure index………………………...………………………......................... vii Ⅰ Introduction…………………….………………………………………....... 1 Ⅱ Literature reviews………….……………………………………………..... 3 1 Paenibacillus………….………………………………………………........ 3 2 Characterization and structure of xylan.….………………………………... 3 3 Xylanase Enzyme System…...………………….………………………..... 4 4 Prebleaching with xylanase………………………………………………... 9 5 Paenibacillus sp. BL11 xylanase………...……………………………....... 10 6 Mutants of endo-1,4-β-xylanase…………………………………................ 11 Ⅲ Objective…………………………………………………………………….. 12 Ⅳ Materials and methods……………………………………………………... 13 1. Overexpression of cloned xylanase…..…………………......................... 13 1.1 Primer design and PCR program optimization…………..……………... 13 1.2 Cloning of PCR products in pXcmI vector and recovery of insert fragment……………………………………………………………….. 14 1.3 Preparation of overexpression vector…………………………………... 15 1.4 Preparation of competent cells…………………………………………. 16 1.5 Ligation, electroporation, transformation and activity assay…………... 17 2. Purification of cloned xylanase…………………………………………. 18 2.1 Condition test of induction....................................................................... 18 2.1.1 Induction at 28oC and 0.1 mM IPTG……………………………….. 18 2.2 Purification and activity assay………………………………………….. 19 3. Xylan hydrolase assay…………………………………………………… 21 3.1 Stain assay of polysaccharide hydrolases………………………………. 21 3.1.1 Staining methods -- Congo red stain………………………………... 21 3.2 SDS - PAGE and Zymography……………...……………………......... 22 3.2.1 SDS-PAGE………………………………………………………….. 22 3.2.2 Zymogram…………………………………………………………... 23 3.3 DNSA (dinitrosalicylic acid) assay...………………………………........ 24 Ⅴ Results…………………………………………………….............................. 28 1. Overexpression of cloned xylanase…………………………………....... 28 v 1.1 Primer set design of xylanase gene and optimal PCR program.……….. 28 1.2 TA cloning, restriction enzyme digestion, and fragment recovery.......... 30 1.3 Restriction enzyme digestion and fragment recovery of pET15b…........ 31 1.4 Ligation, electroporation, and transformation…………………….......... 31 2. Purification of cloned xylanases………………………………………… 32 2.1 Purification of cloned xylanase X, H2 and L2…………………………. 32 2.2 DNSA assay of xylanase H2 and L2 activity…………………………... 35 3. Stablization of xylanases………………………………………………… 38 3.1 Xylanase X with different concentration of polyethylene glycol (PEG). 38 3.2 Stabilization of xylanase X, H2 and L2…………………………..……. 39 VI Discussion and Conclusion……………………………………………… 42 Ⅶ References………………………………………………….…………….. 49 Appendix…………………………………………………………………. 56 | |
dc.language.iso | en | |
dc.title | Paenibacillus campinasensis BL11 木聚醣酶及其變異株之大量表現、純化與熱穩定性探討 | zh_TW |
dc.title | Overexpression, purification and themostability of Paenibacillus campinasensis BL11 xylanase and its mutants | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 杜鎮,王亞男,蕭英倫,劉佳振 | |
dc.subject.keyword | Paenibacillus sp. BL11,xylanase X,xylanase H2,xylanase L2,聚乙二醇, | zh_TW |
dc.subject.keyword | Paenibacillus sp. BL11,xylanase X,xylanase H2,xylanase L2,PEG, | en |
dc.relation.page | 57 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-10-05 | |
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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