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研究生:唐熙卉
研究生(外文):Sei-Hui Tang
論文名稱:Paenibacillussp.isolateBL11之果膠酶分生選殖、大量表現與純化
論文名稱(外文):Molecular cloning, overexpression and purification ofPaenibacillus sp. isolate BL11 pectinase
指導教授:柯淳涵柯淳涵引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:森林環境暨資源學研究所
學門:農業科學學門
學類:林業學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:英文
論文頁數:91
中文關鍵詞:果膠酸鹽酵素親緣樹Paenibacillus sp. BL11
外文關鍵詞:PelXphylogenetic treePaenibacillus sp. BL11
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Paenibacillus sp. BL11 可分泌果膠酶酵素,具有分解果膠的能力,其最適條件在60oC, pH 10.0 時果膠酶活性為10.06 IU/mg 。選殖至大腸桿菌後基因序列比對的結果,可得到ㄧ果膠酶基因。

由Paenibacillus sp. BL11 所選殖出的果膠酶基因是由963 個核苷酸所構成,經由轉錄及轉譯可得到蛋白分子量為38 kDa、其pI 值為pH 9.46 的果膠酶。蛋白質序列分析結果顯示出BL11 的果膠酶其N端胺基酸序列具有一長度為34 個胺基酸的信息胜肽。從活性染的結果中可發現選殖至大腸桿菌中之pelX 為無法有效分泌至胞外的果膠酸鹽酵素 (PelX; pectate transeliminase; EC4.2.2.2)。經由蛋白質序列比對分析,從親緣樹中比較BL11 的果膠酶蛋白質與其他28 株不同菌種的果膠酶蛋白質之關係,與自Bacillus sp. KSM-P15 分離出的Pel-15相似度為99%。

為了提高純化效率,將選殖出來的果膠酶在其C 端胺基酸序列
融合6x His-tag。在37oC 隔夜培養後,將果膠酶利用Ni-NTA agarose的親和力加以純化。純化後的果膠酶最適條件為50oC, pH 10.0 時,活性1,623 IU/mg 。由於其具有良好的活性,因此PelX 在工業上極有發展的潛力。
Paenibacillus, sp. BL11 secreted pectate lyase degrading pectin. The optimum condition was 60oC, pH 10.0, the BL11 crude pectate lyase activity was 10.06 IU/mg. After sequencing and blasting, a pectate lyase gene was received.

The cloned BL11 pectate lyase gene is composed of 963 nucleotides and encodes a protein of 38 kDa. The N-terminal of the enzyme contains a deduced signal peptide of 34 amino acids in length. Zymogram analyses of the BL11 pectin-degrading system revealed that pelX was not secreted
into the extracellular of the bacterium. The cloned BL11 in E. coli was a pectate lyase (pectate transeliminase; EC4.2.2.2), designated pelX. The phylogenetic relationship was aligning with the other 28 species bacterias in pectinase protein, the most similar protein found was Pel-15 from Bacillus sp. KSM-P15E. The degree of similarity was 99%.

In this study, the cloned BL11 pectate lyase has fused on a His-tag for case of purification. The isoelectric point (pI) of PelX was pH 9.46. The BL11 pectate lyase was able to express without additional induction by IPTG under incubation at 37oC overnight in a host of E. coli. The
pectate lyase was purified in Ni-NTA agarose by its affinity. The optimum temperature of pelX was 50oC, pH 10.0, the pectate lyase was 1,623 IU/mg. Because of its good pectate lyase activity, the PelX has potential
for further industrial application.
致謝.................................................... II
Index.................................................. III
Table index............................................ VII
Figure index.......................................... VIII
摘要.................................................... X
Abstract................................................ XI
Ⅰ Introduction...................................... 1
1.1 Pectic substances and pectin...................... 3
1.1.1 Pectic substances................................. 3
1.1.2 Structure of pectin............................... 4
1.2 Pectinases........................................ 5
1.2.1 Machnism of pectinase............................. 5
1.2.2 History of pectinase research..................... 6
1.2.3 Category of pectinase............................. 8
1.3 Pectin degrading enzymes.......................... 12
1.4 Biotechnological applications of pectinases....... 15
1.4.1 Retting of plant fibbers.......................... 15
1.4.2 Pretreatment of pectic wastewaters................ 16
1.4.3 Coffee and tea fermentations...................... 17
1.4.4 Paper and pulp industry........................... 17
Ⅱ Objective......................................... 19
III. Materials and methods............................. 20
3.1. Polygalacturonic acid assays...................... 20
3.2. Bacterial strain identification................... 20
3.2.1 Preparation of bacterial chromosomal DNA.......... 20
3.2.2 DNA electrophoresis............................... 22
3.2.3 TA-Cloning........................................ 22
3.2.4 Plasmid preparation............................... 25
3.3. Polysaccharide hydrolase assay.................... 27
3.3.1 Stain assay of polysaccharide hydrolases.......... 27
3.3.2 SDS-PAGE and zymography........................... 28
3.4. Molecular cloning of xylanase gene................ 30
3.4.1 Restriction enzyme digestion of DNA and recovery of DNA..................................................... 30
3.4.2 Restriction digestion, cipping and recovery of vector DNA.............................................. 32
3.4.3 DNA ligation...................................... 33
3.4.4 Electroporation and transformation................ 34
3.4.5 Replica and activity assay........................ 34
3.4.6 Mapping, Sequencing, and Blasting................. 35
3.5. Overexpression of cloned pectinase................ 35
3.5.1 Primer design and PCR program optimization........ 35
3.5.2 Cloning of PCR product in pOptima vector and recovery of insertfragment...............................36
3.5.3 Preparation of overexpression vector...............37
3.5.4 Ligation, electroporation, transformation and activity assay.......................................... 37
3.6. Purification of cloned pectinase.................. 38
3.6.1 Condition test of induction....................... 38
3.6.2 Pretest of purification condition................. 39
3.6.3 Purification...................................... 41
3.7 Pectinase assay................................... 42
IV. Results and discussion.............................. 45
4.1. Screening and isolation of bacterial strains...... 45
4.1.1 Polygalacturonase activity assay of BL11.......... 45
4.1.2 SDS-PAGE and zymogram of BL11 pectinase........... 46
4.2. Molecular cloning of BL11 pectinase gene.......... 47
4.2.1 Restriction enzyme map and sequences of BL11 16S rDNA.................................................... 47
4.2.2 Preparation of vector............................. 49
4.2.3 DNA Ligation, transformation and clone screening.. 50
4.2.4 Clone mapping, sequencing, and blasting........... 51
4.2.5 Character analysis of pelX-ORF1................... 56
4.3. Polysaccharide hydrolases assay................... 58
4.3.1 Primer set design of pectinase gene and optimal PCR program................................................. 58
4.3.2 TA cloning, restriction enzyme digestion, and fragment recovery....................................... 59
4.3.3 Restriction enzyme digestion and fragment recovery of
pET25b.................................................. 61
4.3.4 Ligation, electroporation, and transformation..... 62
4.3.5 Mutated pelX...................................... 64
4.3.6 The different elements of constructed and mutated pelX.................................................... 66
4.3.7 Activity assay of constructed and mutated PelX.... 68
4.4. Purification of PelX.............................. 70
4.4.1 Induced condition test of PelX.................... 71
4.4.2 The condition of imidazole elution test........... 72
4.4.3 The optimum condition for produce pectate lyase activity of BL11 crude enzyme and PelX.................. 73
V Conclusion............................................ 80
VI. References.......................................... 82
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