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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 柯淳涵 | |
| dc.contributor.author | Ian-Ting Lu | en |
| dc.contributor.author | 呂彥廷 | zh_TW |
| dc.date.accessioned | 2021-06-07T23:54:43Z | - |
| dc.date.copyright | 2013-09-25 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2013-09-12 | |
| dc.identifier.citation | Beg, Q. K., Kapoor, M., Mahajan L., Hoondal, G. S. (2001). Microbial xylanases and their industrial applications: a review. Applied Microbiology and Biotechnology 56(3 - 4):326-338.
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World J Microbiol Biotechnol 27:751–758 Veenhuis, M., Van Dijken, JP., Harder, W. (1980) The significance of peroxisomes in the metabolism of one-carbon compounds in yeasts. Adv. Microb. Physiol. 24, 1-82. Wagner ,L.W., Matheson, N.H., Heisey, R.F., Schneider, K. (1997) Use of a silicone tubing sensor to control methanol concentration during fed batch fermentation of Pichia pastoris. Biotechnology Techniques 11(11):791-795. Wood, T. M.,Bhat, K.M. (1988) Methods for measuring cellulase activities.Methods in Enzymology 160: 87-112. Wong, K. K. Y., L. U. L. Tan and J. N. J. N. S. Saddler (1998). Multiplicity of β - 1, 4 - Xylanase in Microorganisms: Functions and Applications. Microviological Reviews 52(3): 305 - 17. Zhou, C.Y., Li, D.F., Wu, M., Wang, W.(2008) Optimized expression of an acid xylanase from Aspergillus usamii in Pichia pastoris and its biochemical characterization. World J Microbiol Biotech | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17051 | - |
| dc.description.abstract | 木聚醣為木材及植物生質中的重要主成分，而木聚醣水解酶可水解木聚醣，降低其分子量，並廣泛應用於製漿、畜牧、食品產業中。本研究將之胺基酸T44A取代之變異Paenibacillus木聚醣水解酶基因xylX-H1，轉殖於酵母菌Pichia pastoris上。以Pichia pastoris做為蛋白質表現系統，具有將蛋白質正確摺疊、轉錄後修飾並分泌等特性，以利於商業上大量生產。本研究利用Pichia pastoris表現系統表達基因，在原本的DNA上接上引子，引入外來基因的表現載體pPIC9K，再轉殖入Pichia pastoris SMD1168中。本研究以小量搖瓶培養探討重組Pichia pastoris生產木聚醣水解酶(XylX-H1)，並透過活性分析並找出最佳培養條件。結果顯示加入 1% (w/v)甲醇誘導96 hr後，可產生最佳的酵素活性。比較兩株不同載體的轉形株，以pPIC9K為載體的轉形株活性為9171 U/mg，較pPICZaA為載體的轉型株4461 U/mg高。搖瓶培養分析結果以溶氧量20 ppm、起始誘導菌量數OD600 30為最佳培養條件。以相同條件生產五公升發酵槽生產的木聚糖酶活性高達7693 U/mg。而轉殖後的XylX-H1水解樺木木聚醣產物，以木二醣為最多占水解產物42%，木三醣次之占39%。此外，分泌出來的酵素在置於酸性條件pH3.5下1hr後，保有84%的酵素活性，置於於pH2.5下1 hr，仍然可以保持62%的酵素活性，顯示此酵素具有耐酸的特性。未來可望應用於食品以達到腸胃保健功效，或應用於飼料上提升養分消化率。 | zh_TW |
| dc.description.abstract | Xylan is one of the major components of wood and plant biomass. Xylanases hydrolyze xylanase and are widely applied in pulp & paper, animal feeding and food industries. xylX-H1, a T44A amino acid substitution mutant derived from Paenibacillus xylX, has been cloned in Pichia pastoris. Protein expression systems using Pichia pastoris can correctly fold, conduct post-translation modification and secretion. The above systems could be easily expended to a commercial scale. This study constructed a Pichia pastoris expression system, ligate a primer on the original DNA, clone the ligated DNA into the vector pPIC9K and then transform into Pichia pastoris SMD1168. This study investigated production of recombinant xylanase (XylX-H1) by shake flask fermentations, and optimal production conditions were analyzed. Results showed that the optimal enzyme activities were induced by 1% methanol induction after 96 hours. Transformed Pichia strains cloned with pPIC9K and pPICZaA produced 9171 and 4461 and U xylanase/mg protein. Other optimal parameters found were starting dissolved oxygen at 20 ppm and starting OD600 at 30 during shake flask fermentation runs. 5 L fermentation runs produced expressed XylX-H1 at 7693 U/mg. Hydrolyzing birchwood xylan by expressed XylX-H1 for 24 hours produced 42 % xylobiose and 39 % xylotriose. Expressed XylX-H1 retained 84 and 62 % activities after being subjected to pH 2.5 and 3.5 over 1 hour. Hence, it showed XylX-H1 is acid-tolerant and could be gastronomically applied in human and animals for probiotic and enhancing meat yields purposes. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-07T23:54:43Z (GMT). No. of bitstreams: 1 ntu-101-R98625039-1.pdf: 2303402 bytes, checksum: 9411925b44c2cd4258fec3e44818ee94 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 謝誌 I
中文摘要 II Abstract III Contents IV Table Index 1I Figure Index VII Chapter 1 Introduction 1 Chapter 2 Literature reviews 4 2.1 Characterization and structure of xylan 4 2.2 Xylanase Enzyme System 5 2.3 Paenibacillus sp. BL11 xylanase 6 2.4 Protein expression system 7 2.5 Pichia pastoris protein expression system Introduction 8 2.6 Pichia pastoris methanol metabolism path 9 2.7 The performance of the heterologous protein in Pichia pastoris 11 CHAPTER 3 Materials and methods 14 3.1 Objectives 14 3.2 Materials 14 3.2.1 Yeast medium 14 3.3 Yeast expression plasmids and host selection 16 3.3.1 Host strain 16 3.3.2 Pichia pastoris expression 16 3.4 Primer design and PCR program optimization 17 3.4.1 Cloning of PCR products in pOptima vector and recovery of insert fragment 18 3.4.2 Agarose gel electrophoresis detection of PCR product 19 3.4.3 TA cloning 19 3.5 Plasmid DNA transgenic to Pichia pastoris 21 3.5.1 Plasmid DNA transgenic yeast 21 3.5.2 Plasmid DNA 21 3.5.3 Plasmid DNA reorganization 22 3.5.4 Plasmid linearization 22 3.5.5 Transformation to Pichia pastoris SMD1168 22 3.6 Confirm the molecular weight and enzyme activity was detected 23 3.6.1 SDS polyacrylamide gel electrophoresis 23 3.6.2 Zymogram 24 3.6.3 Enzyme assays 24 3.6.4 Effects of pH on xylanase activity and stability 25 3.6.5 Xylan hydrolysis and product analysis 25 3.7 Pichia pastoris yeast fermentation system 26 3.7.1 Pichia pastoris pre-culture 26 3.7.2 Pichia pastoris induction culture 27 3.7.3 Pichia pastoris yeast fermentation system 27 Chapter 4 Results and discussions 29 4.1 Confirm the results of the transgenic 29 4.1.1 The blue-white screening 30 4.1.2 Plasmid DNA quantitative analysis 31 4.1.3 Recovering DNA 32 4.1.4 Plasmid DNA reorganization result . 33 4.2 Confirm P. pastoris to express xylX H1 34 4.2.1 Plasmid linearization 34 4.2.2 Vector express xylX H1 35 4.2.3 Effects of vectors on expressed xylanase activities 39 4.3 Effects of Pichia pastoris induction culture conditions on expressed xylanase activities 41 4.3.1 Effects of starting OD values on expressed activities 41 4.3.2 Effects of amount induced methanol content on expressed activities 42 4.3.3 Effects of dissolved oxygen on expressed activities 43 4.3.4 Effects of acid shock on xylanase activity and stability 46 4.3.5 Xylan hydrolysis product analysis 46 4.4 Production of fermented 47 Conclusions 49 References 51 | |
| dc.language.iso | en | |
| dc.subject | 發酵 | zh_TW |
| dc.subject | 木聚醣? | zh_TW |
| dc.subject | 重組蛋白 | zh_TW |
| dc.subject | xylanase | en |
| dc.subject | recombinant protein | en |
| dc.subject | fermentation | en |
| dc.subject | Paenibacillus | en |
| dc.subject | Pichia | en |
| dc.title | 酵母菌表達小枯草桿菌衍生木聚醣酶 | zh_TW |
| dc.title | Expression system of a paenibacillus- derived
Xylanase in pichia pastoris. | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 杜鎮,劉佳振,施增廉,鄭建中 | |
| dc.subject.keyword | 木聚醣?,重組蛋白,發酵, | zh_TW |
| dc.subject.keyword | Paenibacillus,xylanase,Pichia,fermentation,recombinant protein, | en |
| dc.relation.page | 54 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2013-09-12 | |
| 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 |
| Appears in Collections: | 森林環境暨資源學系 | |
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| ntu-101-1.pdf Restricted Access | 2.25 MB | Adobe PDF |
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