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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李佳音(Chia-Yin Lee) | |
dc.contributor.author | Yi-Fang Tsai | en |
dc.contributor.author | 蔡宜芳 | zh_TW |
dc.date.accessioned | 2021-06-13T03:14:10Z | - |
dc.date.available | 2016-12-31 | |
dc.date.copyright | 2006-08-09 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-08-04 | |
dc.identifier.citation | 林佩勳 ( 2001 )。Variovorax paradoxus Iso1 之N-醯基-D-胺基酸醯基水解酶基因選殖、蛋白質表現、純化、及其特性分析。國立台灣大學農業化學研究所碩士論文。
羅凱尹 ( 2003 )。 Variovorax paradoxus Iso1 之N-醯基-D-胺基酸醯基水解酶操縱子及定額感應因子代謝途徑分析。國立台灣大學農業化學研究所碩士論文。 彭怡禎 ( 2003 )。 Variovorax paradoxus Iso1 之N-醯基-D-胺基酸醯基水解酶之甲硫胺酸殘基功能研究。國立台灣大學農業化學研究所碩士論文。 Allison, A. J. E. Turner and R. Wait. 1995. Degradation of homovanillate by a strain of Variovorax paradoxus via ring hydroxylation. FEMS Microbiol. Lett. 134:213-219 Alonso, J., J. L. Barredo, B. Diez, E. Mellado, F. Salto, J. Garcia and E. Cortes. 1998. D-amino acid oxidase gene from Rhodotorula gracilis ATCC26217. Microbiol. 144: 1095-1101. Anita S., D. Iskandar and N. Bernd. 2005. Single-site oxidation, cysteine 108 to cysteine sulfinic acid, in D-amino acid xidase from Trigonopsis variabilis and its structural and functional consequences. Appl Environ Microbiol. 71:8061-8068 Carreira, S., D. Brun-Achirou, P. Brachet and A. Puigeserver. 1996. Hepatic and renal D-amino acid oxidase activities in the growing rat after ten days of protein undernutrition and refeeding. Reprod. Nutr. Dev. 36: 73-82. Croux C., J.C. Perez., J.L. Fuentes and F Maldonado. 1995. Process for the enzymatic preparation of 7-aminocephalosparanic acid. Biotechnolol. Adv. 13: 270-270 (1). Curti, B., S. Ronchi ans M.S. Pilone., 1992. D- and L-amino acid oxidase. Chemistry and Biochemistry of flavoenzymes. CRC Press, London 3: 69-94. D’Aniello, A., A. Vetere and L. Petrucelli. 1993. Furthur study on the specificity of D-amino acid oxidase and of D-aspartate oxidase and time course for complete oxidation of D-amino acids. Comp. Biochem. Physiol. 105B: 731-734. Don, R. H. and J. M. Pemberton. 1981. Properties of six pesticide degradation plasmids isolated from Alcaligenes paradoxus and Alcaligenes eutrophus. J. Bacteriol. 145:681-686 D’souza, and G. B. Nadkarni. 1987. Coimmobilization of D-amino acid oxidase and catalase by Trigonopsis variabilis in radiation polymerized polyacryamide. J. Biosci. 11: 137-144. Faotto, L., L. Pollegioni, F. Ceciliani, S. Ronchi, and Pilone, M.S. 1995. The primary structure of D-amino acid oxidase from Rhodotorula gracilis. Biotechnol. Lett. 17: 193–198. Fisher, P. R., J. Appleton and J. M. Pemberton. 1978. Isolation and characterization of the pesticide-degrading plasmid pJP1 from Alcaligenes paradoxus. J. Bacteriol. 135:798-804 Friedman, M. 1999. Chemistry, nutrition and microbiology of D-amino acids. J. Food Chem. 47: 3457-3479 Gabler, M., M. Hene and L. Fisher. 2000. Detection and substrate selectivity of new microbial D-amino acid oxidases. Enz. Microb. Technol. 27: 605-611 Gonzalez, F. J., J. Montes, F. Martin, L. M. Carman, E. Ferinan, J. Catalan, M. A. Galan and A. Dominguez. 1977. Molecular cloning of TvDAO1, a gene encoding a D-amino acid oxidase from Trigonopsis variabilis and its expression in Saccharomyces cerevisiae and Kiuyveromyces lactis. Yeast. 13: 1399-1408. Horiike, K.. T. Ishida, H. Tanaka and R. Arai. 2001. Distribution of D-amino acid oxidase and D-serine in vertebrate brains. J. Mol. Catalysis B: Enzymatic. 12: 37-41. Hsu, W. H., H. M. Fu, L. L. Lin, H. R. Chen and W. C, Wang. 2000. Expression of Trigonopsis variabilis D-amino acid oxidase gene in E. coli and characterization of its inactive mutants. Enzyme Microb. Technol. 27: 482-491. Hsu, W. H., W. C. Wang, W. C. Lin and L. L. Lin. 1998. A conserved aspartate is essential for FAD binding and catalysis in the D-amino acid oxidase from Trigonopsis variabilis. FEBS Lett. 436: 119-122. Hwang, T. S., H. M. Fu, L. L. Lin, and W. H. Hsu. .2000. High-level expression of Trigonopsis variabilis D-amino acid oxidase in Escherichia coli using lactose as inducer. Biotechnol. Lett. 22: 655-658. Irene, M. M., J. Navarro-Fernandez, D. Lozada-Ramirez, F. Garcia-Carmona and A. Sanchez-Ferrer. 2006. Maximization of production of His-tagged glycine oxidase and its M261 mutant proteins. Biotechnol. Prog. 22: 647-652. Isogai, T., H. Ono, Y. Ishitani, H. Kojo, Y. Ueda and M. Kohsaka. 1990. Structure and expression of cDNA for D-amino acid oxidase active against cephalosporin C from Fusarium solani. J. Biochem. 108: 1063-1069. Job, V., G. Molla, M. S. and L. Pollegioni. 2002a. Overexpression of a recombinant wild-type and His-tagged Bacillus subtilis glycine oxidase in Escherichia coli. Eur. J. Biochem. 269: 1456-1463. Job, V., G. Molla, M. S. and L. Pollegioni. 2002b. Glycine oxidase from Bacillus subtilis. J. Biol. Chem. 277: 6987-6993. Ka, J. O. and J. M. Tiedje. 1994. Integration and excision of a 2,4-dichlorophenoxyacetic acid-degradative plasmid in Alcaligenes paradoxus and evidence of its natural intergeneric transfer. J Bacteriol 176:5284-9 Kane, J. F. 1995. Effect of rare codon clusters on high level expression of heterologous proteins in Escherichia coli. Curr. Opin. Biotechnol. 6: 494-500. Konno, R. and Y. Yasumura. 1984. Brain and kidney D-amino acid oxidases are coded by a single gene in the mouse. J. Neurochem. 42: 584-586. Kubicek-Pranz M. and M. Rohr. 1985. D-amino acid oxidase from the yeast Trigonopsis variabilis. J. Appl. Biochem. 7: 104-113. Kubst, F., N. Ogasawara, I. Moszer, A. M. Albertini, G. Alloni, V. Azevedo and M. G. Bertero. 1997. The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390: 259-256. La Rue T. A. and J. F. T. Spencer. 1967. The utilization of D-amino acids by yeasts. Can. J. Microbiol. 13: 777-788. Mirella, P. S., F. Sonia and P. Loredano. 2001. Engeneering expression and purification of a His-tagged chimeric D-amino acid oxidase from Rhodotorula gracilis. Enzyme Microb. Technol. 29:407-412. Moffatt, B. A. and Studier, F. W. 1987. Cell 49: 221. Mohammed G.S., O. Shigeru, A. Hiroki. 2005. Catalytic and structural Characteristics of carp hepatopancreas D-amino acid oxidase expressed in Escherichia coli. Comparative Biochemistry and Physiology. 140: 417-425. Molla, G., C. Vegezzi, M. S. Pilone and L. Pollegioni. 1998. Overexpression in Escherichia coli of a recombinant chimeric Rhodotorula gracilis D-amino acid oxidase. Prot. Exp. Purif. 14: 289-294. Mörtl, M., ,K. Diederichs, W. Welte, G. Molla, L. Motteran, G. Andriolo, M. S. Pilone and L. Pollegioni. 2004. Structure-function correlation in Glycine oxidase from Bacillus subtilis. J. Biol. Chem. 279: 29718-29727. Mothet, J. P., A. T. Parent, H. Wolosker, R.O. Brady, Jr., D.J. Linden, C.D. Ferris, M.A. Rogawski and S.H. Snyder. 2000. D-serine is an endogeneous ligand for the glycine of the N-methyl-D-aspartate receptor. Proc. Natl. Acad. Sci. USA. 97:4926-4931. Nakajima, N., D. Conrad, H. Sumi, K. Suzuki, N. Esaki, C. Wandrey and K. Soda. 1990. Continuous conversion to optically pure L-methionine from D-enantiomer contaminated preparations by an immobilized enzyme membrane reactor. J. Ferment. Technol. 70: 322-325. Neilson, J. W., K. L. Josephson, I. L. Pepper, R. B. Arnold , G. D. Di Giovanni and N. A. Sinclair. 1994. Frequency of horizontal gene transfer of a large catabolic plasmid (pJP4) in soil. Appl. Environ. Microbiol. 60:4053- 4058 Nishiya, Y. and T. Imanaka. 1998. Purification and characterization of a novel glycine oxidase from Bacillus subtilis. FEBS Lett. 438: 238-266. Pilone, M.S. 2000. D-amino acid oxidase: new findings. Cell and Molecular Life Sciences. 57: 1732-1747. Pilone, M.S. and L. Pollegioni. 2002. D-amino acid oxidase as an industrial biocatalyst. Biocatal. Biotransformation. 20: 145-159. Pilone, M.S., M.. Vanoni and B. Curti. 1984. D-amino acid oxidase activity in the yeast Rhodotorula gracilis. FEMS Microbiol. Lett. 15: 27-31. Piubelli, L., L. Caldinelli, G. Molla, M.S. Pilone and L. Pollegioni. 2002. Conversion of dimeric D-amino acid oxidase from Rhodotorula gracilis to monomeric from rational mutagenesis approach. FEBS Lett. 526: 43-48. Pollegioni L., A. Falbo and M.S. Pilone. 1992. Specificity and kinetics of Rhodotorula gracilis D-amino acid oxidase. Biochim Biophys Acta. 1120(1):11-6. Pollegioni L., G. Molla, M.S. Pilone. 1997. Cloning, Sequencing and expression in E. coli of a D-amino acid oxidase Cdna from Rhodotorula gracilis active on cephalosporin C. 58: 115-123. Rajendra U., Nagajyothi and S. G. Bhat. 2000. Stabilization of D-amino acid oxidase and catalase in permeabilized Rhodotorula gracilis cells and its application for the preparation of -ketoacids. Biotechnology and Bioengineering. 68: 430-436 Rooney-Vaega, J. N., R. T. Anderson, J. L. Fraga, D. Ringelberg and D. R. Lovley. 1999. Microbial communities associated with anaerobic benzene degradation in a petroleum-contaminated aquifer. Appl. Environ. Microbiol. 65:3056-3063 Sarower, M. G., S. Okada and H. Abe. 2005. Catalytic and structural characteristics of carp hepatopancreas D-amino acid oxidase expressed in Escherichia coli. Comp. Biochem. Physiol. Part B 140: 417-425. Schell, M. J., M. E. Molliver and S. H. Snyder. 1995. D-serine, an endogeneous synaptic modulator: Locolization to astrocytes and glutamate-stimulated release. Proc. Natl. Acad. Sci. USA. 92: 3948-3952. Sentheshanmugganathan, S. and W. J. Nicekerson. 1962. Transaminase and D-amino acid oxidase of Trigonopsis variabilis. J. Gen. Microbiol. 27: 465-471. Serizawa, N., K. Nakagawa, T. Haneishi, S. Kamimura and A. Naito. 1980. Enzymatic conversion of cephalosporin C by D-amino acid oxidase from Trigonopsis variabilis. J. Antibiot. 33: 585-590. Settembre, E. C., P. C. Dorrestein, J. H. Park, A. M. Augustine, T. P. Begley and S. E. Ealick. 2003. Structural and mechanistic studies on ThiO, a glycine oxidase essential for thiamine biosynthesis in Bacillus subtilis. Biochemistry 42: 2971-2981. Smejkal, C. W., T. Vallaeys, F. A. Seymour, S. K. Burton and H. M. Lappin-Scott. 2001. Characterization of (R/S)-mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid]-degrading Alcaligenes sp. CS1 and Ralstonia sp. CS2 isolated from agricultural soils. Environ. Microbiol. 4: 288-93 Studier, F. W. 1991. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J. Mol. Biol. 219: 37-44. Sudhakaran, V. K., B. S. Deshpande, S. S. Ambedkar and J. G. Shewale. 1992. Molecular aspects of penicillin and cephalosporin acylase. Process Biochem. 27: 131-143. Szwajcer E. and K. Mosbach. 1985. Isolation and partial characterization of a D-amino acid oxidase active against cephalosporin C from the yeast Trigonopsis variabilis. Biotechnology Lett. 7: 1−7. Takahashi, S., T. Takahashi, Y. Kera, R. Matsunaga, H. Shibuya and R. H. Yamada. 2004. Cloning and expression in Escherichia coli of the D-aspartate oxidase gene from yeast Cryptococcus humicola and characterization of the recombinant enzyme. J. Biochem.(Tokyo) 135: 533-540. Tishkov, V. I. and S. V. Khoronenkova. 2005. D-amino acid oxidase: structure, catalytic mechanism, and practical application. Biochem. (Moscow) 70: 40-54. Tosa, T., T. Sato, T. Mori, and I. Chibata. 1974. Basic Studies for Continuous Production of L-Aspartic Acid by Immobilized Escherichia coli Cells Appl. Environ. Microbiol. 27: 886-889. Upadhya, R., H. Nagajyothi and S. G. Bhat. 1999. D-amino acid oxidase and catalase of detergent permeabilized Rhodotorula gracilis cells and its potential use for the synthesis of alpha-keto acid. Process Biochem. 35: 7-13. Willems, A., J. De Ley, M. Gillis and K. Kersters. 1991 Comamonadaceae, a new family encompassing the acidovorans rRNA complex, including Variovorax paradoxus gen. nov., comb. nov., for Alcaligenes paradoxus (Davis) 1969. Int. J. Syst. Bacteriol. 41: 445-450 Wolosker, H., K. N. Sheth, M. Takahashi, J. P. Mothet, R. A. Brady, Jr., C. D. Ferris and S.H. Snyder. 1999. Purification of serine racemase: Biosynthesis of the neuronodulator D-serine. Proc. Natl. Acad. Sci. USA. 96: 721-725. Zhang, T., G. Ming and L. Yuan. 2005. Production of active pigment epithelium-derived factor in E. coli. Biotechnol. Lett. 27: 403-407. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/31520 | - |
dc.description.abstract | D型胺基酸氧化酶 (D-amino acid oxidase, EC1.4.3.3, 以下簡稱DAAO ) 具有高度之立體專一性,能夠催化D型胺基酸之脫氨反應,產生alpha-酮酸與過氧化氫。本研究由前人自Variovorax paradoxus ISO1中選殖出的ORF5基因,經由序列分析比對,發現ORF5全長為1251 bp,帶有416個胺基酸,分子量為 45 kDa,與Ralstonia eutropha之putative Glycine/D-amino acid oxidase有51% 的最高相似度。將ORF5此段基因利用不同的表現載體與宿主細胞表現系統,以得到最適之表現條件。在pET21a表現系統中,以大腸桿菌BL21(DE3)pLysS為表現宿主,培養於2X YT培養基,在25℃,0.5 mM IPTG,誘導6小時,以100 mM D-Alanine為基質,可得到DAAO酵素比活性 451 mU/mg ; 而以此為表現條件再添加2% DL-Glutamic acid 時,可提高將近兩倍的DAAO酵素比活性為880 mU/mg。在此條件下,大部分之pET21a-DAAO表現蛋白仍然存在於不可溶部份,只有少部份為可溶性蛋白質,故不利於DAAO之大量純化。在pET41a之表現系統,以大腸桿菌BL21(DE3)pLysS為表現宿主,採用與上述相同表現條件培養及誘導菌體,結果發現所表現之GST-DAAO 融合蛋白幾乎全為可溶性蛋白質,而其DAAO酵素比活性為 450 mU/mg。在蛋白質純化方面,GST-DAAO經由GSTrap column回收,其純化倍率大約為1.93倍,DAAO酵素比活性為790 mU/mg,再經過thrombin 或是enterokinase等酵素截切之後,其比活性可再提高兩倍。探討經由enterokinase及thrombin截切後之DAAO酵素的基質專一性,發現此DAAO對 Glycine有最高的比活性,其次是D-alanine,約為Glycine比活性的25%。推測此酵素有可能為甘胺酸氧化酶的一種。除此之外,此酵素尚對D-Aspartic acid以及D-Arginine有微量的比活性,對於其他受測試之D型或L型的胺基酸均不作用。 | zh_TW |
dc.description.abstract | D-amino acid oxidase (DAAO, EC 1.4.3.3) stereospecifically catalyzes the oxidative reaction of the amino group of D-amino aicd to produce its related alpha-keto acid and the H2O2. This study is to characterize an open reading frame ORF5 sequence from Variovorax paradoxus ISO1 and overexpression its protein to do further study. The ORF5 sequence was analyzed by NCBI web, and found it has 1251 bp, 415 amino acids, 45 kDa molecular weight and a high identity with Glycine/DAAO of Ralstonia eutropha. We subclone the sequence into different expression system and investigate the optimal expression conditions. In pET21a system with host E. coli BL21(DE3)pLysS, the best expression condition is induced at 25℃ for 6hr with 0.5 mM IPTG, and the specific activity of pET21a-DAAO is 451 mU/mg. In the same condition with adding 2% DL-Glu to the medium, the specific activity of pET21a-DAAO raised to 880 mU/mg. Since most of the pET-DAAO are inclusion body fraction, we try to use another expression vector that was pET41a system. In pET41a expression system with host E. coli BL21(DE3)pLysS, induction as the same condition, the GST-DAAO fusion protein is almost totally in soluble form and rear in insoluble fraction, and the specific activity of GST-DAAO is 450 mU/mg. After running through the GSTrap column purification of GST-DAAO fusion protein, the purification fold is about 1.93. The activity of purify GST-DAAO cleavaged by thrombin and enterokinase is 2 fold to GST-DAAO fusion protein. The substrate specificity of DAAO has the highest specific activity to Glycine, and the second is D-Alanine, and D-Aspartic acid and D-Arginine have the fewer specific activity. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T03:14:10Z (GMT). No. of bitstreams: 1 ntu-95-R93623004-1.pdf: 1739052 bytes, checksum: f532eba6f2a89b710aa5127651576b87 (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | 目錄
頁次 中文摘要--------------------------------------------------------i 英文摘要--------------------------------------------------------iii 目錄--------------------------------------------------------------v 表次--------------------------------------------------------------ix 圖次--------------------------------------------------------------xi 附錄圖次、表次----------------------------------------------xii 壹、 前言 ㄧ、Variovorax paradoxus的簡介--------------------------1 二、D型胺基酸氧化酶的簡介------------------------------3 三、甘胺酸氧化酶之介紹------------------------------------9 四、研究緣起與目的-----------------------------------------11 貳、 材料與方法 I. 材料 ㄧ、實驗菌株與質體----------------------------------------13 二、培養基----------------------------------------------------13 三、藥品與試劑----------------------------------------------13 四、實驗中使用的套組-------------------------------------14 五、各種溶液及緩衝溶液 1. DNA瓊脂膠體電泳試劑---------------------------15 2. 蛋白質電泳膠體試劑-------------------------------15 3. 西方氏雜交試劑-------------------------------------17 4. 蛋白質定量試劑及其標準品----------------------17 5. D型胺基酸氧化酶酵素基質專一性測試 相關溶液-----------------------------------------------18 6. D胺基酸氧化酶酵素活性測試相關溶液--------19 7. GST -tagged蛋白質純化使用溶液----------------20 8. His-tagged inclusion body純化使用溶液---------20 六、儀器--------------------------------------------------------20 七、其他--------------------------------------------------------22 II. 實驗方法 ㄧ、本實驗所用的一般DNA技術 1. DNA瓊脂膠體電泳----------------------------------23 2. 質體的製備--------------------------------------------23 3. DNA片段純化---------------------------------------24 4. 接合作用----------------------------------------------24 5. 勝任細胞之製備-------------------------------------24 6. 轉型作用----------------------------------------------25 二、Variovorax paradoxus ISO 1 D型胺基酸氧化酶在 大腸桿菌中的表現 1. 不同載體之建構-------------------------------------25 2. 不同表現載體中D型胺基酸氧化酶的表現 及其粗酵素液之製備-------------------------------26 3. D型胺基酸氧化酶酵素活性之測定-------------27 4. D型胺基酸氧化酶酵素基質專一性分析---------27 5. 以thrombin及enterokinase截切GST融合蛋 白--------------------------------------------------------28 6. SDS-PAGE分析---------------------------------------28 7. 西方免疫雜交分析-----------------------------------29 8. 抗體製備-----------------------------------------------30 9. His-tagged D型胺基酸氧化酶 inclusion body 純化------------------------------------------------------30 10. GST-tagged DAAO重組酵素純化-----------------30 11. 蛋白質定量--------------------------------------------31 参、結果 ㄧ、D型胺基酸氧化酶之序列分析結果------------------32 二、D型胺基酸氧化酶於大腸桿菌系統中的表現------33 三、GST-DAAO蛋白質之純化與thrombin及 enterokinase截切之結果--------------------------------36 四、DAAO基質特異性之分析------------------------------36 肆、討論-------------------------------------------------------38 伍、結論-------------------------------------------------------42 陸、參考文獻------------------------------------------------44 表次 表一、本研究所使用的菌株------------------------------ -56 表二、本研究所使用的質體--------------------------------57 表三、V. paradoxus Iso1 之D型胺基酸氧化酶兩側 推測基因之相對位置及功能分析-----------------58 表四、不同物種來源之DAAO其相似性之比較-------60 表五、pET21a-DAAO 與 pQE-DAAO在 大腸桿菌 BL21(DE3)中表現的比較。------------------------61 表六、以低溫長時間誘導下,DAAO在pET21a/ 大腸桿菌 BL21(DE3)的表現----------------------62 表七、不同IPTG濃度對DAAO在pET21a/ 大腸桿菌 BL21(DE3)表現的影響-----------------63 表八、以不同溫度、時間觀察DAAO在pET21a/ 大腸桿菌 BL21(DE3)pLysS表現-------------------64 表九、以不同IPTG濃度誘導DAAO在pET21a/ 大腸桿菌 BL21(DE3)pLysS表現。----------------65 表十、添加DL-Glu,DL-Ala對pET21a/ 大腸桿菌 BL21(DE3)pLysS表現的影響----------66 表十一、在25℃,誘導時間6小時下,以不同濃度 之IPTG誘導pET41a/大腸桿菌BL21(DE3) pLysS的結果。------------------------------------67 表十二、以低溫長時間誘導下,DAAO在pET41a/ 大腸桿菌BL21(DE3)的表現-------------------68 表十三、添加DL-Glu,DL-Ala對DAAO 對 pET41a/大腸桿菌 BL21(DE3)pLysS表現的 影響--- ----------------------------------------------69 表十四、GST-DAAO融合蛋白純化及以內切酵素 切除GST後之活性結果---------- --------------70 表十五、D型胺基酸氧化酶之基質特異性分析--------71 表十六、本實驗中個別表現系統整合表-----------------72 圖次 圖一、ORF5與其序列相似度最高之蛋白質比對--------72 圖二、D型胺基酸氧化酶之核酸與胺基酸序列 及其上游可能操縱子位置之預測。----------------74 圖三、由Variovorax paeadoxus ISO1中所選殖出之 ORF5與其他物種之D型胺基酸氧化酶之序列 比對-------------------------------------------------------76 圖四、不同物種間D型胺基酸氧化酵素之演化樹排列。 -------------------------------------------------------------81 圖五、確認daao基因之正確大小---------------------------82 圖六、pQE-DAAO與pET21a-DAAO在大腸桿菌 BL21(DE3)中表現的比較-----------------------------83 圖七、pET-DAAO在25℃下,誘導6小時之 表現SDS電泳圖---------------------------------------84 圖八、利用SDS-PAGE及西方漬片法分析偵 測D型胺基酸氧化酶。------------------------------85 圖九、pET41a-DAAO在25℃,誘導時間6小時 下,不同IPTG濃度對其表現之影響。-----------86 圖十、pET41a-DAAO在25℃,誘導時間6小時 下,不同IPTG濃度對其表現之影響。-----------87 附錄表次 附錄表一、本實驗所使用之培養基成分-------------------88 附錄圖次 附錄圖一、D胺基酸氧化酶的作用機制及活性 呈色原理----------------------------------------- 89 附錄圖二、D型胺基酸氧化酶基質專一性測定原理---90 | |
dc.language.iso | zh-TW | |
dc.title | Variovorax paradoxus ISO1 D型胺基酸氧化酶基因大量表現之研究 | zh_TW |
dc.title | The study of overexpression of D-amino acid oxidase from Variovorax paradoxus ISO1 | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 蔡國珍(Kuo-Chen Tsai),余美萱(Mei-Hsuan Yu) | |
dc.subject.keyword | D型胺基酸氧化酶,大量表現, | zh_TW |
dc.subject.keyword | D-amino acid oxidase,expression, | en |
dc.relation.page | 91 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-08-04 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 農業化學研究所 | zh_TW |
顯示於系所單位: | 農業化學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
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ntu-95-1.pdf 目前未授權公開取用 | 1.7 MB | Adobe PDF |
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