(3.238.7.202) 您好!臺灣時間:2021/03/03 22:49
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:林陽森
研究生(外文):Yang-Sen Lin
論文名稱:Rhodosporidium toruloides D型胺基酸氧化酶和幾丁質結合域融合蛋白的生化性質
論文名稱(外文):Biochemical properties of Rhodosporidium toruloides D-amino acid oxidase-chitin binding domain fusion protein
指導教授:官宜靜
指導教授(外文):I-Ching Kuan
學位類別:碩士
校院名稱:大同大學
系所名稱:生物工程學系(所)
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:90
中文關鍵詞:幾丁質鍵結區域D型胺基酸氧化酶幾丁質酶
外文關鍵詞:chitinasechitin-binding domainD-Amino acid oxidase
相關次數:
  • 被引用被引用:0
  • 點閱點閱:82
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
D型胺基酸氧化酶 (D-Amino acid oxidase;DAO) 工業上被應用於製造頭孢菌素類抗生素的前驅物7-amino cephalosporanic acid (7-ACA)。但DAO易因溫度、pH值改變和副產物H2O2氧化傷害,造成蛋白質結構改變、或FAD (flavin adenine dinucleotide)輔酶的脫落,而甚至失去活性。
本研究將Bacillus circulans WL-12幾丁質酶之幾丁質鍵結區域chitin-binding domain (CBD) 的對應基因片段和Rhodosporidium toruloides DAO (RtDAO) cDAO基因融合,並置於大腸桿菌表現生產具有C端CBD的RtDAO,以固定於幾丁質顆粒增加其穩定性。此RtDAO-CBD融合酵素因具有N端His tag,故可以金屬螯合層析純化後進行生化性質檢測。最適反應溫度和TM分別為35和45oC,最適反應pH則為8.0,最佳pH穩定度於pH 7.0,皆與RtDAO相似。當與10 mM H2O2反應時,RtDAO和RtDAO-CBD的半衰期分別為105和75分鐘,當以0.01 mg/ml濃度儲存於4 oC下時,兩者的半衰期分別為7、6天;經過15次重複結凍,RtDAO和RtDAO-CBD分別保有20和50%的殘餘活性。
D-Amino acid oxidase (DAO) has been applied industrially to produce the precusor for cephalosporin antibiotic, 7-amino cephalosporanic acid. However, DAO is vulnerable to changes in temperature, pH as well as oxidative damages by hydrogen peroxide, thus resulting in alteration of the protein structure, the loss of coenzyme FAD (flavin adenine dinucleotide), and even inactvation.
In this study, the corresponding gene fragment for chitinase from Bacillus circulans WL-12 chitin-binding domain (CBD) was fused to Rhodosporidium toruloides DAO (RtDAO) cDAO gene, and was then expressed in E coli to generate the RtDAO fusion protein with C-terminal CBD for immobilization on chitin beads to obtain better stability. Because of having N-terminal His tag, RtDAO-CBD fusion protein was purified by metal chelation chromatography for the analysis of biochemical properties. The optimal temperature and the Tm value were 35 and 45oC, respectively, while the optimal pH was at 8.0 and the best pH stability appeared at pH 7.0. These properties were all similar to those of RtDAO. When reaction with 10 mM hydrogen peroxide, the half-lives of RtDAO and RtDAO-CBD were 105 and 75 min, respectively. At the concentration of 0.01 mg/ml, their half-lives of storage at 4 oC were 7and 6days, respectively. After 15-cycles of freeze-and-thaw, the residual activities of RtDAO and RtDAO-CBD were 20 and 50%, respectively.
目錄
誌謝I
中文摘要I
AbstractIII
目錄V
表索引IX
圖索引X
第一章 前言1
1.1. D型胺基酸氧化酶1
1.1.1 簡介1
1.1.2 生化特性2
1.1.3 生理功能3
1.1.3.1 調控D-serine3
1.1.3.2 調控D-alanine3
1.1.3.3 調節激素分泌4
1.1.4 DAO的應用4
1.1.4.1 D型胺基酸的定量與分析4
1.1.4.2 生產7-ACA5
1.1.4.3 診斷與預防身心失調的疾病與癌症6
1.2.酵素固定化7
1.2.1 固定化技術7
1.2.2 親和力標籤(affinity tag) 8
1.3.幾丁質及幾丁質酶9
1.3.1 幾丁質9
1.3.1.1 簡介9
1.3.1.2 幾丁質的應用11
1.3.2 幾丁質酶11
1.3.2.1 簡介11
1.3.2.2 幾丁質酶之種類12
1.3.2.3 Bacillus circulans WL-12 ChiA114
1.4.研究目的16
第二章 實驗材料與方法17
2.1 實驗材料17
2.1.1 菌種17
2.1.2 質體17
2.2 實驗方法18
2.2.1 重組質體的建構18
2.2.1.1 基因的複製放大18
2.2.1.2 DNA的剪切和黏合19
2.2.1.4 Competent Cell的製備20
2.2.1.5 E. coli 的轉形作用20
2.2.1.6 抽取質體21
2.2.2 DAO於E. coli 中的表現22
2.2.3 以金屬螯合層析法純化22
2.2.4 DAO活性檢測24
2.2.4.1 測定H2O2產量24
2.2.4.2 Pyruvic acid的產量測定25
2.2.5 蛋白質定量 (Bradford assay) 26
2.2.6 SDS-PAGE分析27
2.2.7 固定於幾丁質微珠30
2.2.8 生化性質檢測30
2.2.8.1 溫度對DAO活性的影響30
2.2.8.2 pH質對DAO活性之影響31
2.2.8.3 H2O2耐受性的檢測32
2.2.9 儲存穩定性32
2.2.9.1 重複結凍解凍檢測32
2.2.9.2 保存期32
第三章實驗結果與討論33
3.1重組表現質體的建構33
3.2 HRtDAO-CBD於E.coli中的異源表現33
3.3 HRtDAO-CBD SDS-PAGE分析33
3.4 HRtDAO-CBD的固定化34
3.5 溫度對HRtDAO-CBD活性的影響34
3.6 pH對HRtDAO-CBD活性的影響35
3.7 HRtDAO-CBD的過氧化氫耐受性35
3.8 HRtDAO-CBD重複結凍解凍檢測36
3.9 HRtDAO-CBD儲存穩定性36
第四章 結論37
第五章 圖表38
第六章 參考文獻61
表索引
表1 DAO於生物體內的各種功能38
表2 各種親和力標籤的胺基酸序列和大小39
表3 各種幾丁質酶的特性40
圖索引
圖1 DAO催化反應機制41
圖2 7-ACA的生成機制42
圖3 H2O2生成量測定法之反應式.43
圖4 HRtDAO-CBD表現重組質體的建構流程44
圖5 pRtDAO-CBD 的建構確認45
圖6 HRtDAO cDNA基因的核苷酸和推演出的對應胺基酸序列46
圖7 HRtDAO-CBD cDNA基因的核苷酸和推演出的對應胺基酸序列47
圖8 H2O2標準曲線48
圖9 Pyruvic acid標準曲線49
圖10 BSA標準曲線50
圖11 HRtDAO與HRtDAO-CBD於TB中的活性表現51
圖12 HRtDAO和HRtDAO-CBD SDS-PAGE 分析52
圖13 HRtDAO和HRtDAO-CBD之最適反應溫度53
圖14 HRtDAO和HRtDAO-CBD的熱穩定性54
圖15 HRtDAO和HRtDAO-CBD之最適反應pH55
圖16 HRtDAO和HRtDAO-CBD的pH穩定性56
圖17 HRtDAO和HRtDAO-CBD之H2O2耐受性57
圖18 HRtDAO和HRtDAO-CBD的重複結凍解凍測試58
圖19 HRtDAO和HRtDAO-CBD於4 oC之儲存穩定性59
圖20 HRtDAO和HRtDAO-CBD於25 oC之儲存穩定性60
蔡鎮陽,2009,斑馬魚D型胺基酸氧化酶於大腸桿菌中的異源表現,碩士論文,生物工程研究所,大同大學,台北。

吳東嶺,2003,D型胺基酸氧化酵素在大腸桿菌異源表現的最適化,碩士論文,生物工程研究所,大同大學,台北。

呂卦南,2006,幾丁質與幾丁聚醣之製備與鑑定,康寧學報 8:157-170。

陳榮輝,2001,幾丁質、幾丁聚醣的生產製造、檢測與應用,科學發展月刊 29:776-787。

吳彰哲和黃瀚寧,2010,蝦蟹殼中的寶貝—幾丁質,科學發展月刊 448:12-19

宋思揚、樓士林、李順來,2002,生物技術概論,p.135-141,滄海書局。

葉勤、白濤雄,2005,現代生物技術原理及其應用 ,p.2-16,p.342-350,九州圖書文物有限公司。

Abe, H., N. Yoshikawa, M. G. Sarower and S. Okada. 2005. Physiological function and metabolism of free D-alanine in aquatic animals. Biol. Pharm. Bull. 28:1571-1577.

Aly, S. A., B. D. Jeon and Y. H. Park. 1997. Preparation and evaluation of the chitin derivatives for wastewater treatments. J. Appl. Polym. Sci. 65:1939-1946.

Bhushan, B. and G. S. Hoondal. 1998. Isolation, purification and properties of a thermostable chitinase from an alkalophilic Bacillus sp.
BG-11. Biotechnol. Lett. 20:157–159.

Chumako, I., M. Blumenfeld, O. Guerassimenko, L. Cavarec, M. Palicio, H. Abderrahim,L. Bougueleret, C. Barry, H. Tanaka, P. la Rosa, A. Puech, N. Tahri, A. Cohen-Akenine, S. Delabrosse, S. Lissarrague, F.-P. Picard, K. Maurice, L. Essioux, P. Millasseau, P. Grel, V. Debailleul, A.-M. Simon, D. Caterina, I. Dufaure, K. Malekzadeh, M. Belova, J.-J. Luan, M. Bouillot, J.-L. Sambucy, G. Primas, M. Saumier, N. Boubkiri, S. Martin-Saumier, M. Nasroune, H. Peixoto, A. Delaye, V. Pinchot, M. Bastucci, S. Guillou, M. Chevillon, R. Sainz-Fuertes, S. Meguenni, J. Aurich-Costa, D. Cherif, A. Gimalac, C. Van Duijn, D. Gauvreau,
G. Ouellette, I. Fortier, J. Raelson, T. Sherbatich, N. Riazanskaia, E. Rogaev, P. Raeymaekers, J. Aerssens, F. Konings, W. Luyten, F. Macciardi, P. C. Sham, R. E. Straub, D. R. Weinberger, N. Cohen and D. Cohen. 2002. Genetic and physiological data implicating the new human gene G72 and the gene for D-amino acid oxidase in schizophrenia. Proc. Natl. Acad. Sci. USA. 99:13675-13680.

Cohen-Kupice, R. and I. Chet. 1998. The molecular biology of chitin digestion. Curr. Opin. Biotechnol. 9: 270-277.

Dahiya, N., R. Tewari and G. S. Hoondal. 2006. Biotechnological aspects of chitinolytic enzymes: a review. Appl. Microbiol. Biotechnol. 71:773–782.

Dahiya, N., R. Tewari, R. P. Tiwari and G. S. Hoondal. 2005a. Production of an antifungal chitinase from Enterobacter sp. NRG4 and its
application in protoplast production. World J. Microb. Biot. 21:1611-1616.

Dahiya, N., R. Tewari, R. P. Tiwari and G. S. Hoondal. 2005b. Chitinase
production in solid state fermentation by Enterobacter sp. NRG4 using statistical experimental design. Curr. Microbiol. 51:1–9.

Dahiya, N., R. Tewari, R. P. Tiwari and G. S. Hoondal. 2005c. Chitinase from Enterobacter sp. NRG4: its purification, characterization and reaction pattern. Electron. J. Biotechn. 8:134-145.

D’Aniello, A., M. M. D. Fiore, G. H. Fisher, A. Milone, A. Seleni, S. D’Aniello, A. F. Perna and D. Ingrosso. 2000. Occurrence of D-aspartic acid and N-methyl-D-aspartic acid in rat neuroendocrine tissues and their role in the modulation of luteinizing hormone and growth hormone release. FASEB J. 14:699-714.

Deane, E. E., J. M. Whipps, J. M. Lynch and J. F. Peberdy. 1998. The purification and characterization of a Trichoderma harzianum exochitinase. Biochem. Biophys. Acta 1383: 101-110.

Fang, J., T. Sawa, T. Akaike, K. Greish and H. Maeda. 2004. Enhancement of chemotherapeutic response of tumor cells by a heme oxygenase inhibitor, pegylated zinc protoporphrin. Int. J. Cancer. 109:1–8.

Ferrandon, S., T. Sterzenbach, F. B. Mersha and M-Q. Xu. 2003. A single surface tryptophan in the chitin-binding domain from Bacillus circulans chitinase A1 plays a pivotal role in binding chitin and can be modified to create an elutable affinity tag. Biochim. Biophys. Acta. 1621: 31-40.

Fisher, G., S. Lopez, K. Peterson, T. Goff, I. Philip, R. Gaviria, N. Lorenzo and M. Tsesarskaia. 2007. Is there a correlation between age and D-aspartic acid in human knee cartilage. Amino Acids. 32:27-30.

Frankowski, J., M. Lorito, F. Scala, R. Schmid, G. Berg and H. Bahl. 2001. Purification and properties of two chitinolytic enzymes of
Serratia plymuthica HRO-C48. Arch. Microbiol. 176:421–426.

Gablera, M., M. Henselb and L. Fischerb. 2000. Detection and substrate selectivity of new microbial D-amino acid oxidases. Enzyme and Microbial Technology 27 (2000) 605–611.

Hardt, M. and R. A. Laine. 2004. Mutation of active site in the chitin-binding domain ChBDChiA1 from chitinase A1 of Bacillus circulans alters substrate specificity: use of a green fluorescent protein binding assay. Arch. Biochem. Biophys. 426:286-297

Hashimoto, M., T. Ikegami, S. Seino, N. Ohuchi, H. Fukada, J. Sugiyama, M. Shirakawa and T. Watanabe. 2000. Expression and characterization of
the chitin-binding domain of chitinase A1 from Bacillus circulans
WL-12. J. Bacteriol. 182:3045-3054.

Henrissat, B. and A. Bairoch, 1993. New families in the classification of
glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 293: 781-788.

Ikegami, T., T. Okada, M. Hashimoto, S. Seino, T. Watanabe and M. Shirakawa. 2000. Solution structure of the chitin-binding domain of Bacillus circulans WL-12 chitinase A1. J. Biol. Chem. 275: 13654-13661.

Isel, B., T. Boller and J. M. Neubaus. 1993. The N-terminal cysteine-rich domain of tobacco classI chitinase is essential for chitin binding but not for catalytic or antifungal activity. Plant Physiol. 103:221-226.

Jee, J.-G., T. Ikegami, M. Hashimoto, T. Kawabata, M. Ikeguchi, T. Watanabe and M. Shirakawa. 2002. Solution structure of the fibronectin type III domain of Bacillus circulans WL-12 chitinases A. J. Biol. Chem. 277: 1388-1397.

Jinzhu, S., Y. Qian, L. Beidong and C. Dianfu. 2005. Expression of the chitinaes gene from Trichoderma aureoviride in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 69: 39-43.

Khoronenkova S. V. and V. I. Tishkov. 2008. D-Amino acid oxidase:
physiological role and applications. Biochemistry. 48:359-376.

Kim, C. Y., H. M. Choi and H. T. Cho. 1997. Effect of deacetylation on sorption of dyes and chromium on chitin. J. of Appl. Polym. Sci. 63:725-736.

Leah, R., H. Tommerup, I. Svendsen and J. Mundy. 1991. Biochemical and molecular characterization of three barley seed proteins with antifungal properties. J. Biol. Chem. 19:196-201.

Limon, M. H., E. Margolles-Clark, T. Benitez and M. Penttila. 2001. Addition of substrate-binding domains increases substrate-binding capacity and specific activity of a chitinase from Trichoderma harzianum. FEMS Microbiol. Lett. 198:57-63.

Linder, M., I. Salovuori, L. Ruohonen, T. T. Teeri. 1996. Characterization of a double cellulose-binding domain. Synergistic high affinity binding to crystalline cellulose. J. Biol. Chem. 271:21268-21272.

Mabuchi, N., I. Hashizume and Y. Araki. 2000. Characterization of chitinase excreted by Bacillus cereus CH. Can. J. Microbiol. 46:370-375.

Martineau1, M., G. Baux and J.-P. Mothet . 2006. D-Serine signalling in the brain:friend and foe. TRENDS Neurosci. 29:481-491.

Morimoto, K., S. Karita, T. Kimura, K. Sakka and K. Ohmiya. 1997. Cloning, sequencing, and expression of the gene encoding clostridium paraputrificum chitinase ChiB and analysis of the functions of novel cadherin-like domains and a chitin-binding domain. J. Bacteriol. 179:7306-7314.

Murao, S. T. Kuwada, H. Itoh, H. Oyama and T. Shin. 1992. Purification
and characterization of a novel type of chitinase from Vibrio alginolyticus TK-22. Biosci. Biotech. Bioch. 56:368–369.

Muzzarelli, R.A.A. 1996. Chitosan-based dietary foods. Carbohyd. Polym. 29:309-316.

Nishikawa, T., 2005. Metabolism and functional roles of endogenous D-Serine in mammalian brains. Biol. Pharm. Bull. 28:1561-1565.

Ohno, T., S. Armand, T. Hata, N. Nikaidou, B. Henrissat, M. Mitsutomi, and T. Watanabe. 1996. A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037. J. Bacteriol. 178: 5065-5070.

Park, J. K., K. Morita, I. Fukumoto, Y. Yamasaki, T. Nakagawa,
M. Kawamukai and H. Matsuda. 1997. Purification and characterization
of the Chitinase (ChiA) from Enterobacter sp. G-1. Biosci. Biotech. Bioch. 61:684–689.

Pernot, P., J.-P. Mothet, O. Schuvailo, A. Soldatkin, L. Pollegioni,
M. Pilone, M.-T. Adeline, R. Cespuglio and S. Marinesco. 2008. Characterization of a yeast D-amino acid oxidase microbiosensor for D-Serine detection in the central nervous system. Russ. Chem. Bull. 57:1014-1022.

Pilone, M. S. and L. Pollegioni. 2002. D-amino acid oxidase as an industrial biocatalyst. Biocatal. Biotransfor. 20:145-159.

Pilone, M. S. 2000. D-amino acid oxidase: new findings. Cell. Mol. Life Sci. 57:1732-1747.

Poinar, H. N. and B. A. Stankiewicz. 1999. Protein preservation and DNA retrieval from ancient tissues. Proc. Natl. Acad. Sci. 96:8426-8431.

Pollegioni L., L. Caldinelli, G. Molla, S. Sacchi and M. S. Pilone. 2004.
Catalytic properties of D-amino acid oxidase in cephalosporin C
bioconversion: a comparison between proteins from different sources.
Biotechnol. Prog. 20:467-473.

Pollegioni L., L. Piubelli, S. Sacchi, M. S. Pilone and G. Molla. 2007.
Physiological functions of D-amino acid oxidases: from yeast to humans.
Cell. Mol. Life Sci. 64:1373-1394.

Pollegioni, L., S. Orenzi, E. Rosini, G. L. Marcone, G. Molla, R. Verga, W. Cabrl and M. S. Pilone. 2005. Evolution of an acylase active on
cephalosporin C. Protein Sci. 14, 3064–3076

Roberts, R. L. and E. Cabib. 1982. Serratia marcescens chitinase: one step purification and use for the determination of chitin. Anal. Biochem. 127:402–412.

Schumacher, J., R. Abon Jamra1, J. Freudenberg, T. Becker, S. Ohlraun, A. C. J. Otte, M. Tullius, S. Kovalenko, A. Van Den Bogaert, W. Maier, M. Rietschel, P. Propping, M. M. NOthen and S. Cichon. 2004. Examination of G72 and D-amino-acid oxidase as genetic risk factors for schizophrenia and bipolar affective disorder. Mol. Psychiatry. 9:203- 207.

Shpigel, E., A. Goldlust, G. Efroni, A. Avraham, A. Eshel, M. Dekel and O. Shoseyov. 1999. Immobilization of recombinant heparinase I fused to cellulose-binding domain. Biotechnol. Bioeng. 65:17-23.

Strick, C. A., C. Li, L. Scott, B. Harvey, M. Hajos, S. J. Steyn, M. A. Piotrowski, L. C. James, J. T. Downs, B. Rago, S. L. Becker, A. El-Kattan, Y. Xu, A. H. Ganong, F. D. Tingley, A. D. Ramirez, P. A. Seymour, V. Guanowsky, M. J. Majchrzak, C. B. Fox, C. J. Schmidt, A. J. Duplantier. 2011. Modulation of NMDA receptor function by inhibition of D-amino acid oxidase in rodent brain. Neuropharmacology. 61:1001-1015.

Takigawa, Y., H. Homma, J.-A. Lee, T. Fukushima, T. Santa,
T. Iwatsubo and K. Imai. 1998. D-aspartate uptake into cultured rat pinealocytes and the concomitant effect on L-aspartate levels
and melatonin secretion. Biochem. Biophys. Res. Commun. 248:641-647.

Tanaka, H. and H. J. Phaff. 1976. Enzymatic hydrolysis of yeast cell walls. J. Bacteriol. 89:1570-1580.

Tang, Y., J. Zhao, S. Ding, S. Liu and Z. Yang. 2001. Purification and
properties of chitinase from Enterobacter aerogenes. Wei Sheng Wu Xue Bao. 41:82–86.

Terpe. K. 2003. Overview of tag protein fusions:from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 60:523–533.

Tishkov V. I. and S. V. Khoronenkova. 2005. D-amino acid oxidase: structure, catalytic mechanism, and practical application, Biochemistry.
70:40-54.

Vaidya, R., S. Roy, S. Macmil, S. Gandhi, P. Vyas and H. S. Chhatpar. 2003. Purification and characterization of chitinase from Alcaligenes xylosoxydans. Biotechnol. Lett. 25:715-717.

Wang, J.-Y. and Y.-P. Chao. 2006. Immobilization of cells with surface-displayed chitin-binding domain. AEM. 72:927–931.

Watanabe, T., K. Kobori, K. Miyashita, T. Fujii, H. Sakai, M. Uchida, and H. Tanaka. 1993. Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity. J. Biol. Chem. 268: 18567-18572.

Watanabe, T., Y. Ito, T. Yamada, M. Hashimoto, S. Sekine and H. Tanaka. 1994. The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation. J.
Bacteriol. 176:4465-4472.

Wen, C.M., C. S. Tseng, C. Y. Cheng and Y. K. Li. 2002. Purification,
characterization and cloning of a chitinase from Bacillus sp. NCTU2. Biotechnol. Appl. Bioc. 35:213–219.

Wiwat, C., P. Siwayaprahm and A. Bhumiratana. 1999. Purification and
characterization of chitinase from Bacillus circulans No. 4.1. Curr.
Microbiol. 39:134-140.

Woo, S. L., B. Donzelli, F. Scala, R. Mach, G. E. Harman, C. P. Kubicek, G. D. Sorbo and M. Lorito. 1999. Disruption of the ech42 (endochitinase-encoding) gene affects biocontrol activity in Trichoderma harzianum. Mol. Plant. Microbe. In. 5: 419-429.

Yuli, P. E., M. T. Suhartono, Y. Rukayadi, J. K. Hwang and Y. R. Pyun. 2004. Characteristics of thermostable chitinase enzymes from the Indonesian Bacillus sp. 13.26. Enzyme Microb. Tech. 35:147–153.

Zhang, J. P., Q. X. Chen, Q. Wang and J. J. Xie. 2006. Purification and some properties of β-N-Acetyl-D-glucosaminidase from Viscera of Green Crab (Scylla serrata). Biochemistry (Moscow). 71:55-59.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔