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研究生:陳俊彰
研究生(外文):Chun-Chang Chen
論文名稱:RhizopusoryzaeNBRC4749新型Rhizopuspepsin的純化與特性分析及利用Bacillussubtilis表現StaphylococcusaureusBCRC15205之GlutamylEndopeptidase基因
論文名稱(外文):Purification and Characterization of a New Rhizopuspepsin from Rhizopus oryzae NBRC 4749 and Expression of Staphylococcus aureus BCRC 15205 Glutamyl Endopeptidase Gene in Bacillus subtilis
指導教授:許文輝許文輝引用關係
學位類別:博士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:92
中文關鍵詞:蛋白酶切位專一性
外文關鍵詞:proteasecleavage specificity
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利用陰離子交換樹脂管柱,以快速蛋白液相色層分析的技術(FPLC),從Rhizopus oryzae NBRC 4749的胞外上清液中純化出分泌性的aspartic protease (又稱為 rhizopuspepsin),而純化率可達 45%。這個酵素經由SDS-PAGE的分析,可推估其分子量約為37 kDa;由醣蛋白染色的測定,則可判斷其並非為醣蛋白。 N端序列與LC-MS/MS分析的結果,發現本實驗所純化出的rhizopuspepsin與 R. oryzae 基因體序列資料庫內的hypothetical protein RO3G_12822.1 相符。比較RO3G_12822.1的genomic序列與cDNA 序列,可發現此基因帶有兩個 intron,然而之前所發表的rhizopuspepsin 基因都只帶有一個intron。物種演化的分析也指出本研究所純化出的rhizopuspepsin與之前所發表的rhizopuspepsin有所不同。 純化後的rhizopuspepsin其最適溫度與pH 各為 50 °C 與 pH 3.0;而其半衰期在40 °C的環境下約為3.5小時。此新型rhizopuspepsin對於受質P1位置的hydrophobic 與 positively charged amino acid具有高活性,但是對於Glu、 Pro、Trp、與帶有β-branch 支鏈的aliphatic amino acid則不具有活性。與P1位置的cleavage specificity相較,此酵素對於受質P1’位置上positively charged amino acid不具有活性,但卻對帶有β-branch 支鏈的aliphatic amino acid具有高活性。 本研究亦發現,此rhizopuspepsin在SDS-PAGE上會有不正常緩慢移動的現象。
將Staphylococcus aureus的glutamyl endopeptidase 基因(sspA)選殖於表現載體,並利用Bacillus subtilis來進行表現。當利用wild-type B. subtilis DB2為宿主進行表現時,其所表現出的glutamyl endopeptidase precursor會被活化,並可偵測到活性。可是當宿主換成protease-deficient B. subtilis DB104 (nprE- aprE-)時,其所表現出的glutamyl endopeptidase precursor則無法被活化。雖然B. subtilis DB2所表現出的glutamyl endopeptidase具活性。但在N端pro序列被切除的同時,其C端的His tag也被切除了。因此,利用B. subtilis DB2來表現glutamyl endopeptidase基因,會造成後續酵素純化的困難。本研究亦發現,將glutamyl endopeptidase第68個amino acid由Asp置換成Glu,此protease可在無其他protease的幫助下自行成熟,並且具有活性。
A secretory aspartic protease (also termed as rhizopuspepsin) was purified from Rhizopus oryzae NBRC 4749 by ion exchange chromatography with a yield of 45%. The enzyme was a non-glycoprotein with a molecular mass of 37 kDa as determined by SDS-PAGE analysis. N-terminal sequence and LC-MS/MS analyses revealed that this rhizopuspepsin corresponded to the hypothetical protein RO3G_12822.1 in the R. oryzae genome database. Comparison of genomic and cDNA genes demonstrated that the rhizopuspepsin gene contained two introns, whereas only one intron was reported in other rhizopuspepsin genes. Phylogenetic analysis also indicated that this rhizopuspepsin was distinct from other rhizopuspepsins. The temperature and pH optima for the purified rhizopuspepsin were 50 °C and pH 3.0, respectively, and a half-life of about 3.5 h was observed at 40 °C. This newly identified rhizopuspepsin preferentially cleaved the peptides with hydrophobic and positively charged amino acids in P1 site, but had no activity for the Glu, Pro, Trp, and aliphatic amino acids containing β-branch side chain. In contrast to P1 site, the P1’ site did not accommodate positively charged amino acids for the enzyme. But, it preferentially cleaved the peptides with aliphatic amino acids containing β-branch side chain in P1’ site. In this study, we also noticed that the rhizopuspepsin displayed aberrant mobility in SDS-PAGE analysis.
Staphylococcus aureus glutamyl endopeptidase gene (sspA) was cloned into expressing vector, and expressed in Bacillus subtilis. Active glutamyl endopeptidase could be obtained from the wild-type B. subtilis DB2 expressing the sspA gene, whereas the enzyme remained in the precursor form from protease-deficient B. subtilis DB104 (nprE- aprE-). However, 6His tag was also deleted from the mature glutamyl endopeptidase produced by B. subtilis DB2, leading to difficulty in purification of recombinant glutamyl endopeptidase from culture supernatant. We also found that glutamyl endopeptidase variant with N68E mutation could form mature protein by autocatalysis.
目次
第一章:Rhizopus oryzae NBRC 4749 新型rhizopuspepsin的純化與特性分析
中文摘要 ………………..………………...……………………..…………...…... 2
Abstract…………………………………………….…….………………………… 3
前言……………………………………………..………………………………… 4
一、Rhizopus oryzae……….………………...…………………………………. 4
二、Protease ……………………………………...………………….…………. 5
三、多基因家族的演化……………………………….……….……..………... 8
四、蛋白質在SDS-PAGE上不正常位移的現象…………….…….………… 10
五、研究目的………………………………………………………….………. 10
材料與方法……………………….…………………..…….……………...……… 11
一、菌株、質體與生長條件…………………………….……………….……. 11
二、DNA 操作 ………………………………………………………..………. 11
三、酵素活性分析………………………………………………….…….……. 11
四、Protease的純化…………………………………………………..……….. 12
五、蛋白質分子量的測定…………………………………………….….…… 12
六、Proteases的身份鑑定…………………………………………………….. 12
七、醣蛋白染色…………………………………………………………..…… 13
八、演化分析…………………………………………………………….….… 13
九、酵素特性分析……………………………………..……………..….……. 14
十、Nucleotide序列的登錄編號..……………………..………….…….…….. 15
結果……………………………………………………….………..…………..…. 16
一、從R. oryzae的培養上清液中純化protease ………………...………..…. 16
二、Protease的身份鑑定………………………….………………………..…. 16
三、Rhizopuspepsin 6 的序列分析……………………………………………. 17
四、Rhizopuspepsin的演化與基因結構分析………………………….……... 17
五、Rhizopuspepsin 6的酵素特性……………………………………..……... 18
六、Rhizopuspepsin 6的cleavage specificity ……………………..…………. 18
討論………………………………………………………………………..….….... 20
參考文獻……………………………………………………….….………………. 24
圖表…………….………………………………..………………………………… 38

第二章 利用Bacillus subtilis表現Staphylococcus aureus BCRC 15205 glutamyl endopeptidase
中文摘要 ………………………………………………………………………….. 67
Abstract ………………………………………………………….……..…..……… 68
前言 …………………………………………….……..………………………..… 69
一、Staphylococcus aureus 的glutamyl endopeptidase…..…………………… 69
二、Bacillus subtilis 的基因表現系統…..……………………..……………… 69
三、Protease的cleavage specificity…….…………………………...………… 70
四、研究目的………………………………………………………….…..…… 70
材料與方法 ……………………………………………………………………..… 72
一、菌株、質體與生長條件 ……………………………………….………… 72
二、S. aureus chromosomal DNA 的抽取 …...………………….…………… 72
三、Plasmid DNA 的抽取 ……………………………...…………….……… 72
四、基因選殖 ………..…………………………………………………...…… 73
五、蛋白質的純化與分析 ………….……………………………….………… 73
六、Glutamyl endopeptidase酵素活性分析 …….…….…………...………… 73
結果 …………………………………………………………………..…………… 75
一、S. aureus sspA基因的選殖 ……….……………………………..…..…… 75
二、sspA基因在B. subtilis表現……………….………………….…………… 75
三、利用protease活化S. aureus 的glutamyl endopeptidase …….…………… 76
四、Glutamyl endopeptidase的cleavage specificity …………………...……… 76
討論 ……………………..…………………………………...……………….…… 78
總結 …………………………………………………………………….……….… 80
參考文獻 …………………………………………………………….………….… 81
圖表 …………….……………………………………………………………….… 84
Rhizopus oryzae NBRC 4749 新型rhizopuspepsin
的純化與特性分析
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