請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/52342
標題: Cloning and Overexpression of Aspergillus oryzae Peptidase Gene and Characterization of Its Biochemical Properties
食品工業用特殊胜肽分解酵素基因的選殖、大量表現及生化性質分析
作者: 許文輝
關鍵字: 生物技術
技術發展
摘要: Aspergillus oryzae is widely used in the fermentation industry.This fungus can to secrete many types and high level of proteases to decompose nutrient sources.Leucine aminopeptidase ( LAP )may participate in protein degradation.A.oryzae LAP is commercialized, however, its gene structure and physiological role has never been reported.To obtain a lap gene fragment as probe, LAP was purified from commercial Aspergillus sojae proteases using FPLC and RP-HPLC.The purified LAP was digested with endopeptidase Glu-C or lyslyendopeptidase.Partial LAP fragments were sequenced and back-translated to degenerate primers.A 700bp fragment was amplified by PCR using A.oryzae cDNA as template.The lap genes were then isolated from A.oryzae genomic and cDNA libraries using 700bp fagment as probe.A.oryzae lap gene comprises 1, 320bp with 3introns and encodes a protein consisting of 377amino acids ( 40, 506Da ).The deduced amino acids shows only 33%identity to V.proteolyticus lap.Comparison of the protein sequencing from A.sojae LAP and the deduced amino acid sequence from A.oryzae lap revealed a signal peptide with 77amino acids in A.oryzae LAP.The mature protein has a calculated molecular mass of 36, 280Da which almost matches the molecular mass of 35kDa determined from SDS-PAGE.A TATA box was identified at position -120from the ATG, and a CAAT box was found at position -286.A specific region, -88to -69, may be related to pH regulation.A poly ( A )adding site was found in the 3'region.Three introns are 59, 74, and 56bp in size and located at 155-213, 640-713and 1031-1086, respectively.Except the intron which is the nearest to start codon, others obeys the GT-AG rule.Two putative N-glycosylation sites could be identified in Asn-87and Asn-288of A.oryzae lap.To elucidate the biochemical properties of LAP and overexpress the lap gene in A.oryzae, following research work will be completed in this granted year.1.Large scale preparation and purification of A.oryzae LAP.2.Analysis of biochemical properties of LAP.3.Establish an efficient method for the transformation of lap gene into A.oryzae.Overexpression of lap gene in A.oryzae.
Aspergillus oryzae是醱酵工業上經常使用的菌種.此種真菌能分泌多種蛋白酶 , 以分解環境中的物質, 作為營養來源.Leucine aminopeptidase ( LAP )可能也是扮演相同的角色.在市面上已經有來自A.oryzae 的LAP商品, 但是目前仍未有lap基因序列及其生理意義的相關文獻被發表.為取得lap基因片斷做為探針, 將市售的Aspergillus sojae蛋白酶 , 經FPLC及RP-HPLC純化出LAP, 再分別以endopeptidase Glu-C及lysylendopeptidase處理後, 回收純化胜肽 .經氨基酸定序分析, 將定序所得的氨基酸序列, 設計成degenerate primer, 以A.oryzae cDNA為模板 ( template ), 利用聚合酶 鏈鎖反應( PCR ), 複製出700bp的片段.以此片段作為探針( probe ), 由A.oryzae染色體基因庫及cDNA基因庫中, 分別選殖出lap基因.A.oryzae lap基因的ORF全長為1, 320bp, 帶有3個introns, 可轉譯出377個氨基酸( 40, 506Da ).轉譯出的氨基酸序列, 與V.proteolyticus lap基因具有33%的相似性.將A.sojae LAP蛋白之N端氨酸基序列與A.oryzae lap基因比對, 顯示在A.oryzae lap基因中, 具有由77個氨基酸組成的訊號胜肽 ( signal peptide ), 俾將LAP外泌到胞外.分泌到胞外的LAP, 分子量為36, 280Da, 與利用SDS-PAGE上所測得的分子量( 35kDa )接近.在距ATG-120--114, 有一TATA box, 另有CAAT box位於-286--283.在-88--69位置, 有一個特殊的DNA序列, 可能是pH的調控區域.在ORF的3'端, 有poly ( A )adding site.在ORF內的3個introns, 大小分別為59、74及56bp, 距離ATG 155-213、640-713及1031-1086的位置.除了最靠近轉錄起始點的intron外, 其餘兩個introns的結構, 皆符合GT-AG的原則.lap基因的Asn-87與Asn-288, 可能是會被醣化的位置.為了解LAP的生化性質以及使lap基因能在A.oryzae菌體內大量表現, 未來的一年將執行下列工作: 1.A.oryzae LAP的大量製備及純化.2.LAP生化性質分析.3.建立一個有效的轉形系統, 以便將lap基因轉形入A.oryzae.4.使lap基因在A.oryzae大量表現.
URI: http://hdl.handle.net/11455/52342
其他識別: 90農科-2.1.1-糧-Z4(3)
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=1041758&plan_no=90%E8%BE%B2%E7%A7%91-2.1.1-%E7%B3%A7-Z4%283%29&plan_year=90&projkey=PG9309-1886&target=plan&highStr=*&check=0&pnchDesc=%E9%A3%9F%E5%93%81%E5%B7%A5%E6%A5%AD%E7%94%A8%E7%89%B9%E6%AE%8A%E8%83%9C%E8%82%BD%E5%88%86%E8%A7%A3%E9%85%B5%E7%B4%A0%E5%9F%BA%E5%9B%A0%E7%9A%84%E9%81%B8%E6%AE%96%E3%80%81%E5%A4%A7%E9%87%8F%E8%A1%A8%E7%8F%BE%E5%8F%8A%E7%94%9F%E5%8C%96%E6%80%A7%E8%B3%AA%E5%88%86%E6%9E%90
顯示於類別:分子生物學研究所

文件中的檔案:
沒有與此文件相關的檔案。


在 DSpace 系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。