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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/17502

Title: 解澱粉芽孢桿菌重組幾丁聚醣酶基因之表現及其生化特性
Expression and Characterization of Recombinant Chitosanase Gene from Bacillus amyloliquefaciens
Authors: Yi-Hua Chen
陳逸華
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立臺灣海洋大學:生物科技研究所
Keywords: 解澱粉芽孢桿菌;幾丁聚醣酶;生化特性
Bacillus amyloliquefaciens;chitosanase;characterization
Date: 2003
Issue Date: 2011-07-04
Abstract: The chitosanase gene from B. amyloliquefaciens was cloned by PCR techniques using the specific 5’ and 3’-end primers based on the DNA sequence of the chitosanase from B. amyloliquefaciens csn-UTK (Accession number AB0051574). The full-length chitosanase gene of B. amyloliquefaciens was 858 base pairs and encoded 286 amino acid residues containing the signal peptide of 36 amino acid residues on the N-terminals. The mature BamyCSN was calculated to have a molecular weight of 27.5 kDa. The recombinant chitosanase gene was expressed in E. coli BL21(DE3)pLysS. It was efficiently purified from the host cell lysate by His-tag affinity chromatography and was found enzymatically active. The optimal pH and temperature of BamyCSN was 7.0 and 45 ℃, respectively. Kinetic parameters Km and kcat using the water soluble chitosan as a substrate was 2.12 μg / μl and 2.25x10-5 s-1, respectively. Tween 20 at 1% concentration could stimulate BamyCSN slightly, while most metal ions inhibited the chitosanase activity except 1mM NH4+ activated BamyCSN. By TLC analysis, GlcN, (GlcN)2, (GlcN)3 and (GlcN)4 could be found in the soluble chitosan hydrolyzed by BamyCSN at 37 ℃for 16 h. The major product was (GlcN)4 .
以Bacillus amyloliquefaciens csn-UTK (Accession number AB0051574) 幾丁聚醣酶的基因序列設計B. amyloliquefaciens幾丁聚醣酶基因的5’端及3’端引子,以聚合酶鏈反應 (Polymerase Chain Reaction) 複製出B. amyloliquefaciens幾丁聚醣酶基因全長。 B. amyloliquefaciens幾丁聚醣酶基因全長858個鹼基對,可轉譯出286個胺基酸的蛋白質,其在氮端有36個胺基酸訊息胜肽(signal peptide),分子量為27.5 kDa。此基因於E. coli BL21(DE3)pLysS宿主中表現,以6xHis標記親合性層析可純化具有幾丁聚醣酶活性的重組幾丁聚醣酶。 B. amyloliquefaciens幾丁聚醣酶最適pH為7.0,最適作用溫度為45℃。以水溶性幾丁聚醣為受質,幾丁聚醣酶BamyCSN酵素動力學的Km值為2.12 μg / μl,kcat為2.25x10-5 s-1。1% Tween 20對BamyCSN有些微促進活性,金屬離子大都會抑制,但1 mM NH4+ 促進BamyCSN活性。以水溶性幾丁聚醣為受質,於37℃水解16小時,以薄層色層分析其水解產物有GlcN、(GlcN)2、(GlcN)3與(GlcN)4,主要產物為(GlcN)4。
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M91360007
http://ntour.ntou.edu.tw/ir/handle/987654321/17502
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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