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Biochemical characterizations of C-terminal truncated chitinases from Aeromonas caviae D1 ChiA
|Authors: ||Chia-Yu Hsieh|
|Contributors: ||NTOU:Institute of Bioscience and Biotechnology|
|Issue Date: ||2011-07-04
|Abstract: ||為探討豚鼠氣單胞菌幾丁質□(Ac. D1 ChiA)碳端對酵素活性所扮演的角色，以胰蛋白□及胰凝乳蛋白□對Ac. D1 ChiA進行蛋白質切割，結果產生有活性的小分子蛋白質Ac. D1 ChiA-Try62 K 及 Ac. D1 ChiA-Try59 K。 蛋白質氮端定序及LC-MS-MS分子量分析，找出可能的碳端位置，以聚合□連鎖反應選殖這些幾丁質□衍生小分子於表現載體pET 20b(+)上，並於E. coli Rosetta(DE3)pLys S中表現其蛋白質。以His-Tag affinity column純化後，所得的蛋白質分子大小分別為63.6 kDa及59.8 kDa，再進行與原始分子Ac. D1 ChiA及另一具活性小分子Ac. D1 ChiA-G561之生化特性比較。 生化特性分析包括最適pH與溫度、螢光掃描、酵素動力學分析、受質結合能力分析及受質水解能力分析。結果顯示以可溶性幾丁質為受質時，Ac. D1 ChiA與其衍生物分子Ac. D1 ChiA-Try 62 K , Ac. D1 ChiA-Try 59 K , Ac. D1 ChiA-G561間之最適pH、最適溫度並無顯著差異；但對非可溶性幾丁質之受質，Ac. D1 ChiA所衍生出之小分子蛋白質其幾丁質結合與水解能力隨著修剪幅度增加有顯著的下降。 然而，Ac. D1 ChiA-G561仍保有對非可溶性幾丁質部分結合能力及水解能力，顯示Ac. D1 ChiA之碳端區域與氮端區中的某些芳香族胺基酸有相輔相成的交互作用存在。故此分子的碳端區域角色仍需進一步研究。|
To study the role of the C-terminal domain of Ac. D1 ChiA in the enzymatic hydrolysis of chitin, a systematic C-terminal truncation of Ac. D1 ChiA was approached by proteases digestion. Several smaller derivatives of Ac. D1 ChiA were enzymatically active after SDS-PAGE and zymogram analyses. Both N- and C-termini of these smaller derivatives of Ac. D1 ChiA molecule were determined by N-terminal amino acid sequencing and LC-MS-MS analysis. These C-terminal truncated molecules of Ac. D1 ChiA were cloned by PCR techniques into the expression vector pET 20b(+) and were expressed from E coli. Rosetta(DE3)pLys S. The His-tag affinity column purified recombinant Ac. D1 ChiA-Try 62 K and Ac. D1 ChiA-Try 59 K had the apparent MW’s of 63.6 and 59.8 kDa, respectively. The Ac. D1 ChiA-Try 59 K gene encoded in 1638 bp DNA with 546 amino acid open reading frame was 7 amino acids larger than Ac. D1 ChiA-G561 which was previously found the smallest and enzymatically active molecule of Ac. D1 ChiA in the laboratory. Cloning and biochemical characterizations of Ac. D1 ChiA-Try 59 K were performed. Results obtained from comparison studies of optimum pH and temperature, fluorescence scanning, kinetics analysis, chitin-binding ability and hydrolysis efficiency among Ac. D1 ChiA and its C-terminal truncated derivatives including Ac. D1 ChiA-Try 62 K and Ac. D1 ChiA-G561 molecules indicated that the more extent of C-terminal delection of Ac. D1 ChiA the larger decrease of chitin binding ability and hydrolyzing effeciency toward the insoluble chitin substrate. However, there was some residual enzyme activity and hydrolyzing ability remained in Ac. D1 ChiA-G561 molecule suggesting a some kind of synergistic hydrophobic interactions between the C-terminal domain and the N-terminal aromatic amino acid residues could be existed. Therefore, the role of the C-terminal domain of Ac. D1 ChiA needs to be further investigated.
|Appears in Collections:||[生命科學暨生物科技學系] 博碩士論文|
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