重組果糖轉移酶的特性及功能分析

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Title:	重組果糖轉移酶的特性及功能分析 Characterization and Functional studies of recombinant fructosyltransferase
Authors:	謝宜祐
Contributors:	嘉南藥理科技大學：生物科技系暨研究所江建民
Keywords:	定點突變果糖轉移酶酵素動力學 Fructosyltransferases Enzyme kinetics Site-directed mutagenesis
Date:	2011
Issue Date:	2011-10-25 15:30:09 (UTC+8)
Abstract:	果糖轉移酵素是植物及微生物中重要的酵素，它能將蔗糖上的糖苷鍵水解，並將水解後的果糖轉移到其他受質上，以生產果寡糖或是聚果糖，是植物中調節生理機能的相關酵素，也是生技產業上生產果寡糖的重要酵素。這類酵素目前歸類在糖苷水解酶家族32 (glycoside hydrolase family 32, GH family 32) 之中，除了細菌來源的以外，從麴菌或是植物來的酵素都有不定量且不均勻的糖基化修飾，糖基化的修飾可能與酵素的穩定性有關，首先建立蛋白表現純化系統，並以高效液相層析HPLC及薄層色層分析TLC對於寡糖分子的檢測，這些方法使得我們可以快速判斷酵素的存在與否、分析酵素的各項性質。實驗室已從麴菌Aspergillus japonicus中選殖出果糖轉移酶基因，並轉殖於大腸桿菌tuner(DE3)中，此基因表達後具有生產果寡糖能力，純化水溶液區域的重組果糖轉移酶AjFT，進而分析酵素活性及特性。為取得酵素催化水解醣苷鍵所參與胺基酸，經由序列的比對，與其他β-fructofuranosidase水解型酵素結構對照，可參與催化糖苷鍵水解的胺基酸為D60、D191、E292，進行設計定點突變取得點圖變後之AjFT，並進行酵素活性分析。 Fructosyltransferases (SFTs) is the important enzyme of plant and microorganism. SFTs catalyze the transfer of a fructosyl moiety from sucrose to a water molecule (hydrolysis) or to an acceptor molecule (transferase), yielding fructooligosaccharides (FOS). We have cloned a fructosyltransferases gene from Aspergillus japonicus CB05, and expression this gene in Soluble in Escherichia coli. The recombinant AjFT was purified and its enzymatic characteristic and structural stability was analyzed. The results indicated the AjFT mainly act as sucrose:fructan fructosyltransferase. The optimal temperature of the transferring activity is 60℃ and the optimal pH 5.5. Sequence comparison of the AjFT gene indicated three conserved residues, Asp60, Asp191,and Glu292, proposed to play as nucleophile, intermediate stabilizer, and general acid/base, respectively. Site-directed mutagenesis were performed to obtain D60A, D191A, E292A,E292Q mutants. All the mutant lost their enzymatic activity and thus confirmed the role of these three residues.
Relation:	校內外均一年後公開，學年度：99，92頁
Appears in Collections:	[Dept. of Biotechnology (including master's program)] Dissertations and Theses

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