- 學位論文
Pseudomonas putida GPo1聚羥基烷酸酯合成酶中可改變基質專一性的胺基酸位置之研究
Identification of the amino acid residues that change the substrate specificity of PHA synthase 1 from Pseudomonas putida GPo1
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摘要
本實驗室先前藉由蛋白質工程－區域性半隨機性突變方式(localized semirandom mutagenesis)引入十四個突變點，成左滷NPseudomonas putida Gpo1聚羥基烷酸酯酵素(PHA synthase1；Class II)的酵素基質專一性拓展開來(Sheu and Lee , 2004)。本實驗的目的是藉由建立定點突變的技術，配合廣宿主共表現載體分析平台，轉型入PHA累積菌株Pseudomonas putida GPp104，以礦物鹽培養基加入辛酸鹽(octanoate)為碳源，累積PHA後進行菌體乾燥，利用甲醇分解法將PHA分解為單體，利用氣相層析儀分析各突變株之PHA累積產量與單體組成。藉由這些分析步驟與累積資訊，確認可改變本酵素之基質專一性的胺基酸位置。目前的研究結果指出，在PHA synthase 1中，突變單一位置(L484V)即可將PHA四個碳單體(3-hydroxybutyrate)的組成莫耳分率由野生型酵素的8±2.9 mol﹪顯著提升至36±5.1 mol﹪，為PS-E1中改變基質專一性最重要的點。另一方面，本實驗亦參考文獻引入其他突變點進行分析，發現S325C之突變株可將四個碳單體比率提升至39±2.5﹪mol，S325C/Q481M雙突變點則提升至47±5.0 mol﹪，與PS-E1(48±2.9 mol﹪)相當接近。因此，484與325為影響酵素基質專一性重要的胺基酸位置。在酵素活性方面，S482G、Q481M與A547V等突變均能顯著提高PHA產率為野生株之2-4倍，目前本研究將結合這些突變點，引入低活性但廣基質專一性的突變株作進一步分析。此外，也針對可改變基質專一性的484位置與其所在區域進行飽和突變分析，並將這些與活性及基質專一性相關的突變點，引入class I的PHB synthase作進一步的探討。
並列摘要
In our previously study, the substrate specificity of polyhydroxyalkanoate (PHA) synthase 1 (class II) from Pseudomonas putida GPo1 was successfully altered by localized semi-random mutagenesis. The evolved enzyme PS-E1, in which 14 amino acids had been changed exhibited broad substrate specificity. The promising amino acid positions which change the substrate specificity of PhaC1Pp were investigated by site directed mutagenesis. Mutations at L484V or S325C were remarkably enhanced the short-chain-length monomer composition up to 40 mol% in PHA accumulation experiment. Simultaneously mutated at S325C and Q481M of PhaC1Pp lead to scl monomer composition further enhanced up to 50 mol%. Furthermore, mutations at S482G, Q481M and A547V would increase PHA yields from 10 mol﹪(wild type ) to 20%, 35%, and 37%, respectively. Saturation mutagenesis experiment will be applied to further acquiring more information about the substrate specificity on PHA synthase 1.
參考文獻
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