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研究生:許志行
研究生(外文):Jyh-Shing Hsu
論文名稱:新型擴環酵素基因篩檢及其對penicillinG的定向演化
論文名稱(外文):Screening new expandase gene from actinomycetes and using enzyme directed evolution to increase substrate specificity to penicillin G
指導教授:蔡英傑蔡英傑引用關係
指導教授(外文):Ying-Chieh Tsai
學位類別:博士
校院名稱:國立陽明大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:107
中文關鍵詞:擴環酵素定向演化
外文關鍵詞:DAOCSdirected evolution
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Streptomyces clavuligerus之deacetoxycephalosporin C synthase (DAOCS,expandase,俗稱擴環酵素) 是製藥工業上重要酵素之一,可將penicillin G 擴環成7-aminodeacetoxycephalosporanic acid (一種頭孢菌素原料中間物),此酵素所對應的基因稱為cefE。為了增進擴環酵素對penicillin G的基質特異性,我們使用8種擴環同源酵素以DNA重組法 (DNA shuffling technique) 對此酵素進行定向演化 (directed evolution)。
經大腸桿菌ESS生物檢測,在第一次DNA重組中篩得4個對penicillin G 有較高活性的突變酵素 (chimera)。進一步的以這4個突變酵素進行第二次的DNA重組,結果篩得20個活性明顯提升的突變酵素。選取其中2個不受基質濃度增加而活性下降 (substrate inhibition) 的突變酵素 (FF1及FF8) 測定其動力參數,結果比S. clavuligerus擴環酵素對penicillin G的kcat/Km 參數分別增加了8.5及118倍。
我們綜合evolved chimeras與擴環酵素晶體結構,嘗試找出是哪些可能胺基酸造成kcat/Km的加強。FF8的V246相對於S. clavuligerus擴環酵素有較佳的立體補償 (steric complementarity) 及疏水的結合 (hydrophobic packing) ,因而可能使penicillin G更容易的進入基質結合區。
Lloyd等人由擴環酵素晶體結構及雷射光散射 (dynamic laser light scattering) 實驗推論擴環酵素在溶液中三連體與單體是處於某種平衡的狀態且認為以單體形式存在的擴環酵素才是具有活性。然而從我的超高速分析離心實驗 (analytical ultracentrifugatrion) 數據顯示擴環酵素是以單體存在的。
總結,我們選取同源擴環酵素基因進行對penicillin G專一性的定向演化,並成功的獲得一株kcat/Km比S. clavuligerus 擴環酵素增加118倍的重組擴環酵素,FF8。我們的研究顯示了擴環酵素具有極佳的可塑性,重複進行重組實驗,應可得到活性更高的突變株。
Deacetoxycephalosporin C synthase (expandase) from Streptomyces clavuligerus, encoded by cefE, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G. To improve the substrate specificity to penicillin G, eight cefE homologous genes were directly evolved using the DNA shuffling technique.
After the first round of shuffling and screening using an E. coli ESS bioassay, four chimeras with higher activity were subjected to a second round. Subsequently, twenty clones were found with significantly enhanced activity. The kinetic parameters of two isolates that lack substrate inhibition, showed an 8.5- and a 118-fold increase in the kcat/Km ratio compared to the S. clavuligerus expandase. The 118-fold increased clone, FF8, is the most active obtained to date anywhere.
To identify the putative residues involved in kcat/Km increase, various among different expandases and evolved chimeras were mapped on the crystal structure. Substitution residue A246 with valine in FF8 might result in better complementarity and hydrophobic packing, and therefore enhance penicillin G binding the substrate site.
Based on the crystal structure and dynamic laser light scattering, Lloyd et. al. proposed that in solution apo-DAOCS exists as an equilibrium mixture of monomeric and trimeric forms and implied the active form is the monomeric form. According the analytical ultracentrifugatrion, however, we proved that expandase exist as monomeric form.
In summary, we successfully obtained a 118-fold increased clone, FF8, and our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more active chimeras might be achievable with further rounds.
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