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研究生:林淑梅
研究生(外文):Shu-Mei Lin
論文名稱:用蛋白質工程的方法改良去乙醯頭孢菌素C羥化脢對去乙醯頭孢菌素G的生產
論文名稱(外文):Protein engineering of deacetylcephalosporin C synthase: to improve the substrate specificity towards deacetoxycephalosporin G
指導教授:呂平江
指導教授(外文):Ping-Chiang Lyu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:65
中文關鍵詞:酵素動力學測量任意突變頭孢菌素C
外文關鍵詞:kcat/kmcephalosporinrandom mutagenesisDAOG/DAG
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頭孢菌素之抗菌機制為抑制細菌的細胞壁合成,因其更具廣效性及較少抗藥株,在臨床上的應用非常廣泛;目前的主要生產方法為先使用工業發酵生產前驅物,再經過數個修飾化學官能基的步驟,而得到具藥效的最終產物。因化學合成反應不若生物反應專一,且又有昂貴、較污染等缺點,故如何更加經濟地生產頭孢菌素類抗生素成為現今重要的課題。
DACS為一種需要2-oxoglutarate及二價鐵催化的氧化酵素,來源為放線菌 Streptomyces clavuligerus,對應基因為 cefF,在頭孢菌素C的生合成途徑中,參與催化 DAOC 生成 DAC 的反應。本篇論文的目的在利用蛋白質工程的方法,提高DACS對非天然受質 DAOG 的產物轉換率,以更有效率的方式生成抗生素前驅物 DAG。
我們先使用單點突變的方法產生F268A、R270L、A185Y、V279I、V307A、T308A、T308K、M309L和H310L 等突變株, 但其中只有T308A的催化活性有些微改進。之後,我們利用任意突變,例如加入化學突變劑、合成不特定引子用於單點突變或擾亂聚合脢連續反應等等方法製造大量突變株,先利用反應物和產物毒性強弱的差別,初步篩選其中約 6600 株菌至490株,再個別做小量活性測試,挑出生成產物量比野生株高的 69株,經進一步定序後,確定最佳的突變株分別是:A80G、S218D及A57V。並針對其中活性最好的 A57V,同一點位置再突變成賴胺酸(Lys)、白胺酸(Leu)、異白胺酸(Ile)、麩胺酸(Glu) 和甘胺酸(Gly)。我們純化野生株及這幾株表現較好的酵素,做進一步更精確的動力學分析,發現其中最好的A57E (KM = 0.89±0.02mM, kcat/ KM = 2.59±0.12/Msec) 酵素活性有野生株 (KM = 3.09±0.60mM, kcat/ KM = 1.39±0.29/Msec) 的百分之一百八十六。這項結果顯示從任意突變挑到的位置結合單點突變的策略之可行性。未來甚至可以結合數個有意義的單點突變,以達到提高催化活性的最終目標。

Medicinally useful cephalosporins are produced by fermentation products with further modifications. Although cephalosporins are superior antibiotics compared with penicillins, their distribution is limited because the processes for further chemical modifications are complicated and costly.
DACS, a nonheme-iron(II) and 2-oxoglutarate-dependent oxygenase, catalyzes the 3’-hydroxylation of DAOC in the biosynthesis of cephalosporins. It can convert its natural substrate analogue DAOG to DAG with relatively low yield. The goal of this study is to raise the accessibility of DACS to DAOG via protein engineering techniques.
We began to design mutations via site-directed mutagenesis at the residues near the binding pocket or C terminal, including F268A, R270L, A185Y, V279I, V307A, T308A, T308K, M309L and H310L. Only T308A showed slightly higher activity. Therefore, we applied random mutagenesis such as chemical mutagenesis, site-directed random mutagenesis, and error-prone PCR. After screening 6600 transformants by several steps, we obtained 69 mutants and sequenced them. The three mutants S218D, A80G and A57V showed the highest activities. Furthermore, the A57 was mutated to K, I, E, L and G. Kinetic parameters of the recombinant DACSs were measured by assays under the optimized condition. The enzyme product of the A57E mutant showed the highest activity (kcat/KM = 2.59±0.12/Msec) which was about 1.86 fold of that of the wild type (kcat/KM = 1.39±0.29/Msec). The data showed that the well-developed screening methods could successfully help us to find the desired mutants derived from random mutagenesis and further manipulations at those residues may improve enzymatic activities.

Abstract............... .................I
Abstract (Chinese)..................... II
Acknowledgement........................III
Acknowledgement (Chinese)...............IV
Abbreviations...........................VI
Introduction...........................1-9
Materials and Methods................10-20
Results and Discussion...............21-51
Conclusion..............................52
Appendix.............................53-54
References...........................55-59

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