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研究生:陳麗玲
研究生(外文):Li-Lin Chen
論文名稱:利用基因轉殖與突變技術以改善Rhodosporidiumtoruloides之D-胺基酸氧化酵素活性及分泌特性
論文名稱(外文):Cloning and Mutagenesis of D-amino acid oxidase (DAO) gene from Rhodosporidium toruloides
指導教授:蔡國珍陳任道
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:食品科學系碩士在職專班
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:61
中文關鍵詞:基因轉殖突變
外文關鍵詞:error-prone PCR
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中文摘要
D-amino acid oxidase (DAO)在工業上可應用於生產 -keto acid、L-型胺基酸及半合成Cephalosporin C系抗生素,Rhodosporidium toruloides 生產之DAO屬於胞內酵素,利用物理或化學方法處理菌體,不僅費時耗力還會損失部份酵素。本研究運用重組DNA技術,將外來之amylopullulanase gene的C端重覆區序列嵌入 R. toruloides DAO cDNA中,使E.coli XL1 blue 能穩定表現DAO基因,並改變R. toruloides DAO之分泌特性將DAO分泌到胞外,另利用error-prone PCR (ep PCR) 技術進行突變,以期能增加轉形株之DAO活性。
本研究中構築了兩種重組質體,分別命名為pBDAO3及pBDAOR。質體pBDAO3的構築是利用pBluescript II SK+當作載體,其上含有R. toruloides 的DAO cDNA基因。為了使DAO分泌至胞外,質體所含之amylopullulanase gene的C端重覆區序列以 PCR進行擴增,再將此286 bp片段與載體連結,構築成質體pBDAOR。
將質體 pBDAOR 轉形入E. coli XL1 Blue,經IPTG誘導後進行SDS-PAGE,於膠片上確實可看到DAO 之條帶 (band) 存在。進一步以細胞分劃法 (cell fractionation) 估計,發現DAO酵素有 39% 已由胞內改而為胞外分泌。為了增加胞外之 DAO 活性,利用ep PCR技術進行突變,epPCR之最佳條件,為增加dATP, dCTP及dTTP之濃度至 1.0 mM,增加MgCl2濃度至5 mM,添加0.5 mM MnCl2 及使用3 U Taq polymerase之用量可得到最佳之產物,故利用此條件進行高突變株之篩選,得到之突變株在 37℃反應之 DAO活性較母株E. coli XL1 Blue (pBDAOR) 提高了約20倍。在胺基酸序列比對之結果,突變株 epC11-143 之 Pro-120突變為 Ala-120,推測此位置Ala-120與 DAO 之活性部位有關,所以使epC11-143 喪失了分泌至胞外之 DAO活性。
Abstract
D-amino acid oxidase (DAO) is widely used in industry to produce -keto acid, L- amino acids, and semisynthetic cephalosporin C class antibiotics. Physical and chemical treatments on Rhodosporidium toruloides DAO are not very efficient and will also lose part of the enzyme. Recombinant DNA technique was used in this work to ligate the C-terminal repeat sequence of amylopullulanase gene into R. toruloides cDNA, which induced E. coli XL1 blue to stably express DAO gene and diffuse genetic products extracelluarly for altering the characteristics of R. toruloides DAO. Error-prone PCR (epPCR) technique was also used to increase DAO activity of the transformants through mutation.
Two plasmids were constructed, noted as pBDAO3 and pBDAOR. The pBDAO3 plasmid was built with pBluescript II SK+ as vector, which contains DAO cDNA gene of R. toruloides, to transform pBDAO3 into E. coli DH5,BL21 [DE3] 及 XL1 Blue. DAO gene can express on these plasmids. In order to secrete DAO outside of the cell, PCR was used to amplify the C-terminal repeat sequence of amylopullulanse, and the 286 bp segment was then ligate into the vector to construct the pBDAOR plasmid.
To verify the existence of DAO, transformant was produced by transforming pBDAOR plasmid into E. coli XL1 Blue. DAO protein band was observed on gel prepared by inducing the transformant with IPTG then through SDS-PAGE process. Furthermore, estimated by a cell fractionation method, 39% of the DAO enzyme has changed from intracellular to extracelluar secretion. To increase extracellular activity of DAO, epPCR method was used to screen mutation, and the optimal screening conditions were determined to be 0.2 mM dGTP, 1.0 mM dATP, dCTP and dTTP, 5 mM MgCl2, 0.5 mM MnCl2, and with 3U Taq polymerase. Under the optimal conditions, the DAO activity was increased by a factor of 20 for the transformant reacted at 37 C than that of the original E. coli XL1 blue pBDAOR. Sequencing analysis of these mutants indicated the occurrence of mutant at Pro-120, it is suggested that the residue may play essential structural role in DAO conformation, thereby influencing the extracellular activity of DAO.
目 錄
中文摘要………………………………………………………………… I
Abstract …………………………………………………………………. II
壹、 緒言………………………………………………………………... 1
貳、 文獻整理…………………………………………………………... 2
參、材料與方法
一、材料…………………………………………………………… 14
(1) 菌株與質體……………………………………………….. 14
(2) 藥品………………………………………………………. 14
(3) 培養基及染劑 …………………………………………. 15
二、菌株生長條件………………………………………………… 16
三、重組質體之構築……………………………………………… 16
(1) 質體抽取…………………………………………………. 16
(2) 限制的切割作用………………………………………. 17
(3) DNA回收………………………………………………… 17
(4) DNA的連結……………………………………………… 18
(5) 勝任細胞之製備與轉形…………………………………. 18
四、酵素活性測定………………………………………………... 19
(1) D-胺基酸氧化的活性分析……………………………. 19
(2) Malate dehydrogenase 的活性分析……………………… 20
(3) Alkaline phosphatase 的活性分析……………………….. 20
五、轉形菌株之篩選……………………………………………… 21
(1) 藍白篩選…………………………………………………. 21
(2) 以colony PCR 篩選接合成功之轉形株……………….. 21
六、細胞分劃法…………………………………………………… 22
七、SDS-聚丙烯銨凝膠電泳分析………………………………... 22
八、error-prone PCR………………………………………………. 23
(1) epPCR反應條件之探討…………………………………... 23 (2) epPCR 最適反應條件…………………………………….. 23
九、DAO突變株的篩選………………………………………….. 24
十、序列分析……………………………………………………… 25
肆、結果 一、pBDAO3之構築與表現………………………………………26
二、pBDAOR 之構築與表現…………………………………….. 26
三、轉形株之DAO活性分析……………………………………. 27
四、利用ep PCR技術提高DAO活性…………………………... 28
(1) ep PCR條件的探討…………………………………………. 28
(2) 利用ep PCR構築任意突變 DAO 之質體……………….. 29
(3) 高活性株之篩選……………………………………………. 29
伍、討論………………………………………………………………… 31
陸、結論………………………………………………………………… 35
柒、參考文獻…………………………………………………………… 36
捌、圖表………………………………………………………………… 46
表 次
表一、本研究所使用之菌株與質體…………………………………… 46
表二、PCR 反應使用之引子組……………………………………….. 47
表三、E.coli (pBDAO3) 及 E.coli (pBDAOR) 之 DAO分佈情形…. 48
表四、Error-prone PCR 最適條件的探討………………………..…... 49
表五、epPCR突變株之DAO活性…………………………………….50
圖 次
圖一、DAO 的反應機制……………………………………………… 12
圖二、7-ACA的生產機制…………………………………………….. 13
圖三、pBDAO3質體之構築流程圖…………………………………… 51
圖四、pBDAOR質體之構築流程圖…………………………………... 52
圖五、利用Proofreading PCR 擴增 pBTAP13/EcoR V 的 C 端重覆
區序列產物的電泳圖…………………………………………… 53
圖六、利用Proofreading PCR 擴增 pBTAP13/EcoR的C端重覆區序列
產物的序列……………………………………………………… 54
圖七、以colony PCR篩選轉形株之電泳圖……………………………. 55
圖八、pBTAP13/EcoR V 之 C 端重覆區序列與 pBDAOR 之核
酸序列相似度比較……………………………………………… 56
圖九、E. coli XL1 Blue (pBDAO3) 及 E. coli XL1 Blue (pBDAOR) 的
蛋白質電泳圖…………………………………………………… 57
圖十、error-prone PCR 產物電泳圖…………………………………... 58
圖十一、pBDAO3質體 Hind III/EcoR I片段進行error-prone PCR 之策
略……………………………………………………………… 59
圖十二、利用 96 孔平盤篩選具胞外DAO之高活性突變株………... 60
圖十三、突變株 epC11-143與 R. toruloides DAO cDNA之核酸序
列相似度……………………………………………………… 61
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