請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/21351
標題: Cloning and Expression Trigonopsis variabilis D-Amino Acid Oxidase in Saccharomyces cerevisiae and Escharichia coli
Trigonopsis variabilis D-型胺基酸氧化酵素基因之選殖及在 Saccharomyces cerevisiae 和 Escherichia coli 之表現
作者: Hwang, Tzann-Shing
黃贊勳
關鍵字: D-型胺基酸氧化酵素
選殖
三角變異酵母菌
大腸桿菌
麵包酵母菌
表現
D-amino acid oxidase
cloning
Trigonopsis variabilis
E.coli
出版社: 分子生物研究所
摘要: D-amino acid oxidase (DAO, EC 1.4.3.3) 是工業上用於生產頭孢菌素 類抗生素先驅物的重要酵素。Trigonopsis variabilis DAO 是一醣蛋白 ,由兩個相同的次單位 (subunit) 組成,且需 FAD才具有活性。T. variabilis dao 基因長 1107 鹼基對,並包含有一內子 (intron) 位於 第 25 到第 60 個鹼基對之間。 T.variabilis CCRC 21509 的 dao 染色 體基因的 open reading frame 已被選殖至 Saccharomyces cerevisiae 的表現載體上。攜帶此 dao 染色體基因的 S.cerevisiae 測不到 DAO 的 活性。利用逆錄酵素-聚合酵素連鎖反應 (reverse transcriptase- polymerase chain reaction),直接從 T.variabilis 的總 RNA (total RNA) 將 dao cDNA 基因複製出來,接於質體 pTrc99A 的 Ptac 啟動子後 面。含有此 dao cDNA 基因的 E.coli,在 IPTG 的誘導下,可表現 DAO 的活性。在 E.coli 中可測得的 DAO 活性最高為 0.85 DU/mg,為在 D- alanine 誘導下的 T.variabilis 的 DAO 活性的 18 倍,為在沒 D- alanine 誘導下的 T.variabilis 的 DAO 活性的 30 倍。同時亦發現當 dao cDNA 基因在 E.coli 中表現時, E.coli 的生長不良。由於 Ceph C 及其衍生物,都會被 beta-lactamase 分解。於是將 pTrc99A 接上 kanamycin 的抗藥性基因, 並移除 ampicillin的抗藥性基因, 構築成質 體 pKmA 及 pKmB。將最高表現的 dao cDNA基因選殖入 pKmA,得到重組 質體 pKm-DAO。以高效液相色層分析儀 (HPLC) 分析 pKm-DAO 轉形菌體 的粗萃液中的 DAO 活性, 發現在 IPTG 的誘導下,可測得的 DAO 活性 ,最高可達 0.75 CU/mg。將 dao cDNA 基因選殖至 S.cerevisiae 的表 現載體 pAAH5,得到重組質體 pAAH-cDAO。攜帶 pAAH-cDAO 的 S. cerevisiae,其 DAO 活性達 0.28 DU/mg。以 SDS-PAGE分析 E.coli 及 S.cerevisiae 所生產的 DAO,估計所產生的 DAO 分子量大小約 39.3 KDa。以 native PAGE 配合活性染色法,分析被 dao cDNA 基因轉形的 E.coli 及 S.cerevisiae 的粗萃液,皆在相同位置呈現出 DAO的色帶。 活性染色時,若以 1N NaOH 終止呈色反應,可得到較為典型的色帶。 D-amino acid oxidase (DAO, EC 1.4.3.3) is an important enzyme for producing the precursor of cephem antibiotics in industry. The DAO from Trigonopsis variabilis is a glyco- protein. It consists of two identical subunits and needs FAD for its function. The T.variabilis dao gene is 1,107 bp long and contains an intron between positions 25 to 60. The open reading frame of genomic dao gene from T.variabilis CCRC 21509 was cloned into the expression vector of Saccharomyces cerevisiae. The S.cerevisiae harboring genomic dao gene could not show DAO activity. The dao cDNA gene was prepared by reverse transcriptase- polymerase chain reaction and cloned into Escherichia coli expression vector, pTrc99A, under the control of Ptac promoter. The daocDNA gene could be expressed in E.coli by the IPTG induction and the highest DAO activity was 0.85 DU/ mg. It was about 18-fold higher than that of D-alanine- induced T.variabilis and about 30-fold higher than that of uninduced T. variabilis. It was also found that E.coli could not grow well under the expression of dao cDNA gene, even at basal level expression. Cephalosporin C and its deviratives are sensitive to beta-lactamase. Therefore, the beta-lactamase gene must be removed from the cloning vector used in the genetic manipulation of dao gene. An E.coli expression vector, designated pKm, which uses kanamycin-resistant determinant instead of ampicillin-resistant determinant as selective marker was constructed. The dao cDNA gene was cloned into pKmA. The DAO activity in pKm-DAO-transformed E.coli was about 0.75 CU/mg by IPTG induction. When the dao cDNA gene was cloned into the S. cerevisiae expression vector pAAH5, the DAO activity of S. cerevisiae was 0.28 DU/mg. The molecular weights of the DAO from transformed E.coli and S.cereisiae were estimated about 39.3 KDa by SDS-PAGE analysis. In activity staining, both DAOs had same migration pattern. The banding pattern would be sharp, when 1N NaOH was used to terminate chromogenic reaction.
URI: http://hdl.handle.net/11455/21351
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