Rhodosporidium toruloides D型胺基酸氧化酵素基因在大腸桿菌中之表現

EXPRESSION OF A D-AMINO ACID OXIDEASE GENE FROM RHODOSPORIDIUM TORULOIDES IN ESCHERICHIA COLI

辜健一(Chien-yi Ku)

指導教授 ： 官宜靜

大同大學/工程學院/生物工程學系所/碩士(2005年)

摘要

D-型氨基酸氧化酵素( D-amino acid oxidase )為製造頭胞菌素類抗生素C (cephalosporins)之先驅物7-aminocephalosporanic acid (7-ACA)的重要酵素。以四種不同的表現載體pET23am、pET24am、pET20b和pET26b、兩種培養液LB和TB以及不同濃度誘導劑使Rhodospridum toruloides DAO cDNA基因於Escherichia coli strain BL-21(DE3)異源表現。以LB培養時，當pET23am為表現質體，誘導後3小時可測得胞內DAO最高活性4294 U/l;以pET24am為表現載體時，誘導後約5小時可測得最高胞內DAO活性為2724 U/l;以pET20b和pET26b為表現載體時，活性高峰出現在誘導後2小時，DAO活性分別為571和592 U/l。當培養液置換成TB時，DAO活性可提升5〜10倍。pET23am和pET24am為表現質體時，誘導後18小時有較高DAO胞內活性，分別為50592和39651 U/l;pET20b為表現質體時，誘導後5小時有較高DAO活性3241 U/l;pET26b為表現質體時，在誘導後8小時有較高胞內活性2393 U/l。

關鍵字

D-型氨基酸氧化酵素 ； 頭胞菌素類 ； 表現質體 ； 胞內活性

並列摘要

D-Amino acid oxidase (DAO) is an important enzyme used to produce 7-aminocephalosporanic acid (7-ACA), a starting material for the semi-synthesis of cephem antibiotics. Rhodosporidium toruloides DAO CDNA gene was heterlogously expressed in the E. coli strain BL-21(DE3), using four different expression vectors, pET23am, pET24am, pET20b and pET26b in combination with two media, LB and TB and varying concentration of IPTG. With LB medium and using pET23am as the expression vector, the highest intracellular DAO activity, 4294 U/l was obtained〜3 hours after induction with IPTG. When pET24am as the expression vector instead, the highest intracellular DAO activity, 2724 U/l was observed 5 hours after induction. With pET20b and pET26b as the expression vectors, the highest intracellular DAO activities, 571 and 592U/l, respectively appeared 2 hours after induction. As the culture medium replaced by TB, the DAO activity were increased 5-10 times. Using pET23am and pETET24am as the expression vectors, the higher DAO activity, 50592 and 39651U/l were observed 5 and 18 hours, respectively after induction. When pET24a and pET26b used instead, the higher DAO activity, 3241 and 2393U/l were obtained 18 and 8 hours, respectively after induction.

並列關鍵字

cephalosporins ； DAO ； expression vectors ； Rhodosporidium toruloides

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