D型胺基酸氧化酵素在大腸桿菌異源表現的最適化

OPTIMIZATION OF HETEROLOGOUS EXPRESSION OF D-AMINO ACID OXIDASE GENE IN ESCHERICHIA COLI

李文華(Wen-Hau Li)

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-amino acid oxidase (DAO)是一種以flavin adenine dinucleotide (FAD)為輔基的flavoenzyme，為製造頭孢菌素類抗生素(cephalosporins)的之先驅物 7- aminocephalosporanic acid的重要酵素。
本研究以帶有Yeast Rhodosporidium toruloides或Trigonopsis
variabilis DAO cDNA基因之重組質體的Escherichia coli strain
BL21(DE3)，利用不同培養條件來生產DAO。R. toruloides DAO於TB之甘油濃度為1.6% 時，可有較佳活性為73 U/ml；T. variabilis DAO於甘油濃度為1.2% 時，可有較佳活性達89.7U/ml。於LB培養液中添加sorbitol和glycyl betaine，可使R. toruloides DAO活性增為17.7 U/ml，為不添加時的2.8倍；T. variabilis DAO活性增為20.4 U/ml，為不添加時的3.2倍。於TB培養液中添加sorbitol和glycyl betaine，則未能更加提升活性。當以發酵槽培養表現R. toruloides DAO，於誘導後第12小時開始控制pH = 6.8，可於誘導後約22小時獲得較佳活性50.1 U/ml，而T. variabilis DAO則於誘導後第28小時控制pH = 7.0，可於誘導後36小時達到最佳活性79.7 U/ml。

關鍵字

D型胺基酸氧化酵素 ； 異源表現 ； 大腸桿菌

並列摘要

D-amino acid oxidase (DAO), a flavoenzyme, using flavin adenine dinucleotide (FAD) as its prosthetic group is a key enzyme for the production of 7-aminocephalosporanic acid which is the precursor for synthetic cephalosporins.
In this study, Rhodosporidium toruloides or Trigonopsis variabilis DAO was expressed under different culture conditions by Escherichia coli strain BL21 (DE3) from their cDNA genes. The higher R. toruloides DAO activity of 73 U/ml was obtained using TB containing 1.6% glycerol while the higher T. variabilis DAO activity of 89.7 U/ml was produced using TB containing 1.2% glycerol. The R. toruloides and T. variabilis DAO activities in LB supplemented with sorbitol and betaine were increased 2.8 and 3.2 folds, respectively, in comparsion with those in LB. However, the supplement of sorbitol and betaine did not improve DAO activities in TB. When pH kept constant at 6.8 after induction with IPTG 12 hours, the higher R. toruloides DAO activity of 50.1 U/ml was obtained 22 hours after induction. And the higher T. variabilis DAO activity of 79.7 U/ml was obtained 36 hours after induction when pH kept at 7.0 after induction with IPTG 28 hours.

並列關鍵字

D-amino acid oxidase ； Rhodosporidium torulides ； Trigonopsis variabilis

參考文獻

4. Alonso, J., J. L. Barredo, B. Dze, E. Mellado, F. Salto, J. L. Garca, and E. Corts. 1998. D-amino acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217. Microbiology. 144：1095-1101

6. Bakke, M. and N. Kajiyama. 2004. Improvement in thermal stability and substrate binding of pig kidney D-amino acid oxidase by chemical modification. Appl. Biochem. Biotechnol. 112: 123-131.

7. Benz, F., M. Liersch, J. Nesch and H. J. Treichler. 1971. Methionine metabolism and cephalosporin C synthesis in Cephalosporium acremonium D-amino acid oxidase. Eur. J. Biochem. 20：81-88.

8. Berg, C. P., and F. A. Rodden. 1976. Purification of D-amino acid oxidase from Trigonopsis variabilis. Anal. Biochem. 71：214-222

9. Betancor, L., A. Hidalgo, G. Fernandez-Lorente, C. Mateo, V. Rodriguez, M. Fuentes, F. Lopez-Gallego, R. Fernandez-Lafuente and J. M. Guisan. 2003. Use of phycicochemical tools to determine the choice of optimal enzyme: stabilization of D-amino acid oxidase. Biotechnol. Prog. 19: 784-788.