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

• Main Authors: Wen-Hau Li, 李文華
• Other Authors: I-Ching Kuan
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/45914277085325831593

碩士 === 大同大學 === 生物工程學系(所) === 93 === 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.