High Soluble Expression of d-Amino Acid Oxidase in Escherichia coli Regulated by a Native Promoter

• DOI: 10.1007/s12010-008-8325-x
• Publication Date: 2008-08-11T12:54:08Z
• Authors: Yangqiu Liu; Qiang Li; Hongyu Zhu; Jichu Yang
• Keywords: D-Amino-Acid Oxidase; 0301 basic medicine; 0303 health sciences; 03 medical and health sciences; Fermentation; Escherichia coli; Gene Expression Regulation, Bacterial; Promoter Regions, Genetic; Amidohydrolases; 3. Good health

To express high-active soluble D-amino acid oxidase (DAAO), a constitutive plasmid that is regulated by a native hydantoinase promoter (P(Hase)), was constructed. A D-amino acid oxidase gene (dao) was ligated with the P(Hase) and cloned into pGEMKT to constitutively express protein of DAAO without the use of any inducer such as isopropyl beta-D-1-thiogalactopyranoside which is poisonous to the cells and environment. The ribosome binding site region, host strain, and fermentation conditions were optimized to increase the expression level. When cultivated in a 5-m(3) fermenter, the enzyme activity of JM105/pGEMKT-R-DAAO grown at 37 degrees C was found to be 32 U/mL and increase 16-fold over cells of BL21(DE3)/pET-DAAO grown at 28 degrees C. These results indicate the success of our approaches to overproducing DAAO in soluble form in Escherichia coli.