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

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
DOI: 10.1007/s12010-008-8325-x Publication Date: 2008-08-11T12:54:08Z
ABSTRACT
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.
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