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

摘要

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 A degrees C was found to be 32 U/mL and increase 16-fold over cells of BL21(DE3)/pET-DAAO grown at 28 A degrees C. These results indicate the success of our approaches to overproducing DAAO in soluble form in Escherichia coli.