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  • Title: High soluble expression of D-amino acid oxidase in Escherichia coli regulated by a native promoter.
    Author: Liu Y, Li Q, Zhu H, Yang J.
    Journal: Appl Biochem Biotechnol; 2009 Aug; 158(2):313-22. PubMed ID: 18696261.
    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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