title: High soluble expression of D-amino acid oxidase in Escherichia coli regulated by a native promoter.
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.
authors.affiliation: Department of Chemical Engineering, Institute of Biochemical Engineering, Tsinghua University, Beijing 100084, People's Republic of China.
authors.forename: Yangqiu
authors.initials: Y
authors.lastname: Liu
authors.forename: Qiang
authors.initials: Q
authors.lastname: Li
authors.forename: Hongyu
authors.initials: H
authors.lastname: Zhu
authors.forename: Jichu
authors.initials: J
authors.lastname: Yang
mesh_terms: D000581:Amidohydrolases / Q000235:genetics; D003605:D-Amino-Acid Oxidase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D011401:Promoter Regions, Genetic / Q000235:genetics*
chemical_list: D003605:D-Amino-Acid Oxidase; D000581:Amidohydrolases; C020047:dihydropyrimidinase