High Soluble Expression of <Emphasis Type="SmallCaps">d</Emphasis>-Amino Acid Oxidase in <Emphasis Type="Italic">Escherichia coli</Emphasis> Regulated by a Native Promoter |
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Authors: | Yangqiu Liu Qiang Li Hongyu Zhu Jichu Yang |
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Institution: | (1) Department of Chemical Engineering, Institute of Biochemical Engineering, Tsinghua University, Beijing, 100084, People’s Republic of China |
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Abstract: | To express high-active soluble d-amino acid oxidase (DAAO), a constitutive plasmid that is regulated by a native hydantoinase promoter (PHase), was constructed. A d-amino acid oxidase gene (dao) was ligated with the PHase and cloned into pGEMKT to constitutively express protein of DAAO without the use of any inducer such as isopropyl β-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-m3 fermenter, the enzyme activity of JM105/pGEMKT-R-DAAO grown at 37 °C was found to be 32 U/mL and increase 16-fold over cells
of BL21(DE3)/pET-DAAO grown at 28 °C. These results indicate the success of our approaches to overproducing DAAO in soluble
form in Escherichia coli. |
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Keywords: | d-Amino acid oxidase" target="_blank">d-Amino acid oxidase Hydantoinase promoter Constitutive expression Ribosome binding site Fermentation |
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