title: High-level soluble and functional expression of Trigonopsis variabilis D-amino acid oxidase in Escherichia coli.
abstract: D-Amino acid oxidase is an important biocatalyst used in a variety of fields, and its economically justified level recombinant expression in Escherichia coli has not been established. To accomplish this, after a single Phe54Tyr substitution, fusion proteins of D-amino acid oxidase from Trigonopsis variabilis (TvDAO) with 6 × His-tags were constructed and expressed in E. coli. The effects of his-tags fusing position were revealed. Significant increase in holoenzyme percent and protein solubility made N-terminus tagged TvDAO (termed NHDAO) a suitable choice for TvDAO production. However, reduced cell growth and protein production rates were also observed for the NHDAO bearing strains. To optimize the performance of NHDAO production, changes of culture medium were tested. Finally, a production of 140 U/mL or 3.48 g active enzyme per liter which accounted for 41.4 % of the total protein, and a specific activity of 16.68 U/mg for the crude extract, were achieved in a 3.7 L fermenter in 28.5 h. This indicated a possibility for functional and economical TvDAO expression in E. coli to meet the industrial need.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
authors.forename: Senwen
authors.initials: S
authors.lastname: Deng
authors.forename: Erzheng
authors.initials: E
authors.lastname: Su
authors.forename: Xiaoqiang
authors.initials: X
authors.lastname: Ma
authors.forename: Shengli
authors.initials: S
authors.lastname: Yang
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D003605:D-Amino-Acid Oxidase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004718:Saccharomycetales / Q000201:enzymology* / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D003605:D-Amino-Acid Oxidase