Deng Senwen, Su Erzheng, Ma Xiaoqiang, Yang Shengli, Wei Dongzhi
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.
Bioprocess Biosyst Eng. 2014 Aug;37(8):1517-26. doi: 10.1007/s00449-013-1123-z. Epub 2014 Jan 16.
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.
D-氨基酸氧化酶是一种在多个领域中使用的重要生物催化剂，但其在大肠杆菌中经济合理水平的重组表达尚未实现。为实现这一目标，在进行单个Phe54Tyr替换后，构建了来自可变三角酵母（TvDAO）的D-氨基酸氧化酶与6×His标签的融合蛋白，并在大肠杆菌中进行表达。揭示了His标签融合位置的影响。全酶百分比和蛋白质溶解度的显著增加使N端标记的TvDAO（称为NHDAO）成为TvDAO生产的合适选择。然而，携带NHDAO的菌株也观察到细胞生长和蛋白质生产率降低。为了优化NHDAO的生产性能，测试了培养基的变化。最终，在3.7 L发酵罐中28.5小时内实现了140 U/mL的产量或每升3.48 g活性酶，占总蛋白的41.4%，粗提物的比活性为16.68 U/mg。这表明在大肠杆菌中功能性和经济性地表达TvDAO以满足工业需求是有可能的。
