Liu Yangqiu, Li Qiang, Zhu Hongyu, Yang Jichu
Department of Chemical Engineering, Institute of Biochemical Engineering, Tsinghua University, Beijing 100084, People's Republic of China.
Appl Biochem Biotechnol. 2009 Aug;158(2):313-22. doi: 10.1007/s12010-008-8325-x. Epub 2008 Aug 12.
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.
为表达高活性可溶性D-氨基酸氧化酶（DAAO），构建了一种由天然海因酶启动子（P(Hase)）调控的组成型质粒。将D-氨基酸氧化酶基因（dao）与P(Hase)连接，并克隆到pGEMKT中，以在不使用任何对细胞和环境有毒的诱导剂（如异丙基-β-D-1-硫代半乳糖苷）的情况下组成型表达DAAO蛋白。对核糖体结合位点区域、宿主菌株和发酵条件进行了优化，以提高表达水平。当在5立方米发酵罐中培养时，发现JM105/pGEMKT-R-DAAO在37℃下生长时的酶活性为32 U/mL，比在28℃下生长的BL21(DE3)/pET-DAAO细胞增加了16倍。这些结果表明我们在大肠杆菌中以可溶性形式过量生产DAAO的方法是成功的。
