Yu Huimin, Ma Xianfeng, Luo Hui, Wen Cheng, Shen Zhongyao
Department of Chemical Engineering, Tsinghua University, Beijing 100084, China.
Sheng Wu Gong Cheng Xue Bao. 2008 Jun;24(6):1004-9.
D-amino acid oxidase (DAAO) is one of important industrial enzymes. To increase the solubility and activity of the TvDAAO from Trignoposis variabilis expressed in recombinant Escherichia coli (E. coli), a maltose binding protein (MBP) and Vitreoscilla hemoglobin (VHb) was introduced to fuse with N-terminal of the TvDAAO, respectively. Fusion protein of MBP-TvDAAO was constitutively expressed in JM105/pMKC-DAAO and inductively expressed in JM105/pMKL-DAAO. With respect to the control strain of BL21 (DE3)/pET-DAAO without MBP fusion, the constitutive fusion expression obtained 28% of soluble protein with 3.7 folds of solubility improvement. As for the inductive fusion expression, corresponding results changed to 17% and 1.8 folds, respectively. However, the DAAO activity significantly decreased in the MBP-fusing expression. Fusion protein of VHb-TvDAAO was constructed and inductively expressed in BL21 (DE3)/pET-VDAAO. Its DAAO activity highly reached 3.24 u/mL in flask culture, about 90% increase in contrast to the control without VHb.
D-氨基酸氧化酶（DAAO）是一种重要的工业酶。为了提高在重组大肠杆菌（E. coli）中表达的来自可变三角酵母（Trignoposis variabilis）的TvDAAO的溶解度和活性，分别引入麦芽糖结合蛋白（MBP）和透明颤菌血红蛋白（VHb）与TvDAAO的N端融合。MBP-TvDAAO融合蛋白在JM105/pMKC-DAAO中组成型表达，在JM105/pMKL-DAAO中诱导表达。相对于没有MBP融合的BL21（DE3）/pET-DAAO对照菌株，组成型融合表达获得了28%的可溶性蛋白，溶解度提高了3.7倍。至于诱导融合表达，相应的结果分别变为17%和1.8倍。然而，在MBP融合表达中DAAO活性显著降低。构建了VHb-TvDAAO融合蛋白并在BL21（DE3）/pET-VDAAO中诱导表达。其DAAO活性在摇瓶培养中高达3.24 u/mL，与没有VHb的对照相比增加了约90%。
