[Fusion expression of D-amino acid oxidase from Trignoposis variabilis with maltose binding protein and Vitreoscilla hemoglobin].
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.
Yu
Huimin
H
Department of Chemical Engineering, Tsinghua University, Beijing 100084, China. yuhm@tsinghua.edu.cn
Ma
Xianfeng
X
Luo
Hui
H
Wen
Cheng
C
Shen
Zhongyao
Z
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D003605:D-Amino-Acid Oxidase / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D054793:Truncated Hemoglobins / Q000096:biosynthesis* / Q000235:genetics; D015003:Yeasts / Q000201:enzymology / Q000235:genetics
D001426:Bacterial Proteins; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D054793:Truncated Hemoglobins; C082613:hemoglobin protein, Vitreoscilla; D003605:D-Amino-Acid Oxidase