Construction and application of fusion proteins of D-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase for direct bioconversion of cephalosporin C to 7-aminocephalosporanic acid.
Two fusion proteins of D-amino acid oxidase (DAAO) and glutaryl-7-aminocephalosporanic acid acylase (GLA) were designed to simplify the bioconversion process of cephalosporin C to 7-aminocephalosporanic acid (7-ACA), which is conventionally produced in a two-step enzymatic process. Two recombinant plasmids, pET-DLA and pET-ALD, were constructed to express fusion proteins of DAAO-linker-GLA (DLA) and GLA-linker-DAAO (ALD), respectively. When the recombinant plasmids were expressed in E. coli, the fusion protein DLA was not correctly folded and only DAAO activity could be detected. ALD, however, possessed activities of both DAAO and GLA, which directly catalyze the conversion of cephalosporin C into 7-ACA.
Department of Chemical Engineering, Institute of Biochemical Engineering, Tsinghua University, Beijing 100084, PR China.
Hui
H
Luo
Qiang
Q
Li
Huimin
H
Yu
Zhongyao
Z
Shen
D002511:Cephalosporins / Q000096:biosynthesis* / Q000378:metabolism*; D003605:D-Amino-Acid Oxidase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D010399:Penicillin Amidase / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D002511:Cephalosporins; D011993:Recombinant Fusion Proteins; C025163:cephalosporin C; C030735:7-aminocephalosporanic acid; D003605:D-Amino-Acid Oxidase; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase