title: High activity expression of D-amino acid oxidase in Escherichia coli by the protein expression rate optimization.
abstract: Two categories of expression systems with different promoters and substitutive ribosome binding site region (RBS) were constructed in order to improve the soluble expression of d-amino acid oxidase (DAAO) basing on the hypothesis that the optimal promoter and suitable RBS would provide the recombinant expression system with better matched expression rate, which served as key factor to the heterogenous synthesis of soluble protein. The results showed that with rational promoter recombination and delicate RBS substitution, significant increase of DAAO activity (20-fold) was obtained for strain JM105/pGEMKT-Tac-R-DAAO over the previously constructed strain BL21(DE3)/pET-DAAO. Furthermore, similar optimization strategy proved feasible in the active expression of other enzymes such as glutaryl-7-aminocephalosporanic acid acylase (GCA) and N-Carbamyl-D-amino acid amidohydrolase (D-Case).
authors.affiliation: Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, PR China.
authors.forename: Jixian
authors.initials: J
authors.lastname: Hou
authors.forename: Yangqiu
authors.initials: Y
authors.lastname: Liu
authors.forename: Qiang
authors.initials: Q
authors.lastname: Li
authors.forename: Jichu
authors.initials: J
authors.lastname: Yang
mesh_terms: D003605:D-Amino-Acid Oxidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D003605:D-Amino-Acid Oxidase