title: A novel D-amino acid oxidase from a contaminated agricultural soil metagenome and its characterization.
abstract: A novel D-amino acid oxidase (DAAO) gene designated as daoE was cloned by the sequence-based screening of a plasmid metagenomic library of uncultured microorganisms from contaminated agricultural soil. The deduced amino acid sequence comparison and phylogenetic analysis indicated that daoE and other putative DAAOs are closely related. The putative DAAO gene was subcloned into a pETBlue-2 vector and overexpressed in Escherichia coli Tunner(DE3)pLacI. The recombinant protein was purified to homogeneity. The maximum activity of DaoE protein occurred at pH 8.0 and 37 °C. DaoE recombinant protein had an apparent K m of 2.96 mM, V max of 0.018 mM/min, k cat of 10.9/min, and k cat/K m of 1.16 × 10(4)/mol/min. The identification of this novel DAAO gene demonstrated the importance of metagenomic libraries in exploring new D-amino acid oxidases from environmental microorganisms to optimize their applications.
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, College of Life Science and Technology, Guangxi University, 100 Daxue East Road, Nanning, 530004, Guangxi, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Ou
authors.forename: Yao
authors.initials: Y
authors.lastname: Liu
authors.forename: Jie
authors.initials: J
authors.lastname: Deng
authors.forename: Gao
authors.initials: G
authors.lastname: Chen
authors.forename: Ying
authors.initials: Y
authors.lastname: Yang
authors.forename: Peihong
authors.initials: P
authors.lastname: Shen
authors.forename: Bo
authors.initials: B
authors.lastname: Wu
authors.forename: Chengjian
authors.initials: C
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003605:D-Amino-Acid Oxidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D054892:Metagenome; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D003605:D-Amino-Acid Oxidase