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 2.96mM, V max of 0.018mM/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.
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