Abstract
A predicted sarcosine oxidase (SOX) gene from Thermomicrobium roseum DSM 5159 was expressed, reconstructed, and characterized. Regarding sequence alignment, T. roseum sarcosine oxidase (TrSOX) showed ∼35% similarity with several bacterial SOXs and ∼25% similarity with MTOX from E. coli. Regarding structural modeling and molecular docking, TrSOX interacted with natural flavin cofactors and flavin analogues including flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in the cofactor binding site. The TrSOX gene was built on a plasmid containing a L-rhamnose-inducible promoter and expressed in E. coli BL21 (DE3) as inclusion body. In addition, a soluble mutant (F235 V/F339 L) was identified in a random mutagenesis screening experiment. The inclusion body associated with the native gene were reconstructed with natural or modified ligands. Circular dichroism detection revealed the stable secondary structure of the enzyme, which was unaffected by the ligands. Furthermore, regarding reconstructed TrSOX with a ligand modified at the 7- or 8-position by a halogen atom, the Km, kcat, relative specificity, optimal reaction temperature, and pH adaptability were significantly different compared with refolded TrSOX-FAD, and flavin analogues affected the catalytic properties of this enzyme.
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