Soluble expression of Thermomicrobium roseum sarcosine oxidase and characterization of N-demethylation activity

Abstract

Sarcosine oxidase (SOX) gene from Thermomicrobium roseum DSM 5159 (GeneBank: ACM06094.1) was inserted into a pMA5 plasmid, expressed in Bacillus subtilis WB600 in soluble form, and then was purified. The purified TrSOX revealed a single band at approximately 43 kDa on 10% reducing SDS-PAGE gel, and was further confirmed by linear trap quadropole mass spectrometry (LTQ-MS) analysis. Regarding CD analysis, the secondary structure of TrSOX was composed of 32.7% α-helix, 17.8% β-sheet, 20.6% β-turn, and 28.5% random coil. Nano-DSC analysis showed that the melting temperature (Tm) and the denaturation enthalpy (ΔH) of TrSOX were 100 ℃ and 552.8 kJ/mol, which indicated that the enzyme should be with outstanding thermo stability. Using N-methylglycine (sarcosine) as substrate, the N-methyl group was depleted and hydrogen peroxide was produced, the Km, kcat and kcat/Km of TrSOX were 61.2 ± 4.3 mmol/L, 3.2 ± 0.2 min−1 and 52.2 L·mol−1 min−1, respectively; additionally, TrSOX showed well adaptation and stability in aqueous/solvent biphasic mixture. Using N-methyltryptophan as substrate, the Km, kcat and kcat/Km of TrSOX were 136.5 ± 7.8 mmol/L, 0.27 ± 0.02 min−1 and 1.98 L·mol−1 min−1, respectively. Additionally, TrSOX showed catalytic activity against other N-methyl or N-ethyl compounds; and for most of the N-methyl-L substrates, the enzyme revealed high chiral selectivity (>100).