4.8 Tmm enzyme assay
Cell Lysis: The cells were grown in LB media at 16 °C and induced with 0.5 mM IPTG when the OD reached 0.6. The cells were harvested after overnight growth, resuspended into the lysis buffer, and left for 45 minutes under continuous stirring at 4 °C for homogenisation. The composition of the lysis buffer was 50 mM Tris-HCl buffer (pH 7.5), 250 mM NaCl, 10% Glycerol, 0.1% Triton X-100, and one mM PMSF. After incubation, the cells were sonicated. The cell lysate was centrifuged at 12000 rpm for 1 hour to separate the cell debris.
The Tmm assay mixture contains 1mM trimethylamine, 0.1 mM NADPH, 0.025 mM FMN, 20 µl of crude enzyme in 25 mM tris, and 50 mM NaCl. This assay mixture was incubated for 15 min at 28 °C, and trimethylamine was added to initiate the reaction. Tmm activity was assayed in three ways: the amount of NADPH consumed at 340 nm, trimethylamine consumed, and TMAO product formed in the reaction mixture.
The amount of NADPH consumed was measured at 340 nm (Dixit & Roche, 1984), the amount of trimethylamine consumed was measured by the protocol as reported by Pena-Pereira, Lavilla, & Bendicho, 2010, and the amount of TMAO formed was measured by the method reported in this manuscript.