Isotope enrichment and dilution assay: deuterated and non-deuterated L-Alanine product formation

Deuterated and non-deuterated L-Alanine were synthesized in batch incubating scaffolded AlaDH3 and FDH1 at 1:1 molar ratio at 0.18 µM protein concentration of each in 1 mL of the reaction mixture composed by 75 mM pyruvate, 75 mM deuterated sodium formate, 25 mM sodium formate, 500 mM ammonium chloride, 0.5 mM NADH, 4.4 µM NOX, and 0.15 mM FAD⁺ in nano pure water. Reactions were maintained under orbital agitation at 500 rpm and 25 °C for 1, 2, 4, 8, and 24 h. At different time points samples were withdrawn and analyzed by HPLC-MS to quantify the isotopic abundance of the produced L-Alanine-d-2. Reactions were stopped by tangential filtration at 13100 rcf using an Amicon Ultra-0.5 centrifugal filter units. The reaction samples at different points were diluted and then derivatized by Dansyl method⁶² prior analysis by UPLC-MS (Ultra-Performance Liquid Chromatography-Mass spectrometer). UPLC was performed in a Waters ACQUITY UPLC system with a Acquity BEH C18 column (100 × 2,1 mm/1,7 µm). The gradient elution solvents were A (100 mM Ammonium formate in Water) and B (Acetonitrile) at a flow rate of 300 µL·min⁻¹, with the following gradient 80% A, up to 1% A for 28 min and back to 80% A for the remaining two minutes for a total duration of 30 min. The conversion degree was determined for deuterated or non-deuterated L-Alanine calculating the abundance of both hydrogen isotopes in the analyzed systems by mass spectrometry detection carried out using a time-of-flight mass spectrometer (ESI-TOF) LCT Premier XE from Waters (Milford, MA, USA) with an electrospray ionization source, working in positive/W mode. The MS range acquired was between m/z 50–1000. The capillary and cone voltages were set at 3000 and 50 V, respectively. Desolvation gas temperature was 300 °C and source temperature was 120 °C. The desolvation gas flow was set at 600 L·h^–1 and cone gas flow was set at 50 L·h^–1. For quantification and data analysis, Masslynx v4.1 software was used (Waters, Milford, MA, USA). Dansyl derivatized L-Alanine detected with a mass of 323 g·mol⁻¹ and derivatized deuterated L-Alanine detected with a mass of 324 g·mol⁻¹.