Multiskan go uv spectrometer

Enzyme activity was determined by monitoring the change in absorbance at 340 nm at 30 °C using a MultiSkan GO UV-spectrometer (Thermo Fisher Scientific), which corresponds to the change in the concentration of NADH (Li et al. 2014 (link)). For reductive amination, the reaction mixture (200 μL) contained 20 mM substrate, 0.2 mM NADH, 2 M NH₄Cl/NH₄OH buffer (pH 9.0) and a certain amount of purified enzyme. Enzyme activity unit (U) was defined as the amount of enzyme which catalyzes the production (or consumption) of 1 µmol of NADH per min under the above conditions.

Enzyme activity was measured at 340 nm and at 30 °C using a MultiSkan GO UV-spectrometer (Thermo Fisher Scientific). The standard assay mixture for the oxidative deamination reaction contained 5 mM α-amino acids, 0.1 M Tris-HCl buffer (pH 8.5), and 0.2 mM NAD⁺. The reductive amination reaction was carried out in 1 M NH₄Cl/NH₄OH buffer (pH 9.5) with 5 mM α-keto acids and 0.2 mM NADH. Both reactions were performed in a final volume of 200 μL and initiated by the addition of limiting amounts of enzyme. One unit of enzyme activity (1 U) was defined as the amount of enzyme which catalyzes the production (or consumption) of 1 μmol of NADH per min under the standard assay conditions. Three sets of parallel experiments were performed for each experimental condition. All determinations were performed in triplicate, and error bounds represent ± sd.

Reductive amination activity was determined at a 200-μL scale in 96-well microtiter plates at 30 °C. The rate of initial decrease of the absorbance was measured at 340 nm (indicating NADH consumption) using a MultiSkan GO UV-spectrometer (Thermo Fisher Scientific). The reaction mixture consisted of NH₄Cl/NH₄OH buffer (2 M; pH 9.5), 10 mM 2-OPBA, 0.2 mM NADH, and a certain amount of cell-free extract. One unit of enzyme activity (1 U) was defined as the amount of enzyme oxidizing 1 μM NADH per minute. Kinetic parameters with respect to 2-OPBA (PPA) were evaluated in NH₄Cl/NH₄OH buffer (2 M; pH 9.5) at 30 °C in the presence of 1–20 mM 2-OPBA (PPA) and 0.5 mM NADH with the purified enzymes. Kinetic parameters with respect to NADH were evaluated in NH₄Cl/NH₄OH buffer (2 M; pH 9.5) at 30 °C in the presence of 0.1–0.5 mM NADH and 20 mM 2-OPBA with the purified enzymes (Additional file 1: Table S6). K_m and V_max were calculated in GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA) by nonlinear curve fitting of the initial rate versus substrate concentration data to the Michaelis–Menten equation.