4.7. Kinetics assays

To determine the kinetic parameters for the transamination reaction, the purified proteins (0.1–0.2 μM) were incubated in presence of 100 mM PLP in KP 0.1 M pH 7.4 at 25°C at increasing alanine concentrations (15–500 mM) or glyoxylate concentrations (0.07–5 mM) and a fixed glyoxylate (10 mM) or alanine (500 mM) concentration. The reaction was quenched by adding TCA 10% (v/v), and pyruvate production was measured using a spectrophotometric assay coupled with LDH following the NADH signal at 340 nm. ¹⁰ To determine the activity in crude bacterial cell lysates, a Tecan Spark Microplate Reader (Thermo Fischer Scientific) was used and the cells were grown and lysed as described below under the section “Library Screening.”

To measure the enzymatic activity in HEK293 cellular lysates, 100 μg of lysate were incubated with 0.5 M l‐alanine and 10 mM glyoxylate at 25°C for 20–60 min in 100 mM KP buffer, pH 7.4 in presence of 100 μM PLP. The reactions were stopped by adding TCA 10% (v/v) and pyruvate production was measured using a spectrophotometric assay coupled with lactate dehydrogenase. ⁷²