Overexpression and purification of AncLAAOs

Five genes encoding each of the AncLAAOs were synthesized and cloned into the pET28a vector via the NcoI and XhoI sites by ordering GeneScript. The plasmids were transformed into the BL21(DE3) strain. The strain was cultivated in 1 l of LB broth at 37 °C. The temperature was decreased to 20 °C when the OD₆₀₀ reached 0.5–0.8, and then IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.5 mM. After the cells were grown for approximately 15 h, the cells were collected by centrifugation. The cells were suspended into buffer A (20 mM Tris-HCl [8.0] and 10 mM NaCl). After sonication of the cells, the supernatant was collected by centrifugation at 11,000 g for 40 min. The supernatant was applied to Ni²⁺-sepharose 6 fast flow column (GE Healthcare, Uppsala, Sweden), and the column was washed with 30 mL of buffer A. The samples were eluted utilizing 15 mL of buffer A containing 10, 40, 70, 100, and 300 mM imidazole. The samples containing AncLAAOs (mainly fractions containing 70 and 100 mM imidazole) were utilized for further purification. The eluted samples were concentrated and applied to the Superdex 200 pg column (GE Healthcare, Uppsala, Sweden) equilibrated by buffer A. Purity of AncLAAO was confirmed by SDS-PAGE (Supplementary Fig. 3a). Protein concentration of the purified samples was estimated by measuring the absorption change at 280 nm by UV-Vis spectrometer. The purified AncLAAO was utilized in the following biochemical assay. FAD contents of AncLAAO-N4 and the variants were estimated utilizing the following equation.

Here, the denominator value was calculated by dividing molar absorption coefficient value at 280 nm of AncLAAO-N4 (Ɛ₂₈₀ = 109,000 M/cm), by the value at 450 nm of FAD (Ɛ₄₅₀ = 11,300 M/cm). Abs (280 nm) and Abs (450 nm) were represented as UV-Vis absorption value at 280 and 450 nm region of each samples, respectively.