Effects of heat treatment on the structure, digestive property, and absorptivity of holoferritin

Semirational Design Based on Consensus Sequences to Balance the Enzyme Activity-Stability Trade-Off

“…The supernatant is the crude enzyme solution, which was then purified using gravity flow through an equilibrated 1 mL nickel column (BioRad, CA). The recombinant CuZnSOD was eluted using elution buffer (0.5 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) as previously described , and desalted with a 5 mL HiTrap desalting column (GE Healthcare, Sweden). The enriched enzyme was purified through AKTA chromatography (GE, Uppsala, Sweden), and the sample was loaded into a 24 mL Superdex 200 Increase 10/300 GL gel column and eluted with buffer (20 mM Tris-HCl, pH 7.9, 150 mM NaCl).…”

“…The single-positive clone containing the CuZnSOD gene was selected and inoculated into 5 mL LB medium containing 50 μg/mL kanamycin, and then, the clone was cultured overnight with a rotation speed of 220 rpm at 37 °C. 21 The culture solution was then inoculated at a 1:100 ratio into 500 mL of LB medium and incubated at 220 rpm until the OD 600 reached 0.7, and IPTG at a final concentration of 1 mmol was added to the medium and cultured for 4 h. The cells were resuspended in buffer (10 mM Tris-HCl, 100 mM PMSF, pH 7.9) after the expressed cells were centrifuged at 5000g for 15 min. The cell solution was crushed with an ultrasonic cell disruptor (SCIENTZ, Ningbo, China), then centrifuged at 10,000g for 10 min at 4 °C.…”