E. coli BL21(DE3) cells harbouring recombinant plasmid encoding the appropriate ACCO variant were grown overnight at 37 ℃ and 230 rpm in 4 mL LB media supplemented with 0.05 mg/mL kanamycin (LBkan). Preculture (1.5 mL, 5% v/v) was used to inoculate 28.5 mL TB media supplemented with 0.05 mg/mL kanamycin (TBkan) in a 125 mL Erlenmeyer flask. The culture was incubated at 37 °C and 230 rpm for 2 h to reach an OD600 of approximately 1.5. The culture was then cooled on ice for 20 min and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, final concentrations). Protein expression was performed at 20 ℃ and 200 rpm for 20 h. The cells were then transferred to a conical tube (50 mL) and harvested by centrifugation (4000 rpm, 4 min, 4 °C) using an Eppendorf 5910R tabletop centrifuge.