ONPG

ONPG is a colorimetric and spectrophotometric substrate for detection of β-galactosidase activity. In Vitro: The enzyme displays high hydrolysis ability for ONPG (100%) and moderate activity for its natural substrate lactose (25.7%). However, the hydrolysis ability of the enzyme towards all other chromogenic nitrophenyl analogues is very weak, indicating that Gal308 is a β-galactosidase with narrow substrate specificity. To investigate the kinetic parameters of recombinant enzyme, the Michaelis-Menten constants (K_m), turnover numbers (k_cat), and catalytic efficiencies (k_cat/K_m) of Gal308 for ONPG and lactose are determined. The k_cat and K_m values are 464.7±7.8 s^-1 and 2.7±0.3 mM for ONPG, and 264.2±2.1 s^-1 and 7.1±0.8 mM for lactose, respectively. The k_cat/K_m value of the enzyme for ONPG (172.1 s^-1mM^-1) is 4.6-fold higher than that for lactose (37.2 s^-1mM^-1), which clearly demonstrated that the catalytic efficiency of Gal308 for ONPG is much higher than that for lactose^[1].