Lagerman_2023_Chem.Eng.Sci_277_118804

Amino ester hydrolase (AEH) from Xanthomonas campestris is a promising candidate for beta-lactam synthesis but suffers from low thermostability and rapid deactivation. Wild-type AEH was found to deactivate with an apparent order that varies with temperature from n = 2 at 30 C (close to the temperature of optimum activity Topt) to n = 1.5 at 25 C. AEH features two transitional temperatures: (1) from native N to partially unfolded, inactive intermediate I, and (2) from partially unfolded to fully unfolded entity U at the melting temperature Tm. CD and light scattering data suggest aggregation near Topt. To determine the total turnover number TTN, AEH was deactivated by imposing a temperature gradient and recording instantaneous rates of cephalexin hydrolysis. The TTN increased ~ 5-fold from the wild-type (WT) for the quadruple variant N186D/A275P/E143H/V622I (QVH) at 25 C and 10 nM AEH but varied significantly with AEH concentration and temperature.