Total turnover number prediction of an aggregating biocatalyst: Amino ester hydrolase (AEH)

Abstract

Amino ester hydrolase (AEH) from Xanthomonas campestris is a promising candidate for β-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.