Improved Thermostability of AEH by Combining B-FIT Analysis and Structure-Guided Consensus Method

alpha-amino ester hydrolases (AEHs) catalyze the synthesis and hydrolysis of α-amino β-lactam antibiotics. The AEH enzymes have been shown to feature excellent synthetic capability but suffer from poor thermostability.  AEH from Xanthomonas campestris exhibits an optimal temperature of 25^°C, observed half-life of 5 minutes at 30^°C and a T₅₀³⁰, the temperature at which the half-life is 30 minute, of 27^oC.  To improve the thermostability of the AEH, a modified structure-guided consensus model of seven homologous enzymes was generated along with analysis of the B-values from the available crystal structures of AEH from Xanthomonas citri.  A family of stabilized variants was created including a consensus-driven triple variant, A275P/N186D/V622I. In the third round, independent NNK saturation of two high B-factor sites, K34 and E143, on the triple variant resulted in our best variant, the quadruple mutant E143H/A275P/N186D/V622I, with a T₅₀³⁰ value of 34^oC (7^oC improvement) and 1.3-fold activity compared to wild-type.