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 27oC. 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 34oC (7oC improvement) and 1.3-fold activity compared to wild-type.
Next, we compare different techniques of determining the thermal and process stability of enzymes, using beta-lactam hydrolases. We are able to show that, given a kinetic model of enzyme deactivation, T5030 data can be converted into observed deactivation rate constants kd,obs and thus into half-lives t1/2.
Lastly, PGA from E. coli displays insufficient enantioselectivity towards (R)-phenylglycine derivatives in the synthesis of penicillins. Via site-saturation of selected residues of PGA, we markedly enhanced enantioselectivity.