Expansion of the substrate specificity of an amino acid dehydrogenase to that of an amine dehydrogenase was achieved through several rounds of focused mutagenesis. Application of degenerate codons and a high-throughput screening assay allowed for simplified, rapid evaluation of enzyme variants. Novel activity was achieved toward a number of compounds while maintaining the enzyme’s native enantioselectivity.
Alpha-amino ester hydrolases (E.C. 3.1.1.43) catalyze the synthesis and hydrolysis of α-amino β-lactam antibiotics. To improve AEH thermostability, a modified structure-guided consensus model of seven homologous enzymes was generated along with analysis of the B-values from the available crystal structures. In the third round, independent NNK saturation of two high B-factor sites, K34 and E143, on a consensus-driven triple variant resulted in our best (a quadruple) variant, with a “T-50” value of 34oC (7oC improvement) and 1.3-fold activity compared to wild-type.
Currently, Pen G Acylase only exhibits weak diastereoselectivity with respect to the alpha amino group of rac-phenylglycine methyl ester (rac-PGME) when it is coupled with 6-aminopenicillanic acid to synthesize ampicillin. Four variants with improved selectivity for (R)-ampicillin synthesis were identified, all resulting from a mutation at the beta-24 position.[6] The diastereomeric excess (d.e.R) value of 37% for the wild-type enzyme was improved to a d.e.R value of 98% for our most selective mutant, betaPhe24Ala.