LAUSR

A Pantoea agglomerans-derived phenylalanine aminomutase (PaPAM) was engineered to improve the biocatalytic synthesis of (S)-β-phenylalanine, which is an important precursor of pharmaceuticals and peptidomimetics. A semi-rational design strategy based on a combination of surface-amino-acid engineering and the amino acid preference of the thermozyme was applied to counteract the enzyme trade-off between improving its activity and stability. The surface glycine, lysine and serine of PaPAM were mutated to alanine, arginine and alanine, respectively. A K340R mutant was screened with a 2.23-fold increased activity and 2.12-fold improved half-life at 50 °C over those of the wild-type PaPAM. These improvements resulted from the more stable enzymatic conformation as well as the more rigid inner loop in K340R. When tested in a whole-cell biocatalytic reaction, the (S)-β-phenylalanine volumetric productivity of K340R reached 0.47 g/L·h (1.4-fold greater than that of the wild-type PaPAM), and the conversion rate was improved by 17% compared to that of the wild-type PaPAM. The enzymatic properties of K340R and the resulting (S)-β-phenylalanine production are among the highest reported, and the results indicate that the described strategy is potent for engineering enzymatic stability and activity of PAM.