Improvement of the thermostability and activity of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by engineering C-terminal amino acids

In the current study, a three-tiered mutagenesis strategy was employed to simultaneously improve the thermostability and activity of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) by engineering the last ten amino acids (Met245 similar to Glu254) of its C-terminal region. Initially, truncated mutagenesis results displayed that C-terminal deletions decreased the thermostability and/or activity of HheC. Then ten residues were subjected to single-site saturation mutagenesis, resulting in 20 beneficial single-point variants related to the thermostability or activity of HheC. The results clearly indicated that residues Met252 similar to G1u254 and Trp249 are crucial for regulating enzyme thermostability and activity, respectively. Finally, the beneficial substitutions were combined using efficient multi-site combinatorial mutagenesis approaches, leading to an outstanding variant PX14 (Trp249Pro/Met252Leu/Pro253Asp), which had a 17.8-fold higher half-life and a 4.0-fold higher k(cat) value than that of wild-type HheC. These results indicated that the C-terminal residues play an important role in modulating both the thermostability and activity of HheC. (C) 2015 Elsevier B.V. All rights reserved.