Identification and characterization of a thermostable and cobalt-dependent amidase from Burkholderia phytofirmans ZJB-15079 for efficient synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid.

Enantiomerically pure 3,3,3-trifluoro-2-hydroxy-2-methylpropionic acids are important chiral building blocks for a series of pharmaceuticals. Here, a bacteria strain with 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide-degrading ability was screened and identified as Burkholderia phytofirmans ZJB-15079, from which a novel amidase (Bp-Ami) was cloned and demonstrated to be capable of kinetic resolution of rac-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide to optically pure (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid. Phylogenetic analysis revealed that Bp-Ami was closely located to the acetamidase/formamidase (FmdA_AmdA) family, and it shared high homology with acetamidases. Bp-Ami was found to be the first cobalt-dependent FmdA_AmdA family amidase. The enzyme activity was significantly increased by 37.7-fold in the presence of 1 mM Co2+, with a specific activity of 753.5 U/mg, K m value of 24.73 mM, and k cat /K m value of 22.47 mM-1 s-1. As an enzyme from mesophile, Bp-Ami exhibited extreme thermostability with a half-life of 47.93 h at 80 °C, which was even superior to other reported amidases from thermophiles. The whole cell catalysis of 200 g/L 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide by Escherichia coli harboring Bp-Ami (5 g/L) resulted in 44 % yield and an enantiomeric excess (ee p) of 95 % within 10 min (E = 86). The high substrate tolerance, high specific activity, and extreme thermostability demonstrated the great potential of Bp-Ami for efficient biocatalytic synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid.