Gene Cloning, Expression, and Characterization of a Nitrilase from Alcaligenes faecalis ZJUTB10

Nitrilases are important industrial enzymes that convert nitriles directly into the corresponding carboxylic acids In the current work, the fragment with a length of 1068 bp that encodes the A faecalis ZJUTB10 nitrilase was obtained Moreover, a catalytic triad was proposed and verified by site-directed mutagenesis, and the detailed mechanism of nitrilase was clarified The substrate specificity study demonstrated that the A. faecal's ZJUTB10 nitrilase belongs to the family of arylacetonitrilases The optimum pH and temperature for the purified nitrilase was 7-8 and 40 degrees C, respectively Mg2+ stimulated hydrolytic activity, whereas Cu2+, Co2+, Ni2+, Ag+, and Hg2+ showed a strong inhibitory effect The K-m and nu(max) for mandelonitrile were 474 mM and 15 85 mu mol min(-1) mg(-1) protein, respectively After 30 mm reaction using the nitrilase, mandelonitrile at the concentration of 20 mM was completely hydrolyzed and the enantiomeric excess against (R)-()-mandelic acid was >99% Characteristics investigation indicates that this nitrilase is promising in catalysis applications