Nitrile-metabolizing potential of<i>Bacillus</i>cereus strain FA12; Nitrilase production, purification, and characterization

Abstract

Nitrile-hydrolyzing bacteria have the potential to perform useful biotransformations such as the production of industrially useful acids and amides. In this study, we report a nitrile-degrading bacterium with significant nitrile metabolism. Molecular characterization of 16S rDNA gene characterized this strain as Bacillus cereus. Medium optimization of B. cereus FA12 showed that biomass and nitrilase production was strongly supported by glucose (10 gL− 1) and yeast extract (10 gL− 1). Enzymatic production improved slightly in the pH range from 6.0 to 7.0. The addition of Mg+2, Fe+2, and Na+ supported biomass and nitrilase production; however, other metal ions, Co+2 and Cu+2, inhibited production. The apparent molecular mass of the purified FA12 nitrilase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was about 45 kDa. Nitrilase FA12 shows relatively high activity and stability at pH 7.0 and 40°C. Nitrilase FA12 was marginally inhibited with Ca+ 2 and Co+2, whereas inhibition in the presence of dithiothreitol or DTT was 80%. The pseudo Km (mM) values of resting cells (i.e., treating whole cells as if they were an enzyme) for acetonitrile and acetamide were determined to be 2.36 and 1.81, respectively. Under optimum situations, B. cereus FA12 resting cells produced 83 and 58 (U/mg) acetonitrile/acetamide degrading activity, respectively. Ammonia production from acetamide and acetonitrile by the B. cereus FA12 was maximum after 5 and 7 h of incubation, respectively. These results indicate that B. cereus FA12 resting cells may be used in nitrile biotransformations to produce commercially useful compounds.