Purification and Characterization of Benzonitrilases from Arthrobacter sp. Strain J-1

We found two kinds of benzonitrilases, designated benzonitrilases A and B, in a cell extract of Arthrobacter sp. strain J-1 grown on benzonitrile as a sole carbon and nitrogen source. Benzonitrilases A and B were purified approximately 409-fold and 38-fold, respectively. Purified benzonitrilase A appeared to be homogeneous according to the criteria of polyacrylamide gel electrophoresis. Both the enzymes hydrolyzed benzonitrile to benzoic acid and ammonia without forming benzamide as an intermediate. The molecular weights of benzonitrilases A and B were found to be 30,000 and 23,000, respectively. The subunit molecular weight of benzonitrilase A was the same as its molecular weight. The isoelectric points of benzonitrilases A and B were 4.95 and 4.80, respectively. The optimum temperature and pH, respectively, for benzonitrilase A were 40 degrees C and 8.5, and those for benzonitrilase B were 30 degrees C and 7.5. The K(m) values for benzonitrilases A and B were 6.7 mM and 4.5 mM, respectively. Both the enzymes degraded p-tolunitrile, 4-cyanopyridine, and p-chlorobenzonitrile, but they did not attack aliphatic nitriles or amides. Both the enzymes were inhibited by thiol reagents.