BIOCHEMISTRY AND BIOTECHNOLOGY OF MESOPHILIC AND THERMOPHILIC NITRILEMETABOLIZING ENZYMES

Mesophilic nitrile-degrading enzymes are widely dispersed in the Bacte ria and lower orders of the eukaryotic kingdom. Two distinct enzyme sy stems, a nitrilase catalyzing the direct conversion of nitriles to car boxylic acids and separate but cotranscribed nitrile hydratase and ami dase activities, are now well known. Nitrile hydratases are metalloenz ymes, incorporating Fe-III or Co-II ions in thiolate ligand networks w here they function as Lewis acids. In comparison, nitrilases are thiol -enzymes and the two enzyme groups have little or no apparent sequence or structural homology. The hydratases typically exist as alpha beta dimers or tetramers in which the alpha- and beta-subunits are similar in size but otherwise unrelated. Nitrilases however, are usually found as homomultimers with as many as 16 subunits. Until recently, the two nitrile-degrading enzyme classes were clearly separated by functional differences, the nitrile hydratases being aliphatic substrate specifi c and lacking stereoselectivity, whereas the nitrilases are enantiosel ective and aromatic substrate specific. The recent discovery of novel enzymes in both classes (including thermophilic representatives) has b lurred these functional distinctions. Purified mesophilic nitrile-degr ading enzymes are typically thermolabile in buffered solution, rarely withstanding exposure to temperatures above 50 degrees C without rapid inactivation. However, operational thermostability is often increased by addition of aliphatic acids or by use of immobilized whole cells. Low molecular stability has frequently been cited as a reason for the limited industrial application of ''nitrilases''; such statements notw ithstanding, these enzymes have been successfully applied for more tha n a decade to the kiloton production of acrylamide and more recently t o the smaller-scale production of nicotinic acid, R-(-)-mandelic acid and S-(+)-ibuprofen. There is also a rapidly growing catalog of other potentially useful conversions of complex nitriles in which the regios electivity of the enzyme coupled with the ability to achieve high conv ersion efficiencies without detriment to other sensitive functionaliti es is a distinct process advantage.