Biochemistry and biotechnology of mesophilic and thermophilic nitrile metabolizing enzymes

Abstract

Mesophilic nitrile-degrading enzymes are widely dispersed in the Bacteria and lower orders of the eukaryotic kingdom. Two distinct enzyme systems, a nitrilase catalyzing the direct conversion of nitriles to carboxylic acids and separate but cotranscribed nitrile hydratase and amidase activities, are now well known. Nitrile hydratases are metalloenzymes, incorporating FeIII or CoII ions in thiolate ligand networks where 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 specific and lacking stereoselectivity, whereas the nitrilases are enantioselective and aromatic substrate specific. The recent discovery of novel enzymes in both classes (including thermophilic representatives) has blurred these functional distinctions. Purified mesophilic nitrile-degrading 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 notwithstanding, these enzymes have been successfully applied for more than a decade to the kiloton production of acrylamide and more recently to 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 regioselectivity of the enzyme coupled with the ability to achieve high conversion efficiencies without detriment to other sensitive functionalities is a distinct process advantage.

Biology of Pseudomonas stutzeri

“…As a result, there is considerable interest in these enzymes as industrial catalysts. Therefore, uses include the production of nicotinic acid, R-(Ϫ)-mandelic acid, and S-(ϩ)-ibuprofen and the detoxification of cyanide waste (78).…”

Optimization of nitrilase production from a new thermophilic isolate

“…So interest in enzymes from thermophilic organisms has been greatly stimulated by the fact that these proteins are resistant to chemical and physical denaturation and to proteolysis. 15,16 The recent expansion of nitrile-dependent biotransformation and enzymatic detoxification of nitrile-based herbicides and the instability of mesophilic nitrile metabolizing enzymes have led to the investigation of thermophilic bacteria as an alternative source of these activities. 17,18 We have isolated a gram positive, thin, long filamentous rod shaped thermophilic nitrilase-producing bacterium from the desert region of Rajasthan.…”

Production of Acrylamide using Alginate‐Immobilized E. coli Expressing Comamonas testosteroni 5‐MGAM‐4D Nitrile Hydratase

“…[1a, 2] Wild-type cells with NHase activity such as Rhodococcus sp. N-774, [3] Pseudomonas chlororaphis B23, [4] and Rhodococcus rhodochrous J1 [5] have each been used commercially to convert acrylonitrile to acrylamide.…”